In Vitro and in Vivo Examination of the Cell Surface Glycoprotein CDCP1

In vitro and in vivo examination of the cell surface glycoprotein CDCP1

Andreas Wortmann

Bachelor of Engineering

Institute of Health and Biomedical Innovation and Discipline of Cell and Molecular Biosciences, Faculty of Science and Technology, Queensland University of Technology

A thesis submitted for the degree of Doctor of Philosophy of Queensland University of Technology

‐ 2010 ‐

KEYWORDS

Cancer, chicken embryo experimental metastasis assay, CUB domain- containing protein 1, epithelial to mesenchymal transition, focal adhesion kinase, HeLa cells, metastasis formation, protein kinase C δ, Src family kinase, tyrosine phosphorylation sites, SU6656

ABSTRACT

A number of reports have demonstrated the importance of the CUB domain- containing protein 1 (CDCP1) in facilitating cancer progression in animal models and the potential of this protein as a prognostic marker in several malignancies. CDCP1 facilitates metastasis formation in animal models by negatively regulating anoikis, a type of apoptosis triggered by the loss of attachment signalling from cell-cell contacts or cell-extra cellular matrix (ECM) contacts. Due to the important role CDCP1 plays in cancer progression in model systems, it is considered a potential drug target to prevent the metastatic spread of cancers. CDCP1 is a highly glycosylated 836 amino acid cell surface protein. It has structural features potentially facilitating protein-protein interactions including 14 N-glycosylation sites, three CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five intracellular tyrosine residues.

CDCP1 interacts with a variety of proteins including Src family kinases (SFKs) and protein kinase C δ (PKCδ). Efforts to understand the mechanisms regulating these interactions have largely focussed on three CDCP1 tyrosine residues Y734, Y743 and Y762. CDCP1-Y734 is the site where SFKs phosphorylate and bind to CDCP1 and mediate subsequent phosphorylation of CDCP1-Y743 and -Y762 which leads to binding of PKCδ at CDCP1-Y762. The resulting trimeric protein complex of SFK•CDCP1•PKCδ has been proposed to mediate an anti-apoptotic cell phenotype in vitro, and to promote metastasis in vivo. The effect of mutation of the three tyrosines on interactions of CDCP1 with SFKs and PKCδ and the consequences on cell phenotype in vitro and in vivo have not been examined.

CDCP1 has a predicted molecular weight of ~90 kDa but is usually detected as a protein which migrates at ~135 kDa by Western blot analysis due to its high degree of glycosylation. A low molecular weight form of CDCP1 (LMW- CDCP1) of ~70 kDa has been found in a variety of cancer cell lines. The mechanisms leading to the generation of LMW-CDCP1 in vivo are not well

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Abstract

understood but an involvement of proteases in this process has been proposed. Serine proteases including plasmin and trypsin are able to proteolytically process CDCP1. In addition, the recombinant protease domain of the serine protease matriptase is also able to cleave the recombinant extracellular portion of CDCP1. Whether matriptase is able to proteolytically process CDCP1 on the cell surface has not been examined. Importantly, proteolytic processing of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained portion which suggests that this event leads to initiation of an intracellular signalling cascade.

This project aimed to further examine the biology of CDCP1 with a main of focus on exploring the roles played by CDCP1 tyrosine residues. To achieve this HeLa cells stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F and Y762F were generated. These cell lines were used to examine:

• The roles of the tyrosine residues Y734, Y743 and Y762 in mediating interactions of CDCP1 with binding proteins and to examine the effect of the stable expression on HeLa cell morphology. • The ability of the serine protease matriptase to proteolytically process cell surface CDCP1 and to examine the consequences of this event on HeLa cell phenotype and cell signalling in vitro. • The importance of these residues in processes associated with cancer progression in vitro including adhesion, proliferation and migration. • The role of these residues on metastatic phenotype in vivo and the ability of a function-blocking anti-CDCP1 antibody to inhibit metastasis in the chicken embryo chorioallantoic membrane (CAM) assay.

Interestingly, biochemical experiments carried out in this study revealed that mutation of certain CDCP1 tyrosine residues impacts on interactions of this protein with binding proteins. For example, binding of SFKs as well as PKCδ

Abstract

to CDCP1 was markedly decreased in HeLa-CDCP1-Y734F cells, and binding of PKCδ was also reduced in HeLa-CDCP1-Y762F cells. In contrast, HeLa-CDCP1-Y743F cells did not display altered interactions with CDCP1 binding proteins. Importantly, observed differences in interactions of CDCP1 with binding partners impacted on basal phosphorylation of CDCP1. It was found that HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F displayed strong basal levels of CDCP1 phosphorylation. In contrast, HeLa-CDCP1-Y734F cells did not display CDCP1 phosphorylation but exhibited constitutive phosphorylation of focal adhesion kinase (FAK) at tyrosine 861. Significantly, subsequent investigations to examine this observation suggested that CDCP1-Y734 and FAK-Y861 are competitive substrates for SFK-mediated phosphorylation. It appeared that SFK-mediated phosphorylation of CDCP1- Y734 and FAK-Y861 is an equilibrium which shifts depending on the level of CDCP1 expression in HeLa cells. This suggests that the level of CDCP1 expression may act as a regulatory mechanism allowing cells to switch from a FAK-Y861 mediated pathway to a CDCP1-Y734 mediated pathway. This is the first time that a link between SFKs, CDCP1 and FAK has been demonstrated.

One of the most interesting observations from this work was that CDCP1 altered HeLa cell morphology causing an elongated and fibroblastic-like appearance. Importantly, this morphological change depended on CDCP1- Y734. In addition, it was observed that this change in cell morphology was accompanied by increased phosphorylation of SFK-Y416. This suggests that interactions of SFKs with CDCP1-Y734 increases SFK activity since SFK- Y416 is critical in regulating kinase activity of these proteins. The essential role of SFKs in mediating CDCP1-induced HeLa cell morphological changes was demonstrated using the SFK-selective inhibitor SU6656. This inhibitor caused reversion of HeLa-CDCP1 cell morphology to an epithelial appearance characteristic of HeLa-vector cells.

Significantly, in vitro studies revealed that certain CDCP1-mediated cell phenotypes are mediated by cellular pathways dependent on CDCP1 tyrosine residues whereas others are independent of these sites. For

Abstract

example, CDCP1 expression caused a marked increase in HeLa cell motility that was independent of CDCP1 tyrosine residues. In contrast, CDCP1- induced decrease in HeLa cell proliferation was most prominent in HeLa- CDCP1-Y762F cells, potentially indicating a role for this site in regulating proliferation in HeLa cells. Another cellular event which was identified to require phosphorylation of a particular CDCP1 tyrosine residue is adhesion to fibronectin. It was observed that the CDCP1-mediated strong decrease in adhesion to fibronectin is mostly restored in HeLa-CDCP1-Y743F cells. This suggests a possible role for CDCP1-Y743 in causing a CDCP1-mediated decrease in adhesion.

Data from in vivo experiments indicated that HeLa-CDCP1-Y734F cells are more metastic than HeLa-CDCP1 cells in vivo. This indicates that interaction of CDCP1 with SFKs and PKCδ may not be required for CDCP1-mediated metastasis formation of HeLa cells in vivo. The metastatic phenotype of these cells may be caused by signalling involving FAK since HeLa-CDCP1- Y734F cells are the only CDCP1 expressing cells displaying constitutive phosphorylation of FAK-Y861. HeLa-CDCP1-Y762F cells displayed a very low metastatic ability which suggests that this CDCP1 tyrosine residue is important in mediating a pro-metastatic phenotype in HeLa cells. More detailed exploration of cellular events occurring downstream of CDCP1-Y734 and -Y762 may provide important insights into the mechanisms altering the metastatic ability of CDCP1 expressing HeLa cells.

Complementing the in vivo studies, anti-CDCP1 antibodies were employed to assess whether these antibodies are able to inhibit metastasis of CDCP1 and CDCP1 tyrosine mutants expressing HeLa cells. It was found that HeLa- CDCP1-Y734F cells were the only cell line which was markedly reduced in the ability to metastasise. In contrast, the ability of HeLa-CDCP1, HeLa- CDCP1-Y743F and -Y762F cells to metastasise in vivo was not inhibited. These data suggest a possible role of interactions of CDCP1 with SFKs, occurring at CDCP1-Y734, in preventing an anti-metastatic effect of anti- CDCP1 antibodies in vivo. The proposal that SFKs may play a role in regulating anti-metastatic effects of anti-CDCP1 antibodies was supported by

Abstract

another experiment where differences between HeLa-CDCP1 cells and CDCP1 expressing HeLa cells (HeLa-CDCP1-S) from collaborators at the Scripps Research Institute were examined. It was found that HeLa-CDCP1-S cells express different SFKs than CDCP1 expressing HeLa cells generated for this study. This is important since HeLa-CDCP1-S cells can be inhibited in their metastatic ability using anti-CDCP1 antibodies in vivo. Importantly, these data suggest that further examinations of the roles of SFKs in facilitating anti-metastatic effects of anti-CDCP1 antibodies may give insights into how CDCP1 can be blocked to prevent metastasis in vivo.

This project also explored the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 because it is unknown whether matriptase can cleave cell surface CDCP1 as it has been reported for other proteases such as trypsin and plasmin. Furthermore, the consequences of matriptase-mediated proteolysis on cell phenotype in vitro and cell signalling were examined since recent reports suggested that proteolysis of CDCP1 leads to its phosphorylation and may initiate cell signalling and consequently alter cell phenotype.

It was found that matriptase is able to proteolytically process cell surface CDCP1 at low nanomolar concentrations which suggests that cleavage of CDCP1 by matriptase may facilitate the generation of LWM-CDCP1 in vivo.

To examine whether matriptase-mediated proteolysis induced cell signalling anti-phospho Erk 1/2 Western blot analysis was performed as this pathway has previously been examined to study signalling in response to proteolytic processing of cell surface proteins. It was found that matriptase-mediated proteolysis in CDCP1 expressing HeLa cells initiated intracellular signalling via Erk 1/2. Interestingly, this increase in phosphorylation of Erk 1/2 was also observed in HeLa-vector cells. This suggested that initiation of cell signalling via Erk 1/2 phosphorylation as a result of matriptase-mediated proteolysis occurs by pathways independent of CDCP1. Subsequent investigations measuring the flux of free calcium ions and by using a protease-activated receptor 2 (PAR2) agonist peptide confirmed this hypothesis. These data suggested that matriptase-mediated proteolysis results in cell signalling via a

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Abstract

pathway induced by the activation of PAR2 rather than by CDCP1. This indicates that induction of cell signalling in HeLa cells as a consequence of matriptase-mediated proteolysis occurs via signalling pathways which do not involve phosphorylation of Erk 1/2. Consequently, it appears that future attempts should focus on the examination of cellular pathways other than Erk 1/2 to elucidate cell signalling initiated by matriptase-mediated proteolytic processing of CDCP1.

The data presented in this thesis has explored in vitro and in vivo aspects of the biology of CDCP1. The observations summarised above will permit the design of future studies to more precisely determine the role of CDCP1 and its binding partners in processes relevant to cancer progression. This may contribute to further defining CDCP1 as a target for cancer treatment.

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TABLE OF CONTENTS

Keywords ...... i

Abstract ...... iii

Table of contents ...... ix

List of figures ...... xvii

List of tables ...... xxi

List of abbreviations ...... xxiii

List of publications ...... xxvii

Statement of originality ...... xxix

Acknowledgements ...... xxxiii

CHAPTER 1: Introduction...... 1

1.1 Introduction to CDCP1 ...... 2

1.2 Incidence and hallmarks of cancer ...... 3

1.3 Metastasis formation is a complex multistep process ...... 3

1.4 Identification of CDCP1 and its association with cancer ...... 5

1.5 CDCP1 structural features ...... 6

1.6 CDCP1 interacting proteins ...... 9

1.6.1 Src family kinases (SFKs) ...... 12

1.6.2 Protein kinase C δ (PKCδ) ...... 14

1.7 CDCP1 is poteolytically processed by proteases ...... 15

1.8 Phosphorylation of CDCP1 ...... 17

1.9 CDCP1 expression is dysregulated in human tumours ...... 18

1.10 CDCP1 in leukemia and haematopoetic and progenitor cells ...... 20

1.11 CDCP1 supports cancer progression in model systems ...... 20

1.12 Opportunities and challenges in targeting CDCP1 in cancer ...... 22

1.13 Summary and aims ...... 25

CHAPTER 2: Material and Methods ...... 29

2.1 Reagents ...... 30

2.1.2 Antibodies ...... 30

2.1.3 Vectors ...... 32

2.1.4 Oligonucleotides ...... 32

2.1.5 Enzymes and kits ...... 33

2.1.6 Cell culture reagents and materials ...... 33

2.1.7 Cell lines ...... 33

2.1.8 Buffers ...... 34

2.1.9 Media ...... 35

2.2 Methods ...... 35

2.2.1 General cell culture ...... 35

2.2.2 Cell counting ...... 36

2.2.3 Plasmid DNA isolation, purification and storage ...... 37

2.2.4 Agarose gel electrophoresis ...... 37

2.2.5 Generation of the CDCP1-FLAG expression construct in vector pcDNA 3.1 ...... 37

2.2.6 Transfection of HeLa cells with CDCP1 expression constructs 38

2.2.7 Selection of transfected cells in selection media ...... 40

2.2.8 Generation of monoclonal HeLa cells stably expressing CDCP1, CDCP1-Y734F, -Y743F or -Y762F by isolating single cell clones manually from a culture dish ...... 40

2.2.9 Flow cytometric analysis of CDCP1 cell surface expression levels ...... 40

2.2.10 Generation of monoclonal HeLa cells stably expressing CDCP1, CDCP1-Y734F, -Y743F or -Y762F by fluorescence microscopy...... 40

2.2.11 Generation of polyclonal HeLa cells stably expressing CDCP1, CDCP1-Y734F, -Y743F or -Y762F by fluorescent activated cell sorting (FACS) ...... 41

2.2.12 Generation of monoclonal HeLa cells stably expressing CDCP1-FLAG, CDCP1-Y734F, -Y743F or -Y762F by FACS ...... 42

2.2.13 Generation of whole cell lysates ...... 43

2.2.14 Determination of protein concentration ...... 43

2.2.15 Immunoprecipitation ...... 43

2.2.16 Western blot analysis ...... 44

2.2.17 Isolation of genomic DNA for sequencing ...... 45

2.2.18 PCR amplification of the CDCP1 gene from HeLa-CDCP1-S cells ...... 46

2.2.19 Determination of DNA concentration of purified DNA ...... 47

2.2.20 Sequencing of CDCP1 DNA amplified from genomic DNA ... 47

2.2.21 Adhesion assay using CyQuant NF or crystal violet ...... 48

2.2.22 Chemotactic migration ...... 49

2.2.23 Haptotactic migration ...... 49

2.2.24 Proliferation assay ...... 50

2.2.25 Cell line authentication testing ...... 51

2.2.26 Chicken embryo chorioallantoic membrane (CAM) cell dissemination assay ...... 51

2.2.27 Inhibition of metastasis formation in the chicken embryo CAM assay using anti-CDCP1 antibodies ...... 52

2.2.28 Genomic DNA isolation and real-time Alu PCR for quantitative detection of human tumour cells in the CAM ..... 52

2.2.29 Incubation of cells with the SFK-selective inhibitor SU6656 .. 53

2.2.30 Incubation of cells with SU6656 to examine the effect on the morphology of HeLa cells stably expressing CDCP1 ...... 53

2.2.31 Matriptase treatment of cells ...... 54

2.2.32 Measurement of changes in intracellular Ca2+ ...... 54

2.2.33 Confocal microscopy ...... 55

2.2.34 Cell surface biotinylation ...... 55

2.2.35 Data collection and statistical analysis of in vitro and in vivo experiments ...... 55

CHAPTER 3: Characterisation of CDCP1 expressing HeLa cells ...... 59

3.1 Introduction ...... 60

3.2 Results ...... 62

3.2.1 Generation of HeLa cells stably expressing CDCP1 using four approaches ...... 62

3.2.2 HeLa cells stably expressing CDCP1 display altered cell morphology which requires CDCP1-Y734 ...... 68

3.2.3 Confirmation of CDCP1 expression level and changes on cell morphology in combined HeLa clones ...... 72

3.2.4 Examination of cellular localisation of CDCP1 and co- localisation of CDCP1 with SFKs in CDCP1 expressing HeLa cells ...... 75

3.2.5 Examination of the impact of tyrosine mutations on CDCP1 binding to SFKs and PKCδ ...... 77

3.2.6 CDCP1 is basally tyrosine phosphorylated and phosphorylated on Y734 in stable expressing HeLa cells ...... 78

3.2.7 Binding of SFKs to CDCP1-Y734 reduces phosphorylation of FAK-Y861 depending on the level CDCP1 expression ...... 80

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3.2.8 The SFK-selective inhibitor SU6656 causes distinct effects downstream of SFK inhibition in CDCP1 expressing HeLa cells and reverts the cell morphology caused by CDCP1 ...... 86

3.3 Discussion ...... 90

3.3.1 Generation of HeLa cells stably expressing CDCP1 and CDCP1 tyrosine mutants ...... 90

3.3.2 Some mutations of CDCP1 alter interactions with CDCP1 binding proteins ...... 92

3.3.3 CDCP1 expression causes an EMT-like change in HeLa cell morphology which requires CDCP1-Y734 ...... 93

3.3.4 CDCP1 and FAK are competitive substrates for SFK- mediated phosphorylation ...... 96

CHAPTER 4: Proteolysis of CDCP1 by matriptase ...... 99

4.1 Introduction ...... 100

4.2 Results ...... 104

4.2.1 Matriptase is an efficient proteolytic processor of cell surface CDCP1 and is the most efficient processor amongst three serine proteases ...... 104

4.2.2 Matriptase incubation does not cause cell deadhesion ...... 107

4.2.3 Matriptase-mediated proteolytic processing of CDCP1 causes internalisation of its cell surface-retained portion ...... 107

4.2.4 Cleavage of CDCP1 does not lead to a change in the cellular phenotype of HeLa-CDCP1 cells in vitro ...... 109

4.2.5 Cell signalling via Erk 1/2 activation is not initiated by matriptase-mediated proteolysis of CDCP1 ...... 112

4.3 Discussion ...... 118

4.3.1 Matriptase is an very efficient proteolytic processor of cell surface CDCP1 ...... 119

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4.3.2 Matriptase-mediated proteolysis does not alter the phenotype of CDCP1 expressing HeLa cells and intracellular signalling is not via CDCP1 but via PAR2 ...... 120

CHAPTER 5: Examination of the CDCP1-mediated in vitro and in vivo phenotype of HeLa cells ...... 125

5.1 Introduction ...... 126

5.2 Results ...... 130

5.2.1 CDCP1 expression decreases proliferation and adhesion of HeLa cells ...... 130

5.2.2 CDCDP1 expression increases HeLa cell migration ...... 132

5.2.3 HeLa-CDCP1-Y734F cells are most metastatic in the CAM assay and metastasis formation can be inhibited by an anti- CDCP1 antibody ...... 134

5.2.4 In vivo experiments revealed differences between CDCP1 expressing HeLa cells generated in this project and those from Deryugina et al...... 137

5.2.5 CDCP1 expression reduces proliferation of HeLa-CDCP1-S cells ...... 139

5.2.6 HeLa-CDCP1 and HeLa-CDCP1-S cells are of HeLa cell origin ...... 140

5.2.7 HeLa-CDCP1 and HeLa-CDCP1-S cells express comparable levels of CDCP1 but a C-terminal anti-CDCP1 antibody detects CDCP1 expressed by HeLa-CDCP1-S cells ...... 142

5.2.8 HeLa-CDCP1 and HeLa-CDCP1-S cells exhibit basal phosphorylation of CDCP1-Y734 but expresses different SFKs ...... 148

5.3 Discussion ...... 151

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5.3.1 Certain CDCP1-mediated cell phenotypes in vitro depend on CDCP1 tyrosine residues ...... 151

5.3.2 HeLa-CDCP1 and HeLa-CDCP1-S cells may have been originated from different HeLa cell sublines which may explain observed differences between these two cell lines in vivo ...... 153

5.3.3 Mutations of certain CDCP1 tyrosine residues impact on CDCP1-mediated cell phenotypes in vivo ...... 156

5.3.4 Differential expression of SFKs in HeLa-CDCP1 and HeLa- CDCP1-S cells suggests a possible role for SFKs in mediating an anti-metastatic effect of anti-CDCP1 antibodies in vivo ...... 157

CHAPTER 6: Final conclusions and future directions ...... 161

6.1 Introduction ...... 162

6.2 Findings and discussion ...... 162

6.2.1 The role of CDCP1 and CDCP1 tyrosine mutants in cellular processes in vitro and in vivo ...... 162

6.2.2 The role of proteolytic processing of CDCP1 in cancer progression ...... 168

6.2.3 Verification of findings in other cell lines and other model systems ...... 169

6.2.4 Interaction of CDCP1 with SFKs and possible implications on inhibition of metastasis formation in vivo ...... 172

6.2.5 Focus on CDCP1 in physiology ...... 174

APPENDIX: Larger sized photograps presented in Chapter 3 ...... 174

Appendix 1: Larger sized versions of photograps presented in Figure 3.6 ...... 174

Appendix 2: Larger sized versions of photograps presented in Figure 3.8 ...... 177

Appendix 3: Larger sized versions of photograps presented in Figure 3.9 ...... 180

BIBLIOGRAPHY ...... 189

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LIST OF FIGURES

Figure 1.1: Schematic representation of CDCP1 ...... 7 Figure 1.2: CDCP1 sequence alignment with other species ...... 8 Figure 1.3: Graphical illustration of CDCP1 protein interactions ...... 11 Figure 1.4: Schematic representation of the structure of SFKs ...... 13 Figure 1.5: Speculative overview of CDCP1 in premalignant and cancer cells...... 24

Figure 2.1: Vector map of the pcDNA 3.1 vector ...... 32 Figure 2.2: CDCP1 DNA sequence and illustration of site directed mutagenesis to create CDCP1 tyrosine mutants...... 39 Figure 2.3: Multiple cloning site of the pcDNA 3.1 vector and illustration of the binding site of the BGH-rev primer ...... 47

Figure 3.1: Examination of CDCP1 expression in human cell lines (one of four panels of this figure was generated by Dr Yaowu He). .. 63 Figure 3.2: Selection of HeLa cells stably transfected with the CDCP1 expression construct by fluorescence microscopy yields cell populations with mixed CDCP1 expression (the CDCP1 constructs were generated by Dr MayLa Linn)...... 65 Figure 3.3: A polyclonal population of HeLa cells expressing CDCP1- Y743F lose cell surface expression over time...... 67 Figure 3.4: HeLa cell clones stably expressing CDCP1 display a high and uniform level of CDCP1 expression...... 69 Figure 3.5: Expression analysis of HeLa-CDCP1 clones...... 70 Figure 3.6: Expression of CDCP1 leads to a change in cell morphology in stable expressing HeLa cell clones ...... 71 Figure 3.7: Analysis of CDCP1 expression of combined HeLa cell populations...... 73 Figure 3.8: CDCP1 expression leads to a change in cell morphology in stably expressing HeLa cells which requires CDCP1-Y734 ...... 74

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Figure 3.9: Localisation of CDCP1 and co-localisation of CDCP1 with SFKs in stable CDCP1 expressing HeLa cells ...... 76 Figure 3.10: Examination of interactions between CDCP1, SFKs and PKCδ in stable CDCP1 expressing HeLa cells ...... 78 Figure 3.11: Examination of CDCP1 phosphorylation in stable CDCP1 expressing HeLa cells...... 79 Figure 3.12: Expression of CDCP1 is accompanied by a loss of phosphorylation of FAK-Y861...... 82 Figure 3.13: SFK-mediated phosphorylation of CDCP1-Y734 reduces phosphorylation of FAK-Y861 depending on the level of CDCP1 expression ...... 83 Figure 3.14: Working model: CDCP1 and FAK compete for SFK- mediated phosphorylation in CDCP1 expressing HeLa cells. .. 84 Figure 3.15: CDCP1 does not bind to FAK...... 85 Figure 3.16: Inhibition of SFKs with SU6656 reduces phosphorylation of CDCP1-Y734 in HeLa-CDCP1, HeLa-CDCP1-Y743F and - Y762F cells and phosphorylation of FAK-Y861 in HeLa- vector and HeLa-CDCP1-Y734F cells ...... 87 Figure 3.17: The change in cell morphology caused by CDCP1 expression can be reverted by inhibition of SFKs with SU6656...... 89

Figure 4.1: Matriptase, trypsin and KLK4 cleave CDCP1 to ~70 kDa...... 104 Figure 4.2: Matriptase processes CDCP1 in low nanomolar concentrations to a ~70 kDa product within 60 minutes...... 105 Figure 4.3: Matriptase is an efficient processor of CDCP1 stably expressed by HeLa-CDCP1 cells and endogenously expressing 22Rv1 cells...... 106 Figure 4.4: Matriptase incubation does not cause cell deadhesion...... 107 Figure 4.5: CDCP1 cleavage by matriptase leads to internalisation of the ~70 kDa cell retained portion of CDCP1...... 108 Figure 4.6: CDCP1 cleavage by matriptase does not cause a change in proliferation in CDCP1 expressing HeLa cells...... 110

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Figure 4.7: CDCP1 cleavage by matriptase does not cause change in chemotactic migration in CDCP1 expressing HeLa cells...... 111 Figure 4.8: CDCP1 cleavage by matriptase does not cause a change in adhesion to ECM proteins in CDCP1 expressing HeLa cells .. 112 Figure 4.9: Phosphorylation of Erk-1/2 due to matriptase treatment is not mediated by CDCP1...... 114 Figure 4.10: Calcium flux induced by matriptase is not mediated by CDCP1 but by PAR2 (these experiments were performed with Mark Adams)...... 116 Figure 4.11: Examination of matriptase expression in prostate cancer and prostate epithelial derived cell lines endogenously expressing CDCP1...... 117

Figure 5.1: CDCP1 expression leads to a decrease in proliferation of HeLa cells...... 131 Figure 5.2: CDCP1 expression leads to a decrease in cell adhesion of HeLa cells ...... 132 Figure 5.3: CDCP1 increases migration of HeLa cells towards FCS ...... 133 Figure 5.4: CDCP1 expression reduces dissemination of HeLa cells in the chicken embryo CAM assay (these experiments were performed under guidance from Dr Elena Deryugina)...... 135 Figure 5.5: Examination of the effect of function blocking anti-CDCP1 antibodies on dissemination of HeLa cells stably expressing CDCP1 (these experiments were performed under guidance from Dr Elena Deryugina)...... 136 Figure 5.6: CDCP1 expression reduces proliferation in HeLa-CDCP1 and HeLa-CDCP1-S cells...... 140 Figure 5.7: HeLa-CDCP1 and HeLa-CDCP1-S cells have similar levels of CDCP1 expression...... 143

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Figure 5.8: The anti-CDCP1 antibodies 41-2 and 10D7 are not effected by the FLAG tag encoded at the carboxy terminus of the CDCP1 expression construct or by the amino acid at position 827 (the constructs used in panel B were generated by Dr Yaowu He)...... 145 Figure 5.9: The HeLa-CDCP1-S expression construct has a serine at amino acid position 827 and does not encode a carboxy terminal FLAG tag ...... 147 Figure 5.10: CDCP1 is basally phosphorylated in HeLa-CDCP1 and HeLa-CDCP1-S cells ...... 148 Figure 5.11: HeLa-CDCP1 and HeLa-CDCP1-S cells express different SFKs...... 149 Figure 5.12: Examination of change in expression of CDCP1 in HeLa- CDCP1 and HeLa-CDCP-S cells in response to treatment with the anti-CDCP1 antibody 41-2 ...... 150

Figure 6.1: Consequences of CDCP1 expression on HeLa cell phenotype in vitro and in vivo are mediated by phosphorylation dependent and independent pathways...... 163

LIST OF TABLES

Table 2.1: Specifications of anti-CDCP1 antibodies ...... 31 Table 2.2: Oligonucleotide primers ...... 32

Table 3.1: Summary of four approaches used and the number of clones screened to obtain HeLa cells stably expressing CDCP1 ...... 64

Table 5.1: Examination of the origin of HeLa cells used to generate HeLa- CDCP1 and HeLa-CDCP1-S stable cell lines ...... 142 Table 5.2: Comparison of the STR profiles of HeLa cell sublines and HeLa- CDCP1 and HeLa-CDCP1-S ...... 155

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LIST OF ABBREVIATIONS

AP agonist peptide Bp base pairs BCA bicinchoninic acid assay BSA bovine serum albumin CEA carcinoembryonic antigen CDCP1 CUB domain-containing protein 1 DAPI 4, 6-diamidino-2-phenylindoledihydrochloride hydrate ddH2O double distilled water DMEM Dulbecco’s modified eagles medium DMSO dimethylsulfoxide DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate ECM extracellular matrix EDTA ethylene diamine tetra acetate EGF epidermal growth factor EMT epithelial to mesenchymal transition EpCAM epithelial cell adhesion molecule ERK extracellular regulated kinase FAK focal adhesion kinase FCS fetal calf serum g gram h hour HCl hydrochloric acid HGF hepatocyte growth factor ICAM intercellular adhesion molecule kb kilobase pair(s) kDa kilo Dalton

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KLK kallikrein L litre µg microgram µL microliter µM micromole per litre M mole per litre mL milliliter mM millimole per litre NaCl sodium chloride NaOH sodium hydroxide NCAM neural cell adhesion molecule ng nanogram nM nanomole per litre OD optical density PAR2 protease-activated receptor 2 PBS phosphate-buffered saline PCR polymerase chain reaction PSA prostate specific antigen RPMI-media Roswell Park Memorial Institute media RT room temperature SDS sodium dodecyl sulphate SDS-PAGE sodium dedecyl sulphate-polyacrylamide gel electrophoresis SEM standard error of the mean SFKs Src family kinases SH Src homology TAE tris-acetate EDTA TBS-T tris buffered saline supplemented with 0.1% Tween-20 (v/v) TES tris-EDTA saline TGF transforming growth factor

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Tris tris(hydroxymethyl)aminomethane UV ultraviolet V volts VCAM vascular cell adhesion molecule (v/v) volume per volume (w/v) weight per volume x g G-force

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LIST OF PUBLICATIONS

Publications arising from this PhD program of study.

Publication 1:

Authors (in order of authorship): Andreas Wortmann, Yaowu He, Elena I. Deryugina, James P. Quigley and John D. Hooper

Title: The cell surface glycoprotein CDCP1 in cancer-insights, opportunities, and challenges (Review article).

Journal: IUBMB Life 2009, 61, 723-730.

This review forms the basis of Chapter 1.

Publication 2:

Authors (in order of authorship): Elena I. Deryugina, Erin M. Conn, Andreas Wortmann, Juneth J. Partridge, Tatyana A. Kupriyanova, Veronica C. Ardi, John D. Hooper and James P. Quigley

Title: Functional role of cell surface CUB domain-containing protein 1 in tumour cell dissemination.

Journal: Molecular Cancer Research 2009, 7, 1197-1211.

Reagents from Chapter 3 were used in this publication.

Publication 3:

Authors (in order of authorship): Yaowu He, Andreas Wortmann, Les J. Burke, Janet C. Reid, Mark N. Adams, Ibtissam Abdul-Jabbar, James P. Quigley, Richard Leduc, Daniel Kirchhofer and John D. Hooper

Title: Proteolysis induced amino terminal ectodomain shedding of the integral membrane glycoprotein CUB domain-containing protein 1 (CDCP1) is accompanied by tyrosine phosphorylation of its carboxy terminal domain and recruitment of Src and PKCδ.

Journal: Journal of Biological Chemistry 2010, 285, 26162-26173.

Data from Chapter 4 are reported in this publication.

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STATEMENT OF ORIGINALITY

The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made.

Signed: Andreas Wortmann, BEng

Date: 31/01/2011

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………….. dedicated to Simon Luigart -Lui-. Thank you so much for your genuine friendship and for your support throughout my life.

Shall your beautiful nature, your admirable creations and our wonderful times never be forgotten…………….

ACKNOWLEDGEMENTS

This work was supported by a PhD Scholarship from the Cancer & Bowel Research Trust and travel support granted by the Boehringer Ingelheim Fonds and the Cancer Council of Queensland.

First of all I would like to thank my supervisory team, my principal supervisor Assoc Prof John D. Hooper and my associate supervisor Prof Adrian Herington. I sincerely thank Assoc Prof Hooper for his kind and supportive way and for taking time to discuss not only scientific issues. In countless hours he guided me throughout the PhD journey and helped me understand what it takes to successfully complete my PhD. His motivation, enthusiasm and joy for science were driving forces throughout my PhD. I would like to thank Prof Adrian Herington for constructive suggestions, very helpful feedback and for his time throughout my PhD process.

I would like to thank Dr Elena Deryugina and Prof James Quigley for giving me the opportunity to undertake a research internship in their laboratory at the Scripps Research Institute. Special thanks to Dr Deryugina for her excellent guidance and for enthusiastically sharing her scientific knowledge with me. This was an unforgettable lifetime experience.

Thanks to everyone in the Hooper Group, the Hormone Dependant Cancer Program and the wider community of the Institute of Health and Biomedical Innovation, who created a very pleasant working environment at all times.

I am especially indebted to Aura Quinto, who gave me her love, patience, support and encouragement during my PhD journey and for understanding that I could not have much leisure time during this busy period.

Finally, I can not express in words the most grateful appreciation for the love and the support my family gave me throughout my life and for giving me the opportunity and the freedom to make my dreams come true.

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Chapter 1

Introduction

Chapter 1

1.1 CDCP1

Over the last few years increasing evidence has indicated that CUB domain- containing protein 1 (CDCP1) (Scherl-Mostageer et al., 2001) plays an important role in facilitating metastasis in in vivo models (Uekita et al., 2007; Siva et al., 2008b; Uekita et al., 2008; Deryugina et al., 2009) and alters cell phenotype relevant to cancer progression in vitro (Uekita et al., 2007; Uekita et al., 2008; Deryugina et al., 2009; Miyazawa et al., 2010). In addition, CDCP1 has emerged as a potential diagnostic marker in several human malignancies including lung, kidney, pancreatic and endometrioid cancer (Awakura et al., 2008; Ikeda et al., 2009; Mamat et al., 2010; Miyazawa et al., 2010). Consequently, CDCP1 has been suggested as a potential therapeutic target for cancer treatment (Uekita et al., 2007; Wortmann et al., 2009; Fukuchi et al., 2010). CDCP1 is a cell surface glycoprotein also known as subtractive immunisation associated 135 kDa (SIMA135) (Hooper et al., 2003), gp140 (Brown et al., 2004), and transmembrane and associated with Src kinases (Trask) (Bhatt et al., 2005) and has been assigned the cluster of differentiation (CD) designation CD318.

This thesis examines a number of aspects of the biology of CDCP1 including the effect of stable expression of this protein on the phenotype of cervical cancer derived HeLa cells in vitro and in vivo . In addition, it examines the effect of mutation of CDCP1 tyrosine residues in altering interactions of CDCP1 with binding partners. Furthermore, proteolytic processing of CDCP1 on the cell surface mediated by the serine protease matriptase and the consequences of proteolysis on cell phenotype in vitro and signalling were investigated.

This introductory chapter first summarises briefly general aspects of cancer biology relevant to CDCP1 and describes in detail what is currently known about CDCP1 with a main focus on this protein in cancer progression. In addition, it presents a speculative overview on how CDCP1 could be blocked to prevent the CDCP1-mediated pro-metastatic phenotype in vivo . It then concludes with a brief summary and describes the aims of this project.

Chapter 1

1.2 Incidence and hallmarks of cancer

Cancer related death contributed to 32.8% of all deaths in Australia in 2008 (ABS, 2010) and represents the largest contributor to our disease burden (AIHW, 2010) . In the USA, when deaths are aggregated by age, cancer surpasses heart disease as the leading cause of death for those younger than age 85 (Jemal et al., 2010). Consequently, cancer is one of the biggest socio-economic problems in western civilisations.

The development and progression of cancer is a longterm, multistep process requiring the acquisition of a set of functional capabilities including growth self sufficiency, vascularisation, evasion of immune responses and apoptosis, and metastasis associated processes including invasion and migration, intravasation, transport via circulatory systems, extravasation and establishment at a secondary site (Hanahan and Weinberg, 2000) . The molecular changes driving the acquisition of these capabilities are caused by genetic alterations which result in dysregulated protein expression and changes in cell signalling pathways (Weinberg, 1989) .

One hallmark of malignant tumours is their ability to spread via the circulatory systems (vascular and lymphatic) throughout the body thereby forming metastases. Significantly, formation of metastases is the biggest burden in cancer disease management and the majority of cancer related mortality is due to the formation of metastases at secondary sites, while only about 10% of primary tumours prove to be fatal to affected people (Mehlen and Puisieux, 2006). For patients diagnosed with organ confined cancer which is amenable to surgical removal the survival rates are high, but the same is not true for patients having disseminated cancer. For these patients prognosis is poorer and decreases significantly the later diagnosis occurs (Ott et al., 2009).

1.3 Metastasis formation is a complex multistep process

Metastatic dissemination of tumour cells is a complex process involving a multitude of steps and requires the interplay of multiple cellular proteins in a

Chapter 1 timely sequential manner. Any of the steps required in this process may be rate limiting and failure to complete one step will terminate the metastatic cascade (Bockhorn et al., 2007).

Molecular changes facilitating neoplastic transformation of cells are caused by genetic alterations which result in dysregulated protein expression and changes in cell signalling pathways (Weinberg, 1989) . Once neoplastic transformation of a normal cell has occurred and the this cell has proliferated to a micro tumour of ~1 mm in diameter, an angiogenic cascade has to be initiated which allows the tumour to gain access to the local capillary network and to continue to grow to a sizeable tumour (Fidler, 2002). The development of new blood-vessels sprouting towards the tumour is achieved by induction of the “angiogenic switch” where secretion of pro-angiogenic factors such as fibroblast growth factors (FGFs), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) from tumour cells outbalance the level of angiogenic inhibitors (Bergers and Benjamin, 2003).

