ADAMTS1 IS A PROMOTER OF METASTATIC CELL BEHAVIOUR IN MAMMARY CANCER CELLS
Izza Maria Doreen A. Tan
Robinson Institute School of Paediatrics and Reproductive Health Research Centre for Reproductive Health Discipline of Obstetrics and Gynaecology University of Adelaide, Adelaide Australia
“The ones crazy enough to think they can change the world are the ones that do” - Steve Jobs
“It always seem impossible until it’s done”
-Nelson Mandela
“Everything is okay in the end. If its not okay, its not the end”
- John Lennon
Tan IA II Abstract
Metastatic disease is the primary cause of mortality in breast cancer. It is characterised by the dissemination of cancer cells from the primary site, infiltration into vessel networks and the establishment of new tumour growth in secondary tissues. Several events are required for metastasis to occur, including enhancement of cell-matrix adherence, augmented motility and invasiveness. The extracellular matrix (ECM) environment plays a vital role in the processes involved in metastatic progression and undergoes aberrant remodelling to permit and support the metastatic cascade.
Metalloproteinases are a group of enzymes that play a major role in ECM remodelling. The ADAMTS metalloproteinase family has been implicated in the re-organisation of the tumour microenvironment associated with cancer development and metastatic disease progression. Of the 19 ADAMTS proteases, considerable attention has been devoted to the role of its first member ADAMTS1 in cancer metastasis.
Both exogenous overexpression and upregulation of the endogenous ADAMTS1 gene have been strongly associated with metastatic disease in breast cancer. The MMTV-PyMT transgenic breast cancer model recapitulates in vivo metastasis and ablation of Adamts1 impeded the aggressive advancement and growth of pulmonary metastases. The signalling pathways and mechanistic events through which ADAMTS1 mediates its pro-metastatic effects are currently unknown. The aim of this present study is to therefore identify the causal events imposed by ADAMTS1 to promote breast cancer metastasis, with much focus on its role in matrix adhesion, cell migration and invasion.
Using isolated primary mammary carcinoma cells PyMT/Adamts1+/+, PyMT/Adamts1+/- and PyMT/Adamts1-/- mice, I performed real-time assessment of cell-matrix adhesion, motility and invasion and found diminished capacity of PyMT/Adamts1-/- cells to adhere to matrigel and migrate towards a chemoattractive environment. Consistent with the reciprocal approach, introduction of Adamts1 into the MCF10A breast cell line induced the inverse effect, promoting cell adhesion and motility in cells overexpressing Adamts1. Cell-matrix adhesion is a major cue for the determination of front-rear polarity necessary in cell migration and hence, the influence of ADAMTS1 on cell-matrix adhesion underpinned its effects on breast cancer cell migration. Breast cancer cell invasion was unaffected by loss or gain of Adamts1, suggesting a redundant role for ADAMTS1 in this process.
Tan IA III To unravel the transcriptional differences and mechanistic pathways induced by ADAMTS1, microarray analysis was undertaken with PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours. Remarkably, only 2 differentially regulated genes were identified from our analysis. Further investigation of the most dysregulated gene, BC018473, revealed a non-homologous inheritance of this strain specific gene, which unfortunately prevented conclusions being drawn on the underlying genetic effects attributable to Adamts1 ablation.
This study was the first to present a novel role for ADAMTS1 in the promotion of breast cancer cell adhesion to the ECM. This capacity to dynamically modulate adhesion through ADAMTS1 is important in cell migration and highlights a potential mechanism by which ADAMTS1 promotes breast cancer metastasis.
Tan IA IV Declaration
This thesis contains no material, which has been accepted for the award of any other degree or diploma in other university or tertiary institution. The content of this thesis is an original body of work and does not contain any published material written by another person, except where due reference has been made.
I give consent to this copy of my thesis, when deposited in the University Library, to be made available for loan and photocopying, subject to the provisions of the Copyright Act 1968. I also give consent for the digital version to be made available via the University’s digital thesis repository, the Library catalogue and web search engines, unless permission has been granted by the University to restrict access for a period of time.
The author acknowledges that copyright of published work listed below contained within this thesis resides with the copyright holder/s of the publication.