At a certain time point, single tumour cells or cell clumps detach or are shed from the primary tumour and invade the host stroma and eventually the lymphatic or blood vessels. This requires expression of enzymes capable of facilitating matrix degradation such as collagenase-4 and other matrix metalloproteinases (Tarin et al., 1982; Kanemoto et al., 1990; Rao et al., 1993; Deryugina and Quigley, 2006). Once in the circulatory system the tumour cells may either start to proliferate and thereby form an early metastasis in the vasculature (Wong et al., 2002) or be transported via the circulatory system to distant sites. To achieve this cells need to possess the ability to evade attacks of the immune system, to survive shear forces caused by the circulatory stream and to evade anoikis (Simpson et al., 2008). Anoikis is a form of apoptosis triggered by disruption of anchorage signalling due to the loss of cell-cell or cell-extracellular matrix (ECM) contacts (Hofmann et al., 2007; Chiarugi and Giannoni, 2008). Important negative regulators of anoikis, including the neutrotrophic receptor TrkB and the transmembrane protein CDCP1, have been identified and are discussed

Chapter 1 as potential anti-cancer drug targets (Douma et al., 2004; Desmet and Peeper, 2006; Uekita et al., 2007; Wortmann et al., 2009; Fukuchi et al., 2010). Depending on the repertoire of expressed cell surface molecules circulating cancer cells attach to vascular endothelial cells at distant organs or may get trapped in small capillaries. For example, crucial roles in adhesion of cancer cells to vascular endothelial cells at distant organs have been assigned to integrins, cadherins and adhesion molecules of the immunoglobulin superfamily including intercellular cell adhesion molecule (ICAM), neural cell adhesion molecule (NCAM) and vascular cell adhesion molecule (VCAM) (Glukhova et al., 1995; Maurer et al., 1998). Once attached to vascular endothelial cells at distant sites, the tumour cells start to extravasate into the organ parenchyma, proliferate to the size of micro metastases, initiate the angiogenic cascade and continue to grow to clinically relevant metastatic lesions (Bockhorn et al., 2007).

Importantly, the metastatic cascade is an inefficient multistep process which requires cancerous cells to express and utilise a defined set of cellular proteins in order to complete each rate limiting step. Consequently, each step of this cascade represents a potential point of therapeutic intervention to prevent the metastatic spread of cancer and, therefore, are a measure of controlling cancer disease. Hence, an increased understanding of pathways and proteins involved in this processes, such as CDCP1 , may contribute to improved cancer treatment strategies in the future.

1.4 Identification of CDCP1 and its association with cancer

The independent identification of CDCP1, using several different biased genetic and protein based approaches, provided the initial indications of the potential importance of this protein in cancer progression. In the earliest of these reports in 1996, a processed form of CDCP1 was identified as being tyrosine phosphorylated in response to loss of α6β4 integrin-mediated keratinocyte adhesion to laminin (Xia et al., 1996). A few years later Scherl- Mostageer et al . used a biased mRNA screening approach to show that the CDCP1 gene is highly transcribed in lung and colon cancer derived cell lines

Chapter 1

(Scherl-Mostageer et al., 2001). This study was the first to provide the complete CDCP1 DNA coding sequence and was followed, in 2003, by the first isolation of the complete CDCP1 protein sequence using an in vivo immunological approach biased to identify proteins functionally involved in metastasis (Hooper et al., 2003). Subsequently, two different groups identified CDCP1 as a Src family kinase (SFK) interacting protein in MDA- MB-468 breast cancer cells (Bhatt et al., 2005) and A549 lung adenocarcinoma cells (Uekita et al., 2007). In two other studies aimed at identifying molecules associated with important signalling proteins, CDCP1 was identified as interacting with protein kinase C δ (PKC δ) (Benes et al., 2005) and as a tetraspanin CD9 interacting protein (Andre et al., 2006).

Strengthening the proposal that CDCP1 has a role in cancer progression, its dysregulated expression has been associated with cancer of the lung (Scherl-Mostageer et al., 2001; Ikeda et al., 2009), kidney (Awakura et al., 2008), colon (Scherl-Mostageer et al., 2001; Hooper et al., 2003; Perry et al., 2007), and breast (Scherl-Mostageer et al., 2001; Ikeda et al., 2006). In addition, CDCP1 has been suggested as an independent marker for leukaemia (Buhring et al., 2004).

1.5 CDCP1 structural features

CDCP1 is an 836 amino acid transmembrane glycoprotein with a type-I orientation on the cell surface. This protein contains a 29 residue amino terminal signal peptide and extracellular, transmembrane, and cytoplasmic domains of 636, 21 and 150 amino acids, respectively (Figure 1.1). The extracellular domain contains three regions with low homology to complement protein subcomponents C1r/C1s, urchin embryonic growth factor, and bone morphogenetic protein 1 (CUB) domains, as well as 14 consensus N-glycosylation sites and 20 cysteines likely to be involved in disulﬁde bond formation (Scherl-Mostageer et al., 2001; Hooper et al., 2003; Brown et al., 2004; Bhatt et al., 2005).

Chapter 1

Figure 1.1: CDCP1. Schematic representation of human CDCP1 structural features including signal peptide, CUB-like domains (CUB-L1 to CUB-L3), transmembrane domain, consensus N-glycosylation sites (N) and intracellular tyrosine residues (Y). The cleavage site of matriptase (MT-SP1) is indicated by an arrowhead.

Twelve of the consensus N-glycosylation sites and 19 of the extracellular cysteines are conserved in human, chimpanzee, dog, cow, mouse and rat. N39 is not found in dog, cow, mouse and rat, while N339 is not found in mouse, and C423 is not present in rat (Figure 1.2). The predicted molecular weight of CDCP1, after removal of its signal peptide, is ~90.1 kDa (Hooper et al., 2003), whereas by Western blot analysis the apparent molecular weight is 135–140 kDa (Hooper et al., 2003; Brown et al., 2004; Benes et al., 2005; Bhatt et al., 2005). Deglycosylation using the enzyme N-glycosidase F indicated that between 30 and 40 kDa of the apparent molecular weight is due to the addition of N-linked glycans (Hooper et al., 2003). The roles of these extracellular features have not been deﬁned. Interestingly, CDCP1 also contains a consensus palmitoylation motif adjacent to its transmembrane domain (Hooper et al., 2003) and it is possible that the addition of palmitate will regulate plasma membrane localisation of CDCP1 as well as interactions with other proteins.

Chapter 1

Figure 1.2: CDCP1 sequence alignment with various other species. Alignment of the long CDCP1 protein isoform from human (AAO33397), chimpanzee (XP_001147144), dog (XP_541913), cow (XP_612363), mouse (NP_598735), and rat (NP_001100339). Sequence of the signal peptide is in lower case lettering and mature CDCP1 in upper case. Conserved residues are indicated by an asterisk. Asparagine residues located within consensus N-glycosylation sites and extracellular cysteines are numbered above the amino acid sequence. The transmembrane domain is underlined. Sequence alignment was performed using Clustal W.

Chapter 1

1.6 CDCP1 interacting proteins

An interesting feature of CDCP1 biology is the protein’s ability to bind to a growing number of other proteins. CDCP1 has been reported to interact with the cell/cell adhesion proteins N- and P-cadherin (Bhatt et al., 2005), the cell/matrix adhesion proteins syndecan-1 and -4 (Bhatt et al., 2005), cortactin 1 (Miyazawa et al., 2010), the tetraspanin CD9 (Andre et al., 2006), the cell surface protease matriptase (He et al., 2010) and cell signalling proteins including SFKs (Hooper et al., 2003; Bhatt et al., 2005; Uekita et al., 2007; Alvares et al., 2008) and PKC δ (Benes et al., 2005; Uekita et al., 2007). The best characterised of these binding proteins are SFKs and PKC δ which bind to CDCP1 in a tyrosine phosphorylation-dependent manner. Several groups have shown that, consistent with the presence of ﬁve conserved intracellular tyrosine residues (Figure 1.1), CDCP1 is phosphorylated by SFKs (Hooper et al., 2003; Brown et al., 2004; Benes et al., 2005; Bhatt et al., 2005; Uekita et al., 2007), including Src, Yes, and Fyn (Benes et al., 2005; Bhatt et al., 2005; Uekita et al., 2007). SFK-mediated phosphorylation of CDCP1 occurs when cells lose anchorage signals, when grown in suspension (Uekita et al., 2007; Spassov et al., 2009; Wong et al., 2009), during deadhesion in the mitotic cycle in vitro (Bhatt et al., 2005; Wong et al., 2009) and when cells are shed into the intestinal lumen in vivo (Wong et al., 2009) .

Benes et al. have shown that SFK-mediated phosphorylation of CDCP1 is required for the formation of a SFK•CDCP1•PKC δ multiprotein complex (Benes et al., 2005). As shown in Figure 1.3A, these authors propose that SFK phosphorylation of CDCP1 is initiated at Y734, resulting in SFK binding at this site, promoting additional phosphorylation at Y743 and Y762, and PKC δ recruitment at p-CDCP1-Y762. This was the ﬁrst demonstration of PKC δ as a phosphotyrosine-binding protein (Benes et al., 2005). This observation is signiﬁcant as it indicates that CDCP1 linkage of SFK tyrosine phosphorylation and PKC δ serine/threonine phosphorylation is likely to be important in normal and disease processes. In fact, Uekita et al. have reported that SFK phosphorylation of CDCP1 confers resistance of lung adenocarcinoma cells to anoikis in vitro (Uekita et al., 2007). Importantly, it was also shown that CDCP1-mediated tyrosine phosphorylation of PKCδ is

Chapter 1 required for the observed resistance to anoikis (Uekita et al., 2007). Further support for the anti-apoptotic function of CDCP1 was presented by Deryugina et al. showing that ligation of CDCP1 with an anti-CDCP1 antibody results in increased apoptosis in vitro under pro-apoptotic conditions induced by doxorubicin treatment (Deryugina et al., 2009).

Of further relevance to cancer progression, Uekita et al. have reported that phosphorylation of CDCP1 at Y734 is increased in tumour nodules of gastric cancer 44As3 cells during peritoneal invasion in mice (Uekita et al., 2008). Moreover, this work demonstrated elevated p-CDCP1-Y734 levels in human gastric cancers cells invading the gastric wall in a mouse model as compared to cultured cells in vitro (Uekita et al., 2008). Consistently, in lung cancer patient samples, p-CDCP1-Y734 is largely present in invading tumour cells (Ikeda et al., 2009). Of interest is the finding of Alvarez et al. that ligation of CDCP1 with an activating antibody leads not only to an increase in phosphorylation of CDCP1-Y734, but also to an increase in phosphorylation of SFK-Y416 and to an accumulation of both proteins in detergent resistant membrane clusters (Alvares et al., 2008). These data suggest that engagement of SFKs with CDCP1 leads to auto-activation of SFKs which may cause yet unidentified effects downstream of activated SFKs and may be the cause or a contributing factor to cellular phenotypes mediated by CDCP1.

Chapter 1

Figure 1.3: CDCP1 protein interactions. A, CDCP1 links SFKs and PKC δ. SFKs phosphorylate CDCP1-Y734, initiating SFK binding at this site, which in turn promotes additional phosphorylation at Y743 and Y762 and PKC δ recruitment at p-CDCP1-Y762. B, Serine proteases such as exogenous trypsin, plasmin, and matriptase process CDCP1 from high (135–140 kDa) to low (70–85 kDa) molecular weight species. The question mark (red colour) indicates putative site(s) where trypsin, plasmin, and the endogenous processing enzyme cleave HMW-CDCP1. C, Serine protease-induced phosphorylation of CDCP1-Y734. In addition to cleaving CDCP1, trypsin and plasmin may also induce phosphorylation of CDCP1-Y734. The black question mark indicates that the relative contributions of proteolysis and cell deadhesion to CDCP1 phosphorylation are not known. D, Cell deadhesion/adhesion involving changes in integrin ligation with matrix proteins regulates the phosphorylation state of LMW-CDCP1. Adhesion to laminin-5 via integrins-α6β4 and -α3β1 results in dephosphorylation of LMW- CDCP1. Loss of adhesion to laminin-5 contributes to phosphorylation of LMW-CDCP1.

Chapter 1

1.6.1 SFKs

SFKs comprise nine closely related proteins which possess different patterns of expression (Frame, 2002). Src, Yes and Fyn are expressed in most tissues with elevated levels of individual members in certain cell types (Thomas and Brugge, 1997). Lck, Hck, Lyn, Blk and Fgr and are primarily expressed in cells of haematopoietic origin (Bolen and Brugge, 1997). Yrk is only found in chicken (Thomas and Brugge, 1997). The best characterised SFK member, Src, which was identified three decades ago (Oppermann et al., 1979) is the avian analog of v-Src, the transforming factor which causes sarcomas in chickens transmitted by the Rous sarcoma virus described as early as 1911 (Rous, 1911).

Over-expression and increased activity of Src in human cancers of different epithelial origins is common and proposed to play a key role in cancer progression, particularly at later stages when tumour cells acquire metastatic abilities (Irby and Yeatman, 2000; Wheeler et al., 2009). For example, increased expression and phosphorylation of Src was found in breast cancer tissue (Verbeek et al., 1996) and its importance in the formation of metastasis in in vivo models has been previously described (Boyer et al., 2002; Myoui et al., 2003). The pro-metastatic phenotype caused by Src over- expression and/or activity has been suggested to be mediated by inducing an epithelial to mesenchymal transition (EMT) which leads to a resistance to anoikis, higher cell motility and consequently an increased ability to metastasise in vivo (Rodier et al., 1995; Avizienyte et al., 2005; Avizienyte and Frame, 2005; Galliher and Schiemann, 2006).

SFKs share common structural features and mechanisms of activation. The common domains of the family members comprise Src homology (SH) domains SH4, SH3, SH2, and SH1 (kinase-domain) followed by a short C- terminal regulatory segment (Figure 1.4). The N-terminally located SH4 domain is myristoylated or palmitoylated which allows for anchoring at the plasma membrane thereby facilitating interaction of SFKs with other cellular signalling proteins (Liang et al., 2001). SH2 and SH3 domains mediate intramolecular protein interactions as well as protein-protein interactions and

Chapter 1 the SH1 domain is the site with kinase activity. The unique region localised between SH3 and SH4 domains differs between and defines the individual SFK family members (Frame, 2002; Saito et al., 2010).

Figure 1.4: Schematic representation of the structure of Src family kinases. The SH4 domain becomes myristoylated and palmitoylated which allows anchoring at the plasma membrane thereby facilitates interactions with other proteins. The “unique” domain is a highly variable region unique for the different SFK members. SH3 and SH2 domains mediate intramolecular protein-protein interactions as well as interactions with other proteins. The SH1 domain possesses kinase activity which is primarily regulated by auto-phosphorylation of Y416 and dephosphorylation of Y527.

SFK activity is tightly regulated in normal physiology and activation can be achieved by different mechanisms involving phosphorylation, intra-molecular protein-protein interactions and interactions with binding proteins (Frame, 2002). The regulation of SFK activity by phosphorylation is primarily controlled at two tyrosine residues. Phosphorylation of Y527 causes a closed and inactive confirmation and dephosphorylation leads to a conformational change causing activation; auto-phosphorylation of Y416 is required for maximum kinase activity (Thomas and Brugge, 1997). The regulatory site Y527 is missing in the v-Src gene product thereby causing a constitutively activated Src with a high transformational potential (Frame, 2002).

Another mechanism by which Src can be activated is by displacement of intramolecular interactions mediated by the SH3 and SH2 domains by higher affinity ligands such as the non-receptor tyrosine kinase focal adhesion kinase (FAK) (Thomas et al., 1998; Frame, 2002) which is over-expressed in various human malignancies and plays a role in a variety of cellular processes including proliferation, migration and the regulation of anoikis (Frisch et al., 1996; Hanks and Polte, 1997; Gabarra-Niecko et al., 2003). Importantly, simultaneous inhibition of FAK and SFKs is additive in the

Chapter 1 induction of cell death in colon cancer and neuroblastoma cells, suggesting a role for interaction of these two proteins in regulating cancer cell survival (Golubovskaya et al., 2003; Beierle et al., 2010).

1.6.2 PKC δ

The PKC family comprises three subgroups. Conventional PKCs (c-PKCs) including α, βI, βII and γ require both calcium and diacylglycerol (DAG) for activation. The novel PKCs (n-PKCs) consists of δ, ε, η and θ and are independent of DAG, but require calcium for activation and the third group are the atypical PKCs (a-PKCs) comprising of ζ and ι/λ which are independent of both calcium and DAG (Steinberg, 2004). PKCs in a closed/inactive conformation are located within the soluble fraction of cells and interact only weakly/transiently with the cell membrane. PKCs are activated in response to various stimuli including mitogenic stimuli, inflammatory stimuli and stress and participate in a variety of cellular processes such as proliferation, differentiation, apoptosis and cell migration (Hug and Sarre, 1993).

PKCs are key players in tumourigenic processes. For example, it was found that phorbol esters exert teratogenic effects by activating PKCs (Blumberg, 1988). Subsequently, activity of PKCs has been found to cause a more malignant phenotype in cell lines including those derived from breast and gastric cancer (O'Brian et al., 1985; Schwartz et al., 1993) and elevated levels of PKC expression were detected in breast cancer samples (O'Brian et al., 1989).

PKC δ can act both as a tumour promoter as well as a tumour suppressor and mediates various cellular processes including proliferation, migration, cell cycle regulation and apoptosis (Jackson and Foster, 2004) via interactions with a variety of proteins such SFKs, Abl and IGF1-R (Zang et al., 1997; Shanmugam et al., 1998; Yuan et al., 1998; Sun et al., 2000; Joseloff et al., 2002; Cao et al., 2007). For example, although PKC δ has mainly been described as a negative regulator of proliferation in different

Chapter 1 malignant cell lines, it can also act to positively regulate proliferation (Jackson and Foster, 2004). Similarly, it was observed that PKC δ can both positively and negatively regulate apoptosis (Brodie, 2003).

These reports controversially implicating PKC δ as a positive and negative regulator of the same cellular process are likely to arise because of phosphorylation of distinct tyrosine residues. PKC δ contains 19 phosphorylation sites and at least nine of these are phosphorylated in response to various stimuli (Steinberg, 2004). Further complexity is added due to the ability of PKC δ to translocate to different cell compartments including the Golgi apparatus, mitochondria, nucleus and plasma membrane (Brodie, 2003).

It appears that PKC δ can exert opposing functions depending on the site of tyrosine phosphorylation, its location in the cell and the distinct set of proteins with which it is interacting. For example, Acs et al. reported that PKC δ acts as an inhibitor of proliferation in NIH 3T3 cells, but mutation at Y155 leads to the opposite effect resulting in increased cell proliferation (Acs et al., 2000). SFKs have been shown to phosphorylate PKC δ at different tyrosine residues and inhibition of Src by selective inhibitors prevents PKC δ phosphorylation. In particular, Y311 was found to be crucial for binding of Src to PKC δ and its subsequent phosphorylation (Steinberg, 2004).

1.7 CDCP1 is cleaved by proteases

There are data indicating that 135-140-kDa CDCP1 (further referred to as high molecular weight CDCP1; HMW-CDCP1) is processed through interactions with proteolytic enzymes, to a low molecular weight (LMW) form (Figure 1.3B). For example, trypsin treatment of keratinocytes in vitro generates ~80-kDa CDCP1 via cleavage at, or amino terminal of, E278 (Xia et al., 1996; Brown et al., 2004). In addition, He et al. reported that treatment of 22Rv1 prostate cancer cells, which only produce HMW-CDCP1, with conditioned media from PC3 prostate cancer cells, which produce both HMW- and LWM-CDCP1, induces the production of LMW-CDCP1 (He et al.,

Chapter 1

2010). The identity of the endogenous factor that mediates this processing has not yet been established (He et al., 2010).

Similarly sized endogenously processed CDCP1 species have been observed in cancer cell lines originating from breast, lung, stomach, prostate, bladder, ovary, pancreas, lung, oesophagus, colon and cervix (Bhatt et al., 2005; Uekita et al., 2007; Uekita et al., 2008; Wong et al., 2009; He et al., 2010). Although the cellular mechanisms regulating generation of LMW- CDCP1 have not been defined, a number of reports support the involvement of tryptic serine proteases. Supporting data have come from experiments using the trypsin-fold-specific inhibitor, ecotin, and MDA-MB-468 breast cancer cells stably expressing CDCP1. These cells express both LMW and HMW species from the CDCP1 expression construct, indicating that LMW- CDCP1 is not generated by alternative mRNA splicing. Furthermore, ecotin treatment of these cells resulted in a marked reduction in the level of the LMW form, which is suggestive of serine protease-mediated CDCP1 processing (Bhatt et al., 2005). Similar findings were reported by He et al. who found that treatment of PC3 cells, which endogenously possess both LMW and HMW-CDCP1, with the serine protease inhibitors, PMSF and aprotinin, leads to loss of LMW-CDCP1 (He et al., 2010). Further support that CDCP1 is proteolytically processed has come from experiments showing that the serine protease plasmin, at sub-physiological concentrations, converts HMW-p-CDCP1-Y734 to a ~80-kDa species in vitro and homogenates of epidermis from neonatal laminin-5 deficient mice contain both LMW and HMW CDCP1-pY734 (Brown et al., 2004). The rationale for using laminin-5 deficient mice is that skin blistering occurs in neonates and the epidermis can thus be separated from the dermis without using proteolytic enzymes that would likely cause proteolysis and phosphorylation of CDCP1. Therefore, this approach strongly suggests that LMW-CDCP1 is generated by endogenous mechanisms in vivo (Brown et al., 2004). Also, the catalytic domain of the serine protease matriptase (also known as MT-SP1, ST14 and TADG-15) cleaves the recombinant extracellular domain of CDCP1 at R368 (Figure 1.1) (inadvertently referred to as R369 in (Bhatt et al., 2005)). Of relevance to cancer cell biology is the finding of He et al. that

Chapter 1 an unidentified endogenous factor, present in PC3 prostate cancer cell media can mediate the cleavage of CDCP1 after R368 and K369 (He et al., 2010).

The in vivo relevance of matriptase-mediated processing of CDCP1 is supported by various observations of cellular interactions involving these proteins. First, Bhatt et al. demonstrated that an anti-matriptase antibody immunoprecipitates myc-tagged CDCP1 from over-expressing MDA-MB-468 cells (Bhatt et al., 2005), and, second, endogenous matriptase and CDCP1 have been co-immunoprecipitated from SW480 colon cancer cells using an antibody against the tetraspanin CD9 (Andre et al., 2006). Significantly, He et al. demonstrated that matriptase cleavage of CDCP1 leads to the phosphorylation and the consequent recruitment of SFKs and PKC δ to the cell surface-retained portion of CDCP1 in prostate cancer 22Rv1 cells (He et al., 2010). These data suggest that cleavage of CDCP1 by matriptase and, potentially, other proteases leads to initiation of a cellular signalling event downstream of CDCP1 phosphorylation.

1.8 Phosphorylation of CDCP1

The picture emerging from these reports is that changes in phosphorylation of both LMW- and HMW-CDCP1 are mediated by a number of cellular events including cell adhesion/deadhesion and proteolysis. For example, trypsin treatment of keratinocytes results in loss of HMW-p-CDCP1-Y734 and appearance of LMW-p-CDCP1-Y734. On balance it appears, as proposed by Brown et al. , that proteolytic conversion results in increased phosphorylation of LMW-CDCP1 (Brown et al., 2004), as shown in Figure 1.3C. However, it is likely that another major contributor to the observed increase in phosphorylation of LMW-CDCP1 is the concurrent loss of cellular contact with matrix components during cell deadhesion. For example, in a key experiment, Xia et al. showed that interruption of keratinocyte integrin- α6β4 binding to laminin-5, using an inhibitory anti-laminin-5 monoclonal antibody, increased phosphorylation of LMW-CDCP1 (Xia et al., 1996) (Figure 1.4D, forward arrow). The contribution of cell deadhesion to

Chapter 1 increased phosphorylation of CDCP1 is also indicated by experiments in which phosphorylated LMW- and HMW-CDCP1 were affinity purified more efficiently from A549 cells grown in suspension than in adherent cells, using a Fyn SH2 domain as bait (Uekita et al., 2007).

Although the relative contributions of cell deadhesion and proteolysis to CDCP1 phosphorylation remain to be more exactly deﬁned, it is clear that cell adhesion causes dephosphorylation of CDCP1. This has been shown in experiments examining integrin-mediated readhesion of trypsin-suspended keratinocytes to the basement membrane protein laminin-5. These experiments showed that readhesion onto laminin-5 via integrin-α6β4 and -α3β1 results in dephosphorylation of LMW-CDCP1 (Xia et al., 1996) (Figure 1.3D, reverse pathway). A later report from this group showed that readhesion results in the time-dependent dephosphorylation of LMW-CDCP1 and re-expression of HMW-p-CDCP1-Y734 (Brown et al., 2004). This work also demonstrated that dephosphorylation of Y734 of LMW- and HMW- CDCP1 in keratinocytes occurs with different kinetics; whereas suramin- induced phosphorylation of HMW-CDCP1 disappears within 15 minutes, phosphorylated LMW-CDCP1 persists for longer than 4 hours (Brown et al., 2004).

1.9 CDCP1 expression is dysregulated in human tumours

Dysregulated CDCP1 expression has been associated with a number of cancers and recent reports suggest a functional role in tumour cells that could potentially be targeted to disrupt cancer progression.

A study by Awakura et al. examining 230 renal cell carcinoma (RCC) patient samples demonstrated that CDCP1 was not expressed by nomal kidney cells but was expressed in 77 of the 230 cancer cases (33.5%). CDCP1 expression was significantly associated with indicators of advancing disease (tumour stage, histological grade, and presence of metastases). In addition, CDCP1 expression and metastasis were significant predictors of shorter disease-specific survival and, in patients with localised cancer, CDCP1

Chapter 1 positive staining and tumour stage 2 were signiﬁcant predictors of shorter recurrence-free survival (Awakura et al., 2008). Another study conducted by Ikeda et al. analysed the expression of CDCP1 in 200 lung adenocarcinoma samples and demonstrated important associations of this protein with disease progression (Ikeda et al., 2009). Moderate to high levels of CDCP1 expressing was detected in 60 of the 200 samples. High CDCP1 expression correlated with increased occurrence of lymph node metastasis and tumour relapse with 5-year disease-free and overall survival rates signiﬁcantly lower for patients with high CDCP1 expression (Ikeda et al., 2009). Moreover, high levels of CDCP1 expression were found to correlate with poor overall survival rates in tissue specimens in a cohort of 145 pancreatic cancer cases (Miyazawa et al., 2010).

Several other studies have examined CDCP1 mRNA or protein expression in small numbers of cancer samples. For example, as part of the initial report on the CDCP1 gene and coding sequence, elevated CDCP1 mRNA levels were detected in colon, lung, and breast cancers relative to unmatched normal tissues (Scherl-Mostageer et al., 2001). Further support for increased CDCP1 expression in breast cancer came from a study which, although providing no comparison with normal breast tissue, showed that CDCP1 mRNA levels inversely correlated with methylation of the CDCP1 transcription initiation site in all 25 breast cancer patient samples examined (Ikeda et al., 2006). Consistently, analysis of two breast cancer samples demonstrated that a patient with high CDCP1 mRNA levels had correspondingly high CDCP1 protein levels that correlated with high expression of the proliferation marker Ki67, while a patient with low CDCP1 mRNA levels had correspondingly low CDCP1 and Ki67 protein levels (Ikeda et al., 2006). In another small study, analysis of colon adenocarcinoma and adjacent non-diseased tissue from three patient samples indicated a potential link between more malignant colon cancer cells and CDCP1 staining intensity, although a conclusive association could not be demonstrated from such a small cohort (Hooper et al., 2003). Noting the presence in vivo of CDCP1 shed from the cell surface, these authors proposed that this protein may have utility as a serum marker for colon

Chapter 1 cancer (Hooper et al., 2003). Moreover, analysis of a single patient sample showed an increase of ~2.5 fold in CDCP1 mRNA levels in colon adenocarcinoma compared with adjacent normal tissue, correlating positively with elevated mRNA expression of the current clinical cancer markers carcinoembryonic antigen (CEA) and epithelial cell adhesion molecule (EpCam) (Perry et al., 2007).

1.10 CDCP1 in leukaemia and haematopoietic and progenitor cells

In addition to an association with several solid tumours, CDCP1 expression has also been detected in the erythroleukaemic cell line K562 (Scherl- Mostageer et al., 2001) and in leukemic blast cells from patients with acute lymphoblastic leukaemia, acute myeloid leukaemia and chronic myeloid leukaemia in blast crisis (Buhring et al., 2004). Bone marrow and mesenchymal stem/progenitor cells and neural progenitor cells were also found to express CDCP1 (Conze et al., 2003; Buhring et al., 2004). Signiﬁcantly, normal peripheral blood populations, including B and T cells, monocytes, granulocytes, erythrocytes, and thrombocytes do not express CDCP1 (Buhring et al., 2004). The lack of complete overlap in CDCP1 expression with two current markers of leukaemia (CD34, CD133), coupled with the lack of CDCP1 expression by normal cells from peripheral blood, indicates that CDCP1 may be useful as an additional marker for diagnosis of leukaemia (Buhring et al., 2004).

1.11 CDCP1 supports cancer progression in model systems

Data supporting a functional role for CDCP1 in cancer progression have come from in vitro experiments and animal models. In the ﬁrst of these reports, Uekita et al. identiﬁed tyrosine phosphorylated CDCP1 as a SFK- binding protein in human lung cancer cell lines (Uekita et al., 2007). In these cells, phosphorylated CDCP1 was required to overcome anoikis and permit in vitro anchorage-independent growth. Importantly, although reduction of CDCP1 in A549 lung cancer cells by RNA interference had no effect on

Chapter 1 primary tumour growth in mice, metastasis of these cells to lung after tail vein injection was signiﬁcantly reduced (Uekita et al., 2007). Supporting the suggestion that CDCP1 has a role in cancer progression, a more recent study from this group demonstrated that CDCP1 promoted invasion and peritoneal dissemination in mice of gastric cancer cell lines (Uekita et al., 2008). The authors proposed that CDCP1 mediates these processes through regulation of cell migration and anchorage-independent growth and suggested that suppression of CDCP1 phosphorylation may be useful for modulating cancer metastasis.

The notion that CDCP1 can be targeted to disrupt cancer processes has recently been tested using an anti-CDCP1 monoclonal antibody generated from a phage display combinatorial antibody library (Siva et al., 2008a). In vitro , this antibody inhibited prostate cancer PC3 cell migration and invasion (Siva et al., 2008b). In addition, when either directly conjugated to the cytotoxin saporin or used in conjunction with species appropriate saporin- conjugated secondary antibodies, anti-CDCP1 antibodies induced PC3 cell death in vitro . Furthermore, intravenously administered saporin-conjugated anti-CDCP1 antibody inhibited subcutaneous growth of PC3 cells in mice, while both subcutaneous and intravenous delivery of this antibody–cytotoxin conjugate inhibited metastasis of these cells to lymph nodes (Siva et al., 2008b). Although it is an encouraging finding that targeting of CDCP1 can be employed to eliminate cancer cells, to some extent the ability of a toxin- conjugated antibody to induce the death of cells expressing the cognate cell surface antigen is not surprising. Importantly, it was reported that inhibition of metastasis formation of HeLa cells stably expressing CDCP1 and endogenous CDCP1 expressing PC3 cells can be achieved in the chicken embryo metastasis model as well as in a mouse model by using the anti- CDCP1 monoclonal antibody 41-2 (Deryugina et al., 2009). This finding was confirmed by Fukuchi et al. who employed function-blocking human antibodies against CDCP1, obtained from phage display libraries, to inhibit metastasis formation in a mouse model and the chicken embryo metastasis model (Fukuchi et al., 2010).

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1.12 Opportunities and challenges in targeting CDCP1 in cancer

These reports indicate that CDCP1 mediates tumourigenic and metastatic abilities of tumour cell lines in model systems and that targeting of this molecule can inhibit cancer dissemination in these settings. With clear evidence of upregulation of CDCP1 in lung, kidney and pancreatic cancers (Scherl-Mostageer et al., 2001; Awakura et al., 2008; Ikeda et al., 2009; Miyazawa et al., 2010) and to a lesser extent in breast cancer (Scherl- Mostageer et al., 2001; Ikeda et al., 2006) along with indications of dysregulated expression in colon cancer (Scherl-Mostageer et al., 2001; Hooper et al., 2003; Perry et al., 2007), it is possible that aberrant CDCP1 expression facilitates cancer progression in humans. Accordingly, targeting of CDCP1 in these settings may represent a rational approach to treat certain tumours. However, a key issue potentially hampering this approach is the widespread expression of this molecule in normal tissues. For example, CDCP1 mRNA has been detected in a range of organs, including skeletal muscle, colon, kidney, small intestine, placenta, lung, stomach, oesophagus, and rectum (Scherl-Mostageer et al., 2001; Hooper et al., 2003), while CDCP1 protein has been described in normal epithelial cells of the colon (Hooper et al., 2003), lung, and pancreas, renal tubular subsets, liver hepatocytes (Siva et al., 2008b), epidermis (Alvares et al., 2008), primary cultures of foreskin keratinocytes (Brown et al., 2004), and mesenchymal and neural progenitor cells (Conze et al., 2003; Buhring et al., 2004). In contrast, microvascular endothelial cells and ﬁbroblasts do not express CDCP1 (Hooper et al., 2003). Accordingly, as recognized previously (Buhring et al., 2004; Wortmann et al., 2009), targeting of CDCP1 in cancer may not only eliminate tumour cells but also adversely impact on the survival of normal epithelial cells and stem/progenitor cells. However, this problem is certainly not insurmountable as there are small molecule and monoclonal antibody anticancer drugs targeting widely expressed proteins that are generally well tolerated (Gonzalez-Angulo et al., 2006; Widakowich et al., 2007). These proteins include epidermal growth factor receptors targeted in colon, lung, and head and neck cancers and the human epidermal growth factor receptor 2 (HER2) in breast and other cancers (Dassonville et al.,

Chapter 1

2007). Of relevance, HER2 has been detected in skin, breast, and placenta as well as in epithelial cells of the gastrointestinal, respiratory, reproductive, and urinary tracts (Press et al., 1990) and has been detected on subsets of hematopoietic cells (Leone et al., 2003) and also functions in muscle (Altiok et al., 1995).

Complementing the in vitro and animal based studies focused on understanding the role of CDCP1 in cancer progression, two recent reports have examined epigenetic events leading to dysregulated expression of the CDCP1 gene. From these studies it appears that CDCDP1 expression is inversely correlated with CpG methylation. For example, Ikeda et al. demonstrated that CDCP1 mRNA levels are inversely correlated in 14 cell lines from a range of cancers (leukaemia, mastocytoma, colon, breast, and prostate) with the methylation status of 36 CpG motifs spanning the transcription initiation site of the encoding gene. This report also showed a strong inverse correlation between CDCP1 mRNA levels and methylation at these sites in 25 breast cancer samples (Ikeda et al., 2006). Looking more closely at hematopoietic cell lines, this group also showed that CDCP1 mRNA expression in K562 and Jurkat cells is also inversely correlated to CpG methylation (Kimura et al., 2006). Further work is required to understand the epigenetic events leading to dysregulated CDCP1 expression. Of course, in addition to the possibility that elevated CDCP1 expression, mediated by epigenetic changes, facilitates cancer, there is also the potential that genetic changes in the CDCP1 gene (e.g., amino acid altering polymorphisms) will have a role in cancer progression.

If CDCP1 or CDCP1-mediated signalling pathways are to be targets for cancer therapy, a signiﬁcant challenge is to understand how interactions with other proteins regulate its function. The potential complexity of this task is indicated from the reports summarised earlier that CDCP1-interacting proteins include signalling molecules (SFKs and PKCδ), cell/cell adhesion proteins (N- and P-cadherin), cell/matrix adhesion proteins (syndecan-1 and -4), the tetraspanin CD9, and the serine protease matriptase. These proteins are summarised in Figure 1.5 which also highlights the changes in SFK-

Chapter 1 mediated phosphorylation of CDCP1 and PKC δ binding that occurs during cell deadhesion and adhesion.

Figure 1.5: Speculative overview of CDCP1 in premalignant and cancer cells. Shown are CDCP1 interacting proteins including SFKs, PKC δ, the serine protease matriptase, cadherin cell/cell adhesion proteins, syndecan cell/matrix adhesion proteins, and the tetraspanin CD9. CDCP1 is present on the cell surface as 135-140 kDa and 70-85 kDa species; for simplicity only full length CDCP1 is shown. Cell adhesion (i) involving integrin ligation to the matrix protein laminin-5 induces dephosphorylation of CDCP1. Cell deadhesion (ii) contributes to tyrosine phosphorylation of CDCP1. The accompanying formation of the SFK•CDCP1•PKC δ complex is mechanistically involved in protecting cells from anoikis (Uekita et al., 2007; Uekita et al., 2008). As cell adhesion and deadhesion are important during various stages of cancer, it is possible that strategies targeting CDCP1 that modulate cell adhesion or deadhesion may represent rational approaches to disrupt cancer progression.

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The actions of SFKs (Summy and Gallick, 2003), PKC δ (Mackay and Twelves, 2007), cadherins (Cavallaro and Christofori, 2004; Stemmler, 2008), syndecans (Morgan et al., 2007), and certain tetraspanins (Zoller, 2009) are well known to be important in the aberrant cell adhesion and deadhesion events characteristic of various stages of cancer progression. It also appears that dysregulated matriptase expression may be important in these processes as this protease increases the ability of cell lines derived from a number of cancers to invade through matrix components (List et al., 2006). Thus, located on the cell surface, CDCP1 may represent a key point of molecular convergence in cell adhesion/deadhesion that could be targeted to disrupt these processes and thereby inhibit cancer progression.

1.13 Summary and aims

CDCP1 is a cell surface glycoprotein dysregulated in several cancers with increased expression linked to poor prognosis. CDCP1 has a crucial role in decreasing anoikis in vitro and in vivo which facilitates metastasis formation. Consequently, CDCP1 is considered as a potential target for therapeutic intervention to control the metastatic spread of cancers (Uekita et al., 2007; Wortmann et al., 2009; Fukuchi et al., 2010). The CDCP1-mediated anti- apoptotic phenotype in vitro is mediated by interactions with SFKs and PKC δ at residues including Y734, Y743 and Y762 of CDCP1. It will be important to better understand how signalling events initiated by phosphorylation of these tyrosine residues of CDCP1 contribute to the pro-metastatic phenotype, as well as the cellular effectors in these signalling events. Understanding these signalling events may be beneficial in designing strategies to efficiently block CDCP1 and thereby decrease metastasis formation in vivo.

In addition to the full length form of CDCP1 which has a molecular weight of ~135 kDa, LMW-CDCP1 has been detected in a large number of cancer cell lines of different tissue origin. This suggests a possible role of proteolytic processing of CDCP1 in cancer progression. It has also been proposed that generation of LMW-CDCP1 in vivo is caused by proteolytic processing of CDCP1. Of relevance, trypsin and plasmin are able to proteolytically process

Chapter 1 cell surface CDCP1. Importantly, cleavage of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained fragment (Brown et al., 2004), which suggests that proteolytic processing of CDCP1 may induce cellular signalling. Moreover, the recombinant catalytic domain of matriptase was shown to cleave the recombinant extracellular portion of CDCP1 (Benes et al., 2005), but whether proteolytic processing of CDCP1 by matriptase can occur on the cell surface is not known. Understanding whether proteolytic processing of CDCP1 by proteases impacts on cell signalling, and whether this event can alter cell phenotype is important to gain more insights into the role CDCP1 plays in cancer progression.

This thesis aimed to gain a more comprehensive understanding of the biology of CDCP1. The main focus was to examine cell phenotypes mediated by phosphorylation of CDCP1 tyrosine residues and to examine the effect of proteolytic processing of CDCP1 on cell phenotype and cell signalling. Therefore, the aims of this project were:

Aim 1: To generate and characterise cancer cell lines stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F or Y762F.

For this aim a cancer cell line was employed that does not express CDCP1 endogenously and has low tumourigenic and metastatic potential in in vivo cancer models. These characteristics were important because in Aim 3 these cells were used to examine changes in the metastatic phenotype in vivo and any increase in metastatic ability due to CDCP1 expression could be readily detectable.

Aim 2: To examine whether the serine protease matriptase is able to proteolytically process CDCP1 on the cell surface and whether this alters cell phenotype and induces cell signalling.

For this aim matriptase was used rather than the related serine protease trypsin, as matriptase has a narrower substrate specifity. In addition,

Chapter 1 matriptase-mediated proteolysis does not cause deadhesion of cells. This was important since deadhesion of cells is known to cause phosphorylation of CDCP1 and this may not allow the detection of cellular signalling or a change in cell phenotype caused by proteolytic processing of CDCP1.

After matriptase was identified as an efficient proteolytic processor of CDCP1 the effect of matriptase-mediated proteolysis on cell signalling and cell phenotype in vitro was investigated. In addition, the role of CDCP1-Y734 in these processes was examined.

Aim 3: To examine the effect of CDCP1 expression on in vitro and in vivo processes associated with cancer progression.

For this aim in vitro assays relevant to cellular processes associated with cancer progression including adhesion, proliferation and migration were performed using HeLa-CDCP1, HeLa-CDCP1-Y734F, -Y743F and -Y762F cells. Furthermore, the metastatic ability of the stable CDCP1 expressing cell lines was examined using the chicken embryo chorioallantoic membrane (CAM) intravasation assay. In addition, the impact of mutation of CDCP1 tyrosine residues on the ability of a monoclonal anti-CDCP1 antibody to inhibit metastasis formation was examined.

The chicken embryo CAM assay was used as in vivo model because it allows assessment of the metastatic ability of cells in a shorter time frame and is less labour intensive and more cost efficient than a murine metastasis model.

Chapter 2

Materials and Methods Chapter 2

Materials

2.1 Reagents

2.1.1 General reagents

General reagents were purchased from the following suppliers. PAR2 activating peptide (AP; SLIGKV-NH 2) from AusPep (Parkville, Australia); 40% acrylamide/bis-acrylamide solution-29:1 mixture, temed and ethidium bromide solution from Biorad (Hercules, CA); Nitrocellulose membrane from GE Healthcare Bio-Sciences Pty. Ltd. (Rydalmere, Australia); Platinum Taq DNA polymerase, SU6656, PBS without Mg 2+ and Ca 2+ , polymerase chain

reaction (PCR) grade H 2O, Fura-2, CyQuant-NF reagent, proteinase-K, Prolong Gold, Alexa Fluor 488 phalloidin and DAPI from Invitrogen (Mount Waverley, Australia); EDTA di-sodium salt from Merck Biosciences (Kilsyth, Australia); SYBR Green dye from Molecular Probes (Eugene, OR); sodium dodecyl sulfate from MP-Biochemicals (Sydney Australia); PBS tablets from Oxoid (Adelaide, Australia); EZ-link NHSSS-Biotin and Immunopure immobilized streptavidin from Pierce (Quantum Scientific; Murarrie, Australia); Immobilised Protein A beads, Immobilised Protein G beads, EDTA free protease inhibitor cocktail tablets and Tris-base from Roche (Cromer, Australia); and sodium orthovanadate, sodium fluoride, β-mercaptoethanol, bovine serum albumin (fraction-V) and Whatman blotting paper from Sigma (Sydney, Australia). All other general reagents were purchased from Sigma unless otherwise stated.

2.1.2 Antibodies

A goat anti-CDCP1 antibody specific for the carboxy-terminus of CDCP1 was purchased from Abcam (Saphire Biosciences Pty Ltd.; Waterloo, Australia); rabbit anti-matriptase antibody from Bethyl Laboratories (Quantum Scientific; Murarrie, Australia); mouse anti-Erk 1/2, rabbit anti-phospho Erk1/2, rabbit anti-SFK (Cat.#2108), mouse anti-Src (Cat.#2110) and rabbit anti-phospho SFK-Y416 antibodies from Cell Signaling Technologies (Genesearch Pty Ltd.; Arundel, Australia); Alexa Fluor 488, 568, 647 conjugated secondary

Chapter 2

antibodies and the rabbit anti-phospho FAK-Y861 antibody was purchased from Invitrogen; mouse IgG for CAM assays from Jackson ImmunoResearch (West Grove, U.S.A); mouse anti-phospho tyrosine pY20 antibody from Merck Biosciences; mouse anti-focal adhesion kinase (FAK) antibody from Millipore (North Ryde, Australia); goat anti-CDCP1 antibody specific for the extracellular domain of CDCP1 from R&D-systems (Bio-Scientific Pty. Ltd.; Gymea, Australia); rabbit anti-PKC δ antibody from Santa Cruz Biotechnologies (Applied Medical; Stafford City, Australia); rabbit anti-FLAG tag (DYKDDDDK) antibody from Sigma; goat anti-mouse horseradish- peroxidase (HRP)-conjugated seconday antibody and goat anti-rabbit HRP- conjugated seconday antibody from Pierce (Thermo Fisher Scientific Pty Ltd.; Scoresby, Australia); monoclonal (MAb) mouse anti-CDCP1 antibodies 10D7 and 41-2 were purified as previously described (Brooks et al., 1993; Hooper et al., 2003; Deryugina et al., 2009). During the course of this research project multiple antibodies against CDCP1 were used for different experiments and Table 2.1 provides specifications for all anti-CDCP1 antibodies.

Table 2.1: Specifications of anti-CDCP1 antibodies used in this project.

Chapter 2

2.1.3 Vectors

The pcDNA 3.1 vector system (Figure 2.1) was purchased from Invitrogen and was used to express CDCP1, CDCP1-Y734F, CDCP1-Y743F and CDCP1-Y762F in HeLa cells.

Figure 2.1: Vector map of the pcDNA 3.1 vector. Taken from http://tools.invitrogen.com/content/sfs/ manuals/ pcdna3.1_man.pdf.

2.1.4 Oligonucleotides

Primers used for DNA sequencing and PCR were synthesised by Sigma and are shown in Table 2.2.

Table 2.2: Oligonucleotide primers. Presented are the sequences and specifications of the primers which were used for PCR and sequencing reactions. Tm, theoretical melting temperature.

Primer Target Sequence Size (bp)Tm Comments F-3 CDCP1 5'-CCAGGTGAAGCAGAACATC-3' 19 58.0 binds to CDCP1 at position 1631 F-20 CDCP1 5'-GGAGGTGGAGTCTTACTGC-3' 19 60.0 binds to CDCP1 at position 2104 BGH-rev BGH 5'-TAGAAGGCACAGTCGAGG-3' 18 56.0 binds to the BGH sequence within the multiple cloning side of the pcDNA3.1 vector Alu -sense human-Alu5’-ACGCCTGTAATCCCAGCACTT-3’ 21 61.0 binds human Alu repeat sequence Alu -antisense repeat 5’-TCGCCCAGGCTGGGTGCA-3’ 18 63.0

Chapter 2

2.1.5 Enzymes and kits

Enzymes and kits were purchased from the following suppliers. Puregene DNA purification kit from Genta Systems (Minneapolis, USA); bicinchoninic acid (BCA) kit and Femto super sensitive HRP substrate kit from Pierce; trypsin and the Wizard PCR reaction purification kit from Promega (Sydney, Australia); Pfu Ultra DNA polymerase from Stratagene (La Jolla, USA); QIAprep miniprep kit from Qiagen (Doncaster, Australia); matriptase was generously provided by Dr. Daniel Kirchhofer from the Department of Protein Engineering of Genentech Inc. (South San Franciso, USA) and was previously described (Kirchhofer et al., 2003); Kallikrein4 was purified from the media of Sf9 cells stably transfected with KLK4-pIB/V5-His construct as described previously (Ramsay et al., 2008).

2.1.6 Cell culture reagents and materials

Human fibronectin, human vitronectin, murine laminin-1, murine collagen-IV, growth factor reduced Matrigel, 8 µm Transwell migration inserts and companion 24 well plates were from Becton Dickinson (Eight Mile Plains, Australia); Nucleocounter cassettes, reagent A-100 and reagent B were from chemoMetec (Allerød, Denmark); Dulbecco’s modified eagle medium (DMEM), Opti-MEM medium, Roswell Park Memorial Institute medium-1640 (RPMI-1640), SF9002 serum free media, Versene, fetal calf serum (FCS), penicillin, streptomycin and Lipofectamine were from Invitrogen; Geneticin was from Merck Biosciences; 6, 12 and 96 well plates, T25 and T75 flasks were from Nunc (Thermo Fisher Scientific Pty Ltd.; Scoresby, Australia); black walled 96 well plates were from Perkin Elmer (Melbourne, Australia); crystal violet solution was from Sigma.

2.1.7 Cell lines

The non-tumourigenic (RWPE1) and the tumourigenic (RWPE2) prostate epithelial derived cell lines, the prostate cancer derived cell lines LNCaP,

Chapter 2

PC3, DU145, 22Rv1, the colon carcinoma derived cell lines SW480, SW620, CaCo2, HCT116 and HT29 and the cervical cancer derived cell lines HeLa and Caski were obtained from ATCC (Manassas, USA).

2.1.8 Buffers

If a pH value for a buffer is stated, the baseline value was obtained using a labChem-TPS pH electrode (TPS Pty Ltd.; Brisbane, Australia), before addition of either 1 M HCl or 2 M NaOH to obtain the desired pH. The following buffers were used:

Agarose gel loading dye: 0.09% (w/v) bromophenol blue, 0.09% (w/v) xylene cyanol FF, 60% (v/v) glycerol, 60 mM EDTA, 1M Tris (pH 8.0). This buffer was used in Section 2.2.4.

Phosphate buffered saline (PBS): sodium chloride 8 g/L, potassium chloride 0.2 g/L, di-sodium hydrogen phosphate 1.15 g/L, potassium di-hydrogen phosphate 0.2 g/L, pH 7.3. This buffer was used in Section 2.2.9, 2.2.10, 2.2.11, 2.2.12, 2.2.13, 2.2.16, 2.2.17, 2.2.21, 2.2.22, 2.2.23, 2.2.24, 2.2.25, 2.2.26, 2.2.29, 2.2.30, 2.2.32, 2.2.33, 2.2.34.

Cell lysis buffer: PBS supplemented with 1% Chaps (w/v), 2 mM MgCl 2 and

1x protease inhibitor cocktail. Sodium orthovanadate (Na 3VO 4) and 10 mM sodium fluoride (NaF) were added for phosphorylation state specific protein analysis at concentrations of 2 mM and 10 mM, respectively. This buffer was used in Section 2.2.13, 2.2.15.

Cell lysis buffer for cell surface biotinylation: 150 mM NaCl, 20 mM HEPES, 1 mM EDTA and 1% (v/v) Triton X-100. This buffer was used in Section 2.2.34.

Tris-buffered saline + Tween 20 (TBS-T): 100 mM Tris, 137 mM NaCl, 0.1% (v/v) Tween-20, pH 7.5. This buffer was used in Section 2.2.16.

Tris-acetate EDTA (TAE): 40 mM Tris-acetate, 1 mM EDTA, pH 8.0. This buffer was used in Section 2.2.4.

Chapter 2

Tris-EDTA-Saline (TES): 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8.0. This buffer was used in Section 2.2.7.

Extracellular medium: 121 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl 2, 1.8 mM

CaCl 2, 5.5 mM glucose, 25 mM HEPES, pH 7.4. This buffer was used in Section 2.2.32.

6x Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS- PAGE) loading dye: 430 mM Tris pH 6.8, 18% (w/v) SDS, 0.2% (w/v) bromophenol blue, 30% (v/v) glycerol, 15% (v/v) β-mercaptoethanol. This buffer was used in Section 2.2.15, 2.2.16, 2.2.34.

10x SDS-PAGE Running buffer: 250 mM Tris, 1.92 M glycine, 1% (w/v) SDS, pH 8.3, diluted to 1x solution with double distilled water (ddH 2O) before use. This buffer was used in Section 2.2.16.

Protein transfer buffer: 25 mM Tris, 192 mM glycine, 20% (v/v) methanol. This buffer was used in Section 2.2.16.

2.1.9 Media

The following medium was used to propagate XL10 competent bacterial cells transformed with the CDCP1/pcDNA-3.1 constructs. Luria-Bertani (LB) bacterial growth medium: 1% (w/v) Bacto-Tryptone, 0.5% (w/v) Bacto-yeast extract and 0.5% (w/v) NaCl, pH 7.0.

2.2 Methods

2.2.1 General cell culture

All cell lines used during the course of this project were cultured and maintained in a cell culture incubator in a 5% CO 2 containing humidified atmosphere at 37°C. All cell lines were passaged ex clusively non- enzymatically twice weekly using Versene (0,48 mM EDTA in PBS), when cells had reached a confluency of ~80%. HeLa, Caski, SW-480, SW-620,

Chapter 2

CaCo-2, HT-29, HCT-116 were cultured in DMEM supplemented with 10% FCS, 100 units/mL penicillin and 100 µg/mL streptomycin. All stably transfected HeLa cells were maintained in DMEM supplemented with 10% FCS, 100 units/mL penicillin, 100 µg/mL streptomycin and 700 µg/mL G418 (Geneticin). The prostate cancer cell lines 22Rv1, DU-145, PC3, LNCaP, RWPE-1 and RWPE-2 were maintained in RPMI-1640 medium supplemented with 10% FCS, 100 units/mL penicillin and 100 µg/mL streptomycin. All cell lines were periodically tested for Mycoplasma contamination and were regularly subjected to examination by light microscopy to assess cell morphology, cell viability and potential contaminations. No contamination with mycoplasma or contamination of any other kind was detected during the period of this research project.

2.2.2 Cell counting

Cells were grown to a confluency of ~80%, deadhered with Versene (5 mL for a T75 flask or 2.5 mL for a T25 flask), pelleted at 200 x g for 5 minutes and resuspended in medium (10 mL for a T75 flask or 5 mL for a T25 flask). For cell counting the cell solution (200 µL) was transferred into an Eppendorf tube and 200 µL solution A-100 (pH 1.25, cell membrane disruption and disaggregating solution containing propidium iodide [PI] for DNA staining) was added and vortexed for 5 seconds followed by addition of 200 µL solution B (pH raising solution for effective staining by PI) and another vortexing step. This solution was immediately transferred into a nucleocounter cassette and analysed on a Nucleocounter NC-100 (chemoMetec; Allerød, Denmark). The detected amount of cells per mL was calculated according to the dilution factor and the cell number was adjusted to meet experimental requirements.

Chapter 2

2.2.3 Plasmid DNA isolation, purification and storage

For the isolation and purification of DNA, which was used for transfection of HeLa cells, a QIAprep Spin Miniprep kit was used following the instructions of the manufacturer. The quality of the plasmid DNA was assessed by agarose gel electrophoresis and the DNA concentration was determined using a Nanodrop spectrophotometer device (Thermo Fisher Scientific). The isolated plasmid DNA was stored in aliquots at -80°C until transfections were performed.

2.2.4 Agarose gel electrophoresis

DNA was separated according to size in a 1% (w/v) agarose gel. Ethidium bromide solution was added to a final concentration of 0.5 µg/mL prior to casting of the gel. DNA samples were diluted 1:5 in loading dye (Section 2.1.8), loaded on the gel and separated in TAE buffer (Section 2.1.8) at 90 volts for 45-90 minutes depending on fragment size. The DNA bands were visualised in darkness using an ultraviolet (UV) light transilluminator and photographed using a Syngene UV system (Gene Works; Adelaide, Australia).

2.2.5 Generation of the CDCP1-FLAG expression construct in vector pcDNA 3.1

The CDCP1 DNA sequence was cloned from DNA reverse transcribed from total RNA isolated from the prostate cancer cell line PC3. The primers used for the PCR were 5’-gaattcGGCCGAGGCGTCCCGAG-3’ (lower case letters indicate an EcoRI site used for cloning) and 5’-ctcgagTTACTTGTCGTC ATCGTCCTTGTAGTC TTCTGCTGGCTCCATGGGCTCC-3’ (lower case letters indicate a Xho1 site used for cloning; underlined is DNA encoding a FLAG epitope (DYKDDDDK)). This work was performed by Dr Nigel Bennett. Constructs encoding CDCP1-FLAG-Y734F, -Y743F or -Y762F (Figure 2.2) were generated by site directed mutagenesis using Pfu Ultra DNA

Chapter 2

polymerase. All constructs were sequenced to confirm validity. This work was performed by Dr MayLa Linn. These CDCP1-FLAG expression constructs are referred to as CDCP1, CDCP1-Y734F, CDCP1-Y743F and CDCP1-Y762F.

2.2.6 Transfection of HeLa cells with CDCP1 expression constructs

HeLa cells were seeded in antibiotic free DMEM in 6 well plates to allow for 80% confluency at the start of the transfection. The next day, two Eppendorf tubes were filled with 750 µL of Opti-MEM medium and 1 µg of purified DNA was added to one tube and 3 µL of Lipofectamine was added to the other tube and incubated for 5 minutes at room temperature. After five minutes, the contents of the two tubes were combined, mixed by pipetting and incubated for 15 minutes at room temperature. During the incubation time, cells were washed once with 3 mL Opti-Mem and 1.5 mL Opti-MEM was added into each well followed by addition of 1.5 mL Opti-MEM/DNA/Lipofectamine solution. After 8 hours, the Opti-MEM/DNA/Lipofectamine solution was removed and 3 mL of antibiotic free DMEM supplemented with 10% FCS was added and the cells were maintained for 24 hours at 37°C before further experimental procedures were performed.

Chapter 2

Figure 2.2: CDCP1 DNA sequence and illustration of site directed mutagenesis to create CDCP1 tyrosine mutants. Shown is the CDCP1 DNA sequence starting at the transmembrane domain and encoding amino acid position 666 through to the end of the CDCP1 protein and the attached FLAG tag after amino acid position 836. Illustrated are the codons which were altered by site directed mutagenesis to encode phenylalanine to create the CDCP1 tyrosine mutant constructs. Underlined after amino acid position 836 is the FLAG tag and * represents the stop codon.

Chapter 2

2.2.7 Selection of transfected cells in selection media

The media of the transfected cells were removed and replaced with DMEM containing 700 µg/mL of G418. Media were replaced every two days. After ten days, the G418 resistant cell populations were subjected to further experimental procedures.

2.2.8 Generation of monoclonal HeLa cells stably expressing CDCP1, CDCP1-Y734F, -Y743F or -Y762F by isolating single cell clones manually from a culture dish

After selection in G418 containing media, the cells were deadhered, counted, pipetted multiple times to achieve single cell suspension and 100 cells were seeded in 10 cm diameter culture dishes in 10 mL of G418 (700 g/ml) containing DMEM supplemented with 10% FCS. The cells were allowed to form colonies for 10-14 days until these were visible by eye. Colonies were isolated using a 200 µL pipette tip, disaggregated by pipetting multiple times, transferred into wells of a 24 well plate and then serially expanded into a T25 flask format. The cell surface expression of CDCP1 was then assessed by flow cytometry.

2.2.9 Flow cytometric analysis of CDCP1 cell surface expression levels

Cells were grown to a confluency of 70-90%, deadhered and 1x10 6 cells were incubated for 30 minutes on ice with 50 µL of MAb 10D7 in PBS (3 µg/mL). Cells were washed with 5 mL cold PBS, centrifuged for 5 minutes at 200 x g at 4°C and the cells were incubated in 50 µL of a 1:750 dilution of a goat anti-mouse Alexa Fluor 488 secondary antibody in PBS for 30 minutes on ice. The cells were washed with 5 mL cold PBS and pelleted by centrifugation at 200 x g at 4°C for 5 minutes. The supernatant was discarded and the cell pellet was resuspended in 500 µL PBS and a

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minimum of 10,000 cells were analysed using a Cytomics FC-500 MPL flow cytometer (Beckman Coulter; Gladesville, Australia).

2.2.10 Generation of monoclonal HeLa cells stably expressing CDCP1, CDCP1-Y734F, -Y743F or -Y762F by fluorescence microscopy

After selection in G418 containing media, the cells were deadhered and counted followed by seeding cells into 96 well plates by limiting dilutions to a theoretical cell number of 0.1 cells per well. Wells containing a single cell were identified by light microscopy and marked 3 hours after cell seeding. The cells were allowed to form colonies for 10-14 days while changing the media every 3-4 days. In order to identify CDCP1 expressing monoclonal populations, the media of the 96 well plates was removed and the wells which were identified to contain a monoclonal cell population were incubated with 5 µg/mL of the anti-CDCP1 antibody 41-2 in PBS for 15 minutes at room temperature. After 15 minutes, the antibody/PBS solution was removed, the cells were washed once with 200 µL PBS and stained with a goat anti-mouse Alexa Fluor 488 conjugated secondary antibody in PBS (1:750 dilution) for 15 minutes at room temperature. The secondary antibody solution was removed, the cells were washed once with 200 µL PBS then 200 µL phenol- red free DMEM supplemented with 10% FCS was added. The cell surface expression of the clones was immediately assessed by fluorescence microscopy using an excitation wavelength of 485 nm and emission was detected at 525 nm. Clones showing a positive stain for CDCP1 were serially expanded into a T25 flask format.

2.2.11 Generation of polyclonal HeLa cells stably expressing CDCP1, CDCP1-Y734F, -Y743F or -Y762F by fluorescent activated cell sorting (FACS)

HeLa cells were transfected in a T75 flask and stable expressing cells were selected for 10 days in G418 containing media. The cells were deadhered,

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pelleted by centrifugation at 200 x g for 5 minutes, resuspended in PBS (1 mL) supplemented with 5 µg/mL MAb 41-2 and incubated on ice for 20 minutes. After incubation, cells were washed with 10 mL of ice cold PBS and centrifuged at 200 x g for 5 minutes at room temperature. The cells were resuspended in a solution containing a goat anti-mouse Alexa Fluor 488 antibody in PBS (1:750 dilution) and incubated for 20 minutes on ice followed by another washing step with PBS (10 mL) and centrifugation at 200 x g for 5 minutes at room temperature. The cells were then resuspended in PBS (1 mL) supplemented with 2% FCS and transported on ice to the cell sorting facility at the Princess Alexandra Hospital in Brisbane. Cells (15,000) displaying a mean fluorescence intensity (MFI) of greater than 40 were collected into 5 mL sterile cell culture tubes at room temperature in G418 containing DMEM. The cells were serially expanded into a T25 flask format.

2.2.12 Generation of monoclonal HeLa cells stably expressing CDCP1- FLAG, CDCP1-Y734F, -Y743F or -Y762F by FACS

After selection in G418 containing media, the cells were deadhered, pelleted at at 200 x g for 5 minutes at room temperature and the pellet was resuspended in 1 mL PBS supplemented with 5 µg/mL of MAb 41-2 and incubated on ice for 20 minutes. The cells were washed with cold PBS (10 mL), centrifuged at 200 x g for 5 minutes at room temperature and the pellets were resuspended in a goat anti-mouse Alexa Fluor 488 secondary antibody solution in PBS (1:750 dilution), incubated for 20 minutes on ice followed by another wash step with cold PBS (10 mL) and centrifugation at 200 x g for 5 minutes at room temperature. The cells were resuspended in PBS (1 mL) supplemented with 2% FCS and transported on ice to the cell sorting facility at the Princess Alexandra Hospital. CDCP1 expressing single cells displaying an MFI>50 were sorted in 96 well plates at room temperature and serially expanded into a T25 flask format.

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2.2.13 Generation of whole cell lysates

Cell culture media was removed, the cells were washed once with cold PBS before addition of lysis buffer supplemented with protease inhibitor cocktail (Section 2.1.8), and, if the phosphorylation state of a protein was to be investigated, 2 mM Na 3VO4 and 10 mM NaF was also added. The cells were lysed for 20 minutes on ice and vortexed every 5 minutes. Whole cell lysates were cleared by centrifugation at 18,000 x g at 4°C for 20 minutes and transferred into a new Eppendorf tube. The protein concentration of lysates was determined using a microbicinchoninic acid (BCA) assay kit.

2.2.14 Determination of protein concentration with a microbicinchoninic acid (BCA) kit

BCA standards were prepared in ddH 2O at concentrations of 2, 1, 0.5, 0.25, 0.125 and 0.063 mg/mL, aliquoted into 25 µL aliquots and stored at -80°C until used. Whole cell lysates were diluted 1:10 in ddH 2O and 20 µL of the sample, standards and a water control were added in duplicates into the wells of a 96 well plate and 200 µL of freshly prepared working solution (solution A 50:1 solution B, supplied in the BCA kit) was added to the wells and incubated at 37°C. After a 30 minute incubation period plates were allowed to cool to room temperature and absorbance was detected at 560 nm using a Benchmark Plus microplate spectrophotometer (Bio-Rad; Sydney, Australia).

2.2.15 Immunoprecipitation

Total protein (500 µg) was pre-cleared for 2 hours at 4°C with end to end rotation with 40 µL of pre-washed immobilised Protein A or G beads, depending on the antibody species used for the immunoprecipitation. The protein beads were pelleted for 1 minute at 830 x g at 4°C and the pre- cleared lysates were transferred into a new Eppendorf tube and 5 µg MAb 10D7 or 3 g anti-FLAG antibody were added for the immunoprecipitation.

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After incubation overnight at 4°C with end to end r otation, 40 µL of pre- washed immobilised protein beads were added and incubated at 4°C for 4 hours with end to end rotation. The protein beads were pelleted by centrifugation at 830 x g for 30 seconds, the supernatant was discarded and 750 µL lysis buffer (Section 2.1.8) was added followed by another centrifugation step. This washing step was repeated twice more and the samples were either stored at -80°C for future West ern blot analysis or 30 µL of 2 x SDS loading dye (Section 2.1.8) was added immediately, the samples boiled for 10 minutes and loaded on a SDS-polyacrylamide gel for Western blot analysis (Section 2.2.16).

2.2.16 Western blot analysis

Immunoprecipitated proteins or total proteins (10 to 50 µg), depending on the antibody used for analysis and as stated in the figure legend, were combined with 6 x reducing SDS-loading buffer (Section 2.1.8) to dilute the loading buffer to 1 x concentration in the sample of interest. If the anti-CDCP1 antibodies 41-2 or 10D7 were used for analysis, the loading dye did not contain β-mercaptoethanol. The sample were boiled for 10 minutes at 95°C, briefly centrifuged in a benchtop centrifuge and loaded into the wells of a 10 or 15 well SDS-polyacrylamide gel. The proteins were separated according to size under a constant voltage of 90-115 volts until the dye front ran off the bottom of the gels in a Biorad Western blot apparatus containing 1 x running buffer (Section 2.1.8).

The transfer apparatus was assembled using two Western blot sponges, two pieces of blotting paper and one piece of nitrocellulose membrane pre- soaked in 1 x transfer buffer (Section 2.1.8). One gel at a time was placed in between the nitrocellulose membrane and one layer of blotting paper and Western blot sponge on either side, locked into a transfer cassette and placed into a BioRad transfer apparatus. A cooling block was placed into the transfer tank and the proteins were transferred in 1 x transfer buffer onto the

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nitrocellulose membrane overnight at 40 volt or for 3 hours at 90 volts in the cold room.

After transfer, the membranes were blocked with 5% skim milk or 5% bovine serum albumin (BSA) overnight or for 90 minutes at room temperature on a rotational shaker. The membranes were incubated with the primary antibodies for 90 minutes at room temperature or overnight at 4°C on a rotational shaker. Primary antibodies were used at dilutions recommended by the manufacturer and MAb 10D7 was used at a concentration of 5 g/ml in 1% skim milk TBST. After primary antibody incubation, the membranes were washed 4 x 5 minutes at room temperature with PBS or TBS-T, according to the instructions of the antibody manufacturer. The membranes were then incubated with an appropriate species specific secondary HRP- conjugated antibody for 90 minutes at room temperature (1:3000 dilution in the same buffer as the primary antibody). The membranes were washed again 4 x 5 minutes and protein bands were visualised using Femto super sensitive HRP substrate according to the instructions of the manufacturer and exposed to X-ray films (Imaging Solutions; Underwood, Australia) which was developed using an AGFA CP 1000 automated X-ray film processor (Agfa-Gevaert Ltd .; Scoresby, Australia). To examine total protein loading Western blot analysis was performed using anti-GAPDH or anti-tubulin antibodies according to the instructions of the manufacturer.

2.2.17 Isolation of genomic DNA from HeLa cells expressing CDCP1 (HeLa-CDCP1-S) generated by Deryugina et al. or stably transfected with empty Vector (HeLa-vector-S)

Cells were grown to a confluency of 70-90% in T75 flasks, deadhered and pelleted by centrifugation at 200 x g for 5 minutes at room temperature. Cell pellets were resuspended in 10 mL cold PBS and again centrifuged at 200 x g for 5 minutes. This washing step was repeated once more. Cell pellets were resuspended in 300 µL Tris-EDTA-Saline (TES) and 20 µL of a 20% SDS solution and 50 µL proteinase-K (1 mg/mL) was added and the cells were incubated overnight at 37°C. The next day 100 µL 5 M NaCl was added 45

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and the solution was vortexed vigorously for 15 seconds followed by centrifugation at 575 x g for 15 minutes. The supernatant was transferred into a new Eppendorf tube and the DNA was precipitated with 2 x volumes of 100% ethanol and looped out using a 200 µL pipette tip. The DNA was transferred into an Eppendorf tube containing 1 mL 70% ethanol and pelleted by centrifugation at 12000 x g for 5 minutes. The ethanol solution was removed, the pellet was briefly air dried and resuspended in PCR grade H 2O.

2.2.18 PCR amplification of the CDCP1 gene from HeLa-CDCP1-S cells

The following reagents were combined in a sterile 200 µL PCR reaction tube: 0.25 µL Pfu Ultra , 5 µL of 10 x PCR buffer including Mg 2+, 100 ng genomic DNA, 1 µL of 10 mM dNTP mix (0.2 mM), 2.5 µL 10 µM primer mixture (0.5 µM) and PCR-grade water was added to a total volume of 50 µL. The specifications of the CDCP1 specific primer and the vector specific primer which were used in this PCR reaction to amplify CDCP1 are described in Table 2.2. The binding site of the BGH-rev primer within the multiple cloning site of the pcDNA3.1 vector is shown in Figure 2.3.

CDCP1 was amplified by using a touch down PCR protocol which included DNA denaturation at 95°C for 30 seconds, primer ann ealing at 60°C for 30 seconds and an elongation step at 72°C for 1 minute . The primer annealing temperature was continuously decreased every 3 cycles in one degree steps to a temperature of 55°C. An additional 15 cycles w ere performed at 55°C and the PCR reaction volume was kept at 4°C overnig ht. The resulting PCR product was purified with the Wizard PCR product purification kit according to the instructions of the manufacturer and the DNA concentration of the purified PCR product solution was determined. A small amount (3 µL) was subjected to agarose gel electrophoresis, as described in Section 2.2.4. Two negative controls were used for this PCR reaction; one reaction did not contain genomic DNA and the other contained genomic DNA isolated from HeLa-vector-S cells.

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Figure 2.3: Multiple cloning site of the pcDNA 3.1 vector and illustration of the binding site of the BGH-rev primer. Sequence of the multiple cloning site of the pcDNA 3.1 vector with the target sequence of the BGH-rev primer underlined in red (modified from http://tools.invitrogen. com/ content/ sfs/ manuals/ pcdna3.1_man.pdf).

2.2.19 Determination of DNA concentration of purified DNA

The DNA concentration was determined using a Nanodrop spectrophotometer device after water was used as a blank. The purity of the DNA was assessed by using the ratio of the absorbance of 260 nm / 280 nm, where a ratio of greater than 1.8 indicates that the DNA solution has a satisfactory purity and suitable for downstream applications.

2.2.20 Sequencing of CDCP1 DNA amplified from genomic DNA

Purified PCR product (50 ng) from the touchdown PCR reaction was combined with 9.6 pmol of the CDCP1-specific primer F-20 (Table 2.2) and the volume was adjusted to a total of 12 µL with PCR grade water. The sample was sent to the Australian Genome Research Facility in Brisbane where sequencing was performed. The primer used for this sequencing

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reaction binds to CDCP1 at nucleotide 2104 and allows coverage of DNA encoding CDCP1 amino acid positions 827-836 (Table 2.2).

2.2.21 Adhesion assay using CyQuant NF or crystal violet

Black walled 96 well plates were coated with 50 µL extracellular matrix (ECM) proteins diluted in PBS for 3 hours at room temperature. The following ECM components were used: laminin-1, collagen-I, collagen-IV and fibronectin at a concentration of 10 µg/mL and vitronectin at a concentration of 5 µg/mL. After three hours, the ECM/PBS solution was removed and the plate was washed twice with 200 µL/well of PBS. The plate was blocked for 1 hour at 37°C with 100 µL of 2% BSA/PBS solution and washed three times with 200 µL PBS. Cells were deadhered, washed with PBS, counted and the cell number was adjusted to 0.3x10 6 cells/mL in serum free DMEM. Cell suspensions (100 µL) were added to the wells of the plate and the cells were allowed to adhere for 30 minutes at 37°C.

If CyQuant NF was used for the adhesion assay, the cell solution was removed and the plate was washed three times with 200 µL PBS to remove unbound cells. CyQuant-NF solution was prepared according to the instructions of the manufacturer and 100 µL/well was added and incubated for 45 minutes at 37°C. The relative number of adhe red cells was assessed by detecting emission at 520 nm using a fluorescent plate reader. The CyQuant NF dye binds to cellular DNA and consequently the level of fluorescence at 520 nm after excitation at 485 nm is proportional to the number of cells per well in the linear range of the assay for any given cell line. Wells coated with ECM-protein solution without addition of cells and uncoated wells with addition of cells served as negative controls.

If crystal violet solution was used for the assay, the cell solution was removed, the plate was washed three times with 200 µL PBS and 100 µL of ice cold methanol was added and the cells were fixed for 15 minutes at room temperature. The methanol was aspirated and 100 µL/well of 0.5% crystal violet solution was added and incubated for 15 minutes at room temperature.

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The crystal violet solution was aspirated and the plate was washed six times with ddH 2O (250 µL) and the plate was briefly air dried at room temperature. A 10% acetic acid solution (100 µL/well) was added to the wells to extract the crystal violet dye and the relative amount of adhered cells was assessed by detecting the absorbance at 595 nm on a plate reader. The PBS used for the adhesion assays was without Ca 2+ and Mg 2+ .

2.2.22 Chemotactic migration

Cells were grown to a confluency of ~70-80%, then deadhered, washed with PBS, counted and the cell number adjusted to 1x10 6 cells/mL in serum free media. DMEM supplemented with 5% FCS (500 µL) was pipetted into each well of a 24 well plate and 8 µm Transwell inserts were then placed into the wells. Cell solutions (100 µL) were added onto the Transwell inserts and allowed to migrate towards the FCS gradient at 37°C . After 24 hours the media in the wells was collected into Eppendorf tubes and the cell culture inserts were placed into 500 µL of 0.05% trypsin solution for 20 minutes at 37°C. The underside of the insert was thoroughly wa shed with the trypsin solution and combined with the media in the Eppendorf tube and centrifuged at 830 x g for 5 minutes. The supernatant was carefully removed and the cell pellet was resuspended in 50 µL serum free media, thoroughly mixed by pipetting and 20 µL of the cell solution was transferred into a haemocytometer to count the number of migrated cells per well.

2.2.23 Haptotactic migration

ECM components were diluted to 10 µg/mL in PBS and 200 µL were added to the wells of a 24 well plate and 24 well plate Transwell inserts were placed into the wells to allow coating of the underside of the inserts for 3 hours at room temperature. The ECM components used were collagen-IV, fibronectin, laminin-1 and the basement membrane mimetics Matrigel at a concentration of 10 µg/mL. After the incubation period, the wells of a new 24 well plate

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were filled with 500 µL PBS and the inserts were dipped into two wells to wash the underside of the inserts. Cells were deadhered, washed with PBS, counted and the cell number adjusted to 1x10 6 cells/mL in serum free DMEM. The wells of a 24 well plate were filled with 500 µL of serum free DMEM and the coated 24 well plate Transwell inserts were placed into the wells. Cell solution (100 µL) was immediately added into the Transwell inserts and cells were allowed to migrate at 37°C. After 48 hours, the media in the wells was collected into Eppendorf tubes and the cell culture inserts were placed into 500 µL of 0.05% trypsin solution for 20 minutes at 37°C. The underside of the insert was thoroughly washed with the trypsin solution and combined with the media in the Eppendorf tube and centrifuged at 830 x g for 5 minutes at room temperature. The supernatant was carefully discarded and the cell pellet was resuspended in 50 µL serum free media and the total number of migrated cells was counted using a haemocytometer.