1. de Arao Tan I, Ricciardelli C & Russell DL (2013). “The metalloproteinase ADAMTS1: A comprehensive review of its role in tumorigenic and metastatic pathways.” International Journal Cancer.133(10):2263-76
Tan IA V Acknowledgements
I first would like to express my gratitude for the guidance and support provided by my supervisors, Dr Darryl Russell and Dr Carmela Ricciardelli throughout my PhD, especially during the writing of this thesis. I thank them for granting me the opportunity to undertake a PhD study with them and for the knowledge and expertise they have shared with me. I am grateful for the time and effort they have devoted to help me succeed and overcome the challenges I’ve confronted in my study.
Thank you to all the past and present staff in the Discipline of Obstetrics and Gynaecology for their friendly, smiling faces and technical assistance. I’d like to especially acknowledge Ms Kate Frewin, who has taught me many of the experimental protocols performed in this project. Also, to fellow postgraduate students in the MSN Level 2 office, whom I’ve shared the peaks and throughs of my PhD journey with – thank you for creating a friendly, happy and comforting environment.
The studies conducted in my project were financially supported by grant funding from the National Health and Medical Research Council. I would like to thank the Australian government and the Robinson Institute for supporting my postgraduate scholarships, and the Discipline of Obstetrics and Gynaecology and the Research Centre for Reproductive Health for funding travel opportunities.
Last but not the least, undertaking my PhD would have been next to impossible if without the advice, help and moral support of my family, in particular my parents, Antonio and Cynthia, my siblings Harold and Rhea, my nephew, Rhys and my grandmother, Erlinda. Thank you for keeping me well fed, entertained and distracted. I especially would like to acknowledge and thank my parents whose hard work and perseverance has given me a better life in Australia. I am eternally grateful for your unconditional love, patience and guidance. Anything I’ve achieved and will achieve is inspired by both of you.
Tan IA VI Publications arising from thesis
1. de Arao Tan I, Ricciardelli C, Russell DL (2013). “The metalloproteinase ADAMTS1: A comprehensive review of its role in tumorigenic and metastatic pathways.” International Journal of Cancer.133(10):2263-76
2. de Arao Tan I, Frewin K, Ricciardelli C & Russell DL. “ADAMTS1 promotes the adhesion of mammary cancer cells to structural proteins that make up the extracellular matrix and basement membrane that in turn promotes cancer cell migration”. In preparation
Tan IA VII Abstracts arising from thesis
1. de Arao Tan I, Frewin K, Williams ED, Opeskin K, Pritchard MA, Ingman W, Ricciardelli C & Russell DL. “Role of the protease Adamts1 in breast cancer growth and metastasis” Australian Society for Medical Research, SA conference, Adelaide, SA, June 2009
2. de Arao Tan I, Frewin K, Williams ED, Opeskin K, Pritchard MA, Ingman W, Ricciardelli C & Russell DL. “Role of the protease Adamts1 in breast cancer growth and metastasis” Society for Reproductive Biology, Adelaide, SA, 2009.
3. de Arao Tan I, Frewin K, Williams ED, Opeskin K, Pritchard MA, Ingman W, Ricciardelli C & Russell DL. “The role of Adamts1 in breast cancer progression and metastasis” Matrix Biology Society of Australia and New Zealand, Adelaide, SA, 2009.