2.2.24 Proliferation assay

Cells were grown to a confluency of 70-80%, then deadhered, washed with PBS, counted and the cell number adjusted to 3.5x103 cells/mL in DMEM supplemented with 10% FCS. Cell solutions (200 µL) were added into the wells of a black 96 well plate. After 24, 48, 72, 96 and 120 hours, one plate was removed from the incubator, the media was aspirated and 100 µL of CyQuant NF solution was added to the wells and incubated for 45 minutes in the cell culture incubator. To ensure exponential growth of cells prior to measurement the cell confluency in the wells was assessed by phase contrast microscopy to confirm confluency of <90% before addition of CyQuant NF. The relative number of cells per well was assessed by detecting the emission at 520 nm using a fluorescence plate reader.

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2.2.25 Cell line authentication testing

Cells were allowed to grow to a confluency of ~80% in a T75 flask before being deadhered, washed twice with PBS and pelleted by centrifugation at 200 x g for 5 minutes at room temperature. The cell pellet was snap frozen in liquid nitrogen and sent on dry ice to the Australian Cell Bank Facility where examination of cell line authenticity was performed. A short tandem repeat sequence (STR) profile was generated by amplifying polymorphic repeat regions of the genomic DNA and the PCR product was separated by gel electrophoresis alongside with size standards. The size of the PCR product of the amplified regions is unique for each cell line and was used to confirm the authenticity of the cell lines (Masters et al., 2001; Parson et al., 2005).

2.2.26 Chicken embryo chorioallantoic membrane (CAM) cell dissemination assay

Fertilized white Leghorn eggs (Charles River, North Franklin, USA) were incubated in a humidified rotary incubator at 38°C for 12 days. The position of the chorioallantoic membrane (CAM) vein was assessed and marked with a pencil by holding the egg against a light source and a small area of the egg shell was removed carefully using a MultiPro rotary tool (Dremel; Racine, USA), without causing a haemorrhage on the membrane to allow best possible visibility of the vein through the window of the egg shell. Cells were grown to a confluency of ~80% then deadhered, washed with PBS, counted and the cell number adjusted to 1x10 6 cells/mL in serum-free DMEM. Cell solutions (50 µL) were injected into the CAM vein using a 1 mL syringe and a 30 g needle. In between injections, the syringe was turned upside down to ensure distribution of cells within the media/cell solution. The embryos were then incubated in a humidified stationary incubator at 38°C. Five days after the injection of the cells, the chicken embryos were sacrificed and three portions of the lower CAM were harvested, distant from the side of cancer cell injection. The CAM portions were washed with PBS, transferred into pre-

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labelled Eppendorf tubes, placed on dry ice and stored at -80°C for later isolation of genomic DNA as described below.

These experiments were performed in the laboratory of Prof James Quigley at the Scripps Research Institute in La Jolla, California, USA.

2.2.27 Inhibition of metastasis formation in the chicken embryo CAM assay with anti-CDCP1 antibodies

The experimental procedures were identical to those described in the previous paragraph (Section 2.2.26) with the only modification that the cells were incubated for 45 minutes with 50 µg/mL of the anti-CDCP1 antibodies 41-2 (Brooks et al., 1993; Deryugina et al., 2009), 10D7 (Deryugina et al., 2009) or control mouse IgG (Jackson ImmunoResearch; West Grove, USA) in serum free DMEM on ice before injection of the cells into the CAM vein.

These experiments were performed in the laboratory of Prof James Quigley at the Scripps Research Institute in La Jolla, California, USA.

2.2.28 Genomic DNA isolation and real-time Alu PCR for quantitative detection of human tumour cells in the CAM

Harvested portions of the lower CAM were homogenised using a tissue homogeniser (IKA; Wilmington, USA) and genomic DNA was extracted using the Puregene DNA purification kit according to the instructions of the manufacturer. Human Alu sequences were amplified by real-time PCR using 30 ng of genomic DNA as template in a 10 µL reaction containing 2 mmol/L

MgCl 2, 200 µm NTP mixture, 0.4 unit of Platinum Taq polymerase, 1:105 dilution of SYBR Green dye, and 0.4 µm of each Alu sense and Alu antisense primer. PCR conditions included polymerase activation at 95°C for 4 minutes followed by 30 cycles at 95°C for 30 seco nds, 63°C for 30 seconds, and 72°C for 30 seconds. The specification s of the primers used are presented in Table 2.2. Each assay included a negative control (water), a

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positive control (human genomic DNA), and the experimental samples in duplicate. To determine the number of tumour cells present in each tissue sample a standard curve was generated by quantitative amplification of genomic DNA extracted from a serial dilution of HEp3 cells ranging from 1x10 3 to 1x10 5 cells mixed with 1x10 6 chicken embryo fibroblasts (Zijlstra et al., 2002).

These experiments were performed in the laboratory of Prof James Quigley at the Scripps Research Institute in La Jolla, California, USA.

2.2.29 Incubation of cells with the SFK-selective inhibitor SU6656

Cells were grown to ~80% confluency, and then treated with SU6656 (Blake et al., 2000) diluted in DMEM supplemented with 10% FCS to final concentrations of 0.1, 0.27, 1, 2.7 and 10 µM for 60 minutes at 37°C. After the incubation period the DMEM/SU6656 solution was removed, the cells were washed once with cold PBS and whole cell lysates were prepared in the presence of phosphatase inhibitors and analysed by Western blot analysis (Section 2.2.16).

2.2.30 Incubation of cells with SU6656 to examine the effect on the morphology of HeLa cells stably expressing CDCP1

Cells were deadhered, washed with PBS, counted and seeded at 2x10 5 cells in T25 flasks. The cells were allowed to adhere to the cell culture plastic for 30 minutes before addition of SU6656 solution to a final concentration of 5 µM or 0.018% DMSO (v/v) as vehicle control followed by a 48 hour incubation period in the cell culture incubator. After 48 hours, the resulting cell morphology was assessed by phase contrast microscopy using an inverted microscope.

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2.2.31 Matriptase treatment of cells

Cells were seeded at 1.6x10 5 per well for HeLa-CDCP1 or 2.5x10 5 per well for 22Rv1 cells in 12 well plates and allowed to attach for 12 hours in DMEM supplemented with 10% FCS in the cell culture incubator. After 12 hours, the media was removed, the cells washed once with serum free DMEM and 500 µL of serum free DMEM was added and the cells were serum starved for 24 hours. The next day aliquots of matriptase in serum free DMEM were prepared to final concentrations of 1, 5 or 10 nM. Culture media was removed and cells were washed once with fresh serum-free media and one aliquot of serum-free media containing matriptase was added to the cells and incubated for 1, 5, 10 or 30 minutes at 37°C. At ea ch time point the DMEM/matriptase solution was removed and cells washed once with serum free DMEM. Whole cell lysates were then prepared in the presence of protease and phosphatase inhibitors and analysed by Western blot analysis (Section 2.2.16).

2.2.32 Measurement of changes in intracellular Ca 2+

Cells were grown to a confluency of ~80%, washed once with PBS, detached, resuspended at 4x10 6 cells/mL in extracellular medium (Section 2.1.8) containing 0.2% (w/v) BSA and then loaded with the fluorescence indicator Fura-2 acetoxymethyl ester (1 µM) at 37°C for 60 minutes. The cells were then pelleted by centrifugation at 150 x g for 5 minutes followed by resuspension in extracellular medium (without BSA) at a concentration of 2x10 6 cells/mL for fluorescence measurements. The ratio of fluorescence at 510 nm after excitation at 340 and 380 nm was monitored using a fluorescence plate reader. Agonist treatments were trypsin (10 nM), matriptase (20 nM) and PAR2-AP (AP; SLIGKV) (100 µM).

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2.2.33 Confocal microscopy

HeLa cells stably expressing CDCP1, CDCP1-Y734F, -Y743F or -Y762F were allowed to adhere at 37°C to sterile cover sli ps in wells of a 24 well plate in DMEM supplemented with 10% FCS. After two days the media was removed, the cells were washed with cold PBS then fixed with 4% paraform- aldehyde in PBS for 15 minutes at room temperature followed by two washing steps with 500 µL PBS. The cells were then permeabilised with 0.1% Triton X-100 in PBS for 15 minutes at room temperature while gently agitating. After permeabilisation, the Triton X-100 solution was removed, washed twice with 500 µL PBS then blocked with 500 µL of 0.5% BSA/PBS solution for 15 minutes at room temperature. The BSA/PBS solution was removed, the cover slips washed three times with 500 µL PBS and incubated with the mouse anti-CDCP1 antibody 41-2 (5 µg/mL) for 45 minutes at room temperature followed by incubation with the rabbit anti-Src antibody (1:100 dilution). The cells were washed twice with PBS and incubated with species specific Alexa Fluor conjugated secondary antibodies (1:750 dilution) for 30 minutes at room temperature followed by two more washing steps with 500 µL PBS. The cells were then incubated with Alexa Fluor 488 phalloidin and 4',6-diamidino-2-phenylindole (DAPI) (1:1000 dilution) in PBS for 10 minutes at room temperature to stain actin and the nucleus, respectively. Cells were washed two more times with PBS, the cover slips were allowed to air dry for 15 seconds then mounted on glass slides using Pro-long gold. Cover slips incubated with the secondary antibodies, Alexa Fluor 488 phalloidin and DAPI only were prepared to assess non-specific binding of the secondary antibodies. Cells were imaged using a Leica SP5 confocal microscope (Leica Microsystems; Sydney, Australia). Images were processed and displayed using Corel Draw (Corel Pty Ltd.; Sydney, Australia).

2.2.34 Cell surface biotinylation

Cells were grown to a confluency of ~80% in T75 flasks, washed three times with 10 mL of ice cold PBS then incubated on ice in 1.5 mL solution of PBS

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containing NHSSS-biotin at 1.22 mg/mL. The cells were incubated with the NHSSS-biotin solution for 1 hour at 4°C on a rotati onal shaker. After the incubation period, the cells were washed three times with 10 mL ice cold PBS then lysed with 500 µL biotinylation lysis buffer (Section 2.1.8). The lysate was transferred into an Eppendorf tube, centrifuged at 100 x g for 1 minute at 4°C to remove intact cells and the supern atant was carefully transferred into a new Eppendorf tube and an aliquot of the lysate was collected for future analysis. Streptavidin beads (50 µL) were added to the cleared lysates, incubated on ice for 15 minutes while vortexing the solution every 5 minutes then centrifuged at 1800 x g for 5 minutes at 4°C. The supernatant (cytosolic fraction, 25 µL) was transferred into a new Eppendorf tube and 5 µL of 6 x SDS loading dye (Section 2.1.8) was added. SDS loading dye (30 µL diluted to 1 x in ddH 2O) was added to the streptavidin bead pellet (biotinylated fraction) and the samples were boiled at 95°C for 10 minutes before loading on a SDS-polyacrylamide gel for Western blot analysis (Section 2.2.16).

2.2.35 Data collection and statistical analysis of in vitro and in vivo data

In vitro experiments : All experiments were performed a minimum of three times in replicates. The numbers of replicates per experiment and the number of performed experiments are indicated in the figure legend. Data are presented as mean of the means of the replicates per experiment ± SEM. P-value was calculated using the Student’s t-test with a 95% confidence interval using the means of the replicates for each experiment. P- values <0.059 are indicated in the figure and p-values <0.05 are considered statistically significant.

In vivo experiments : All experiments were performed at least twice with a minimum of 6 embryos per experiment and cell line or treatment group. The number of performed experiments is indicated in the figure legend and the total numbers of embryos are indicated in the bars of the figures. Data are presented as mean ± SEM calculated from data from pooled experiments. P- value was calculated using the Student’s t-test with a 95% confidence

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interval of pooled experiments using the number of cancer cells detected in the chorioallantoic membrane (CAM) in each embryo and cell line or treatment group. P-values <0.059 are indicated in the figure and p-values <0.05 are considered statistically significant.

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Generation and characterisation of HeLa cells stably expressing CDCP1 Chapter 3

3.1 INTRODUCTION

The cell surface glycoprotein CDCP1 has gained attention in recent years because of its role in metastasis formation in in vivo models (Uekita et al., 2007; Siva et al., 2008b; Uekita et al., 2008; Deryugina et al., 2009; Fukuchi et al., 2010), its potential as a prognostic marker in lung, kidney, endometrioid and pancreatic cancer (Awakura et al., 2008; Ikeda et al., 2009; Mamat et al., 2010; Miyazawa et al., 2010) and its interaction with the cell signalling proteins PKC δ (Benes et al., 2005; Bhatt et al., 2005; Uekita et al., 2007) and SFKs (Hooper et al., 2003; Brown et al., 2004; Bhatt et al., 2005; Uekita et al., 2007). CDCP1 contains features potentially relevant in mediating protein-protein interactions such as 14 N-glycosylation sites, three domains with low homology to CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five conserved intracellular tyrosine residues (Scherl-Mostageer et al., 2001; Hooper et al., 2003; Brown et al., 2004; Bhatt et al., 2005). Little is known about whether these features contribute to the role CDCP1 plays in metastasis. One of the most interesting aspects is the tyrosine phosphorylation-dependent binding of PKC δ and SFKs.

Hooper et al. were the first to show that CDCP1 is phosphorylated by SFKs by employing the SFK-selective inhibitor PP2 which causes reduction in phosphorylation of CDCP1 (Hooper et al., 2003). This finding was confirmed by Brown et al. and Bhatt et al. who provided evidence that SFKs phosphorylate CDCP1 and that this phosphorylation can be inhibited by employing the SFK-selective inhibitors PP2 and SU6656 (Brown et al., 2004; Bhatt et al., 2005). The first report providing insight into how the interaction between SFKs and CDCP1 occurs was provided by Benes et al. This group employed tyrosine mutants of CDCP1-Y734, -Y743 and -Y762 and kinase domain defective constructs of PKC δ and Src to show that SFK-mediated phosphorylation of CDCP1 is required for formation of a SFK•CDCP1•PKC δ multiprotein complex. These authors proposed that SFK phosphorylation of CDCP1 is initiated at Y734 resulting in SFK binding at this site, promoting additional phosphorylation at Y743 and Y762, and PKC δ recruitment at CDCP1-Y762; this was the ﬁrst demonstration of PKC δ as a

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phosphotyrosine-binding protein (Benes et al., 2005). This proposal was underpinned by data showing that mutation of CDCP1-Y734 abolishes SFK binding to CDCP1 as well as PKC δ binding and that a single mutation of Y762 leads to a decrease in binding of PKC δ to CDCP1. In contrast, the mutation of CDCP1-Y743 did not have an effect on interactions between these three proteins.

Uekita et al. reported that SFK-mediated phosphorylation of CDCP1 confers resistance in lung adenocarcinoma cells in vitro to a type of apoptosis, anoikis, initiated by loss of cell contact with the ECM (Uekita et al., 2007). The formation of the trimeric protein complex Src•CDCP1•PKC δ and the phosphorylation of PKC δ-Y311 was shown to be crucial for the anti-apoptotic role mediated by CDCP1 in A549 lung cancer cells (Uekita et al., 2007). For example, this group reported that CDCP1 phosphorylation increases strongly after cells were grown in suspension for a period extending up to 48 hours. This phosphorylation of CDCP1 initiated by the loss of anchorage signalling decreases to a low basal phosphorylation level shortly after the cells were allowed to re-adhere to the cell culture plastic. In addition, mutation of CDCP1-Y734, as well as knock down of CDCP1, leads to a significant increase in apoptosis of cells grown in suspension (Uekita et al., 2007). More relevant to an in vivo situation is the finding that phosphorylation of CDCP1- Y734 is increased in tumour nodules of gastric cancer 44As3 cells during peritoneal invasion in mice, as is the phosphorylation of SFK-Y416 in these cells (Uekita et al., 2008).

Despite the insights gained by these groups into the functional role mediated by the tyrosine phosphorylation sites of CDCP1, little data is available on the role of these tyrosine residues in influencing the cellular phenotype in vitro. Furthermore, no work has been done to explore the roles of these tyrosine residues in metastatsis in in vivo models.

To increase the understanding of the functional relevance of the tyrosine residues of CDCP1 in mediating distinct cellular phenotypes, one aim of this PhD project was to generate cells stably expressing CDCP1 or CDCP1 mutated to phenylalanine at Y734, Y743 or Y762. For the purpose of this

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study, the aim was to stably express CDCP1 in a cell line that has undetectable levels of endogenous CDCP1. After screening of a range of cell lines by Western blot analysis, HeLa cells were selected for stable expression of CDCP1 or the mutant CDCP1-Y734F, CDCP1-Y743F or CDCP1-Y762F expression constructs. This chapter explores the effects of this stable expression on HeLa cells and the impact of mutation of Y734, Y743 and Y762 on interactions of CDCP1 with its binding partners. In Chapter 4, these cells are used to explore the effect of proteolytic processing of CDCP1 by matriptase and Chapter 5 examines the effect of CDCP1 expression on the cellular phenotype in vitro and in vivo .

3.2 RESULTS

3.2.1 Generation of HeLa cells stably expressing CDCP1 using four different approaches

To identify human cell lines which lack CDCP1 expression and can be used as a cell line for stable transfection with CDCP1, a panel of prostate, colon, cervical and kidney cancer cell lines were screened by Western blot analysis. As shown in Figure 3.1, CDCP1 was detected in 17 of the 19 analysed cell lines. Cervical carcinoma HeLa and kidney carcinoma A704 cells did not show any detectable endogenous CDCP1. Of the 17 CDCP1 expressing lines, seven expressed LMW-CDCP1 in addition to HMW-CDCP1. Amongst these cells are PC3, LNCaP, and DU145 prostate cancer cells, HCT116 and CaCo2 colon cancer cells, CaSki cervical cancer cells, and HK-2 and ACHN kidney cells. It has been previously proposed that proteolytic processing by certain serine proteases including trypsin and plasmin may generate LMW- CDCP1 in vivo (Brown et al., 2004; Bhatt et al., 2005) . In Chapter 4, the ability of the serine protease matriptase as a proteolytic processor of CDCP1 in cancer cells will be examined.

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Figure 3.1: Examination of CDCP1 expression in human cell lines. A panel of prostate, colon, cervical and kidney cancer cell lines were passaged exclusively non-enzymatically and whole cell lysates were collected and analysed by Western blot analysis with an anti-CDCP1 antibody raised against the c-terminus of CDCP1. Anti-GAPDH Western blot analysis was performed to examine protein loading. The kidney cancer cell line blot was performed by Dr Yaowu He.

It was decided to utilise HeLa cells as a cell line for stable transfection with CDCP1 because in addition to their lack of CDCP1 expression they are a low metastatic cell line in the chicken embryo chorioallantoic membrane (CAM) intravasation assay (Zijlstra et al., 2002; Blancafort et al., 2005). This will be of advantage for later in vivo experiments when examining the effect of CDCP1 expression on metastasis formation since an increase in the ability of HeLa cells to metastasise due to CDCP1 expression should be readily detectable.

During the course of this project, four different methods were used to attempt to generate CDCP1 expressing HeLa cells as summarised in Table 3.1 . These approaches were:

• Manual isolation of CDCP1 expressing monoclonal cell populations. • Selection of clones by fluorescence microscopy. • FACS to select mixed polyclonal cell populations. • FACS to select monoclonal cell populations.

In the first approach, transfected HeLa cells, which had been selected for 10 days in selection media, were seeded in very low numbers in cell culture dishes, and single cell clones were manually isolated and expanded. This

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approach was chosen because it promised to lead to stable expressing monoclonal populations without the need to use FACS, which was not easily accessible to us at that time. However, using this methodology it was not possible to obtain HeLa cell clones expressing high or medium levels of CDCP1 (Table 3.1). Clones expressing low levels of CDCP1 or clones which displayed heterogeneous CDCP1 expression were identified, but these were not further characterised (data not shown). HeLa cells stably transfected with the empty vector were obtained by this approach.

Table 3.1: Summary of four approaches used and the number of clones screened to obtain HeLa cells stably expressing CDCP1. HeLa-vector cells are not shown in this table and five monoclonal empty vector expressing populations were obtained when clones were picked manually (1). MFI, mean fluorescence intensity; N/A, not applicable; –, numbers not listed.

Methodology Type of Clones Clones Clones Clones applied CDCP1 screened by screened obtained obtained construct fluorescence by flow expressing expressing microscopy cytometry high levels medium of CDCP1 levels of (MFI>115) CDCP1 (MFI>40 <115) 1. Manual selection of CDCP1 N/A 76 0 0 clones 2. Identification of CDCP1 expressing CDCP1 180 28 0 0 cells by fluorescence microscopy 3. Cell sorting for polyclonal CDCP1 N/A 12 0 0 populations All N/A 546 11 46 constructs CDCP1 N/A - 2 5 4. Cell sorting CDCP1 - N/A - 3 3 for monoclonal Y734F populations CDCP1 - N/A - 3 19 Y743F CDCP1 - N/A - 3 19 Y762F

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In the second approach, colonies were screened by fluorescence microscopy to identify CDCP1 expressing HeLa cells. In this approach, cells were plated by limiting dilutions into 96 well plates. To identify CDCP1 expressing HeLa cell clones, colonies were stained in the 96 well plates with an anti-CDCP1 antibody and an Alexa Fluor 488 labelled secondary antibody, followed by visualisation of CDCP1 positive clones by fluorescence microscopy (Figure 3.2A and B). This approach was chosen with the aim to identify CDCP1 expressing clones at an early stage which eliminates the need to invest effort and time in expanding cell clones expressing the resistance marker, but not CDCP1. Using this methodology, some low CDCP1 expressing clones (data not shown) and clones which did not display homogenous expression were identified, as suggested by dual peaks apparent from flow cytometry analysis (an example is shown in Figure 3.2C).

Figure 3.2: Selection of HeLa cells stably transfected with the CDCP1 expression construct by fluorescence microscopy yields cell populations with mixed CDCP1 expression. HeLa cells were transfected with the CDCP1 construct and seeded into 96 well plates by limiting dilution in selection media. After 14 days, the clones were assessed by light- and fluorescent-microscopy as well as by flow cytometry. A, C lone F4 after 14 days of growth in a 96 well plate assessed by light microscopy. B, Clone F4 was stained with the anti-CDCP1 antibody 10D7 and an appropriate Alexa Fluor 488 labelled secondary antibody and expression of CDCP1 on the cell surface was assessed by fluorescence microscopy. Bar = 100 m. C, Clone F4 was expanded into a T25 flask and 1x10 6 non-permeabilised cells were stained with MAb 10D7 and a species appropriate Alexa Fluor 488 labelled secondary antibody and cell surface expression was assessed by flow cytometry.

As the first two attempts failed to generate high CDCP1 expressing HeLa cells, FACS was employed to isolate high expressing HeLa-CDCP1 cells as

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polyclonal populations. FACS analysis was performed in collaboration with Dr Ibtissam Abdul Jabbar at the Diamantina Institute at the Princess Alexandra Hospital, Brisbane. Cells were stained with the anti-CDCP1 antibody 10D7 and an Alexa Fluor 488 labelled secondary antibody and the stained cells were subjected to FACS. Cells which displayed strong staining for CDCP1 were sorted to obtain stable over-expressing polyclonal cell populations. Populations were expanded into a T75 flask format and cell surface expression was assessed by flow cytometry 14 days after FACS was performed. At this time point, mixed cell populations of high CDCP1 expressing cells and at least one cell population showing weak CDCP1 expression were identified. This occurred for HeLa cells stably expressing CDCP1, CDCP1-Y734F, -Y743F and -Y762F and an example of flow cytometric analysis for HeLa-CDCP1-Y743F cells is shown in Figure 3.3A.

Polyclonal populations were passaged and maintained for a further 4 weeks and the cell surface expression of CDCP1 was reassessed by flow cytometry. At this later time point, the amount of CDCP1 positive cells had dropped markedly for each of the CDCP1, CDCP1-Y734F, -Y743F and - Y762F cells. Figure 3.3B shows that over this 4 weeks period, expression of CDCP1-Y743F had dropped from ~60% to ~20%. Representative flow cytometry histograms of HeLa-vector and prostate cancer PC3 cells which were used as negative and positive control for this experiment are shown in Figure 3.3C.

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Figure 3.3: A polyclonal population of HeLa cells expressing CDCP1- Y743F lose cell surface expression over time. HeLa cells were transfected with the CDCP1-Y743F construct and selected for 10 days in G418 containing media. Cells were stained with the anti-CDCP1 antibody 10D7 and an appropriate Alexa Fluor 488 labelled secondary antibody and high expressing cells were isolated by FACS. The obtained polyclonal population was expanded and maintained in selection media and the CDCP1 cell surface expression was re-assessed by flow cytometry 14 days after FACS A or 6 weeks after FACS B. C, Representative flow cytometry histograms of HeLa cells stably transfected with empty vector and PC3 cells, which served as negative- and positive control, respectively.

The fourth approach to generate HeLa cells stably expressing CDCP1, CDCP1-Y734F, -Y743F and -Y762F involved selection of monoclonal cell populations by FACS after selection of G418 resistant cells. Transfected cells were stained using the anti-CDCP1 antibody 10D7 and an Alexa Fluor 488 secondary antibody and highly positive CDCP1 expressing single HeLa cell clones were sorted into 96 well plates in selection media. The single cells were maintained, expanded and the expression of CDCP1 was assessed at the time when the cells had been expanded into a T25 flask format.

As shown in Figure 3.4 and summarised in Table 3.1, two high expressing HeLa-CDCP1 clones were identified and three clones for each of HeLa- CDCP1-Y734F, -Y743F and -Y762F. Each of the clones displayed similar levels of expression of CDCP1, as determined by flow cytometry analysis

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(Figure 3.4A) and Western blot analysis (Figure 3.4B). Despite further screening, it was not possible to identify a third clone expressing CDCP1 uniformly at about the same level as the other two clones (Figure 3.5A). A third clone (CDCP1 #3) with lower and less uniform expression of CDCP1 is shown as an example in the right panel of Figure 3.5A. In addition to high expressing HeLa-CDCP1, HeLa-CDCP1-Y734F, -Y743F and -Y762F clones, multiple clones displaying various levels of CDCP1 expression were collected for each cell line (Table 3.1). Some of these HeLa-CDCP1 clones were employed to examine the biology of CDCP1 which is described later in this chapter.

3.2.2 HeLa cells stably expressing CDCP1 display altered cell morphology which reverts to an epithelial morphology on mutation of CDCP1-Y734

It was noted that over expression of CDCP1, CDCP1-Y743F and -Y762F dramatically altered the cell morphology of HeLa cells. Both isolated HeLa cell clones expressing CDCP1 display an elongated and fibroblastic cell morphology (Figure 3.6A) which is clearly distinguishable from the cobble stone cell morphology of each of the three HeLa-vector cell clones (Figure 3.6B). Significantly, each of the three HeLa-CDCP1-Y743 and HeLa-CDCP1- Y762 cell clones also display an elongated and fibroblastic cell morphology (Figure 3.6C and D) very similar to the cell morphology observed for HeLa- CDCP1 cells (Figure 3.6A). In contrast, HeLa-CDCP1-Y734F cells display an epithelial and cobble stone cell morphology (Figure 3.6E) characteristic of HeLa-vector cells (Figure 3.6B). This data suggests that CDCP1-Y734 is required to mediate a change in HeLa cell morphology caused by stable expression of CDCP1.

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Figure 3.4: HeLa cell clones stably expressing CDCP1 display a high and uniform level of CDCP1 expression. A, Flow cytometry analysis of HeLa cells stable transfected with CDCP1, CDCP1-Y734F, -Y743F and - Y762F or the empty vector. B, Western blot analysis of the same cells shown in panel A using the anti-CDCP1 antibody 10D7. Anti-GAPDH Western blot analysis was performed to examine protein loading. Cell lysates of HeLa- vector and HeLa-CDCP1 cell clones were resolved on the same gel, transferred on the same membrane and exposed to the same film.

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Figure 3.5: Expression analysis of HeLa-CDCP1 clones. HeLa cells were transfected with the CDCP1 construct and selected for 10 days in selection media. Cells were stained with the anti-CDCP1 antibody 10D7 and a secondary Alexa Fluor 488 labelled antibody. High expressing clones were collected by FACS into 96 well plates. The clones were expanded into T25 flask format and the expression of CDCP1 was examined. A, Cells (1x10 6) were stained with MAb 10D7 and an Alexa Fluor 488 labelled secondary antibody and cell surface expression was assessed by flow cytometry. The CDCP1 expressing HeLa cell clones were examined on the same day and with identical flow cytometry settings. B, Representative histograms of HeLa- vector and PC3 cells, which served as negative and positive control, respectively. C, Whole cell lysates were prepared and analysed by Western blot analysis using an anti-FLAG antibody. Anti-GAPDH Western blot analysis was performed to examine protein loading.

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Figure 3.6: Expression of CDCP1 leads to a change in cell morphology in stable expressing HeLa cell clones. HeLa cells were transfected with CDCP1 (A) , empty vector (B), CDCP1-Y743F (C), CDCP1-Y762F (D) or CDCP1-Y734F (E) and selected for 10 days in G418 containing media. In order to select expressing clones, cells were stained with the anti-CDCP1 antibody 10D7 and a secondary Alexa Fluor 488 labelled antibody and high expressing clones were collected by FACS into 96 well plates. The clones were expanded into T25 flask format and the cell morphology examined by light microscopy. Bar = 100 µm. For larger sized photographs please refer to Appendix 1.

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3.2.3 Confirmation of CDCP1 expression level and changes on cell morphology in combined HeLa clones

To facilitate further characterisation of CDCP1, the high expressing monoclonal cells for each CDCP1 construct were combined to create cell populations that could be used to examine the cell biology of CDCP1. Two HeLa-CDCP1 clones were combined (further referred to as HeLa-CDCP1) and the three clones of each HeLa-CDCP1-Y734F, -Y743F and -Y762F were combined (further referred to as HeLa-CDCP1-Y734F, HeLa-CDCP1-Y743F or HeLa-CDCP1-Y762F). These combined cell populations were used for all further experiments described in the remainder of this thesis.

After combination of the monoclonal populations for each CDCP1 construct, the expression level of CDCP1 in the combined CDCP1 expressing HeLa cells was reassessed by flow cytometry and by Western blot analysis. The high and uniform level of cell surface and total expression of CDCP1 for the combined clones is shown in Figure 3.7 and is the same as was apparent for the monoclonal populations (Figure 3.4). Also apparent from Figure 3.7 is that CDCP1 expression in these cell lines was ~4 times higher than in endogenous expressing prostate cancer PC3 cells. The changed cell morphology towards a spindle-shaped, elongated and fibroblastic appearance was reconfirmed in the combined populations of HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F cells four weeks after combination of the clones (Figure 3.8). Moreover, the cell morphology of HeLa-CDCP1-Y734F cells displaying epithelial cell morphology characteristic of HeLa-vector cells was also reconfirmed (Figure 3.8). Significantly, these morphological differences persisted throughout the course of this project.

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Figure 3.7: Analysis of CDCP1 expression of combined HeLa cell populations. Three high expressing HeLa cell clones were combined for HeLa-CDCP1-Y734F, -Y743F or -Y762F and HeLa-vector and two clones were combined for HeLa-CDCP1 cells to create representative cell populations to further characterise CDCP1. The level of expression of CDCP1 was examined 14 days after the clones were combined. A, Non- permeabilised cells (1x10 6) cells were stained with the anti-CDCP1 antibody 10D7 and a secondary Alexa Fluor 488 labelled antibody and cell surface expression was assessed by flow cytometry. PC3 cells were used as positive control (left bottom panel). All HeLa cell lines were examined on the same day and with identical flow cytometry settings. B, Whole cell lysates were analysed by Western blot analysis using MAb 10D7. Anti-GAPDH Western blot analysis was performed to examine protein loading.

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Figure 3.8: CDCP1 expression leads to a change in cell morphology in stably expressing HeLa cells which requires CDCP1-Y734. Three high CDCP1 expressing HeLa cell clones were combined for HeLa-vector, HeLa- CDCP1-Y734F, -Y743F or -Y762F and two clones were combined for HeLa- CDCP1. The morphology of the combined cell populations was assessed by light microscopy 4 weeks after the clones were combined. Bars = 100 µm. For larger sized photographs please refer to Appendix 2.

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3.2.4 Cellular localisation of CDCP1 and co-localisation of CDCP1 with SFKs is unaltered by mutation of CDCP1-Y734, -Y743 and -Y762 in CDCP1 expressing HeLa cells

Confocal microscopy was carried out to examine the cellular localisation of CDCP1 and SFKs, which are known to be CDCP1 interacting proteins (Brown et al., 2004; Bhatt et al., 2005; Uekita et al., 2007). Cells were seeded on sterile glass cover slips in 24 well plates and allowed to adhere for 48 hours. The cells were then stained with the monoclonal mouse anti- CDCP1 antibody 41-2, a rabbit anti-SFK antibody followed by species specific Alexa Fluor labelled secondary antibodies. CDCP1 is predominantly located on the cell surface with lower levels of cytoplasmic signal also apparent (Figure 3.9). This localisation is independent of mutation of CDCP1-Y734, -Y743 or -Y762. Importantly, strong co-localisation of CDCP1 with SFKs at the plasma membrane was observed in CDCP1 expressing HeLa cells as suggested by the strong purple staining in the merged pictures, and this is also unaffected by mutation of CDCP1 at Y734, Y743 or Y762 (Figure 3.9). Also apparent in Figure 3.9, is the elongated and fibroblastic-like appearance of HeLa-CDCP1, HeLa-CDCP1-Y743F and - Y762F cells, but not in HeLa-CDCP1-Y734F cells.

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Figure 3.9: Localisation of CDCP1 and co-localisation of CDCP1 with SFKs is unaltered by mutation of CDCP1-Y734, Y743 or Y762 in CDCP1 expressing HeLa cells. Cells were stained with antibodies for CDCP1 and SFKs and species specific fluorescently labelled secondary antibodies were used to assess the cellular localisation of the two proteins with a confocal microscope. Actin was stained using phalloidin Alexa Fluor 488 and the nucleus was stained with DAPI. ( I), HeLa-vector cells; ( II), HeLa-CDCP1 cells; ( III), HeLa-CDCP1-Y734F cells; ( IV), -Y743F cells or ( V), -Y762F cells. Nonspecific binding of secondary antibodies was examined using slides where cells were incubated with only the secondary antibodies. No nonspecific binding was detected for the goat anti mouse Alexa Fluor 568 antibody (secondary for CDCP1) and barely detectable background staining was observed for the goat anti rabbit Alexa Fluor 647 (secondary to SFK). DAPI nuclear staining is shown in the top left corner of the phalloidin (actin) picture. Bar = 25 µm. For larger sized photographs please refer to Appendix 3.

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3.2.5 Examination of the impact of tyrosine mutation on CDCP1 binding to SFKs and PKC δ

Immunoprecipitation experiments were performed followed by Western blot analysis to examine the impact of mutation of CDCP1 tyrosine residues on the ability of this protein to bind to SFKs and PKCδ. As expected, anti-SFK Western blot analysis of proteins immunoprecipitated from HeLa-CDCP1 cell lysates with the anti-CDCP1 antibody 41-2 showed that CDCP1 and SFK interact (Figure 3.10A). The anti-SFKs antibody used for the Western blot analysis was generated with a sequence that includes the Y527 site of c-Src, which is highly conserved among the SFK members, and is, therefore, expected to cross react with multiple SFKs. Moreover, interaction of CDCP1 with PKC δ (Figure 3.10B) was confirmed by anti-PKC δ Western blot analysis of CDCP1 immunoprecipitated from HeLa-CDCP1 cell lysate using an anti- FLAG antibody (Figure 3.10B). Parallel experiments using lysates from HeLa-CDCP1-Y734F, -Y743F and -Y762F cells demonstrated that mutation of Y734 almost completely abolished binding of SFK and PKC δ to CDCP1 and mutation of Y762 caused a minor decrease in binding of PKC δ to CDCP1 (Figure 3.10B). No change in interaction between CDCP1 with SFKs and PKC δ was observed in HeLa cells expressing CDCP1-Y743F.

Alvares et al. recently showed ligation of CDCP1 with an activating anti- CDCP1 antibody causes an increase in phosphorylation of p-SFK-Y416 (Alvares et al., 2008). Auto-phosphorylation of SFK-Y416 due to binding of SFK with its target proteins has also previously been reported (Xing et al., 2000; Arias-Salgado et al., 2003; Yadav and Miller, 2007). These reports led us to examine whether stable expression of CDCP1 and loss of binding of SFKs to CDCP1-Y734 has an effect on the phosphorylation of SFK-Y416. Therefore, whole cell lysates were subjected to Western blot analysis with an anti-phospho SFK-Y416 antibody. This antibody may cross react with other SFKs phosphorylated at this tyrosine residue. As shown in Figure 3.10C, increased phosphorylation levels of SFK-Y416 were observed in all CDCP1 expressing HeLa cells but not in HeLa-CDCP1-Y734F cells as compared to

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HeLa-vector cells. A particularly strong increase in phosphorylation of SFK- Y416 was observed in HeLa-CDCP1-Y762F cells (Figure 3.10C).

Figure 3.10: Examination of interactions between CDCP1, SFKs and PKC δ in stable CDCP1 expressing HeLa cells. Lysates of CDCP1 expressing HeLa cells were collected in the presence of phosphatase inhibitors and 500 µg of total protein was subjected to immunoprecipitation using the anti-CDCP1 antibody 41-2 A or an anti-FLAG antibody B. A, Immunoprecipitated proteins were analysed by Western blot analysis using anti-SFK and anti-phospho SFK antibodies. B, Binding of PKC δ to CDCP1 was assessed by Western blot analysis using an anti-PKC δ antibody. Anti-FLAG Western blot analysis was performed to examine loading of immunoprecipitated protein. C, Whole cell lysates were subjected to Western blot analysis using antibodies against p-SFK-Y416, SFK and FLAG tag. Anti-tubulin Western blot analysis was performed to examine protein loading. Ratio of total SFK to phosphorylated SFK-Y416 was calculated by densitometry analysis from two independent experiments using Image-J. The ratio of total to phospho SFK is presented as fold change in comparison to HeLa-vector cells where the ratio was set to 1. Bars are mean and SEM (n=2).

3.2.6 CDCP1 is basally tyrosine phosphorylated and phosphorylated on Y734 in stable expressing HeLa cells

As CDCP1 is known to be phosphorylated at Y734, Y743 and Y762 (Benes et al., 2005), the phosphorylation of this protein was examined in stable

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CDCP1 expressing HeLa cells. To examine total tyrosine phosphorylation of CDCP1, immunoprecipitation was performed on lysates from HeLa-vector, HeLa-CDCP1, HeLa-CDCP1-Y734F, -Y743F or -Y762F cells using the anti- CDCP1 antibody 10D7. Subsequent, anti-phospho tyrosine Western blot analysis indicated that CDCP1 is basally tyrosine phosphorylated in all CDCP1 expressing cells, but not in HeLa-CDCP1-Y734F cells (Figure 3.11A). This finding demonstrates the importance of CDCP1-Y734 in facilitating SFK-mediated tyrosine phosphorylation of CDCP1. To specifically examine phosphorylation at CDCP1-Y734, immunoprecipitation was performed on the same lysates using an anti-FLAG antibody followed by Western blot analysis with an anti-phospho FAK-Y861 antibody that also reacts strongly with p-CDCP1-Y734 (Brown et al., 2004). As shown in Figure 3.11B, this analysis showed that CDCP1-Y734 is basally phosphorylated and that Western blot analysis with the p-FAK-Y861 antibody produces a similar signal to Western blot analysis with the anti-p-tyrosine antibody. HeLa- CDCP1-Y743F and -Y762F cells display tyrosine phosphorylation and phosphorylation of CDCP1-Y734 on a comparable level to HeLa-CDCP1 cells with a marginally higher level in HeLa-CDCP1-Y762F cells (Figure 3.11A and B). These data indicate that phosphorylation at CDCP1-Y734 in not altered by mutations of the tyrosine residues Y743 and Y762 of CDCP1.

Figure 3.11: Examination of CDCP1 phosphorylation in stable CDCP1 expressing HeLa cells. Immunoprecipitation was performed on lysates collected in the presence of phosphatase inhibitors. A, Anti-phospho tyrosine antibody (pY20) Western blot analysis of proteins immunoprecipitated using anti-CDCP1 antibody 41-2. B, Anti-p-FAK-Y861 antibody Western blot analysis of proteins immunoprecipitated using an anti-FLAG antibody. The anti-p-FAK-Y861 antibody also detects p-CDCP1-Y734 (Brown et al., 2004). Anti-FLAG Western blot analysis was performed to examine loading of immunoprecipitated protein.

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3.2.7 Binding of SFKs to CDCP1-Y734 reduces phosphorylation of FAK-Y861 depending on the level of expression of CDCP1

In immunoprecipitation experiments it was demonstrated that CDCP1 is basally phosphorylated in HeLa cells stably expressing CDCP1 (Figure 3.11). In addition, it was shown in Figure 3.10, that SFKs bind to CDCP1 and that mutation of CDCP1-Y734 greatly decreases the binding of SFKs to CDCP1. This decrease in binding of SFKs to CDCP1 leads to a loss of CDCP1 tyrosine phosphorylation in HeLa-CDCP1-Y734F cells (Figure 3.11). These data agree with published findings of multiple groups reporting that SFKs bind to CDCP1-Y734 (Benes et al., 2005; Uekita et al., 2007; Alvares et al., 2008) and phosphorylate this protein (Hooper et al., 2003; Brown et al., 2004; Wong et al., 2009). Notably, all studies examining the SFK mediated phosphorylation of CDCP1 found that CDCP1 phosphorylation occurs upon deadhesion of cells (Uekita et al., 2007; Spassov et al., 2009; Wong et al., 2009), indicating that CDCP1 acts in response to cellular processes related to cell adhesion/deadhesion. Interestingly, Brown et al. reported that deadhesion of human foreskin keratinocytes, which causes increased phosphorylation of LMW-CDCP1, is accompanied by a decrease in phosphorylation of FAK-Y861 (Brown et al., 2004), a protein well established for its role in cell adhesion related processes (Schlaepfer et al., 1999). Readhesion of these cells had the opposite effect causing a decrease in phosphorylation of CDCP1 and an increase in phosphorylation of FAK- Y861 (Brown et al., 2004). This report suggests that CDCP1 and FAK are differentially phosphorylated by SFKs depending on the state of cell adhesion (Brown et al., 2004), where the state of phosphorylation of one of these proteins influences the state of phosphorylation of the other. Whether SFK mediated phosphorylation of one of these proteins can influence the phosphorylation of the other protein independently of adhesion/deadhesion has not been examined. As the high level of basal phosphorylation found in HeLa-CDCP1 cells mirrors the phosphorylation of CDCP1 reported to occur due to cell deadhesion, and as HeLa cells are known to express and phosphorylate FAK (Yano et al., 2004), whether the high level of basal phosphorylation of CDCP1 impacts on the state of phosphorylation of FAK-

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Y861 in HeLa cells was examined. To address this question, the anti-p-FAK- Y861 antibody utilised in Figure 3.11B was now employed on whole cell lysates rather than on immunoprecipitated proteins.

As shown in Figure 3.12A, this antibody detects p-FAK-Y861 in HeLa-vector cells suggesting that FAK-Y861 is basally phosphorylated in this cell line. In contrast, in HeLa-CDCP1 cells this antibody only detects CDCP1-Y734. Interestingly, when CDCP1-Y734 was no longer available for phosphorylation in HeLa-CDCP1-Y734F cells, p-FAK-Y861 was again detected by this antibody, suggesting that CDCP1-Y734 and FAK-Y861 compete for phosphorylation by SFKs. Consistently, the anti-p-FAK-Y861 antibody detected phosphorylated CDCP1-Y734 in lysates from HeLa- CDCP1-Y743F and HeLa-CDCP1-Y762F cells. These data are supported by Western blot analysis using an anti-p-tyrosine antibody (pY20) that detects phosphorylated tyrosine residues (Figure 3.12B), which also indicates that CDCP1-Y734 is the predominant tyrosine phosphorylation site of CDCP1. Finally, Western blot analysis using an antibody that detects total FAK demonstrated that the examined cell lines express FAK at about the same level (Figure 3.12C).

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Figure 3.12: Expression of CDCP1 is accompanied by a loss of phosphorylation of FAK-Y861. Whole cell lysates were collected in the presence of phosphatase inhibitors and subjected to Western blot analysis with antibodies against A, phospho tyrosine (pY20), B, phospho FAK-Y861 or C, total FAK. Anti-FLAG and anti-GAPDH Western blot analysis was performed to examine protein loading.

To examine whether CDCP1 and FAK are competitive substrates for SFK- mediated phosphorylation, HeLa cells were transiently transfected with the CDCP1 construct, and after 24 hours whole cell lysates were subjected to Western blot analysis with the anti-p-FAK-Y861 antibody. As shown in Figure 3.13A, two bands were observed in the lysates of the transiently transfected HeLa cells, one representing p-CDCP1-Y734 and the other representing p- FAK-Y861. Since cells transiently transfected with a lipid based transfection system are likely to contain transfected as well as untransfected cells, it was questioned whether the absolute level of expression of CDCP1 could be the critical determinant causing a shift from SFK-mediated phosphorylation of FAK to phosphorylation of CDCP1. To address this question, four stable expressing HeLa-CDCP1 cell clones were examined. These clones displayed low to high protein expression levels of CDCP1, as determined by flow cytometry and Western blot analysis (Figure 3.13B and C). Significantly, it was found that the cell clone expressing CDCP1 at a barely detectable level (#4) displayed only phosphorylation of FAK-Y861, the two medium CDCP1 expressing clones (#5, #6) displayed phosphorylation of CDCP1-

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Y734 as well as phosphorylation of FAK-Y861 and the cell clone expressing CDCP1 at a high level (#1, described earlier in Figure 3.4 and 3.5) exhibited exclusive phosphorylation of CDCP1-Y734 (Figure 3.13C).

Figure 3.13: SFK-mediated phosphorylation of CDCP1-Y734 reduces phosphorylation of FAK-Y861 depending on the level of CDCP1 expression. A, HeLa cells were transiently transfected with the CDCP1 construct and whole cell lysates were collected in the presence of phosphatase inhibitors 24 hours after the transfection and subjected to Western blot analysis with an anti-CDCP1 antibody 10D7 and an anti-p-FAK- Y861 antibody. B, Stably expressing CDCP1 clones displaying various levels of CDCP1 expression were identified by flow cytometry using MAb 10D7 and an Alexa Fluor 488 conjugated secondary antibody. C, Stable expressing HeLa-CDCP1 cell clones were collected and analysed by Western blot analysis with an anti-FLAG or anti-p-FAK-Y861 antibody. Anti-GAPDH or anti-tubulin Western blot analysis was performed to examine protein loading

These data further support the proposal that CDCP1 and FAK are competitive SFK substrates and increasing levels of CDCP1 expression causes sequestration of SFKs, facilitating SFK-mediated phosphorylation of CDCP1-Y734 which prevents phosphorylation of FAK-Y861 in stable CDCP1 expressing HeLa cells. A working model to illustrate the relationship between

Chapter 3

SFK-mediated phosphorylation of FAK-Y861 and CDCP1-Y734 is presented in Figure 3.14.

Figure 3.14: Working model: CDCP1 and FAK compete for SFK- mediated phosphorylation in CDCP1 expressing HeLa cells. A working model to illustrate the relationship between SFK-mediated phosphorylation of FAK-Y861 and CDCP1-Y734. ( I) In adherent HeLa cells without CDCP1 expression SFK-mediated basal phosphorylation of FAK is observed. ( II) If CDCP1 is expressed at equal levels to FAK, SFK mediates the phosphorylation of FAK-Y861 as well as CDCP1-Y734. ( III) In HeLa cells which express CDCP1 at a high level, SFKs are auto-phosphorylated and SFK-mediated phosphorylation shifts towards the phosphorylation of CDCP1-Y734 causing a change in cell morphology ( IV) . Mutation of CDCP1-Y734 which prevents binding and auto-phosphorylation of SFK-Y416 and consequent phosphorylation of CDCP1-Y734 allows SFK-mediated phosphorylation of FAK-Y861 and no change in cell morphology occurs (V) .

In this working model, SFK-mediated basal phosphorylation of FAK is observed in cells which do not express CDCP1. In HeLa cells which express CDCP1 and FAK at equal levels, SFKs mediate the phosphorylation of FAK- Y861 as well as CDCP1-Y734. If HeLa cells express CDCP1 at a high level, SFKs are auto-phosphorylated at Y-416 and SFK-mediated phosphorylation shifts towards the phosphorylation of CDCP1-Y734 which leads to an elongated and fibroblastic cell morphology. In contrast, in HeLa-CDCP1- Y734F cells which lack binding of SFKs to CDCP1 no auto-phosphorylation

Chapter 3

of SFK-Y416 and phosphorylation of CDCP1-Y734 occurs. In these cells, SFKs mediate the phosphorylation of FAK-Y861, but not of CDCP1-Y734 and these cells maintain the cobble stone epithelial cell morphology characteristic of HeLa-vector cells.

An alternative hypothesis to explain the data shown in Figure 3.13B and C is that CDCP1 binds to FAK and thereby blocks the binding site for SFKs on FAK. To examine this hypothesis immunoprecipitation experiments with an anti-FLAG antibody were performed and the immunoprecipitated proteins were subjected to Western blot analysis with an antibody against FAK. As shown in Figure 3.15, FAK could not be detected after immunoprecipitation with an anti-FLAG antibody in HeLa-CDCP1 cells as well as in HeLa- CDCP1-Y734F cells. Western blot analysis performed on whole cell lysates demonstrated that HeLa-CDCP1 and HeLa-CDCP1-Y734F possess about the same amount of FAK protein. These data further strengthen the proposal that CDCP1 and FAK are competitive substrates for tyrosine phosphorylation mediated by SFKs.

Figure 3.15: CDCP1 does not bind to FAK. Whole cell lysates were collected in the presence of phosphatase inhibitors and analysed by Western blot or 500 µg of protein was subjected to immunoprecipitation with an anti- FLAG antibody. Whole cell lysates and the immunoprecipitated proteins were subjected to Western blot analysis using anti-FLAG and anti-FAK antibodies. Protein loading in the whole cell lysate was examined by anti-tubulin Western blot analysis. The cell lysates and the immunoprecipitated proteins were resolved on the same gel, transferred on the same membrane and exposed to the same film.

Chapter 3

3.2.8 The SFK-selective inhibitor SU6656 causes distinct effects downstream of SFK inhibition in HeLa cells stably expressing CDCP1 and tyrosine mutants and reverts the cell morphology caused by CDCP1 expression

To confirm that phosphorylation of CDCP1 in HeLa cells is mediated by SFKs, the cells were incubated with various concentrations of the SFK selective inhibitor SU6656 (Blake et al., 2000). As shown in Figure 3.16A, incubation with this inhibitor leads to a dose-dependent decrease of phosphorylation of CDCP1-Y734 in HeLa-CDCP1 cells. When SU6656 was used at a concentration of 10 µM the phosphorylation of CDCP1 decreases to a barely detectable level. This suggests that phosphorylation of CDCP1- Y734 is facilitated by SFKs in HeLa cells expressing CDCP1. In HeLa- CDCP1-Y743F and HeLa-CDCP1-Y762F cells, incubation with SU6656 also causes a dose-dependent decrease in phosphorylation of CDCP1-Y734 to barely detectable levels at an inhibitor concentration of 10 µM (Figure 3.16B and C). These data indicate that phosphorylation of CDCP1-Y734 is facilitated by SFKs in these two cell lines as it is observed in HeLa-CDCP1 cells (Figure 3.16A). In contrast, incubation with SU6656 leads to a dose- dependent decrease in phosphorylation of FAK-Y861 in HeLa-CDCP1- Y734F cells (Figure 3.16D). At an inhibitor concentration of 10 µM, no phosphorylation of FAK-Y861 can be detected. This suggests that SFKs facilitate the phosphorylation of FAK-Y861 in HeLa-CDCP1-Y734F cells. In HeLa-vector cells, SFK inhibition also leads to a decrease in phosphorylation of FAK-Y861 (Figure 3.16E), as was observed in HeLa-CDCP1-Y734F cells (Figure 3.16D). In summary, the data from these experiments suggest that phosphorylation of CDCP1-Y734 is facilitated by SFKs in HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F cells whereas in HeLa-CDCP1-Y734F and HeLa-vector cells SFKs mediate the phosphorylation of FAK-Y861.

Chapter 3

Figure 3.16: Inhibition of SFKs with SU6656 reduces phosphorylation of CDCP1-Y734 in HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F cells and phosphorylation of FAK-Y861 in HeLa-vector and HeLa-CDCP1-Y734F cells. Stable CDCP1 expressing HeLa cells were incubated with the SFK- selective inhibitor SU6656 at concentrations ranging from 100 nM to 10 M, as indicated above the Western blots. After 60 minutes whole cell lysates were collected in the presence of phosphatase inhibitors. The lysates were analysed by Western blot analysis with the anti-p-FAK-Y861 antibody which cross reacts with p-CDCP1-Y734 A-E. Protein loading was examined by Western blot analysis with antibodies against GAPDH or Tubulin and FLAG tag.

Chapter 3

To examine whether SFK-mediated phosphorylation of CDCP1 is the cause for the observed change in cell morphology (Figure 3.6 and 3.8), HeLa- vector, HeLa-CDCP1 and HeLa-CDCP1-Y734F cells were incubated with SU6656 at a concentration of 5 µM for 48 hours and cell morphology was assessed by phase contrast microscopy. As shown in Figure 3.17, it was found that incubation of HeLa-CDCP1 cells with SU6656 reverts the elongated and fibroblastic-like appearance of these cells back to the classical epithelial morphology of the HeLa-vector and HeLa-CDCP1-Y734F cells. Significantly, this suggests that SFK activity is required to maintain this elongated and fibroblastic cell morphology in HeLa-CDCP1 cells. In addition, it was noted that incubation with SU6656 also induced further spreading of HeLa-vector and HeLa-CDCP1-Y734F cells. Although the mechanism of this spreading is not known, it is possible that this is caused by altered FAK signalling as a result of SFK inhibition. The role of FAK signalling in effective cell spreading, particularly on ECM proteins has previously been described (Richardson et al., 1997; Brown et al., 2005).

Chapter 3

Figure 3.17: The change in cell morphology caused by CDCP1 expression can be reverted by inhibition of SFKs with SU6656. Stable expressing HeLa-CDCP1, HeLa-CDCP1-Y734F and HeLa-vector cells were seeded at 0.2x10 6 cells in T25 flasks. The cells were allowed to adhere for 30 minutes before addition of SU6656 to a final concentration of 5 µM or 0.19% DMSO (v/v) as vehicle control. The cells were incubated for 48 hours at 37°C, 5% CO 2 in a humidified atmosphere and the morphology of the cells was assessed by phase contrast microscopy under 20x magnification using an Olympus-XX41 microscope.

Chapter 3

3.3 DISCUSSION

CDCP1 expressing HeLa cell lines were generated with the aim of examining the roles of three conserved tyrosine phosphorylation sites in the function of this protein. The generated HeLa cells stably expressed CDCP1 or one of three mutated forms of CDCP1 where a tyrosine residue at amino acid position 734, 743 or 762 had been mutated to phenylalanine. HeLa-vector cells were generated as controls.

The key findings of the experiments described in this chapter are:

• High and uniform CDCP1 and CDCP1 tyrosine mutant expressing cell lines were generated. • Mutation of tyrosine residues alter the interactions of CDCP1 with binding proteins and alter tyrosine phosphorylation of CDCP1. • CDCP1 expression causes an EMT-like change in HeLa cell morphology which requires CDCP1-Y734. • CDCP1 and FAK are competitive substrates for SFK-mediated phosphorylation.

3.3.1 Generation of HeLa cells stably expressing CDCP1 and CDCP1 tyrosine mutants

Four different methodologies were employed in order to generate these stable and homogenous expressing HeLa-CDCP1 cells. The first two approaches involved isolation of single cell clones from cell culture dishes and from 96 well plates after cell seeding by limiting dilution. These two approaches failed to generate highly and homogenously expressing HeLa- CDCP1 cells. The third approach involved isolation of polyclonal cell populations by FACS. It was observed that these polyclonal cell populations lost CDCP1 expression within a few weeks after FACS. Therefore, it was proposed that expression of CDCP1 causes a decrease in HeLa cell proliferation, and consequently low CDCP1 expressing cells or non expressing cells would overgrow the high CDCP1 expressing cells over time.

Chapter 3

This proposal was confirmed at a later stage of this project when proliferation rates of the stable CDCP1 expressing HeLa cells were found to be significantly decreased in comparison to the HeLa-vector cells (Chapter 5, Figure 5.1). This finding is also likely to explain why the first attempts to isolate monoclonal cell populations had failed. In the first two approaches, the cells were seeded into cell culture dishes and 96 well plates and once the cell colonies were big enough to be seen by eye they were isolated, expanded and screened for CDCP1 expression. These isolated clones were resistant to the selection drug G418, but did not express CDCP1. This might be a consequence of the higher rate of proliferation of cell clones not expressing CDCP1, because these cells will form visible colonies earlier than CDCP1 expressing cells. At the time when clones were isolated multiple barely visible cell colonies were noticed which were discarded after the largest and most visible colonies were isolated. It is possible that these small colonies represented CDCP1 positive cell clones. Therefore, these approaches might have been successful if colonies had been isolated at a later time point. This would have allowed CDCP1 expressing cells to proliferate until these cells could have formed easily visible colonies.

Since isolation of stable CDCP1 expressing cells with the first three approaches failed, FACS was used to generate stable CDCP1 expressing monoclonal HeLa cell populations. This approach allowed the isolation of multiple high and homogenously expressing cell clones. To create mixed high expressing cell populations for the assessment of CDCP1 biological function, two monoclonal populations for HeLa-CDCP1 cells and three monoclonal populations for HeLa-CDCP1-Y734F, -Y743F and -Y762F and HeLa-vector cells were combined. The combined HeLa cell populations were subjected to a variety of experiments described in this chapter and in Chapter 4 and Chapter 5 of this thesis. The performed experiments in these chapters included:

• Examination of the effect of CDCP1 expression on HeLa cell morphology by microscopy (Chapter 3).

Chapter 3

• Biochemical analysis to examine interaction of CDCP1 with the CDCP1 binding proteins SFKs and PKC δ (Chapter 3). • Examination of the ability of the serine protease matriptase to proteolytically process CDCP1 on the cell surface (Chapter 4). • In vitro examination of the resulting cell phenotype caused by matriptase-mediated proteolysis of CDCP1 (Chapter 4). • Examination of the effect of CDCP1 expression on cell phenotype in cellular processes associated with cancer progression in vitro including proliferation, adhesion and migration (Chapter 5). • In vivo assays to examine the metastatic phenotype caused by CDCP1 and to examine the effect of anti-CDCP1 function-blocking antibodies to inhibit metastasis formation (Chapter 5).

3.3.2 Some mutations of CDCP1 alter interactions with CDCP1 binding proteins

CDCP1 is known to have five conserved tyrosine residues at amino acid position Y707, Y734, Y743, Y762 and Y806. Three of these tyrosine residues (Y734, Y743 and Y762) have previously been shown to play a role in mediating interactions of CDCP1 with its described binding partners; SFKs and PKC δ (Benes et al., 2005). Tyrosine residue 734 was found to be the site where SFK binds to CDCP1 to mediate further phosphorylation of Y743 and Y762 which subsequently leads to binding of PKCδ to Y762 (Benes et al., 2005). The main focus of the biochemical experiments to characterise the different CDCP1 expressing cell populations was to examine the interaction of CDCP1 with its binding partners (SFKs and PKC δ) and to examine the effect of mutation of the three tyrosine residues. These experiments indicate that HeLa-CDCP1-Y734F cells are most distinguishable from HeLa-CDCP1 cells. In contrast, HeLa-CDCP1-Y743F and -Y762F did not show obvious changes in interaction of CDCP1 with its binding partners.

Specifically, no differences between HeLa-CDCP1 and HeLa-CDCP1-Y743F cells on a molecular level could be detected. It can be concluded that Y743

Chapter 3

does not have a crucial role in mediating interactions between CDCP1 and SFKs and PKC δ. This is consistent with findings of Benes et al. who did not observe any change in interactions between CDCP1 and SFK and PKC δ in human osteosarcoma U2-OS cells transiently transfected with a CDCP1- Y743F expression construct (Benes et al., 2005). However, this tyrosine phosphorylation site might be important in interactions with yet unidentified binding partners of CDCP1. This is suggested by observed differences in cell phenotype in vitro and in vivo between this cell line and HeLa-CDCP1 cells, as described in Chapter 5 of this thesis. More extensive biochemical studies are required to provide clarity about the role of this tyrosine residue in mediating interactions with other CDCP1 binding proteins.

A marked decrease in binding of PKC δ to CDCP1 was not detected in HeLa- CDCP1-Y762F cells. In contrast, binding of PKC δ was completely abolished in HeLa-CDCP1-Y734F cells. This suggests that a single mutation at Y762 is not sufficient to diminish binding of PKC δ, but that binding of SFKs at Y734 is required to facilitate PKC δ binding to CDCP1-Y762. This suggests that CDCP1-Y762 is not the only site which can facilitate interaction between CDCP1 and PKC δ as previously reported (Benes et al., 2005). CDCP1-Y743 did not appear to be a site where interaction between CDCP1 and PKC δ can occur, as there was no detectable change in the level of PKC δ after immunoprecipitation with an anti-CDCP1 antibody in HeLa-Y743F cells. Hence, it is possible that one of the other potential tyrosine phosphorylation sites (Y707, Y806) of CDCP1 is important in mediating interactions between CDCP1 and PKC δ. Alternatively, it is possible that PKC δ is in direct contact with SFKs and that CDCP1-Y762 functions to increase binding of PKC δ to CDCP1.

3.3.3 CDCP1 expression causes an EMT-like change in HeLa cell morphology which requires CDCP1-Y734

It was noticed that CDCP1 expression leads to a change in cell morphology in HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F cells while the

Chapter 3

morphology of HeLa-CDCP1-Y734F cells is unaltered in comparison to HeLa-vector cells. The cell morphology of these three cell lines is more elongated and fibroblastic-like which suggests a possible EMT in these cells induced by CDCP1 expression. Whether, this cell morphology does indeed represent an EMT needs to be explored by examining markers commonly used to detect this change in phenotype, for example vimentin, fibronectin, twist or snail (Lee et al., 2006). Interestingly, this change in cell morphology was accompanied by an increase in phosphorylation of SFK-Y416 in these cell lines. This suggests that the change in cell morphology is mediated by SFKs binding to CDCP1-Y734 which leads to phosphorylation of CDCP1- Y734 and increased phosphorylation of SFK-Y416. This hypothesis is supported by two experimental observations. Firstly, it was found that CDCP1 is basally phosphorylated in all HeLa-CDCP1 expressing cells except for HeLa-CDCP1-Y734F cells and this is accompanied by an increase in phosphorylation of SFK-Y416. Secondly, inhibition of SFK with the specific inhibitor SU6656 reverts the elongated and fibroblastic-like appearance of HeLa-CDCP1 cells back to the morphology of the HeLa-vector and HeLa- CDCP1-Y734F cells. It appears that engagement of SFKs with CDCP1-Y734 leads to auto-phosphorylation of SFKs. Consequent increased SFK activity may allow for subsequent phosphorylation of other CDCP1 tyrosine residues. This is consistent with the observation of increased phosphorylation of SFK- Y416 in all cell lines except HeLa-CDCP1-Y734F cells. This is also consistent with a previous report showing that increased SFK phosphorylation occurs due to SFK binding to target proteins (Xing et al., 2000; Arias-Salgado et al., 2003; Yadav and Miller, 2007) which can facilitate further phosphorylation of its binding partners (Shvartsman et al., 2007).

Whether it is the increased phosphorylation of CDCP1, or SFKs, or the interaction of these proteins, which causes the change in cell morphology the CDCP1 expressing HeLa cells was not examined during this project. Although, inhibition of SFKs leads to a decrease in CDCP1 phosphorylation, which might be the reason for the reversion of the cell morphology, it is possible that there are effects on other signalling pathways downstream of SFK inhibition which may play a role in HeLa cell morphology.

Chapter 3

Published reports support the observation that CDCP1 expression can induce an EMT-like phenotype. For example, Bhatt et al. described that CDCP1 expression in the breast cancer cell line MDA-MB-468 leads to rounded cells growing in a loosely adhered and semi-suspended state (Bhatt et al., 2005). Moreover, Bonuccelli et al. discovered that a mutated form of caveolin-1 (P132L), which causes a more metastatic, migratory and invasive phenotype is accompanied by up-regulation of CDCP1 gene expression (Bonuccelli et al., 2009). The resulting invasive and migratory phenotype could be blocked by inhibitors of the EGF, TGF-β and HGF mediated pathways. This is of interest because EGF and TGF-β have been reported to induce an EMT in multiple studies (Janda et al., 2002; Docherty et al., 2006; Zhang et al., 2006; Uttamsingh et al., 2007; Lee et al., 2008). Of further interest is the report of Borczuk et al. who found that knock down of the type II TGF-β receptor leads to a decrease in CDCP1 expression in an bronchioloalveolar adenocarcinoma cell line (Borczuk et al., 2005).

In addition, SFK phosphorylation and activation has been well established to play an important role in mediating an EMT-like transition in a variety of cell lines including those derived from breast cancer, hepatocellular carcinoma, rat bladder carcinoma and gastric cancer (Rodier et al., 1995; Avizienyte and Frame, 2005; Galliher and Schiemann, 2006; Humar et al., 2007; Yang et al., 2009). Moreover, SFK inhibition reverting an EMT-like phenotype back to an epithelial cell morphology has been described previously (Yang et al., 2009; Liu and Feng, 2010). Therefore, it can be speculated that the increased phosphorylation of SFKs, which is caused by interaction with CDCP1-Y734F, mediates the EMT-like cell morphology seen in HeLa-CDCP1 cells.

Alternatively, it is possible that the activated state of FAK-Y861 in HeLa- CDCP1-Y734F cells does not allow for a change towards an EMT-like morphology despite CDCP1 expression, since FAK is known to play an important role in effective cell spreading, particularly on ECM proteins (Owen et al., 1999). However, countering this possibility is the finding that SFK inhibition with SU6656 reverts the observed elongated and fibroblastic-like phenotype without inducing phosphorylation of FAK in HeLa-CDCP1 cells

Chapter 3

and inhibition of SFKs leads to a decrease in phosphorylation of FAK-Y861 without changing the cell morphology of HeLa-CDCP1-Y734F cells.

3.3.4 CDCP1 and FAK are competitive substrates for SFK-mediated phosphorylation

It was found that expression of CDCP1 can completely prevent the phosphorylation of FAK-Y861 observed by us in untransfected HeLa cells (Figure 3.13A) and HeLa-vector cells (Figure 3.12B). In contrast, HeLa- CDCP1-Y734F cells, which have markedly reduced binding of SFKs to CDCP1 (Figure 3.10A) and consequently no SFK-mediated phosphorylation of CDCP1-Y734 (Figure 3.11), only exhibit phosphorylation of FAK-Y861 while displaying comparable levels of CDCP1 (Figure 3.7B) and total FAK protein (Figure 3.12C).

The data presented here indicate that this “switch” between SFK-mediated phosphorylation of FAK and CDCP1 depends on the expression level of CDCP1 (Figure 3.13C) which is illustrated in Figure 3.14. The equilibrium between SFK-mediated phosphorylation of FAK and CDCP1 appeared to be pushed completely towards the phosphorylation of CDCP1 in HeLa-CDCP1 cells. In these cells the observed level of cell surface expression is ~4 times higher than the CDCP1 level in the endogenous CDCP1 expressing prostate cancer cell line PC3. This prostate cancer cell line is one of the highest endogenous CDCP1 expressing cell lines which were analysed in this project amongst a panel of colon, prostate, cervical and kidney cancer cell lines (Figure 3.1). From this data it can be proposed that CDCP1-Y734 can sequester SFK from FAK-Y861, which leads to phosphorylation of CDCP1 and prevents the phosphorylation of FAK in HeLa-CDCP1 cells. These findings suggest that the level of expression of CDCP1 may regulate cellular functions by allowing the cells to switch between two different pathways, either mediated via FAK-Y861, or via CDCP1-Y734 or by utilising both. This may lead to distinct cellular phenotypes depending on the level of expression of these two substrates.

Chapter 3

Significantly, an increase of CDCP1 expression has been reported by Uekita et al. in A549 cells grown in suspension (Uekita et al., 2007). CDCP1 expression increased marginally between 3 and 6 hours and became markedly up-regulated between 12 and 48 hours after cell detachment (Uekita et al., 2007). The increase in expression observed by Uekita and colleagues was accompanied by an increase in phosphorylation of CDCP1, which supports the hypothesis that the level of expression of CDCP1 can act as a cellular regulatory mechanism, possibly to regulate anoikis.

Of note, it has also been reported by Liu et al. that growth of A549 cells in suspension leads to an increase in phosphorylation of FAK-Y861, between 30 minutes and 4 hours after detachment (Liu et al., 2008). Additionally, an increase in phosphorylation of FAK-Y397 and SFK-Y416 was observed between 30 minutes and 24 hours after cell detachment. These events were proposed to mediate the decrease in anoikis observed in A549 cells (Liu et al., 2008). Accordingly, it can be speculated that auto-phosphorylated SFKs phosphorylate FAK-Y861 and CDCP1-Y734 in a time-dependent manner and this interplay between SFKs, FAK and CDCP1 can mediate protection from anoikis. In this working model, FAK becomes phosphorylated shortly after cells lose attachment, and CDCP1 expression increases gradually and sequesters SFK from FAK. This allows CDCP1 to execute its anti-apoptotic function via SFK-mediated phosphorylation at CDCP1-Y734, which leads to recruitment of PKC δ and formation of the trimeric protein complex SFK•CDCP1•PKC δ, which mediates an anti-apoptotic phenotype.

Although speculative, this model is supported by several recent reports. The important role of FAK-Y861 and FAK-Y397 phosphorylation in preventing cells from undergoing anoikis has been described (Frisch et al., 1996) and requires FAK phosphorylation within 30 minutes after cell detachment (Liu et al., 2008). Moreover, tyrosine dephosphorylation of FAK is known to precede the cleavage of FAK by caspases in cells undergoing apoptosis induced by the nephrotoxicant dichlorovinylcysteine (DCVC) in renal epithelial cells (van de Water et al., 1999).

Chapter 3

It is likely that anti-apoptotic functions in a given cell line are not mediated by a single effector protein, but rather by concerted action of different molecules in a time-dependent manner. It is possible that the actions of both FAK and CDCP1 are required for anti-apoptotic effects regulated by SFKs. Of relevance, both FAK and CDCP1 have been shown to play crucial roles in protecting cells from undergoing anoikis and the requirement of SFKs in the interaction with both proteins has been described (Golubovskaya et al., 2003; Uekita et al., 2007; Liu et al., 2008; Beierle et al., 2010). Importantly, it was the first time that a connection between these two proteins has been described in a cancer cell line.

Further studies are required to dissect the functional roles of interactions between SFKs, FAK and CDCP1 in an anti-apoptotic cascade as well as, the signalling pathways downstream of these events and the consequence on the cellular phenotype. In particular, further work is required to examine whether these events occur in cells that endogenously express CDCP1.

Chapter 4

Examination of the membrane anchored serine protease matriptase as a cellular processor of CDCP1

Chapter 4

4.1 INTRODUCTION

CDCP1 is a 836 amino acid transmembrane protein with a predicted molecular weight, after removal of its signal peptide, of ~90 kDa, but is usually seen as a ~135 kDa protein due to its high degree of glycosylation (Hooper et al., 2003). LMW-CDCP1, with a molecular weight of ~70 kDa, by Western blot analysis, has been detected in a variety of cancer cell lines including those derived from prostate, cervical, breast and lymphoid cancers (He et al., 2010). Similarly, colon, pancreatic, oesophageal and lung cancer cell lines possess a LMW form of CDCP1 with a reported molecular weight of ~85 kDa (Wong et al., 2009).

The cellular mechanisms which lead to and regulate the generation of LMW- CDCP1 are not well understood. It appears that LMW-CDCP1 can be generated by endogenous mechanisms, as shown by the appearance of phosphorylated LMW-CDCP1 in response to treatment with the polysulfonated, membrane impermeable naphthylurea suramin and several of its analogues in adherent keratinocytes (Brown et al., 2004). Significantly, the high molecular weight form of CDCP1 (HMW-CDCP1) as well as LMW- CDCP1 were found in neonatal mouse epidermis, suggesting that the generation or expression of the low molecular weight form occurs in vivo (Brown et al., 2004).

Some of the published data suggest involvement of an endogenous tryptic serine protease in generating LMW-CDCP1. For example, trypsin treatment of human foreskin keratinocytes (Brown et al., 2004), MCF10A breast epithelial cells (Spassov et al., 2009) and 22Rv1 prostate cancer cells (He et al., 2010) leads to generation of LMW-CDCP1 with reported molecular masses of ~80 kDa, ~85 kDa and ~70 kDa, in the three cell lines respectively. Furthermore, it was found that the trypsin fold serine protease, plasmin, at sub-physiological concentrations, can cleave CDCP1 to a product with a molecular weight of ~80 kDa in cultured keratinocytes (Brown et al., 2004). Moreover, it was reported that the catalytic domain of the serine protease matriptase can cleave the recombinant extracellular portion of CDCP1 (Bhatt et al., 2005). Further data supporting the hypothesis that an

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endogenous serine protease cleaves CDCP1 in vivo arises from studies employing protease inhibitors. He et al. demonstrated that an EDTA free protease inhibitor cocktail, the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) and high concentrations of the serine protease inhibitor aprotinin prevents the cleavage of cell expressed CDCP1 (He et al., 2010). Moreover, Bhatt et al. used the serine protease inhibitor ecotin in MDA-MB- 468 cells transiently expressing CDCP1 to reduce the levels of LMW-CDCP1 in this breast cancer cell line. Of note, this cell line transiently transfected with a CDCP1 encoding expression vector, produces HMW-CDCP1 as well as LMW-CDCP1 which suggests that the generation of LMW-CDCP1 is not due to alternative mRNA splicing (Bhatt et al., 2005).

Although several studies have provided evidence that cell surface anchored CDCP1 can be cleaved by trypsin and plasmin and that the recombinant extracellular portion of CDCP1 can be cleaved by the catalytic domain of matriptase, the in vivo relevance and the consequences of CDCP1 cleavage have not been examined. However, several recent reports suggest that matriptase-mediated processing of CDCP1 may be relevant in vivo . First, Bhatt et al. demonstrated that an anti-matriptase antibody immunoprecipitates myc-tagged CDCP1 from stably expressing breast cancer MDA-MB-468 cells (Bhatt et al., 2005). Second, endogenous matriptase and CDCP1 have been coimmunoprecipitated from SW480 colon cancer cells using an antibody against the tetraspanin CD9 (Andre et al., 2006).

The potential importance of proteolytic processing of CDCP1 is indicated by the observation that the cell surface-retained portion of CDCP1 becomes phosphorylated in response to CDCP1 cleavage. For example, it was reported that proteolytic processing of CDCP1 by trypsin, and the generation of LMW-CDCP1 by endogenous mechanisms, in response to suramin treatment, leads to the phosphorylation of the cell surface-retained portion of CDCP1 (Brown et al., 2004). This suggests that induction of intracellular signalling due to CDCP1 cleavage may occur. Although many cancer cell lines, including those derived from prostate, pancreatic, lung, breast,

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oesophageal and colon cancer, possess LMW-CDCP1 (Wong et al., 2009; He et al., 2010), the relevance of CDCP1 cleavage in cancer progression is not understood. Of interest is the finding of Hooper et al. who detected the cell surface shed form of CDCP1 in the lumen of normal colonic crypts and colon adenocarcinoma cells, which suggests that cleavage of CDCP1 leads to shedding of the intact cleavage product in vivo and this may also act as a mediator of cell signalling events (Hooper et al., 2003). Significantly, it has been proposed that the cell surface shed form of CDCP1 may be detectable in blood or body fluids of cancer patients and be of prognostic or diagnostic value (Hooper et al., 2003; He et al., 2010). Importantly, the relevance of protease-mediated cleavage and ectodomain shedding of cell surface proteins in the regulation of cellular events has previously been described (Hayashida et al., 2010; Sabbota et al., 2010).

It is of relevance to study proteolytic processing of CDCP1 in order to gain a better understanding of the consequences of this event on cancer cell phenotype and cell signalling. Importantly, there are well established examples of cell surface proteins which are proteolytically processed and play an important role in altering cancer cell phenotype. An example of such cell surface proteins includes the protease activated receptor (PAR) family which comprises four members PAR1-4. PARs are differentially activated by various proteases including thrombin, trypsin, hepsin, matriptase and matrix metalloproteinases (Takeuchi et al., 2000; Arora et al., 2007). Expression of PARs has been detected in various human malignancies (Elste and Petersen, 2010) and these receptors may play important roles in facilitating cancer progression (Camerer, 2007). For example, over-expression of PAR1 in melanoma was found to correlate with metastasis in patients, and with increased cell motility and invasiveness in vitro (Silini et al., 2010). Significantly, activation of PARs by proteases has been associated with cellular processes relevant to cancer progression. For example, activation of PAR1 by α-thrombin increases proliferation and invasion of gastric cancer derived cell lines (Fujimoto et al., 2010). Another example of a class of cell surface proteins which are proteolytically processed are the Notch receptors (Mumm and Kopan, 2000). These transmembrane proteins can be cleaved

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by the ADAM (containing a disintegrin and metalloprotease) family (Zolkiewska, 2008). Proteolysis of Notch receptors on the cell surface represents the first step of activation which eventually results in translocation of an intracellular portion of these receptors to the nucleus resulting in transcriptional activation of target genes (Brou et al., 2000). Patho- physiological Notch signalling has been implicated in progression of various human malignancies (Allenspach et al., 2002). Significantly, it was reported that activation of Notch signalling confers a transformed phenotype in human melanocytes (Pinnix et al., 2009) and alters cellular processes including adhesion, proliferation and spheroid formation in primary melanoma cells (Liu et al., 2006).

In an attempt to elucidate whether proteolytic processing of CDCP1 plays a role in altering cellular processes associated with cancer progression the ability of the serine protease matriptase to cleave cell surface anchored CDCP1 was examined. HeLa-vector and HeLa-CDCP1 cells were used to examine whether matriptase-mediated proteolytic processing of CDCP1 leads to a change in cellular phenotype in vitro. To investigate proliferation, migration and adhesion in vitro, cells were incubated with matriptase and assayed and compared to untreated cells. In addition, whether proteolytic processing of CDCP1 causes initiation of intracellular signalling was examined by Western blot analysis of lysates from matriptase-treated cells using antibodies against phosphorylated Erk 1/2. Moreover, whether matriptase-mediated proteolysis of CDCP1 initiates cell signalling was examined by employing the calcium flux assay. HeLa-CDCP1-Y734F cells were included in these experiments to examine whether CDCP1-Y734 is critical in mediating a change in cell phenotype and signalling in response to matriptase-mediated proteolysis of CDCP1.

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4.2 RESULTS

4.2.1 Matriptase can process cell surface expressed ~135 kDa CDCP1 to its ~70 kDa cleavage product and is the most efficient processor amongst three serine proteases tested

To assess whether matriptase can process cell surface anchored CDCP1 and to delineate the concentration at which efficient cleavage takes place, HeLa-CDCP1 cells were incubated with the serine proteases matriptase, KLK4 and trypsin (as a positive control) at a concentration of 50 nM. The cells were incubated for 1, 6 and 24 hours, whole cell lysates were prepared and subjected to Western blot analysis with an anti-CDCP1 C-terminal antibody. As shown in Figure 4.1, it was found that incubation with matriptase for one hour is sufficient to cleave all HMW-CDCP1 to its ~70 kDa form. The cleavage of CDCP1 by matriptase is more efficient than cleavage of CDCP1 by trypsin, since after one hour of incubation with trypsin some full length CDCP1 remained detectable by Western blot analysis. In contrast, KLK4 is an inefficient cleaver of CDCP1, as shown by the fact that after a 24 hour incubation period only ~50% of HMW-CDCP1 is processed to its LMW form.

Figure 4.1: Matriptase, trypsin and KLK4 cleave CDCP1 to ~70 kDa. HeLa-CDCP1 cells were incubated with matriptase, trypsin or KLK4 at a concentration of 50 nM for 1, 6 or 24 hours at 37°C . At the different time points whole cell lysates were prepared and examined by Western blot with the anti-CDCP1 C-terminal antibody. Anti-GAPDH Western blot analysis was performed to examine protein loading.

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Since it was found that matriptase at a concentration of 50 nM can cleave all CDCP1 within 1 hour, the concentration of matriptase was reduced to low nanomolar concentrations. The aim was to further delineate the concentrations at which matriptase can process CDCP1. Therefore, endogenous CDCP1 expressing 786-O kidney cancer cells were incubated with matriptase at concentrations ranging from 1 nm to 50 nm. After 60 minutes whole cell lysates were prepared and subjected to Western blot analysis with the anti-CDCP1 C-terminal antibody. As shown in Figure 4.2 it was found that after 60 minutes all CDCP1 was cleaved at a concentration of 10 nM or higher. At 1 nM and 5 nM about ~40% and ~80% of CDCP1 were cleaved within 60 minutes. This suggests that recombinant matriptase efficiently cleaves cell surface anchored CDCP1 within 60 minutes.

Figure 4.2: Matriptase processes CDCP1 in low nanomolar concentrations to a ~70 kDa product within 60 minutes. The kidney cancer cell line 786-O was incubated at 37°C with t he indicated concentrations of a freshly prepared matriptase solution in serum free media. After a 60 minute incubation period whole cell lysates were prepared and analysed by Western blot analysis using the anti-CDCP1 C-terminal antibody. Anti-GAPDH Western blot analysis was performed to examine protein loading..

To examine the kinetics of cleavage of CDCP1 by matriptase, HeLa-CDCP1 cells and the endogenous CDCP1 expressing prostate cancer cell line 22Rv1 were incubated with recombinant matriptase at concentrations of 1, 5 and 10 nM for 1, 5, 10 and 30 minutes. At the different time points whole cell lysates were generated and subjected to Western blot analysis with the anti-CDCP1 C-terminal antibody. As shown in Figure 4.3 it was found that after a 30

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minute incubation period with a matriptase concentration of 1 nM a small amount of CDCP1 has been cleaved, at 5 nM most CDCP1 had been cleaved and at 10 nM the entire amount of CDCP1 has been converted to the ~70 kDa form. These data support the proposal that matriptase is a very efficient proteolytic processor of cell surface anchored CDCP1 capable of cleaving all CDCP1 within 30 minutes at a concentration of 10 nM. The calculated matriptase concentration which allows cleavage of 50% of CDCP1 in HeLa-CDCP1 cells (IC-50) was determined by densitometry analysis of three independent experiments and is 4.0 +/- 1.8 nM.

Figure 4.3: Matriptase is an efficient processor of CDCP1 stably expressed by HeLa-CDCP1 cells and endogenously expressing 22Rv1 cells. HeLa-CDCP1 (A) and 22Rv1 (B) cells were incubated with 1, 5 or 10 nM of recombinant matriptase for 1, 5, 10 and 30 minutes. At the different time points whole cell lysates were prepared and subjected to Western blot analysis with the anti-CDCP1 C-terminal antibody. Anti-GAPDH Western blot analysis was performed to examine protein loading.

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4.2.2 Matriptase incubation does not cause cell deadhesion

As the aim was to study the effects of proteolytic processing of CDCP1 by matriptase on cell phenotype and signalling and it is known that deadhesion of cells leads to phosphorylation of CDCP1 (Brown et al., 2004), it was examined whether incubation with matriptase leads to cell deadhesion. As shown in Figure 4.4 it was found that incubation of matriptase (10 nM) for 30 minutes did not cause deadhesion of endogenous CDCP1 expressing 22Rv1 cells. This suggests that matriptase is a suitable protease to study the effect of proteolytic processing of CDCP1 independent of cellular events initiated by cell deadhesion.

Figure 4.4: Matriptase incubation does not cause cell deadhesion. 22Rv1 prostate cancer cells were incubated with recombinant matriptase (10 nM) at 37°C. After 30 minutes the cells were examin ed by light microscopy. Bar = 100 µm.

4.2.3 Cleavage by matriptase causes internalisation of the cell surface- retained portion of CDCP1

To assess whether proteolytic processing of CDCP1 leads to internalisation of its cell surface-retained portion, cell surface biotinylation experiments were performed using endogenously expressing 22Rv1 prostate cancer cells. The cells were incubated for 30 minutes with matriptase (10 nM) and cell surface biotinylation was performed (as described in Chapter 2.2.34) on the matriptase treated cells, as well as on untreated control cells. Cell lysates

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were fractionated into a biotinylated cell surface fraction and a non- biotinylated fraction using streptavidin beads.

As shown in Figure 4.5 (and Figure 3.1) 22Rv1 cells do not possess basal levels of LMW-CDCP1 and incubation with matriptase (10 nM) for 30 minutes cleaves all CDCP1 in this cell line (Figure 4.5). In untreated cells HMW- CDCP1 is localised at the cell surface (biotinylated fraction) as well as in the cytosol. After a 30 minute incubation period with matriptase, CDCP1 was completely processed to LMW-CDCP1 which was detected both at the cell surface (biotinylated fraction) as well as the cytosol. These data suggest that HMW-CDCP1 at the cell surface is rapidly processed to LMW-CDCP1 which is then internalised, and that cytosolic HMW-CDCP1, in response to internalisation of LMW-CDCP1, is translocated to the cell surface where it is also proteolytically processed by matriptase. This suggests a dynamic transport of cytosolic HMW-CDCP1 to the cell surface and LMW-CDCP1 from the cell surface into the cytosol. This may be initiated in response to proteolysis of CDCP1 on the cell surface or may occur constantly as part of cellular processing of CDCP1.

Figure 4.5: CDCP1 cleavage by matriptase leads to internalisation of the ~70 kDa cell retained portion of CDCP1. 22Rv1 cells were incubated with matriptase (10 nM) for 30 minutes at 37°C. Cell sur face biotinylation was performed on the matriptase treated cells and on untreated cells. The biotinylated and cytosolic fractions were analysed by Western blot analysis with the anti-CDCP1 C-terminal antibody. GAPDH analysis was performed to examine protein loading and to ensure that the biotinylated fractions were free of cytosolic protein contamination.

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4.2.4 Cleavage of CDCP1 does not lead to a change in the cellular phenotype of HeLa-CDCP1 cells

To examine whether matriptase cleavage of CDCP1 leads to a change in the cellular phenotype a variety of in vitro assays on untreated and matriptase treated cells including proliferation, migration and adhesion were performed. To perform the proliferation assay, HeLa-vector, HeLa-CDCP1 and HeLa- CDCP1-Y734F cells were incubated with matriptase at a concentration of 10 nM for 30 minutes. The matriptase treated cells and untreated control cells were then seeded into 96 well plates, incubated at 37°C and every 24 hours for five days, one plate removed and the relative number of cells in the wells was assessed.

As shown in Figure 4.6 and described in Chapter 5 it was found that CDCP1 expression leads to a decrease in proliferation of HeLa-CDCP1 cells (Figure 5.1 and 5.6) and HeLa-CDCP1-Y734F cells (Figure 5.1) in comparison to HeLa-vector cells. Proteolytic processing of CDCP1 by matriptase did not cause a change in proliferation in any of the three HeLa cell lines in comparison to untreated control. These data suggest that matriptase treatment does not have an effect on proliferation of HeLa cells expressing CDCP1 or CDCP1-Y734F. The data presented in Figure 4.6 shows the results after a five day proliferation period.

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Figure 4.6: CDCP1 cleavage by matriptase does not cause a change in proliferation. HeLa-vector, HeLa-CDCP1 and HeLa-CDCP1-Y734F cells were either untreated (light grey bars) or treated with matriptase (10 nM) (dark grey bars). After 30 minutes cells were deadhered, counted and seeded at a density of 700 cells in 200 µL media in 96 well black plates. Every 24 hours after seeding the relative number of cells/well was assessed using CyQuant NF by detecting the emission at 520 nm on a fluorescence plate reader. For ease of graphical presentation the data shown is the detected emission after a five day proliferation period. Three independent experiments were performed with each sample in triplicate. Presented are the means of the means of each experiment (n=3). Bars are ± standard error of the mean (SEM). P-value was calculated using Student’s t-test with 95% confidence interval.

To assess whether cell motility changed in response to matriptase treatment HeLa-vector, HeLa-CDCP1 and HeLa-CDCP1-Y734F cells were treated with matriptase (10 nM) for 30 minutes at 37°C. These ce lls and untreated controls were then subjected to a chemotactic Transwell migration assay towards a concentration gradient of 5% FCS.

As shown in Figure 4.7 and fully described in Chapter 5 (Figure 5.2) it was observed that expression of CDCP1 and CDCP1-Y734F rendered HeLa cells with a more than two fold increased migratory ability. Matriptase treatment caused an insignificant increase in migration of HeLa-vector cells in comparison to untreated controls. In HeLa-CDCP1 and HeLa-CDCP1-Y734F cells matriptase treatment did not cause a change in the ability of cells to

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migrate in comparison to untreated controls. These data suggest that proteolytic processing of CDCP1 does not cause a change in cell motility of HeLa cells expressing CDCP1 and CDCP1-Y734F.

Figure 4.7: CDCP1 cleavage by matriptase does not cause change in chemotactic migration in CDCP1 expressing HeLa cells. HeLa-vector, HeLa-CDCP1 and HeLa-CDCP1-Y734F cells were either untreated (light grey bars) or treated with matriptase (10 nM) (dark grey bars). After 30 minutes cells were deadhered, counted and the cell number was adjusted to 1x10 6 cells/mL. Transwell inserts were placed into wells of a 24 well plate filled with 500 µL DMEM supplemented with 5% FCS and 100 µL of the cell solution was added into the insert. The cells were allowed to migrate for 24 hours and the migrated cells were counted using a haemocytometer. The experiment was performed four times in duplicate. Presented are the means of the means of the experiments (n=4). Bars are ± SEM. P-value was calculated using Student’s t-test with 95% confidence interval.

In a further attempt to identify changes in the cellular phenotype in response to CDCP1 cleavage by matriptase, adhesion assays were performed using a variety of ECM proteins. Cells were incubated with a 10 nM solution of matriptase, deadhered and allowed to adhere to the ECM proteins for 30 minutes at 37°C.

Proteolytic processing of CDCP1 by matriptase did not alter adhesion to fibronectin, vitronectin, laminin-1 and collagen-IV of HeLa-vector cells and HeLa cells expressing CDCP1 or CDCP1-Y734F in comparison to untreated

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controls (Figure 4.8). These data suggest that proteolytic processing of CDCP1 does not lead to altered cell adhesion in CDCP1 expressing HeLa cells.

Figure 4.8: CDCP1 cleavage by matriptase does not cause a change in adhesion to ECM proteins. HeLa-vector, HeLa-CDCP1 and HeLa-CDCP1- Y734F cells were either untreated (light grey bars) or treated with matriptase (10 nM) (dark grey bars). After 30 minutes cells were deadhered, counted, and cell number was adjusted to 0.25x10 6 cells/mL. Black walled 96 well plates were coated for 3 hours at room temperature with fibronectin (A) , collagen-IV (C) or laminin-1 (D) at 10 ug/mL or vitronectin (B) at 5 µg/mL. Cells (25000 per well) were added into the wells of the 96 well black plate and incubated at 37°C. After 30 minutes the relativ e number of attached cells was determined using CyQuant NF by detecting the emission at 520 nm on a fluorescent plate reader. This experiment was performed 3 times in triplicate. Presented are the means of the means of the experiments (n=3). Each cell line is normalised to its untreated control which was set to 1. Bars are SEM.

4.2.5 Initiation of an intracellular signalling cascade via Erk 1/2 activation is not initiated by proteolysis of CDCP1 by matriptase

Whether matriptase-mediated proteolysis leads to an induction of intracellular signalling in CDCP1 expressing HeLa cells was examined next. To address this question, two different approaches were employed. First,

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whether proteolytic processing of CDCP1 causes an increase in phosphorylation of Erk 1/2 was examined by Western blot analysis. Second, whether matriptase-mediated proteolytic processing causes a flux of free calcium was examined using the calcium flux assay.

To examine whether CDCP1 cleavage leads to an intracellular signalling cascade via Erk 1/2 phosphorylation, HeLa-vector, HeLa-CDCP1 and HeLa- CDCP1-Y734F cells were incubated with matriptase at concentrations of 1, 5 and 10 nM for 1, 5, 10 and 30 minutes. At the indicated time points whole cell lysates were collected in the presence of phosphatase inhibitors and subjected to Western blot analysis with antibodies against phospho Erk 1/2, total Erk 1/2, FLAG tag and tubulin as loading control. As shown in Figure 4.9A matriptase-mediated proteolytic processing of CDCP1 in HeLa-CDCP1 cells was accompanied by an increase in phosphorylation of Erk 1/2 in a dose-dependent manner while total Erk 1/2 was unchanged (Figure 4.9A). This increase in phosphorylation of Erk 1/2 in response to matriptase- mediated proteolytic processing of CDCP1 was also observed in HeLa- CDCP1-Y734F cells (Figure 4.9B). Of note, an increase in phosphorylation of Erk 1/2 in response to matriptase treatment was also detected in HeLa- vector cells (Figure 4.9C). These data suggest that the increase in phosphorylation of Erk 1/2 observed in HeLa-CDCP1 and HeLa-CDCP1- Y734F cells in response to matriptase treatment is not mediated by CDCP1.

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Figure 4.9: Phosphorylation of Erk 1/2 due to matriptase treatment is not mediated by CDCP1. HeLa-CDCP1 (A) , HeLa-CDCP1-Y734F (B) and HeLa-vector cells (C) were untreated (-) or treated with matriptase in serum free DMEM at concentrations of 1, 5 and 10 nM for 1, 5, 10 and 30 minutes at 37°C. At the different time points whole cell ly sates were prepared in the presence of phosphatase inhibitors and subjected to Western blot analysis with the indicated antibodies. Anti-GAPDH Western blot analysis was performed to examine protein loading..

To further assess whether cleavage of CDCP1 can initiate intracellular signalling calcium flux experiments were performed. The measurement of a flux of free calcium to assess the induction of a signalling event in response to proteolytic processing of cell surface proteins has previously been described (Ramsay et al., 2008). PC3 cells were used as a positive control cell line for this assay. It was found that matriptase at a final concentration of 20 nM resulted in a similar response in calcium flux in HeLa-vector, HeLa-

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CDCP1 and HeLa-CDCP1-Y734F cells (Figure 4.10A). This suggests that proteolysis by matriptase initiates an intracellular signalling event which is not mediated by CDCP1. As expected PC3 cells showed a strong response to matriptase treatment in this experiment as these cells are known to express cell surface localised protease-activated receptor 2 (PAR2) (Ramsay et al., 2008) and PAR2 is a known proteolytic target of matriptase (Uhland, 2006).

To examine whether matriptase-induced changes in Ca2+ concentration were also mediated by PAR2 expressed by HeLa cells (Sanchez-Hernandez et al., 2008), the PAR2 activating peptide (PAR2-AP) was employed as an agonist in the calcium flux assays. The PAR2-AP has previously been shown to initiate a calcium flux in PAR2 expressing cells (Ramsay et al., 2008) which leads to the internalisation of PAR2. As shown in Figure 4.10B, incubation with the PAR2-AP leads to a flux of Ca 2+ in all three HeLa cell lines to a similar extent suggesting that these three HeLa cell lines are equally responsive to PAR2 signalling. This indicated that initiation of a cellular signalling event in response to matriptase treatment may be mediated by PAR2 rather than by CDCP1 in HeLa cells. To examine this possibility, sequential treatment of HeLa cells with PAR2-AP followed by matriptase treatment was performed. In this experimental setting it was found that cells treated first with PAR2-AP became unresponsive to subsequent treatment with matriptase (Figure 4.10C). These data suggest that HeLa-CDCP1 cells respond to matriptase via a pathway mediated by PAR2 activation rather than by a CDCP1 initiated signalling cascade.

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Figure 4.10: Calcium flux induced by matriptase is not mediated by CDCP1. PC3, Hela-vector, HeLa-CDCP1 and HeLa-CDCP1-Y734F cells (4x10 6 cells/mL) were loaded with the fluorescence indicator Fura-2 (1 µM) at room temperature for 60 minutes. Cells were then resuspended at a concentration of 2x10 6 cells/mL for fluorescence measurements. The ratio of fluorescence at 510 nm after excitation at 340 and 380 nm was monitored using a fluorescent plate reader. Single agonist treatments were matriptase (20 nM) (A, C) and PAR2-AP (100 µM) (B, C) . Displayed data are representative of experiments performed in triplicate and repeated on two independent occasions. The arrows indicate the time of agonist addition. These experiments were performed with Mark Adams, another PhD student in the Hooper laboratory.

As there were no changes detectable in cellular phenotype or signalling mediated by CDCP1 in response to proteolysis by matriptase and it was demonstrated that other proteases such as trypsin and KLK4 can process CDCP1 (Figure 4.1), it was questioned whether expression of matriptase correlates with the abundance of LMW-CDCP1 in endogenous CDCP1 expressing cells. To address this question Western blot analysis of a panel of

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endogenous CDCP1 expressing prostate cancer cell lines was performed. As shown in Figure 4.11, it was found that matriptase expression does not correlate with the appearance of LMW-CDCP1 in endogenous CDCP1 expressing prostate cancer cell lines. This is demonstrated by the finding that matriptase expression is detected in both cells which possess LMW-CDCP1 and cells which possess HMW-CDCP1 only. Importantly, in the prostate cancer cell line DU145 which possesses a strong CDCP1 ~70 kDa band, no matriptase expression could be detected. These data indicated that matriptase is not the only endogenous proteolytic processor of CDCP1 in these cultured cancer cells.

Figure 4.11: Examination of matriptase expression in prostate cancer and prostate epithelial derived cell lines endogenously expressing CDCP1. Whole cell lysates were analysed with the anti-CDCP1 C-terminal antibody and an anti-matriptase antibody. Anti-GAPDH Western blot analysis was performed to examine protein loading..

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4.3 DISCUSSION

CDCP1 has a theoretical molecular weight of ~90 kDa but is detected with a molecular weight of ~135 kDa by Western blot analysis due to its high degree of glycosylation. Interestingly, a shorter form of CDCP1 with a reported molecular weight of ~70 kDa (He et al., 2010) or ~85 kDa (Wong et al., 2009) is found in lysates of a variety of cancer cell lines. It was reported that LMW-CDCP1 can be produced by an endogenous mechanism in response to suramin treatment in cultured keratinocytes (Brown et al., 2004). Importantly, involvement of tryptic serine proteases in the cleavage of CDCP1 has been previously suggested. For example, it was found that recombinant trypsin and plasmin can cleave cell surface anchored CDCP1 to a cleavage product with a molecular weight of ~80 kDa (Brown et al., 2004) and treatment with the protease inhibitor ecotin can greatly decrease the abundance of LMW-CDCP1 in stably expressing MDA-MB-468 breast cancer cells (Bhatt et al., 2005). In addition, Bhatt et al. reported that the catalytic domain of matriptase can cleave the recombinant extracellular portion of CDCP1. However, whether matriptase is able to cleave intact CDCP1 on the cell surface of cancer cells has not been established. Moreover, consequences on cell phenotype induced by proteolytic processing of CDCP1 have not been investigated previously.

In this chapter it was examined whether matriptase is able to proteolytically process cell surface localised CDCP1 and whether this proteolytic event impacts on cell phenotype and intracellular signalling.

Matriptase was chosen because it has a narrower substrate specifity (Uhland, 2006) compared to trypsin, which cleaves a large number of cell surface proteins (Olsen et al., 2004) and, therefore, is likely to elicit off target effects. In addition, trypsin treatment leads to deadhesion of cells (Brown et al., 2004) and any detected consequences on cell phenotype in response to trypsin treatment may be induced by cell deadhesion rather than proteolytic processing of CDCP1.

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The key findings of this chapter are:

• The serine protease matriptase is an efficient proteolytic processor of cell surface CDCP1. • Matriptase-mediated proteolysis of CDCP1 does not alter the phenotype of CDCP1 expressing HeLa cells in vitro in proliferation, adhesion and migration. • Proteolysis by matriptase leads to initiation of intracellular signalling which is not mediated via CDCP1, but by PAR2.

4.3.1 Matriptase is a very efficient proteolytic processor of cell surface CDCP1 and generates LMW-CDCP1 with a molecular weight of ~70 kDa

It was found that matriptase is a very efficient proteolytic processor of cell surface expressed CDCP1. It is able to cleave CDCP1 at low nanomolar concentrations to its ~70 kDa cleavage product with a calculated IC-50 of 4.0 +/- 1.8 nM. This suggests that proteolytic processing of CDCP1 by matriptase may occur in tumours in vivo where the concentration of this protease has been estimated at 13-24 ng/mg of tissue protein (Oberst et al., 2001) . Interestingly, it was found that the tryptic serine protease KLK4 is also able to cleave CDCP1. Although KLK4 cleavage is less efficient than matriptase-mediated CDCP1 proteolysis it may indicate that these two proteins interact in vivo and may have a role in cancer since KLK4 is known to be over-expressed in several human malignancies including prostate cancer (Dong et al., 2001; Zhang et al., 2009).

Different molecular weights have been reported for LMW-CDCP1 ranging from ~70 kDa to ~85 kDa (Brown et al., 2004; Bhatt et al., 2005; Wong et al., 2009; He et al., 2010). During the course of this study data was generated which suggest that this difference in molecular weight of LMW-CDCP1 is likely to be an artefact which may arise as a result of different molecular weight markers for protein sizing, different percentages of polyacrylamide gels, and different levels of glycosylation of CDCP1 or other post- translational modifications of CDCP1. This is based on two published

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observations and the findings presented in Figure 4.1. First, He et al. showed that cleavage of CDCP1 by trypsin produces a LMW-CDCP1 which migrates at the same molecular weight as LMW-CDCP1 which is seen endogenously in PC3 cells (He et al., 2010). Second, Wong et al . presented a Western blot analysis of cancer cell lines including PC3 cells, that showed that LMW- CDCP1 expressed by each cell migrates at the same molecular weight as the LMW-CDCP1 of PC3 cells. Although, these authors quantified the molecular weight of LMW-CDCP1 as ~85 kDa, it is clear that this is the same molecular weight of ~70 kDa described by He et al. Third, it was found that cleavage of CDCP1 by three different serine proteases, namely matriptase, trypsin and KLK4, produces a LMW-CDCP1 species with a molecular weight of ~70 kDa (Figure 4.1).

4.3.2 Matriptase-mediated proteolysis does not alter the phenotype of CDCP1 expressing HeLa cells and intracellular signalling initiated by this serine protease is not caused by proteolysis of CDCP1

As matriptase efficiently cleaves CDCP1 it was examined whether this event impacts upon in vitro cellular processes associated with cancer progression including proliferation, migration and adhesion. However, the generated data indicated that matriptase proteolysis does not cause a significant change in the cellular phenotypes via a CDCP1-mediated pathway . It is possible that variation in the way these in vitro assays were performed may produce different results. The cells were treated with matriptase (10 nM) for 30 minutes, and were then deadhered before being examined in in vitro assays. Based on the observation that deadhesion initiates phosphorylation of CDCP1, which is likely to be required for initiation of intracellular signalling, it is possible that deadhesion following matriptase treatment may initiate intracellular signalling in CDCP1 expressing HeLa cells. This deadhesion induced signalling event may counteract the initiation of cellular events induced by proteolytic processing of CDCP1 and may, therefore, not allow for the detection of differences between matriptase treated cells and the untreated control. Therefore, it would be advisable for future experiments to

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deadhere cells first then allow them to adhere to the cell culture plastic (proliferation assay) or to the Transwell inserts before performing matriptase treatment. This approach will allow examination of events induced by proteolytic processing of CDCP1 by matriptase without potential side effects due to cell deadhesion.

To examine whether cleavage of CDCP1 by matriptase initiates signalling two intracellular signalling cascades were examined. First, phosphorylation of Erk 1/2 was analysed by Western blot analysis, and second calcium flux assays were performed to measure changes in free Ca2+. These assays have previously been extensively used to study initiation of signalling due to proteolysis of the cell surface proteins protease activated receptor (Bohm et al., 1996a; Bohm et al., 1996b; Kahn et al., 1999; Wang et al., 2002) . It was found that Erk 1/2 phosphorylation, as detected by Western blot analysis is increased in response to matriptase treatment in a dose-dependent manner, but this can be seen in HeLa-CDCP1 cells, HeLa-CDCP1-Y734F cells as well as in HeLa-vector cells. This finding suggests that the initiation of cellular signalling involving Erk 1/2 is not mediated by proteolytic processing of CDCP1. Moreover, an equal response to matriptase treatment of HeLa- vector, HeLa-CDCP1 and HeLa-CDCP1-Y734F cells was detected in calcium flux assays. By employing a PAR2 specific activating peptide in the calcium flux assay it was demonstrated that HeLa-vector cells and CDCP1 expressing HeLa cells respond to matriptase treatment via a signalling event initiated by PAR2 activation rather than by proteolysis of CDCP1. It remains to be examined whether matriptase-mediated proteolysis of CDCP1 induces intracellular signalling via other pathways.

It is worth noting that HeLa-CDCP1 cells may have certain limitations for the study of cellular events arising from matriptase-mediated proteolysis of CDCP1. This is based on findings from He et al. and data of the biochemical characterisation of CDCP1 expressing HeLa cells, as described in Chapter 3. He et al. demonstrated that proteolysis of CDCP1 leads to phosphorylation of the cell surface-retained portion of CDCP1 and consequent recruitment of SFKs and PKC δ (He et al., 2010). This suggests that proteolysis of CDCP1

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may cause intracellular signalling via activation of SFKs. Importantly, the 22Rv1 prostate cancer cell line which was used for this experiment does not display basal phosphorylation of CDCP1 (He et al., 2010). In contrast, HeLa- CDCP1 cells display strong basal phosphorylation of CDCP1 and basal co- localisation of CDCP1 with SFKs and PKC δ (Figure 3.7, 3.10 and 3.11). It is possible that this high level of basal phosphorylation of CDCP1 in HeLa- CDCP1 cells does not allow for initiation of cell signalling via CDCP1 as a consequence of matriptase-mediated proteolysis. This suggests a possible limitation in using this cell line to study cellular events which require initiation of CDCP1 phosphorylation in response to proteolysis of CDCP1.

Interestingly, it was also found that matriptase expression does not correlate with the appearance of the ~70 kDa form of CDCP1 in prostate cancer cell lines endogenously expressing CDCP1. As shown in Figure 4.11, matriptase expression is observed in cells which possess LMW-CDCP1, but also in cells which possess HMW-CDCP1 only. Importantly, in the prostate cancer cell line DU145 which possesses a strong ~70 kDa band, no matriptase expression can be detected. This suggests that matriptase is not the endogenous processor in CDCP1 expressing cell lines or that matriptase is one of a number of proteases capable of proteolytically processing CDCP1. This is also supported by the observation that trypsin and the tryptic serine protease KLK4 are able to proteolytically process cell surface CDCP1.

In conclusion, it was shown that matriptase is an efficient proteolytic processor of cell surface localised CDCP1. It appears that this protease can be used to further study the effects of CDCP1 cleavage in vitro . However, to study changes in cellular phenotypes as a consequence of increased phosphorylation of CDCP1 due to its proteolytic processing, it will be beneficial to employ a cell line that endogenously expresses CDCP1 and is not phosphorylated under basal conditions. In addition, such a cell line would ideally not express other cell surface serine protease substrates such as PAR2, and it will also be important to use a CDCP1 knockdown cell line as control. By using such cell lines it may be possible to detect changes in cell phenotype and cellular signalling involving an increase in SFK-mediated

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CDCP1 phosphorylation as a consequence of proteolytic processing of CDCP1 by matriptase and other proteases.

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Examination of the effect of expression of CDCP1 and CDCP1 tyrosine mutants on the phenotype of HeLa cells in vitro and in vivo

Chapter 5

5.1 INTRODUCTION

The metastatic spread of cancer cells represents the biggest problem in the management of cancer disease. The majority of cancer related mortality is due to manifestation of metastasis at secondary sites (Mehlen and Puisieux, 2006). For patients diagnosed with organ confined cancer which is amenable to surgical removal, survival rates are high. However, the same is not true for patients with disseminated cancer. For these patients prognosis is poorer and drops away significantly the later diagnosis occurs (Ott et al., 2009). Once a cancer has spread, current treatment options have limited efficiency and new avenues for treatment are desperately needed. Metastatic dissemination of malignant tumour cells is a complex multistep process which involves a multitude of cellular proteins (Bacac and Stamenkovic, 2008). A better understanding of cellular pathways and proteins facilitating metastasis may contribute to improved treatment strategies in the future. In this respect the identification and characterisation of proteins facilitating metastasis is crucial.

CDCP1 is a cell surface protein which has been shown to play an important role in metastasis formation in animal models. This protein was identified by Hooper et al. to be expressed on the cell surface of metastatic human cancer Hep-3 cells in contrast to the low metastatic version of this cell line (Hooper et al., 2003). This finding suggested that CDCP1 may play a key role in facilitating metastasis formation in vivo . Since then, several reports have indicated that CDCP1 is indeed important in metastasis in in vivo models. Uekita and co-workers reported that knock down of CDCP1 in lung cancer A549 cells and two gastric cancer cell lines leads to a significant decrease of metastases in mice (Uekita et al., 2007; Uekita et al., 2008). Additionally, Deryugina et al. reported that expression of CDCP1 in HeLa cells leads to a more metastatic phenotype in the chicken embryo metastasis model and in mice (Deryugina et al., 2009).

Importantly, it has been reported that targeting of CDCP1 with anti-CDCP1 antibodies in a variety of cancer cell lines leads to a decrease in metastasis in in vivo models. For example, Siva et al. reported that targeting CDCP1

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expressed by the prostate cancer cell line PC3 with an anti-CDCP1 antibody conjugated with the cytotoxin saporin inhibits primary tumour growth, as well as metastasis formation in mice (Siva et al., 2008b). Furthermore, Deryugina et al. demonstrated that ligation of CDCP1 with two anti-CDCP1 antibodies, generated by a subtractive immunisation approach against highly metastatic Hep3 cells (Brooks et al., 1993; Hooper et al., 2003), leads to a significant decrease in metastasis formation in mice, as well as in the chicken embryo metastasis model (Deryugina et al., 2009). In addition, Fukuchi et al. used function-blocking human antibodies against CDCP1 to inhibit metastasis formation of CDCP1 expressing HeLa cells and PC3 cells in mice and in the chicken embryo metastasis model (Fukuchi et al., 2010). The mechanisms by which anti-CDCP1 antibodies exert their anti-metastatic effects are not understood. It is possible that internalisation of an CDCP1•antibody complex mediates the anti-metastatic effect of anti-CDCP1 antibodies. It has been demonstrated that CDCP1•antibody complex internalisation occurs, using anti-CDCP1 antibodies coupled to the cytotoxin saporin. Incubation of CDCP1 expressing cancer cells with saporin conjugated anti-CDCP1 antibodies resulted in delivery of this cytotoxin to the cell interior followed by cell killing and this is indirect evidence of CDCP1•antibody complex internalisation (Siva et al., 2008b; Fukuchi et al., 2010).

The pro-metastatic phenotype initiated by CDCP1 has been reported to be mediated through an anti-apoptotic phenotype. Deryugina et al. showed that ligation of CDCP1 with an anti-CDCP1 antibody caused an increase in apoptosis in vitro under pro-apoptotic conditions induced by doxorubicin (Deryugina et al., 2009). This in vitro finding was supported by the observation in vivo that CDCP1 expressing HeLa cells treated with an anti- CDCP1 antibody undergo apoptosis after extravasation through CAM capillaries in the chicken embryo metastasis model (Deryugina et al., 2009). Similar reports were presented by Uekita et al. who demonstrated that knock down of CDCP1 leads to a significant increase in apoptosis in A549 lung cancer cells grown in suspension (Uekita et al., 2007).

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The phosphorylation of CDCP1 by SFKs has been suggested to be an integral event in the pro-metastatic function of CDCP1 expressing cancer cells. Importantly, it was reported by Uekita et al. that phosphorylation of CDCP1 at Y734 is increased in tumour nodules of gastric cancer 44As3 cells during peritoneal invasion in mice (Uekita et al., 2008). Moreover, this work also demonstrated elevated p-CDCP1-Y734 levels in human gastric cancer cells invading the gastric wall (Uekita et al., 2008). Consistently, in lung cancer patient samples p-CDCP1-Y734 is largely present in invading tumour cells (Ikeda et al., 2009).

Another reported CDCP1 interacting protein, PKC δ, binds to CDCP1-Y762 after SFK binding and phosphorylation of CDCP1-Y734. Importantly, it was suggested that formation of a SFK•CDCP1•PKC δ trimeric complex plays a critical role in mediating the CDCP1 induced anti-apoptotic phenotype in vitro (Uekita et al., 2007). It was reported that tyrosine phosphorylation of PKC δ- Y311 is required for the observed resistance to anoikis in vitro (Uekita et al., 2007). Of further interest, is the observation that binding of PKC δ to CDCP1 and the phosphorylation of PKC δ-Y311 increased the ability of cells to degrade ECM proteins and to migrate and invade, which translates into a more metastatic phenotype (Miyazawa et al., 2010).

Two studies also suggest that CDCP1-Y734 is crucial for CDCP1-mediated in vitro phenotypes. Uekita et al. reported that transient expression of CDCP1-Y734F did not increase invasion and migration in a gastric cancer cell line in contrast to CDCP1 expression which significantly increased migration and invasion (Uekita et al., 2008). The importance of CDCP1- Y734 in cell migration was also examined by Miyazawa et al. These authors used pancreatic cancer cells endogenously expressing CDCP1, transfected with a CDCP1-siRNA and a CDCP1-Y734F rescue construct to demonstrate that CDCP1-Y734F expressing cells do not display an increased ability to migrate in a Transwell assay, in contrast to cells transfected with a rescue construct for CDCP1 (Miyazawa et al., 2010).

The relevance of phosphorylation of CDCP1-Y743 in cancer has also recently been examined. Wong et al. used an antibody raised against

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CDCP1-Y743 to show that phosphorylation of this tyrosine is increased in cancer tissue samples of different origin and various histological grading, while no phosphorylation of CDCP1-Y743 was detectable in normal epithelial tissues (Wong et al., 2009).

Of relevance to this project, no data has yet been published examining the functional consequence of single mutations of the conserved tyrosine residues at position 734, 743 and 762 of CDCP1 on metastasis in vivo . Furthermore, no data is currently available giving insight into how phosphorylation of Y743 and Y762 contributes to in vitro cellular phenotypes. To explore the consequences of the mutations of these tyrosine residues on cell phenotype, the cell lines described in Chapter 3 were subjected to a variety of in vitro and in vivo assays. As described in this chapter the in vitro assays were focused on examining cellular processes relevant to cancer progression including proliferation, adhesion and migration.

To study the metastatic ability of the stable CDCP1 expressing HeLa cells the experimental chicken embryo metastasis assay was performed in the laboratory of collaborators at the Scripps Research Institute who are leading experts in the use of this animal model (Zijlstra et al., 2002; Deryugina and Quigley, 2008). The chicken embryo model is an in vivo model that allows the study of tumour formation, angiogenesis and tumour cell dissemination. It offers some unique advantages over murine in vivo models to study the complex, multistep process of metastasis formation. It is less costly and labour intensive and results can be obtained in a relatively short time frame. Another advantage of this model is the possibility of repetitive experimental manipulations to study the effects on tumour cell phenotype in vivo . The chorioallantoic membrane (CAM) of chicken embryos provides support for the growth of inoculated tumour cells in a naturally immunodeficient environment (Deryugina and Quigley, 2008). In the study of “spontaneous” tumour cell dissemination, tumour cells are inoculated onto the CAM and dissemination of tumour cells from the spot of inoculation into the CAM, extravasation from the CAM capillaries, intravasation into distant parts of the CAM as well as intravasation into other organs can be examined. In the

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“experimental” study of tumour cell dissemination, tumour cells are injected intravenously into the CAM vein and extravasation from the CAM capillaries and dissemination into the CAM are assessed (Zijlstra et al., 2003; Deryugina and Quigley, 2008). In this research project the experimental chicken embryo metastasis assay was performed because HeLa cells have low tumourigenic and low metastatic ability and do not metastasise in the spontaneous setting of this assay.

5.2 RESULTS

5.2.1 CDCP1 expression decreases proliferation and adhesion of HeLa cells

HeLa-vector, HeLa-CDCP1, HeLa-CDCP1-Y734F, -Y743F and -Y762F cells were used in an assay to determine whether CDCP1 impacts on proliferation of HeLa cells. Not only is it important to perform this assay to examine whether CDCP1 can influence this cellular process, but the outcome of this experiment can also be taken into consideration for in vivo metastasis formation experiments where a difference in proliferation is likely to be reflected in the results of these assays. As shown in Figure 5.1 expression of CDCP1, caused an ~35% decrease in proliferation of HeLa-CDCP1 cells in comparison to HeLa-vector cells. Proliferation of HeLa-CDCP1-Y734F and - Y743F cells was similarly reduced while proliferation of HeLa-CDCP1-Y762F cells was ~50% lower than HeLa-vector cells.

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Figure 5.1: CDCP1 expression leads to a decrease in proliferation of HeLa cells. Cells were seeded at a density of 700 cells in 200 µL media per well of a 96 well plate. Every 24 hours after seeding one plate was incubated with CyQuant NF solution for 45 minutes and the relative number of cells per well assessed by detecting emission at 520 nm. Presented are the means of the means of three experiments each having 3 replicates (n=3). Bars are ± SEM. P-value was calculated with Student’s t-test with 95% confidence interval.

CDCP1 has previously been reported to decrease adhesion to various ECM proteins (Deryugina et al., 2009). Accordingly, assays to examine whether any of the CDCP1 tyrosine mutants can mediate a change in cell adhesion were performed. For these assays a variety of ECM proteins were used, including collagen-I, fibronectin and laminin-1 and the basement membrane matrix, Matrigel. As shown in Figure 5.2 it was found that adhesion to ECM components and to Matrigel is greatly reduced in HeLa-CDCP1 cells, as well as in all the tyrosine mutants in comparison to HeLa-vector cells. The ability of HeLa-CDCP1 cells to adhere to the examined ECM components and to Matrigel was reduced between 65% and 80% in comparison to HeLa-vector cells. The adhesion of the three CDCP1 tyrosine mutants was also greatly decreased with a decrease of as much as 70% in comparison to HeLa-vector cells. Of note, all tyrosine mutants displayed a trend towards a slightly higher adhesion than HeLa-CDCP1 cells with the highest adhesion consistently apparent for HeLa-CDCP1-Y743F cells. The adhesion of HeLa-CDCP1-

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Y743F cells is particularly high to fibronectin where the adhesion is ~80% that of HeLa-vector cells whereas HeLa-CDCP1 cells have an adhesion level of ~35% that of HeLa-vector cells for this ECM protein.

Figure 5.2: CDCP1 expression leads to a decrease in cell adhesion. Cells were seeded in 96 well plates pre-coated with 10 µg/mL of (A) collagen-I, (B) fibronectin, (C) laminin-1 and (D) Matrigel. Cells were allowed to adhere for 30 minutes, the plates were washed and adhered cells stained with a 0.5% crystal violet solution. The relative number of adhered cells was determined by detecting the OD at 560 nm. Presented are the means of the means of four experiments each having 3 replicates (n=4). Bars are ± SEM. P-value was calculated with Student’s t-test with 95% confidence interval. The data presented is normalised to the vector control for each ECM component (Vector=1).

5.2.2 CDCDP1 expression increases HeLa cell migration

CDCP1 has been reported to increase migration of transiently transfected lung and gastric cancer cells in a p-CDCP1-Y734 dependent manner (Uekita et al., 2007; Uekita et al., 2008). To assess the effects of stable expression of CDCP1 and the three CDCP1 tyrosine mutants on cell migration chemotactic Transwell migration assays towards a concentration gradient of 5% FCS were performed. As shown in Figure 5.3 it was found that CDCP1

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leads to a more than ~2 fold increase in migration of HeLa-CDCP1 cells compared to HeLa-vector cells. Migration of CDCP1 tyrosine mutant expressing cells was increased to a similar extent as was observed for HeLa- CDCP1 cells.

Figure 5.3: CDCP1 increases migration of HeLa cells towards FCS. Cells (100 µL) at a density of 1x10 6 cells/mL in serum free DMEM were plated out into Transwell inserts in wells of a 24 well plate filled with 500 µL DMEM supplemented with 5% FCS. After 24 hours the number of migrated cells was counted using a haemocytometer. The experiment was performed four times in duplicate. Presented are the means of the means of the experiments (n=4). Bars are SEM. P-value was calculated with the Student’s t-test with a 95% confidence interval.

The in vitro data from the proliferation, adhesion and migration assays presented in Figure 5.1, 5.2 and 5.3 suggest that the decrease in proliferation and adhesion and the increase in migration due to CDCP1 expression in HeLa cells is largely independent of the three examined CDCP1 tyrosine residues Y734, Y743 and Y762. However, the marked decrease in proliferation of HeLa-CDCP1-Y762F cells and the higher rate of adhesion of HeLa-CDCP1-Y743F cells to fibronectin suggests a potential role for these two tyrosine residues in altering proliferation and adhesion of HeLa cells.

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5.2.3 HeLa-CDCP1-Y734F cells are most metastatic in the chicken embryo metastasis assay and metastasis formation can be inhibited by an anti-CDCP1 antibody

It was examined next whether CDCP1 mutants Y734F, Y743F and Y762F can mediate a change in metastasis formation in vivo using the experimental chicken embryo metastasis model. In this assay the cells are injected intravenously into the CAM vein of 12 day old chicken embryos and after 5 days the embryos are sacrificed and three distant portions of the lower CAM are harvested, combined and subjected to genomic DNA extraction. The extent of HeLa cell dissemination in the CAM is detected by a PCR approach assessing the number of human Alu repeats versus the amount of chicken GAPDH (Zijlstra et al., 2002; Deryugina et al., 2009).

As shown in Figure 5.4, it was found that CDCP1 expression causes a ~50% decrease in dissemination of HeLa-CDCP1 cells in this in vivo assay. HeLa- CDCP1-Y734F and HeLa-CDCP1-Y743F cells display ~30% increased ability to disseminate in the CAM in comparison to HeLa-CDCP1 cells. In contrast, HeLa-CDCP1-Y762F cells display a very low level of metastasis; displaying ~60% lower metastasis than HeLa-CDCP1 cells. Although these data did not achieve statistical significance, it suggests that CDCP1 expression causes a decrease in metastasis formation of HeLa cells and mutation of CDCP1-Y734 and -Y743 leads to an increase in the ability of HeLa cells to disseminate into the CAM.

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Figure 5.4: CDCP1 expression reduces dissemination of HeLa cells in the chicken embryo CAM assay. Cells (5x10 4) were injected intravenously into the CAM vein within 45 minutes after the cells were deadhered. After 5 days three portions of the lower CAM were harvested and the level of cell dissemination was assessed by PCR analysis of the extracted genomic DNA. Presented are means of pooled data of 2-3 independent experiments where a total number of 14 to 24 embryos were used. Bars are standard error. The number of embryos used per cell line is indicated in the bars of the graph. These experiments were performed under guidance from Dr Elena Deryugina.

To assess whether there are any differences in inhibition of metastasis formation by an anti-CDCP1 antibody between the CDCP1 and the CDCP1 tyrosine mutant expressing cells in vivo , the cells were incubated with the anti-CDCP1 antibody 41-2 at 50 µg/mL before injection into the CAM vein (Deryugina et al., 2009). It was found that dissemination of HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F cells was unaffected by this antibody (Figure 5.5A). In contrast, dissemination of HeLa-CDCP1-Y734F cells was reduced by ~60% due to treatment with the anti-CDCP1 antibody 41-2. The same results for HeLa-CDCP1 and HeLa-CDCP1-Y734F cells were obtained using an increased concentration of MAb 41-2 (Figure 5.5B) and another function-blocking anti-CDCP1 antibody 10D7 (Figure 5.5C) (Deryugina et al., 2009) .

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Figure 5.5: Examination of the effect of function-blocking anti-CDCP1 antibodies on dissemination of HeLa cells stably expressing CDCP1, CDCP1-Y734F, -Y743F and -Y762F. Cells were either untreated (grey bars) or treated with the anti-CDCP1 antibody 41-2 (black bars) (50 µg/mL A, 100 µg/mL or 200 µg/mL B or the anti-CDCP1 antibody 10D7 at a concentration of 50 µg/mL C). Cells were incubated with the antibody for 45 minutes on ice and 50 µL of the cell solution was injected into the CAM vein. A, Presented are means of pooled data of 2-3 independent experiments were a total number of 16 to 33 embryos was used for each treatment group. P-value was calculated using the Student’s t-test with 95% confidence interval. B and C, Data presented is from one experiment were a total number of 6 to 9 chicken embryos were used for each treatment group. Bars are SEM. The number of embryos used per cell line and treatment is indicated in the bars of the graph. These experiments were performed under guidance from Dr Elena Deryugina.

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5.2.4 In vivo experiments revealed differences between the CDCP1 expressing HeLa cells generated in this project and those from Deryugina et al.

The finding that HeLa-vector cells are more metastatic than HeLa-CDCP1 cells was unexpected, as CDCP1 has been reported to significantly increase metastasis formation of CDCP1 expressing HeLa cells in the chicken embryo (Deryugina et al., 2009). However, the data presented in Figure 5.4 did not achieve statistical significance. Importantly, while reviewing the data of Deryugina et al. it was noticed that HeLa-CDCP1-Y734F cells, which were generated for this study and provided for the purpose of the publication of Deryugina et al ., displayed a ~50% higher metastatic ability than the HeLa cells generated by Deryugina et al. (Deryugina et al., 2009), referred here to as HeLa-CDCP1-S cells. This is consistent with data generated in this project (Figure 5.4 and 5.5), where it was found that HeLa-CDCP1-Y734F cells possess a higher metastatic ability in comparison to HeLa-CDCP1 cells to a similar extent. This indicated that rather than HeLa-CDCP1 cells possessing low metastatic ability, HeLa-vector cells display a high level of metastasis formation in this in vivo assay.

Significantly, neither anti-CDCP1 antibody (41-2 nor 10D7) caused a decrease in dissemination of HeLa-CDCP1 cells in the chicken embryo metastasis assay using antibody concentrations as high as 200 µg/mL. These observations are in strong contrast to Deryugina et al. who published that dissemination of HeLa-CDCP1 cells can be reduced by ~70% using the anti-CDCP1 antibodies 41-2 and 10D7 at 50 µg/mL in this animal model (Deryugina et al., 2009). Importantly, a statistically significant decrease of ~50% in metastasis formation was observed when HeLa-CDCP1-Y734F cells were treated with these antibodies which is consistent with the finding of Deryugina et al. , who reported the same decrease in metastasis formation for HeLa-CDCP1-Y734F cells using MAb 41-2 (Deryugina et al., 2009).

The difference in the ability of the anti-CDCP1 antibodies 41-2 and 10D7 to decrease metastasis formation of HeLa-CDCP1 cells generated in this

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project (HeLa-CDCP1) and those employed in the work of Deryugina et al. (HeLa-CDCP1-S), was unexpected and seems difficult to explain given that both CDCP1 expressing cell lines were generated from HeLa cells stably transfected with a CDCP1 construct cloned into a pcDNA-3.1 vector backbone. The only known difference between the CDCP1 construct used in this project and the construct used in the work of Deryugina et al . is the FLAG tag at the carboxy-terminus of CDCP1 which was encoded by the CDCP construct used for this project. In an attempt to find possible explanations for the observed difference in the ability of the anti-CDCP1 antibodies to decrease metastasis formation of the two sets of CDCP1 expressing HeLa cell lines, HeLa-CDCP1-S and HeLa-vector-S cells were obtained from Deryugina et al. and the following in vitro assays and biochemical experiments were performed.

• Comparison of proliferation rates between HeLa-vector and HeLa- CDCP1 cells with the proliferation rates of HeLa-vector-S and HeLa- CDCP-S cells. • Generation of a short tandem repeat sequence (STR) profile to examine whether the two cell lines originated from HeLa cells. • Comparison of CDCP1 expression levels by Western blot analysis and examination of cell surface expression by flow cytometry. • Examination of differences in binding of the anti-CDCP1 antibodies 41-2 and 10D7 to the CDCP1 protein expressed by HeLa-CDCP1 and HeLa-CDCP1-S cells. • Comparison of the basal level of phosphorylation of CDCP-Y734 by Western blot analysis between HeLa-CDCP1 and HeLa-CDCP1-S cells. • Examination of the level of expression of Src and other SFKs expressed by HeLa-CDCP1 and HeLa-CDCP1-S cells by Western blot analysis. • Sequencing of the CDCP1 DNA encoding the intracellular portion of CDCP1-S.

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5.2.5 CDCP1 expression reduces proliferation of HeLa-CDCP1-S cells

As described above and shown in Figure 5.1 it was found that CDCP1 expression causes a decrease in proliferation of HeLa-CDCP1 cells, but importantly CDCP1 had not been previously reported to play a role in proliferation. Therefore, it was examined whether this observed decrease in proliferation might be particular to HeLa-CDCP1 cells which might have been caused by integration of the CDCP1 expression construct into a chromosomal locus which would cause a decrease in proliferation. Because the in vivo metastasis formation assays described above are performed over a period of 5 days, any difference in proliferation is likely to be reflected in the results of these experiments. Hence, the proliferation rate of HeLa- CDCP1-S cells in comparison to the cellular counterpart stably transfected with empty vector (HeLa-vector-S cells) was examined alongside HeLa- CDCP1 and HeLa-vector cells.

As shown in Figure 5.6 proliferation of HeLa-CDCP1-S cells was decreased by ~20% in comparison to the HeLa-vector-S cells while a decrease of proliferation of HeLa-CDCP1 cells compared to HeLa-vector cells of ~30% was observed. Of note, HeLa-CDCP1-S cells display a ~20% higher rate of proliferation in comparison to HeLa-CDCP1 cells. Although this difference is not statistically significant, it may partly serve to explain a difference in the metastatic ability of these two CDCP1 expressing cell lines. Importantly, it strengthens the finding that CDCP1 expression leads to a decrease in proliferation of HeLa cells (Figure 5.1).

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Figure 5.6: CDCP1 expression reduces proliferation in HeLa-CDCP1 and HeLa-CDCP1-S cells. Cells were seeded at a density of 700 cells in 200 µL media per well of a 96 well plate. Every 24 hours after seeding one plate was incubated with CyQuant NF solution for 45 minutes and the relative number of cells per well assessed by detecting emission at 520 nm. Presented are the means of the means of three experiments each having 3 replicates (n=3) after a proliferation period of 120 hours. Bars are SEM. P-value was calculated with Student’s t-test with 95% confidence interval.

5.2.6 HeLa-CDCP1 and HeLa-CDCP1-S cells are of HeLa cell origin

Due to the difference in the ability of an anti-CDCP1 antibody to decrease metastasis formation of HeLa-CDCP1 and HeLa-CDCP1-S cells the possibility was examined that one of the cell lines was not derived from HeLa cells but represents in fact another epithelial cell line. This is possible since cross-contamination of cell lines is known to occur and it is estimated that up to 36% of all cell lines used in research are not the cell line claimed to be (Masters et al., 2001; Lacroix, 2008). To address this possibility cell pellets of untransfected HeLa cells, HeLa-vector, HeLa-CDCP1 and HeLa-CDCP1-S cells were collected. The cell pellets were sent to the Australian Cell Bank facility where a short tandem repeat sequence (STR) profile was generated to examine the HeLa cell identity of both CDCP1 expressing cell lines. Using this methodology, STR repeat sequences are amplified by PCR using commercially available primer sets and the PCR products are resolved by gel

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electrophoresis alongside a size standard and the length of the PCR product is expressed as a numerical code. Cell lines are considered to be identical if the STR profiles match each other in >80% of the examined loci (Masters et al., 2001; Yoshino et al., 2006).

As shown in Table 5.1, the obtained STR profile of both CDCP1 expressing cell lines matched the STR profile of untransfected HeLa cells in 15 out of 16 examined STR loci (93% match). It is worth noting that HeLa-CDCP1-S cells possess a difference in the STR profile in the D21S11 locus in comparison to untransfected HeLa cells. This indicates that these cells are possibly to have originated from a HeLa cell subline, namely HeLa-P3, which possess this STR profile (Masters et al., 2001). However, this observed difference could also be due to a mutation, as it is known that mutations can occur in this locus (Parson et al., 2005). Also of note, HeLa-CDCP1 cells possess a difference in the vWA locus in comparison to untransfected HeLa cells which may be due to a mutation since HeLa-CDCP1 cells originated from this parental HeLa cell line. It is also possible that incorporation of the CDCP1 expression construct occurred at sites in the HeLa cell genome which alters the STR profile in comparison to the parental HeLa cells. In summary, the results show that both CDCP1 expressing cell lines originated from HeLa cells, but it is possible that HeLa-CDCP1-S cells originated from a different HeLa subline.

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Table 5.1: Examination of the origin of HeLa cells used to generate HeLa-CDCP1 and HeLa-CDCP1-S stable cell lines. HeLa-CDCP1, HeLa- CDCP1-S, HeLa-vector and untransfected HeLa cell pellets were sent on dry ice to the Australian Cell Bank Facility for short tandem repeat sequence (STR) profiling. The STR profile was generated by employing commercially available primer sets to amplify polymorphic repeat regions in the genome. The resulting PCR products were resolved on a gel along with a size standard and are presented as a numerical code which corresponds to the length of the PCR product.

5.2.7 HeLa-CDCP1 and HeLa-CDCP1-S cells express comparable levels of CDCP1 but a C-terminal anti-CDCP1 antibody has a stronger affinity to detect CDCP1 expressed by HeLa-CDCP1-S cells

To examine whether the difference in the ability of the anti-CDCP1 antibody 41-2 to decrease metastasis formation of HeLa-CDCP1 and HeLa-CDCP1-S cells is due to differences in expression levels of CDCP1 these cells were examined by Western blot analysis (Figure 5.7A) and flow cytometry (Figure 5.7B and C). It was found that the total CDCP1 protein (Figure 5.7A) and the level of cell surface expression (Figure 5.7B and C) is comparable in HeLa- CDCP1 and HeLa-CDCP1-S cells.

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Figure 5.7: HeLa-CDCP1 and HeLa-CDCP1-S cells have similar levels of CDCP1 expression. A, Lysates from HeLa-CDCP1 and HeLa-CDCP1-S cells were subjected to Western blot analysis with the anti-CDCP1 antibody 10D7. Anti-GAPDH Western blot analysis was performed to examine protein loading. B and C, Cells (1x10 6) were stained with 3 µg/mL of the anti-CDCP1 antibody 10D7 followed by a species specific Alexa Fluor 488 conjugated secondary antibody. The level of cell surface expression of CDCP1 was analysed by flow cytometry. D, Representative flow cytometry histogram for HeLa-vector cells (left) and PC3 cells (right) which served as negative and positive controls, respectively.

During the course of investigation to compare the two different CDCP1 expressing HeLa cells anti-CDCP1 antibodies from different suppliers were used. Interestingly, when Western blot analysis of both cell lines was performed with the anti-CDCP1 C-terminal antibody from Abcam (ab1377, also used and described in Chapter 4) a very strong signal for HeLa-CDCP1- S cells, but not for HeLa-CDCP1 cells (Figure 5.8A) was detected . This was particularly obvious for short exposure times. In contrast, an anti-CDCP1 antibody, obtained from R&D systems (#AF2666) which is a polyclonal antibody raised against the entire extracellular portion of CDCP1, detected CDCP1 at about the same level expressed in HeLa-CDCP1 and HeLa- CDCP1-S cells. This observation appeared to be of significance because it was noticed that there are different CDCP1 DNA sequences deposited in Genbank coding for either asparagine (accession# AF468010.1) or serine (accession# AK026622.1) at amino acid position 827. Importantly, the anti- CDCP1 C-terminal antibody was raised against amino acids of the

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intracellular domain of CDCP1 spanning the last thirteen C-terminal amino acids from position 824 to 836. The antigen used to generate this antibody contains an asparagine at position 827 and the construct employed for this project used to generate CDCP1 expressing cell lines encodes serine at this position. Due to the difference in the ability of this antibody to detect CDCP1 in the two different sets of CDCP1 expressing HeLa cells (Figure 5.8A) it was considered to be likely that HeLa-CDCP1-S cells encode asparagine at position 827.

It is possible that this difference at amino acid 827 or the FLAG epitope encoded at the carboxy terminus by the CDCP1 expression construct used for this project could cause a difference in the ability of the anti-CDCP1 antibodies 41-2 or 10D7 to bind to the CDCP1 extracellular portion. This could potentially explain the difference in inhibition of metastasis formation in vivo between the two different CDCP1 expressing cell lines. To address this question constructs encoding CDCP1 with and without a carboxy terminal FLAG tag and with asparagine or serine at amino acid position 827 were generated.

The generation of these constructs by site directed mutagenesis was performed by Dr Yaowu He in the Hooper laboratory. These constructs were used to transiently transfect HeLa cells and whole cell lysates were collected in the presence of phosphatase inhibitors and subjected to Western blot analysis with the anti CDCP1 antibodies 41-2, 10D7, the anti-CDCP1 C- terminal antibody from Abcam, the anti-CDCP1 antibody from R&D-systems and an anti-p-FAK-Y861 antibody. Because the anti-CDCP1 antibody from R&D systems is raised against the entire extracellular portion of CDCP1 it is expected to equally recognise CDCP1 with and without the intracellular FLAG tag and with serine or asparagine at position 827. This antibody served, therefore, as control for the transfection efficiency in the transiently transfected HeLa cells for the different constructs. As shown in Figure 5.8B it was found that the C-term anti-CDCP1 antibody most strongly detected CDCP1 with asparagine at position 827 (lane 2), as was expected since the antigen used to generate this antibody contained asparagine at position 827.

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The protein containing a serine at position 827 was detected at lower intensity (lane 5) as was CDCP1 containing an asparagine at position 827 and a carboxy terminal FLAG epitope (lane 3). In contrast, there was no signal detectable from cells expressing CDCP1 containing a serine at position 827 and a carboxy terminal FLAG tag, which is equivalent to the CDCP1 protein encoded by the construct used to generate HeLa-CDCP1 cells (lane 4). Importantly, the intensities of the bands obtained using the anti-CDCP1 antibodies 41-2 and 10D7 and an anti-p-FAK-Y861 antibody paralleled the signal intensity of the anti-CDCP1 antibody from R&D-systems which served as a control for transfection efficiency of the different CDCP1 encoding constructs.

Figure 5.8: The anti-CDCP1 antibodies 41-2 and 10D7 are not effected by the FLAG tag encoded at the carboxy terminal of the CDCP1 expression construct or by the amino acid at position 827. A, Lysates from HeLa- vector, Hela-CDCP1 and HeLa-CDCP1-S cells were subjected to Western blot analysis using two different anti-CDCP1 antibodies. B, HeLa cells were transfected with constructs encoding CDCP1 with and without a FLAG tag and with serine or asparagine at amino acid position 827. Whole cell lysates were generated in the presence of phosphatase inhibitors 24 hours after the transfection and subjected to Western blot analysis with four different anti- CDCP1 antibodies and a p-FAK-Y861 antibody. Anti-GAPDH Western blot analysis was performed to examine protein loading. The constructs used in panel B were generated by Dr Yaowu He.

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In summary, it appears that there is no difference in the ability of the anti- CDCP1 antibodies 41-2 and 10D7 to bind to CDCP1 expressed by HeLa- CDCP1 and HeLa-CDCP1-S cells, because the intensity of the bands detected in Figure 5.8B with these antibodies paralleled the intensity of bands obtained using the anti-CDCP1 antibody from R&D-systems. There also was no detectable difference in the level of phosphorylation of CDCP1 because the intensity of the bands of the p-FAK-Y861 antibody paralleled the band intensity of the anti-CDCP1-antibody from R&D-systems. This experiment confirmed the finding described in Chapter 3 (Figure 3.13) where it was observed that transient transfection of HeLa cells with a CDCP1 expression construct leads to the detection of two bands by Western blot analysis using an anti-p-FAK-Y861 antibody; the higher molecular weight band represents phosphorylated CDCP1-Y734 and the lower molecular weight band represents phosphorylated FAK-Y861.

As described above there was strong indication that HeLa-CDCP1-S cells have an asparagine at amino acid position 827, as suggested by the finding that the C-terminal anti-CDCP1 antibody detects CDCP1 from HeLa-CDCP1- S cells but not from HeLa-CDCP1 cells (Figure 5.8A). To deliver experimental evidence for the hypothesis that HeLa-CDCP1-S cells contain an asparagine at position 827 and to confirm that these cells lack a carboxy terminal FLAG tag, genomic DNA from HeLa-vector-S and HeLa-CDCP1-S cells was isolated and CDCP1 encoding DNA was amplified by PCR. The primers used for this experiment, which are described in Table 2.2, are specific for the CDCP1 DNA from nucleotide 1637 (forward primer) and for sequence within the bovine growth hormone (BGH) sequence in the pcDNA 3.1 vector. The PCR product was subjected to analysis by agarose gel electrophoresis as shown in Figure 5.9A. The remainder of the PCR product was purified and sequenced using a sequencing-primer which binds to CDCP1 sequence from nucleotide 2104. This approach was designed to generate sequencing data of the region encoding amino acid position 827 as well as the region encoding a carboxy terminal FLAG tag.

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An alignment of the sequence obtained from HeLa-CDCP1-S cells with the sequence from CDCP1 construct used in this project is shown in Figure 5.9B.

Figure 5.9: The HeLa-CDCP1-S expression construct has a serine at amino acid position 827 and does not encode a carboxy terminal FLAG tag. Genomic DNA from HeLa-CDCP1-S cells was isolated and CDCP1 DNA sequence was amplified by PCR. A small amount of the PCR product was analysed on an ethidium bromide stained agarose gel A. B, Sequence of the CDCP1 construct aligned with the sequence of CDCP1-S. The red bold letters represent the DNA sequence encoding serine at amino acid position 827. The black frame represents the DNA sequence encoding a stop codon after amino acid position 836 in HeLa-CDCP1-S cells. Underlined in blue is the FLAG tag in HeLa-CDCP1 cells and the blue frame represents DNA sequence encoding for the stop codon after the FLAG tag in HeLa-CDCP1 cells.

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The results of the sequence analysis confirmed that the HeLa-CDCP1-S construct has a stop codon after position 836, whereas the construct used for this project encodes for another 8 amino acids representing the FLAG tag (Figure 5.9B). Unexpectedly, it was found that both constructs have a serine at amino acid position 827, indicating that the difference in the ability of the anti-CDCP1 C-terminal antibody to bind to CDCP1 expressed in HeLa- CDCP1 and HeLa-CDCP1-S cells is solely due to the presence of the FLAG tag the construct used in this project.

5.2.8 HeLa-CDCP1 and HeLa-CDCP1-S cells are basally phosphorylated at CDCP1-Y734, but each express different SFKs

To further characterise the two sets of CDCP1 expressing HeLa cells it was examined whether there is a difference in the level of basal phosphorylation of CDCP1. Therefore, whole cell lysates of stable CDCP1 expressing HeLa cells and HeLa-CDCP1-S cells were collected in the presence of phosphatase inhibitors and subjected to Western blot analysis with an anti-p- FAK-Y861 antibody. As shown in Figure 5.10 it was found that HeLa- CDCP1-S cells display basal phosphorylation of CDCP1 on a level comparable with the CDCP1 expressing cell lines generated for this project.

Figure 5.10: CDCP1 is basally phosphorylated in HeLa-CDCP1 and HeLa-CDCP1-S cells. Cells were collected in the presence of phosphatase inhibitors and examined by Western blot analysis using an anti-p-FAK-Y861 antibody and an appropriate species specific secondary antibody. Anti- GAPDH Western blot analysis was performed to examine protein loading.

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As CDCP1 is phosphorylated by SFKs the different HeLa cell lines were examined by Western blot analysis using two different anti-SFK antibodies; one detects multiple SFKs (CST #2108) and another is specific for Src (CST #2110). The anti-Src antibody #2110 was raised against a 110 amino acid sequence spanning the unique domain of Src which has lowest homology to the unique domains of other SFKs. As shown in Figure 5.11 the Src specific antibody detected Src exclusively in HeLa-CDCP1-S cells at the expected molecular weight of ~60 kDa but this protein was not detectable in HeLa- CDCP1, HeLa-CDCP1-Y734F, -Y743F and -Y762F cells.

In contrast, when using the anti-SFK antibody which cross-reacts with multiple SFKs, a band of about the same intensity was observed in each of the CDCP1 expressing HeLa cells as well as in HeLa-CDCP1-S cells. These results suggest that HeLa-CDCP1-S cells express Src and possibly other SFKs whereas HeLa-CDCP1 cells do not express Src but do express other SFKs.

Figure 5.11: HeLa-CDCP1 and HeLa-CDCP1-S cells express different SFKs. Lysates from HeLa-vector, HeLa-CDCP1 and HeLa cells expressing three different CDCP1 tyrosine mutants and HeLa-CDCP1- S cells were prepared in the presence of phosphatase inhibitors and subjected to Western blot analysis using either a Src specific antibody (A) or an antibody that detects multiple SFKs (B). Anti-GAPDH Western blot analysis was performed to examine protein loading.

As it is known that SFKs are functionally involved in receptor/ligand complex internalisation (for example, internalisation of the epidermal growth factor receptor (EGFR) after binding of its ligand (EGF) (Ware et al., 1997)) and it is possible that the anti-metastatic effect of anti-CDCP1 antibodies is caused by internalisation of the CDCP1•antibody complex (Siva et al., 2008b;

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Fukuchi et al., 2010), whether the level of expression of CDCP1 changed in HeLa-CDCP1 and HeLa-CDCP1-S cells was examined after incubation of cells with the anti-CDCP1 antibody 41-2. In addition, it may be that the FLAG tag which was used in HeLa-CDCP1 but not in HeLa-CDCP1-S cells influences CDCP1•antibody internalisation. As shown in Figure 5.12 it was found by Western blot analysis that CDCP1 levels in HeLa-CDCP1 cells were unchanged after 30 minutes of treatment with 41-2. In contrast, there was a marked reduction (~50%) in CDCP1 levels expressed by HeLa- CDCP1-S cells treated under the same conditions. This may be mediated by internalisation and degradation of the CDCP1•antibody complex.

Figure 5.12: Examination of change in expression of CDCP1 in HeLa- CDCP1 and HeLa-CDCP-S cells in response to treatment with the anti- CDCP1 antibody 41-2. Cells were either untreated (-) or treated with (+) with 41-2 (50 µg/mL) for 30 minutes at 37°C. Lysates wer e prepared and analysed by Western blot analysis with the anti-CDCP1 antibody 10D7. Anti- GAPDH Western blot analysis was performed to examine protein loading.

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5.3 DISCUSSION

In an attempt to gain further insight into the role played by CDCP1 in cellular processes relevant to cancer progression, HeLa cells stably expressing CDCP1, CDCP1-Y734F, -Y743F or -Y762F were used in adhesion, proliferation and migration assays to explore the cellular phenotype mediated by CDCP1 in vitro. In addition, the metastatic ability of these CDCP1 expressing cell lines in vivo was examined using the chicken embryo metastasis assay. HeLa cells were chosen for this approach for two reasons. First, because these cells do not express CDCP1 endogenously (Figure 3.1) and second, because HeLa cells are poorly metastatic in the chicken embryo metastasis assay (Zijlstra et al., 2002; Blancafort et al., 2005) and an increase in the metastatic ability of these cells due to CDCP1 expression should be readily detectable in in vivo studies.

The key findings from this chapter are:

• Certain CDCP1-mediated cell phenotypes in vitro depend on CDCP1 tyrosine residues. • HeLa-CDCP1 and HeLa-CDCP1-S cells may have originated from different HeLa cell sublines which may explain the observed difference between these two CDCP1 expressing cell lines in vivo. • Mutation of certain CDCP1 tyrosine residues impacts on CDCP1- mediated cell phenotypes in vivo . • Differential expression of SFKs in HeLa-CDCP1 and HeLa-CDCP1-S cells suggests a possible role for SFKs in mediating the anti- metastatic effect of anti-CDCP1 antibodies in vivo.

5.3.1 Certain CDCP1-mediated cell phenotypes in vitro depend on CDCP1 tyrosine residues

Interestingly, it was found that expression of CDCP1 significantly decreased proliferation of HeLa cells (Figure 5.1). This was unexpected, as no previous reports had identified a role for CDCP1 in mediating a change in proliferation.

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However, it is worth noting that all published CDCP1 related proliferation data have been obtained by stable knock down of CDCP1. The generated data indicated that the decreased rate of proliferation is not mediated via a signalling pathway which requires increased phosphorylation of CDCP1 on Y734 because it was found that HeLa-CDCP1-Y734F cells, which lack binding of SFKs to CDCP1 (Figure 3.10) and do not undergo phosphorylation of CDCP1 (Figure 3.11), show a decrease in proliferation similar to HeLa- CDCP1 cells. Although HeLa-CDCP1-Y762F cells possess the lowest rate of proliferation it appears that PKC δ binding to CDCP1 does not cause a decrease in proliferation of HeLa cells. This is based on the observation that HeLa-CDCP1-Y734F cells, which have markedly decreased interaction with PKC δ (Figure 3.10), have proliferation rates similar to HeLa-CDCP1 cells but higher than HeLa-CDCP1-Y762F cells.

It is unlikely that the genomic site of incorporation of the CDCP1 expression construct caused the observed reduction in proliferation of these cells. This is based on the finding that approximately the same decrease was observed in all CDCP1 expressing cells including HeLa-CDCP1-S cells generated by Deryugina et al . (Deryugina et al., 2009).

The results of the adhesion assays support published findings that CDCP1 plays a role in altering adhesion to ECM proteins (Deryugina et al., 2009) . This decrease in adhesion was observed for all examined ECM proteins, including fibronectin, collagen-I, laminin-1 and the basement membrane matrix mimetic Matrigel. There is a trend towards higher adhesion of the CDCP1 tyrosine mutant expressing cells in comparison to HeLa-CDCP1 cells. In particular, a higher adhesive capacity of HeLa-CDCP1-Y743F cells was observed in comparison to HeLa-CDCP1 cells towards fibronectin. It remains to be examined whether this difference in adhesion is due to an altered expression of integrins or other cell/matrix or cell/cell adhesion proteins in these cell lines.

Consistent with published studies that CDCP1 pro-metastatic effects are mediated by an increase in cell motility (Uekita et al., 2008), a ~2 fold increase in the ability of CDCP1 expressing cells to migrate towards an FCS

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gradient was observed. This increase in migration is not mediated by the three examined CDCP1 tyrosine residues because an increase in migration was observed in all CDCP1 expressing cells. In contrast, Uekita et al. and Miyazawa et al. have reported that expression of CDCP1, but not CDCP1- Y734F, leads to an increase in migration in gastric and pancreatic cancer cell lines (Uekita et al., 2007; Miyazawa et al., 2010). These authors used transiently expressing CDCP1 lung cancer cells or endogenous CDCP1 expressing gastric cancer cells transfected with a CDCP1-siRNA construct and a CDCP1-Y734F rescue construct (Uekita et al., 2007; Miyazawa et al., 2010). These different observations suggest that CDCP-Y734 plays a role in migration in some cell lines, but not in HeLa cells and that phosphorylation of CDCP1-Y734 is not required for increased migration of HeLa cells mediated by CDCP1.

5.3.2 HeLa-CDCP1 and HeLa-CDCP1-S cells may have originated from different HeLa cell sublines which may explain observed differences between these two cell lines in vivo

It was surprising to find that HeLa-CDCP1 cells were less metastatic in the chicken embryo metastasis model than HeLa-vector cells, although this data did not achieve statistical significance. These data disagree with data published by Deryugina et al. who found that HeLa-CDCP1-S cells are more metastatic in the chicken embryo assay than the HeLa-vector-S cells (Deryugina et al., 2009). Moreover, it was unexpected to find that metastasis of HeLa-CDCP1 cells could not be inhibited by an anti-CDCP1 antibody. In contrast, it was published by Deryugina et al. that the anti-CDCP1 antibodies 41-2 and 10D7 can decrease metastasis in HeLa-CDCP1-S cells by ~70% (Deryugina et al., 2009). The differences between HeLa-CDCP1 cells and HeLa-CDCP1-S cells are difficult to reconcile, as both CDCP1 expressing cells were generated from HeLa cells stably transfected with a CDCP1 expression construct in the pcDNA 3.1 vector backbone.

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To address this issue experiments were performed to examine whether both cell lines are in fact of HeLa cell origin since cross-contamination of cell lines is a recognised problem and concerns have been raised that up to 36% of all cell lines used in research are not the claimed cell line (Masters et al., 2001; Lacroix, 2008). To this end a short tandem repeat (STR) profile was generated to examine the HeLa cell identity of the two cell lines. The method of STR profiling was developed in the mid 1990s initially for forensic applications and has since been recognised as a useful method to examine the authenticity of cell lines (Masters et al., 2001; Yoshino et al., 2006). This methodology employs commercially available primer sets to amplify polymorphic repeat regions in the genome. The resulting PCR products are resolved on a gel along with a size standard and presented as a numerical code which corresponds to the length of the PCR product. Cell lines are considered identical if they match each other in 80% or more of the examined STR loci (Masters et al., 2001; Yoshino et al., 2006). The STR analysis indicated that the two Hela-CDCP1 expressing cell lines are of HeLa cell origin, as both CDCP1 expressing cell lines match the STR profile of the untransfected HeLa cells in 15 out of 16 profiled STR loci.

Problematically, one question can not be adequately addressed using this technology and this is whether the two HeLa cells originated from the classical parental HeLa cell line or from other established sublines of HeLa cells. In May 2010 seven different HeLa cell sublines were available at ATCC and another five in the Cellbank of the Japanese Collection of Research Bioresources (JCRB) and many more HeLa cell line contaminants have been described including the two well known cell lines chang-liver and Hep-2 (Lacroix, 2008). For a majority of the HeLa cell sublines commercially available from ATCC and JCRB, the STR profiles have been reported. Some of the STR profiles of these sublines are not distinguishable from the classical HeLa cell line, including HeLa-H1 and HeLa-229, whereas others are distinguishable at one or more loci including HeLa-S3, HeLa-P3, HeLa- TG and HeLa-AG (Table 5.2).

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Table 5.2: Comparison of the STR profiles of HeLa cell sublines and HeLa-CDCP1 and HeLa-CDCP1-S. Shown are STR profiles provided by ATCC and JCRB for HeLa cells and HeLa cell derived sublines. The two columns on the right show part of the STR profile of the CDCP1 expressing HeLa cells used during the course of this PhD research project. For the full STR profile please refer to Table 5.1. * STR loci profiled for these cell lines published by Masters et al ., 2001. Boxes filled with blue indicate a difference in the STR profile of the cell line to the classical HeLa cell line (HeLa-CCL-2). --, STR profile has not been examined or published.

The obtained STR profile showed a difference in the D21S11 loci for HeLa- CDCP1-S cells in comparison to untransfected HeLa cells. The difference in this locus suggests that the cell line used by Deryugina et al. may have originated from a subline of HeLa cells, namely HeLa-P3 (Table 5.2). This cell line was established almost forty years ago by continuous culturing of HeLa cells in protein- and lipid-free chemically defined synthetic media (Takaoka and Katsuta, 1971) and is the only HeLa cell subline with a loss of 28 in this locus (Masters et al., 2001). These data suggest that the two CDCP1 expressing HeLa cell lines (HeLa-CDCP1 and HeLa-CDCP-S) were derived from different HeLa sublines. Although indicative of a difference in the two examined CDCP1 expressing HeLa cells, it is not conclusive since mutations in these loci can occur (Parson et al., 2005). This is shown by the difference in the vWA locus between HeLa-CDCP1 and untransfected HeLa cells. HeLa-CDCP1 cells originated from HeLa cells and are, therefore, expected to possess an STR profile identical to that of HeLa cells. Importantly, additional support for the proposal that the two CDCP1

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expressing HeLa cells originated from different sublines comes from the observation that these two cell lines express different SFKs (Figure 5.11).

5.3.3 Mutations of certain CDCP1 tyrosine residues impact on CDCP1- mediated cell phenotypes in vivo

In experiments examining the impact of CDCP1 tyrosine mutations on the metastatic phenotype in vivo it was found that certain CDCP1 tyrosine residues have an impact on the ability of CDCP1 expressing HeLa cells to metastasise. For example, was observed that HeLa-CDCP1-Y762F cells have a low metastatic ability in comparison to all other CDCP1 expressing HeLa cells. This may be caused by the greatly decreased rate of proliferation observed for this cell line (Figure 5.1), but a direct effect of this mutation in affecting the ability of these cells to metastasise is possible. Further investigations are required to elucidate differences in interacting proteins between HeLa-CDCP1 cells and HeLa-CDCP1-Y762F cells and to assess the effects downstream of mutation of this tyrosine residue. In contrast, HeLa-CDCP1-Y734F and HeLa-CDCP1-Y743F cells were more metastatic than HeLa-CDCP1 cells (Figure 5.5 and 5.6). The underlying molecular mechanisms mediating the increased ability of HeLa-CDCP1-Y743F cells to metastasise in this in vivo model could not be established due to time limitations. However, in biochemical investigations no differences could be detected between this cell line and HeLa-CDCP1 cells while examining the phosphorylation of CDCP1, interactions between CDCP1 with SFK and PKC δ, the phosphorylation of SFKs induced by CDCP1 expression and the cell morphology displayed by these cells.

The increased metastatic ability of HeLa-CDCP1-Y734F cells which lack any detectable phosphorylation of CDCP1 (Figure 3.11) and which lack interaction between CDCP1-Y734F and SFK or PKC δ (Figure 3.10) is of particular interest. This finding, challenges the accepted mechanism by which CDCP1 causes a pro-metastatic phenotype. This mechanism proposes that phosphorylation of CDCP1-Y734 induced by SFKs is essential

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for CDCP1 mediated metastasis. Data presented in this thesis also suggest that CDCP1-mediated metastasis is not dependent of the formation of the SFK•CDCP1•PKC δ complex in HeLa cells .

Further experimental work is required to confirm the role of CDCP1-Y734 in metastasis. The observations of differential phosphorylation of CDCP1-Y734 and FAK-Y861 do suggest a potential reason why HeLa-CDCP1-Y734 cells are more metastatic than HeLa-CDCP1 cells. As described in Chapter 3 (Figure 3.11) it was found that HeLa-CDCP1-Y734F cells possess basal phosphorylation of FAK-Y861. In contrast HeLa-CDCP1 cells demonstrate basal phosphorylation of CDCP1-Y734. Due to this difference in basal phosphorylation of CDCP1 and FAK, it seems possible that the increased metastatic ability of HeLa-CDCP1-Y734F cells may be due to the increased phosphorylation of FAK-Y861, as FAK has been shown to be important in cancer formation in in vivo models (Duxbury et al., 2004; Roberts et al., 2008). However, the data presented here indicate that the metastatic phenotype of HeLa-CDCP1-Y734F cells is not mediated by the phosphorylation of FAK alone; it rather suggests that crosstalk between CDCP1 and FAK causes this phenotype since metastasis formation of these cells can be significantly decreased in the chicken embryo CAM assay by using the anti-CDCP1 antibodies 41-2 and 10D7 (Figure 5.5), which provides evidence for the important role of CDCP1 in mediating this phenotype.

5.3.4 Differential expression of SFKs in HeLa-CDCP1 and HeLa- CDCP1-S cells suggests a possible role for SFKs in mediating an anti- metastatic effect of anti-CDCP1 antibodies in vivo

While examining the consequences of CDCP1 tyrosine mutants on the ability of an anti-CDCP1 antibody to inhibit metastasis formation in the chicken embryo metastasis model, it was found that the ability of the anti-CDCP1 antibody 41-2 to inhibit metastasis formation seems to negatively correlate with the observed increased phosphorylation of SFK-Y416 in HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F cells (Figure 3.10) . This suggests that

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phosphorylation of CDCP1 by SFKs other than Src prevents the anti- metastatic effects of the anti-CDCP1 antibodies 41-2 and 10D7.

This is based on the following in vivo observations. It was found that HeLa- CDCP1-S cells, which express Src (Figure 5.12) can be inhibited in their metastatic ability (Deryugina et al., 2009). In contrast, Hela-CDCP1 cells and HeLa-CDCP1-Y743F and -Y762F cells, which express SFKs other than Src (Figure 5.12) and possess high levels of phosphorylation of these SFKs (Figure 3.10), are insensitive to inhibition by 41-2 in vivo . However, it is apparent that HeLa-CDCP1-Y734F cells, which also express SFKs other than Src (Figure 5.12), but which possess low levels of phosphorylation of these SFKs (Figure 3.10), can be significantly inhibited in their metastatic ability in vivo (Figure 5.5). This suggests that increased phosphorylation of SFKs other than Src may prevent inhibition of metastasis formation by an anti-CDCP1 antibody. This may be due to a difference in internalisation and a consequent decrease in CDCP1 density on the cell surface of CDCP1 expressing HeLa cells. Interestingly, it was found that treatment with the anti- CDCP1 antibody 41-2 leads to a decrease in CDCP1 levels in HeLa-CDCP1- S cells but not in HeLa-CDCP1 cells in vitro .

Although it speculates that the anti-metastatic action of anti-CDCP1 antibodies is due to internalisation of the CDCP1•antibody complex, there is a report supporting the hypothesis of differential roles of SFKs in internalisation of cell surface associated proteins. For example, it was observed that EGF induced endocytosis of Flotillin-1 and Flotillin-2 does not occur in SFK null cells, which lack expression of Src, Fyn and Yes, whereas expression of Fyn, but not Src or Yes restored endocytosis of these proteins. These data suggest that Fyn is required for endocytosis of these proteins whereas Src and Yes were not necessary to mediate this process (Riento et al., 2009).

Future studies are needed to examine whether the anti-metastatic effects of the anti-CDCP1 antibodies 41-2 and 10D7 are in fact due to internalisation of the CDCP1•antibody complex and how different SFKs and their state of phosphorylation may play a role in this process. To further examine this

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possibility it will be necessary to transfect parental HeLa cell lines, used in this project with a CDCP1 construct without a FLAG tag. These cells may then be used to ensure that the observed results are not influenced by the FLAG tag of CDCP1 used in HeLa-CDCP1 cells and consequently to confirm that the differences in CDCP1•antibody complex internalisation are due to differentially expressed SFKs. This may give important insights into how the CDCP1 mediated pro-metastatic phenotype can be prevented by using anti- CDCP1 antibodies and may be valuable in designing effective strategies to block the pro-metastatic activity of CDCP1 in vivo that has been reported in other studies (Uekita et al., 2007; Siva et al., 2008b; Uekita et al., 2008; Deryugina et al., 2009).

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Final conclusions and future directions

Chapter 6

6.1 INTRODUCTION

In this study, HeLa cells stably expressing CDCP1 or the tyrosine mutants CDCP1-Y734F, -Y743F or -Y762F were generated. These cell lines were used to study interactions between CDCP1 and its binding partners and the effect of CDCP1 expression on cell morphology. Moreover, the cellular phenotype caused by expression of CDCP1, CDCP1-Y734F, -Y743F and - 762F in cellular processes associated with cancer progression in vitro and in vivo were examined. Also examined was the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 and the consequences on cell phenotype and signal transduction via this protein.

6.2 FINDINGS AND DISCUSSION

6.2.1 The role of CDCP1 and CDCP1 tyrosine mutants in cellular processes in vitro and in vivo

As summarised in Figure 6.1, the data presented in this thesis suggest that cellular processes mediated by CDCP1 can be regulated by pathways which are either dependent or independent of phosphorylation at CDCP1-Y734, - Y743 and -Y762.

For example, the observed changes in HeLa cell morphology towards an elongated and fibroplastic appearance depend on CDCP1-Y734. This is suggested by the finding that change in cell morphology occurs in HeLa- CDCP1, HeLa-CDCP1-Y734F and -Y762F cells, but not in HeLa-CDCP1- Y734F cells. Interestingly, this change in cell morphology is accompanied by an increase in phosphorylation of SFK-Y416. These data suggest a possible role for CDCP1-Y734 in inducing an EMT in cancer cells which may be caused by phosphorylation of CDCP1 or by increased phosphorylation of SFK-Y416 due to SFK binding to CDCP1-Y734.

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Figure 6.1: Consequences of CDCP1 expression on HeLa cell phenotype in vitro and in vivo are mediated by phosphorylation dependent and independent pathways. Illustration of the effects of expression of CDCP1 and CDCP1 tyrosine mutants on phosphorylation of CDCP1, binding and phosphorylation of SFKs and binding of PKC δ. The in vitro effects of expression of CDCP1 and CDCP1 tyrosine mutants on HeLa cell phenotype are summarised: -, decrease in comparison to HeLa-vector cells 1-25%; - -, 25-50%; - - -, >50%. +, increase in comparison to HeLa-vector cells 1-25%; ++, 25-50%; +++, >50%. *, p=<0.05 for the observed difference in comparison to HeLa-vector cells. In vivo effects of expression of CDCP1 and tyrosine mutants on HeLa cell phenotype. HeLa-CDCP1 cells are used as reference to illustrate the effects of mutations of tyrosine residues on metastasis = 1. ↓, decrease in comparison to HeLa-CDCP1 cells 1-25%; ↓↓ , 25-50%; ↓↓↓ , >50%. ↑, increase in comparison to HeLa-vector cells 1-25%; ↑↑ , 25-50%; ↑↑↑ , >50%. ■, p=<0.05 for the observed difference in comparison to HeLa-CDCP1 cells. X, no change observed using MAb 41-2 in the chicken embryo CAM assay; ‡ = decrease of metastasis >50% using MAb 41-2 in the chicken embryo CAM assay; ^, p=<0.05 for decrease of metastasis using MAb 41-2 as compared to IgG treated control cells.

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An EMT is commonly characterised by changes in cellular markers characteristic of a more mesenchymal cell phenotype. An example of markers used to detect an EMT is upregulation of N-cadherin, fibronectin, snail and twist, or downregulation of E-cadherin, desmoplakin and cytokeratin (Lee et al., 2006). Due to time constraints this study did not examine any of these markers and it remains, therefore, unclear as to whether this change in cell morphology can be classified as an EMT. Importantly, the involvement of increased Src activity in mediating an EMT in various cell types has previously been described (Avizienyte and Frame, 2005; Humar et al., 2007; Yang et al., 2009). As it was demonstrated that the change in HeLa cell morphology induced by CDCP1 parallels the increase in phosphorylation of SFK-Y416, it may indicate that the observed morphological changes indeed represent an EMT. This suggests a possible mechanism by which CDCP1 can alter cell phenotype in vitro and in vivo, but further experiments examining EMT markers are needed to clarify this possibility.

HeLa cell motility was found to be increased and this was independent of phosphorylation of CDCP1 at the examined tyrosine residues Y734, Y743 and Y762. This suggests that the CDCP1-mediated increase in cell motility may occur via pathways independent of the currently known CDCP1 binding proteins, SFKs and PKC δ. This is very exciting, as it suggests that future efforts may unravel yet unidentified CDCP1-mediated cellular pathways which impact on cell migration. In contrast, proliferation appears to be mediated by cellular pathways partly depending on CDCP1 phosphorylation at Y762. This is suggested by the finding that the CDCP1-mediated decrease in proliferation of HeLa cells is most prominent in CDCP1-Y762F cells. Future efforts are needed to delineate the cellular mechanism mediating this decrease in HeLa cell proliferation. From the data generated during the course of this study, it can be concluded that the low rate of proliferation for HeLa-CDCP1-Y762F cells is not likely to be due to altered binding of PKC δ at this site. This conclusion is based on the observation that HeLa-CDCP1- Y734F cells do not exhibit a lower rate of proliferation in comparison to HeLa-CDCP1 cells, although these cells display markedly reduced binding of

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PKC δ to CDCP1 (Figure 3.10B). This suggests that the decrease in proliferation of HeLa-CDCP1-Y762F cells may be due to a difference in binding of proteins other than PKC δ at CDCP1-Y762. Alternatively, it may be that the decrease in proliferation of HeLa-CDCP1Y762F cells is caused by altered phosphorylation of PKC δ rather than by altered binding of PKC δ to CDCP1. Importantly, Uekita and colleagues showed that PKC δ-Y311 is phosphorylated in endogenously CDCP1 expressing A549 lung cancer cells and knockdown of CDCP1 abolishes the phosphorylation of PKC δ-Y311 (Uekita et al., 2007). This published finding suggests that interactions of CDCP1 with PKC δ cause phosphorylation of PKC δ-Y311. Of relevance, it is known that PKC δ has multiple phosphorylation sites and many of these are phosphorylated, mediating complex downstream signalling including the regulation of proliferation (Jackson and Foster, 2004). Moreover, an involvement of CDCP1-Y762 in regulating phosphorylation of PKC δ could also serve as a possible explanation for the low metastatic ability of HeLa- CDCP1-Y762F cells in vivo . Uekita et al. demonstrated that phosphorylation of PKC δ-Y311 is important in mediating an anti-apoptotic phenotype in vitro , which is likely to translate into a greater metastatic phenotype in vivo (Uekita et al., 2007). Future experiments examining the phosphorylation of PKC δ in HeLa-CDCP1-Y762F cells may provide answers for the observed in vitro and in vivo phenotype of these cells.

It also appears that adhesion of CDCP1 expressing HeLa cells to fibronectin and the basement matrix Matrigel depends on CDCP1-Y743. This is based on the observation that HeLa-CDCP1-Y743F cells have a mostly restored ability to adhere to fibronectin in contrast to the marked decreased adhesion of HeLa-CDCP1 cells. It remains to be examined whether expression of integrins and other cell-adhesion molecules is altered in stable CDCP1 expressing HeLa cells. If the observed decrease in adhesion of HeLa- CDCP1 cells is due to altered expression of these cell adhesion molecules it may be possible that CDCP1-Y743 in fact plays a role in regulating the expression of these proteins.

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Data from the in vivo experiments using the chicken embryo metastasis model indicated that CDCP1 expression causes a decrease in metastasis of HeLa cells compared to HeLa-vector cells. This was unexpected and does not agree with published data showing that expression of CDCP1 in a HeLa cell line (HeLa-CDCP1-S) causes an increase in the ability of these cells to metastasise (Deryugina et al., 2009). Moreover, it was found in this PhD project that HeLa-CDCP1 cells can not be inhibited in their metastatic ability by an anti-CDCP1 antibody. This is also in contrast to findings by Deryugina et al. where a decrease in metastasis formation of ~70% was observed using an anti CDCP1 antibody (Deryugina et al., 2009). In an attempt to find explanations for these differences between HeLa-CDCP1 cells and HeLa- CDCP1-S cells a series of biochemical experiments were performed and are described in Chapter 5. Data from these experiments suggest that HeLa- CDCP1 cells and HeLa-CDCP1-S cells may have originated from different HeLa cell sublines which may explain the observed differences in vivo . Further experiments are required to obtain an understanding of the molecular basis causing these differences. Of note, the data comparing the metastatic ability of HeLa-vector cells to HeLa-CDCP1 cells did not obtain statistical significance. These experiments were performed in a time constrained overseas research field trip in the laboratory of Prof James Quigley at the Scripps Research Institute. Therefore it was not possible to repeat the experiments, presented in Figure 5.5, often enough to achieve statistical significance. Consequently, the difference between HeLa-vector cells and HeLa-CDCP1 cells found in this project is somewhat inconclusive and requires further in vivo experiments to increase sample size and to achieve statistical significance.

Importantly, a higher metastatic ability of HeLa-CDCP1-Y734F cells in comparison to HeLa-CDCP1 cells did achieve statistical significance in the experiments performed in this research project (Figure 5.6) and was also found in experiments performed and published by Dr Deryugina (Deryugina et al., 2009). This finding suggest a potentially interesting pathway involving signalling downstream of CDCP1-Y734 because these cells possess a higher metastatic ability than HeLa-CDCP1 cells despite the lack of

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Chapter 6 detectable phosphorylation of CDCP1 or interactions of CDCP1 with SFKs and PKC δ (Figure 3.10). Notably, HeLa-CDCP1-Y734F cells were found to be the only CDCP1 expressing cell line which possesses basal levels of phosphorylation of FAK-Y861. This increased phosphorylation of FAK-Y861 may explain the highly metastatic and migratory phenotype, despite the lack of CDCP1 phosphorylation and interaction of CDCP1 with SFKs and PKC δ. Importantly, metastasis formation of this cell line can be significantly decreased using an anti-CDCP1 antibody in vivo ((Figure 5.5 and (Deryugina et al., 2009)). This suggests that CDCP1 can play a role in inducing the pro- metastatic phenotype, independent of its phosphorylation and formation of a trimeric complex of SFK•CDCP1•PKC δ.

This observed metastatic phenotype of HeLa-CDCP1-Y734F cells contrasts the accepted mechanism, which requires SFK-mediated phosphorylation of CDCP1 to induce a pro-metastatic phenotype. This opens new and exciting avenues implicating CDCP1 in facilitating a metastatic cancer cell phenotype via yet undefined mechanisms. The data presented here suggest one possible mechanism; cross talk of CDCP1 with phosphorylated FAK-Y861. Significantly, HeLa-CDCP1-Y734F cells are the only CDCP1 expressing cell line displaying phosphorylation of FAK-Y861. Interestingly, it was found that CDCP1 is able to sequester SFKs from FAK-Y861 depending on the level of expression of CDCP1. It appears that the level of expression of CDCP1 can act as a regulatory mechanism regulating two distinct SFK-mediated pathways. Both of these SFK-mediated pathways, on the one hand involving phosphorylation of CDCP1-Y734, and on the other, phosphorylation of FAK- Y861, may cause a pro-metastatic cell phenotype. Importantly, CDCP1 and FAK were shown to play important roles in regulating anoikis, which is likely to translate into a more metastatic cell phenotype (Frisch et al., 1996; Uekita et al., 2007; Liu et al., 2008), but this is the first time that a possible link between these proteins has been found resulting in a pro-metastatic cancer cell phenotype. Although, the mechanisms of interaction between SFKs, CDCP1 and FAK and consequences on cell phenotype need to be further examined, this finding may be a key to new exciting knowledge about the biology of CDCP1.

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6.2.2 The role of proteolytic processing of CDCP1 in cancer progression

The serine protease matriptase was found to be a very efficient proteolytic processor of cell surface localised CDCP1 being able to rapidly convert all HMW-CDCP1 to LMW-CDCP1 at low nanomolar concentrations. This suggests that this proteolytic event may be of relevance in the generation of the low molecular weight form of CDCP1 in cancer cells in vivo .

Interestingly, the generated data suggested that matriptase-mediated induction of cell signalling events via the phosphorylation of Erk 1/2 in CDCP1 expressing HeLa cells is not mediated by proteolysis of CDCP1. Instead it was found that the detected phosphorylation of Erk 1/2 in response to matriptase treatment is caused by the activation of PAR2. These data suggest that induction of cell signalling as a consequence of CDCP1 proteolysis may occur via pathways other than by phosphorylated Erk 1/2. The data also indicate that treatment of CDCP1 expressing HeLa cells with matriptase does not alter HeLa cell phenotype in vitro. Interestingly, it was found that matriptase expression does not correlate with the abundance of LMW-CDCP1 in prostate cancer cell lines endogenously expressing CDCP1. These data suggest that matriptase may be one of a number of proteases that facilitate conversion of CDCP1 to its LMW form in cultured prostate cancer cell lines. Alternatively, it may be that matriptase is not the endogenous proteolytic processor of CDCP1 in vivo .

Future research needs to identify the endogenous proteolytic processor and examine the effect on cellular processes induced by CDCP1 proteolysis including the effect on the metastatic ability of cancer cells in vivo . If proteolytic processing of CDCP1 is shown to increase the metastatic ability of cancer cells it may suggest that proteolysis and signalling via CDCP1 is an important mechanism which facilitates the metastatic spread of cancers. This would suggest that cancer cells exhibiting LMW-CDCP1 may depend on cell signalling induced by proteolysis of CDCP1 to acquire a metastatic phenotype. This knowledge may help to differentiate between tumours that depend on proteolysis-induced signalling via CDCP1 and tumours that

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Chapter 6 express CDCP1, but do not rely on CDCP1-mediated signalling to acquire a metastatic phenotype. This would suggest that tumours displaying LMW- CDCP1 may be more susceptible to CDCP1 inhibition in vivo . Moreover, if the endogenous cleaver of CDCP1 specifically cleaves CDCP1 it may represent a potential target to indirectly block the pro-metastatic phenotype of CDCP1.

6.2.3 Verification of findings in other cell lines and other model systems

Although new and exciting data was generated which elucidates so far undefined aspects of the biology of CDCP1, it will be necessary to verify some aspects of these data. It is worth noting that most of the data was obtained using CDCP1 expressing HeLa cells and it will be necessary to confirm findings by using other cell lines. The data generated in this study suggest that there may be some limitations in translating results obtained from stable CDCP1 expressing HeLa cells into other cell lines. One reason for this is that HeLa cells are one of only a few reported cancer cell lines which do not endogenously express CDCP1. For example, in this project 19 cancer cell lines originating from four different tissues were examined by anti-CDCP1 Western blot analysis and CDCP1 expression was detected in 17 cell lines (89.5% of the examined cell lines) (Figure 3.1). Similarly, Wong et al. examined the expression of CDCP1 by Western blot analysis in 51 cancer cell lines from nine different tissues and detected CDCP1 expression in 43 cell lines (84.3% of the examined cell lines) (Wong et al., 2009). This shows that CDCP1 expression is common in cancer cell lines of various tissue origins. Therefore, a lack of endogenous CDCP1 expression may indicate that non-CDCP1 expressing cells do not possess adequate cellular machinery to process CDCP1. Another reason that findings from this study need to be examined in other cell lines is that stable CDCP1 expressing HeLa cells display high levels of basal CDCP1 phosphorylation. In addition, constant interaction between CDCP1, SFKs and PKC δ was detected in CDCP1 expressing HeLa cells. This high level of basal phosphorylation

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Chapter 6 might be particular to stable CDCP1 expressing cells which do not express CDCP1 endogenously. This was also reported by Wong et al. , based on unpublished data, who found that cells of unrevealed origin employed to express CDCP1 showed strong basal level of CDCP1 phosphorylation (Wong et al., 2009). In contrast, in cancer cell lines expressing CDCP1 endogenously, only ~40 % of all cell lines of various tissue origins exhibit basal phosphorylation of CDCP1 (Wong et al., 2009). For future projects with the aim of studying the induction of cellular signalling cascades which require an increase in CDCP1 phosphorylation, it might be advisable to use cells endogenously expressing CDCP1. One experimental approach would be to use a number of cell lines which endogenously express high levels of CDCP1 and stably knockdown CDCP1 using siRNA. Another approach would be to employ cell lines which express a low level of CDCP1 to generate stably CDCP1 expressing cells. These approaches would allow studying the biology of CDCP1 in cells which are likely to possess the cellular machinery required to utilise CDCP1.

The role of tyrosine residues in certain cellular processes (including CDCP1- Y734 as a competitive SFK substrate, CDCP1-Y762 in proliferation or CDCP1-Y743 in adhesion) could be studied in cancer cell lines endogenously expressing CDCP1 by employing membrane permeable peptides (Carrigan and Imperiali, 2005). This approach could involve masking the tyrosine residue of interest on endogenously expressed CDCP1 and thereby eliminating interactions with binding proteins and thus signalling initiated at this site. This would be achieved by incubating cells with peptides possessing favourable amino acid sequence, for example a highly lipophilic moiety and an overall net positive charge, which allows efficient cellular peptide uptake (Carrigan and Imperiali, 2005). This approach would allow examination of the effects on cell phenotype and signalling mediated at a certain tyrosine residue in an endogenously CDCP1 expressing cell line which is likely to utilise CDCP1 signalling. An alternative approach to study the induction of cellular events by CDCP1 tyrosine residues could involve the use of membrane permeable phospho peptides (Dunican and Doherty, 2001). In contrast to the above described approach this could be used to

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Chapter 6 study CDCP1-mediated signalling in non CDCP1 or low CDCP1 expressing cell lines. This approach would involve stable transfection of cells with an expression construct encoding for CDCP1 without relevant tyrosine residues. These stable transfected cells could then be incubated with membrane permeable phospho peptides (Dunican and Doherty, 2001). These short phospho peptides penetrate the plasma membrane and mimic a phosphorylated tyrosine site of CDCP1 by displaying an appropriate binding sequence for interacting proteins. This approach would allow studying signalling initiated by binding partners interacting at the phosphorylated site of CDCP1 which is mimicked by the employed phospho peptide sequence. Another approach to study signalling initiated at certain CDCP1 tyrosine residues in endogenously CDCP1 expressing cells could involve stable knockdown of CDCP1 and introduction of a CDCP1 rescue construct encoding for the CDCP1 tyrosine mutant of interest (Miyazawa et al., 2010). Data gathered by the above described approaches will indicate whether the effects observed in this project using HeLa cells are applicable to other cell types and whether these observations could serve as the basis to design strategies to block the pro-metastatic CDCP1 cell phenotype in vivo .

Also worthwhile considering are possible limitations of the generated in vivo data because only one animal model was employed in this study. In particular the higher metastatic phenotype of HeLa-CDCP1-Y734F cells in comparison to HeLa-CDCP1 cells needs to be verified. These cells lack all aspects reported to be important in mediating the CDCP1 cell phenotype, including phosphorylation of CDCP1 and interaction of CDCP1 with SFKs and PKC δ. It is important to examine whether the same high level of metastasis formation of HeLa-CDCP1-Y734F cells would have occurred in an in vivo metastasis model other than in the experimental CAM intravasation assay. In this assay early steps of the metastatic cascade such as dissemination from the primary tumour are not of relevance for cancer cells to metastasise because cells are injected directly into the CAM vein. Worth noting is that interactions of CDCP1 with PKCδ were found to be important to render cells with the ability to degrade extracellular matrix (Miyazawa et al., 2010), which is an important ability for cancer cells to

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Chapter 6 disseminate from the primary tumour in vivo . Therefore, it is possible that metastasis formation of cells expressing CDCP1-Y734F may be lower in comparison to CDCP1 expressing cells in another animal model. The in vivo data obtained could be verified in a spontaneous version of chicken embryo metastasis assay. In this approach tumour cells would be inoculated onto the CAM and active intravasation of cancer cells into the CAM capillaries is required. Moreover, a mouse model of subcutaneous tumour growth could be employed to measure the ability of cells to disseminate from the primary tumour and to metastasise to distant sites.

Confirmation of a highly metastatic phenotype of HeLa-CDCP1-Y734F cells in the above animal models would suggest that CDCP1 facilitates metastasis formation through yet undefined pathways independent of SFKs and PKC δ. It is possible that the pathway involving FAK-Y861, which was revealed in this study, may contribute to this phenotype. This is significant, as it allows a focused investigation into one possible mechanism mediating the observed metastatic in vivo phenotype of these cells.

6.2.4 Interaction of CDCP1 with SFKs and possible implications for the in vivo anti-metastatic effects of anti-CDCP1 antibodies

Interestingly, differences in the ability of anti-CDCP1 antibodies to inhibit metastasis formation of HeLa-CDCP1 and HeLa-CDCP1-S cells in vivo were observed . Investigations to provide explanations for this finding suggest the possible involvement of differentially expressed and phosphorylated SFKs in mediating the anti-metastatic effect of these antibodies. For example, it was found that HeLa-CDCP1-S cells, which express Src, can be inhibited in their metastatic ability. In contrast, Hela-CDCP1, HeLa-CDCP1-Y743F and - Y762F cells, express SFKs other than Src and exhibit increased phosphorylation of these SFKs. These cell lines are unresponsive to inhibition by an anti-CDCP1 antibody in vivo . However, HeLa-CDCP1-Y734F cells which also express SFKs other than Src, but exhibit low levels of p- SFK-Y416 can be significantly inhibited in their ability to metastasise (Figure

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5.5). This suggests that high levels of phosphorylation of SFKs other than Src may inhibit the anti-metastatic action of anti-CDCP1 antibodies.

These observed differences of an anti-CDCP1 antibody to inhibit metastasis formation in vivo of HeLa-CDCP1 and HeLa-CDCP1-S cells may be due to differences in CDCP1 levels following treatment with these antibodies. For example, it was found that anti-CDCP1 antibody treatment resulted in a marked reduction of CDCP1 levels in HeLa-CDCP1-S cells, but not in HeLa- CDCP1 cells (Figure 5.12). This decrease may be caused by internalisation of the CDCP1•antibody complex as internalisation of a CDCP1•antibody complex has been previously described (Siva et al., 2008b; Fukuchi et al., 2010). It is possible that differences in internalisation may be regulated by the differentially expressed and phosphorylated SFKs as the role of distinct SFKs in mediating internalisation of transmembrane proteins has previously been described (Riento et al., 2009). For example, it was described that EGF-induced flotillin internalisation does not occur in SYF cells which lack expression of Src, Fyn and Yes. In these cells the internalisation of flotillins can be restored by expression of Fyn but not Src or Yes and this internalisation occurs in a Fyn tyrosine phosphorylation-dependent manner (Riento et al., 2009). This indicates that different SFKs can exert distinct functions in mediating internalisation of transmembrane proteins which also depends on the phosphorylation of these proteins. Whether distinct SFKs mediate internalisation of CDCP1•antibody complex remains to be examined. Future experiments could involve knockdown of distinct SFKs in CDCP1 expressing cells. These cells could then be incubated with anti-CDCP1 antibodies followed by examination of internalisation of CDCP1•antibody complex and examination of anti-metastatic effects of anti-CDCP1 antibodies in vivo . These experiments could indicate whether distinct SFKs mediate internalisation of CDCP1•antibody complex and importantly whether this correlates with an anti-metastatic response of CDCP1 expressing cells as a result of an anti-CDCP1 antibody treatment. Insights gained in such experiments may be important to delineate the role of CDCP1 as a potential drug target. If differentially expressed and phosphorylated SFKs would be shown to mediate an anti-metastatic response due to CDCP1 inhibition it

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Chapter 6 may suggest that certain tumours would be more sensitive to anti-CDCP1 targeted approaches. Therefore, use of anti-CDCP1 based therapies against CDCP1 positive tumours showing appropriate SFKs expression may achieve improved tumour response rates.

6.2.5 Focus on CDCP1 in physiology

The crucial role of CDCP1 as a mediator of anti-apoptotic and pro-metastatic phenotypes in animal models is becoming well established (Uekita et al., 2007; Siva et al., 2008b; Uekita et al., 2008; Deryugina et al., 2009). Although these findings suggest that CDCP1 is a potential drug target to prevent the metastatic spread of cancers (Wortmann et al., 2009) there are still many unknowns. These unknowns include the role of distinct SFKs in regulating CDCP1, the functional consequences of cleavage of CDCP1 in cancer progression in vivo and the signal transduction pathways downstream of CDCP1 that create the cellular phenotype observed in this study require further elucidation.

In addition, the physiological role of CDCP1 is unknown and it remains an open question about whether targeting of CDCP1 would be an effective strategy to reduce cancer progression or whether this approach would prove too toxic. On the one hand CDCP1 expression has been found in a variety of normal tissues as well as in progenitor blood/bone marrow stem cells (Conze et al., 2002; Buhring et al., 2004). In particular, the expression of CDCP1 in progenitor bone marrow cells is of concern as this may indicate an essential role of CDCP1 in this early cell lineage that would be disrupted with toxic side effects by anti-CDCP1 targeted approaches. On the other hand the recent report that CDCP1 knockout mice develop normally with no gross abnormalities (Tang et al., 2010) may indicate that this protein can be used as a target to treat cancer. However, further research will be required to confirm this finding and to delineate the role of CDCP1 in normal physiology.

If further insights indicate that targeting of CDCP1 will not disrupt essential physiological processes, this protein may be a valuable target for therapeutic

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Chapter 6 intervention given its crucial role in metastasis formation in preclinical animal models and the demonstrated efficacy of anti-CDCP1 antibodies in disrupting these processes (Siva et al., 2008b; Deryugina et al., 2009). Therefore, creating effective strategies to inhibit CDCP1 might contribute to improved outcomes to inhibit the metastatic spread of cancers in humans. The insights I was able to provide during the course of this PhD research project will be of value for pursuing these efforts.

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Appendix 1

Appendix 1: Larger version of photographs presented in Figure 3.6 A

Larger version of photographs presented in Figure 3.6 B

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Appendix 1

Larger version of photographs presented in Figure 3.6 C

Larger version of photographs presented in Figure 3.6 D

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Appendix 1

Larger version of photographs presented in Figure 3.6 E

Figure legend to Figure 3.18: Expression of CDCP1 leads to a change in cell morphology in stable expressing HeLa cell clones. HeLa cells were transfected with CDCP1 (A) , empty vector (B), CDCP1-Y743F (C), CDCP1- Y762F (D) or CDCP1-Y734F (E) and selected for 10 days in G418 containing media. In order to select expressing clones, cells were stained with the anti- CDCP1 antibody 10D7 and a secondary Alexa Fluor 488 labelled antibody and high expressing clones were collected by FACS into 96 well plates. The clones were expanded into T25 flask format and the cell morphology examined by light microscopy. Bar = 100 µm.

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Appendix 2

Appendix 2: Larger version of photographs presented in Figure 3.8

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Appendix 2

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Appendix 2

Figure legend to Figure 3.19: CDCP1 expression leads to a change in cell morphology in stably expressing HeLa cells which requires CDCP1- Y734. Three high CDCP1 expressing HeLa cell clones were combined for HeLa-vector, HeLa-CDCP1-Y734F, -Y743F or -Y762F and two clones were combined for HeLa-CDCP1. The morphology of the combined cell populations was assessed by light microscopy 4 weeks after the clones were combined. Bars = 100 µm.

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Appendix 3

Appendix 3: Larger version of photographs presented in Figure 3.9 (I)

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Larger version of photographs presented in Figure 3.9 (II)

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Appendix 3

Larger version of photographs presented in Figure 3.9 (III)

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Larger version of photographs presented in Figure 3.9 (IV)

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Larger version of photographs presented in Figure 3.9 (V)

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Appendix 3

Figure legend to Figure 3.9: Localisation of CDCP1 and co-localisation of CDCP1 with SFKs is unaltered by mutation of CDCP1-Y734, Y743 or Y762 in CDCP1 expressing HeLa cells. Cells were stained with antibodies for CDCP1 and SFKs and species specific fluorescently labelled secondary antibodies were used to assess the cellular localisation of the two proteins with a confocal microscope. Actin was stained using phalloidin Alexa Fluor 488 and the nucleus was stained with DAPI. ( I), HeLa-vector cells; ( II), HeLa- CDCP1 cells; ( III), HeLa-CDCP1-Y734F cells; ( IV), -Y743F cells or ( V), - Y762F cells. Nonspecific binding of secondary antibodies was examined using slides where cells were incubated with only the secondary antibodies. No nonspecific binding was detected for the goat anti mouse Alexa Fluor 568 antibody (secondary for CDCP1) and barely detectable background staining was observed for the goat anti rabbit Alexa Fluor 647 (secondary to SFK). Bar = 25 µm.

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