4. de Arao Tan I, Frewin K, Ricciardelli C & Russell DL. “The metalloproteinase Adamts1 increases the adhesive capacity of mammary epithelial cancer cells” 7th International Conference on Proteoglycans/Matrix Biology Society of Australia and New Zealand Annual Meeting, Manly, NSW, October 2011
5. de Arao Tan I, Frewin K, Ricciardelli C & Russell DL. “The metalloproteinase Adamts1 increases the capacity of mammary cancer cells to adhere to extracellular components”. Faculty of Health Sciences Postgraduate Conference, Adelaide, SA, August 2011
6. de Arao Tan I, Frewin K, Ricciardelli C & Russell DL. “The metalloproteinase Adamts1 increases the capacity of mammary cancer cells to adhere to extracellular components”. Research Centre for Reproductive Health and Centre for Cancer Stem Cell Research Conference. Adelaide, SA, November 2011
Tan IA VIII Table of Contents
Title page
Abstract ...... III Declaration ...... V Acknowledgements ...... VI Publications arising from thesis ...... VII Abstracts arising from thesis ...... VIII Table of contents ...... IX List of figures ...... XIII List of tables ...... XV Abbreviations ...... XVI
CHAPTER 1 – Introduction
1.1. Introduction ...... 3 1.2. The synthesis, structure and protein interactions of ADAMTS1 ...... 5 1.3. Epigenetic downregulation of ADAMTS1 in primary cancers ...... 7 1.4. ADAMTS1 expression in cancer ...... 9 1.4.1. ADAMTS1 is downregulated as prostate cancer becomes castrate resistant ...... 9 1.4.2. ADAMTS1 promotes hepatocellular carcinoma by aggravating liver fibrosis ...... 12 1.4.3. Upregulated expression of ADAMTS1 promotes breast cancer progression ...... 15 1.5. ADAMTS1 in metastatic cancer ...... 16 1.6. ADAMTS1-mediated pathways in cancer development and metastasis ...... 17 1.6.1. Angiogenesis ...... 17 1.6.2. Cell proliferation ...... 19 1.6.3. Cell survival ...... 20 1.6.4. Cell migration and invasion ...... 23 1.7. Conclusion ...... 25
Tan IA IX 1.8. Hypotheses and aims ...... 26
CHAPTER 2 – ADAMTS1 enhances breast cancer cell adhesion to the extracellular matrix
2.1. Introduction ...... 29 2.2. Materials and methods ...... 30 2.2.1. Animals ...... 30 2.2.2. DNA extraction ...... 31 2.2.3. PCR genotyping and gel electrophoresis ...... 31 2.2.3.1. MMTV-PyMT ...... 31 2.2.3.2. ADAMTS1 ...... 32 2.2.4. Isolation and propagation of mammary cancer cells ...... 34 2.2.5. Viral transduction of 2756 knockout primary mammary cancer cells ...... 34 2.2.6. Generation of MCF10A-Adamts1 and MCF10A-GFP clones ...... 35 2.2.7. Quantitative real-time RT-PCR ...... 36 2.2.8. Immunocytochemistry ...... 37 2.2.9. Quantification of cytokeratin-positive mammary epithelial carcinoma cells ...... 37 2.2.10. Real-time cell based assay ...... 38 2.2.10.1. In vitro proliferation assay ...... 38 2.2.10.2. In vitro adhesion assay ...... 38 2.2.11. Statistics ...... 39 2.3. Results ...... 41 2.3.1. Isolated primary cells from Adamts1+/+, Adamts1+/- and Adamts1-/- PyMT mammary tumours were predominantly mammary epithelial cancer cells ...... 41 2.3.2. PyMT/Adamts1-/- primary cells exhibited reduced adhesion to Matrigel™ compared with Adamts1+/+, Adamts1+/- primary mouse mammary cancer cells ...... 45 2.3.3. Induced Adamts1 expression in knockout primary mammary carcinoma cells did not affect cell adhesion due to poor transduction efficiency ...... 48 2.3.4. Lentiviral transduction of Adamts1 in MCF10A mammary epithelial cells promoted cell adhesion to matrigel ...... 51 2.4. Discussion ...... 55
Tan IA X
CHAPTER 3 – ADAMTS1 accelerates mammary cancer cell migration does not alter mammary cancer cell invasion
3.1. Introduction ...... 60 3.2. Materials and methods ...... 62 3.2.1. Cell culture ...... 62 3.2.2. Quantitative real-time RT-PCR ...... 62 3.2.3. Real time cell-based assay ...... 62 3.2.3.1. In vitro migration assay ...... 63 3.2.3.2. In vitro invasion assay ...... 63 3.2.4. Statistics ...... 67 3.3. Results ...... 67 3.3.1. Loss of Adamts1 impeded mammary carcinoma cell migration ...... 67 3.3.2. The loss of Adamts1 did not alter the invasive capacity of Adamts1-/- 1omMCC ..... 70 3.3.3. Adamts1 overexpression in non-transformed MCF10A cells promotes cell migration over 45hrs ...... 73 3.3.4. Adamts1 overexpression in MCF10A cells had no effect on cell invasion ...... 76 3.4. Discussion ...... 79
CHAPTER 4 – Differential gene expression analysis of mammary tumours derived from PyMT/Adamts1+/+ and PyMT/Adamts1-/- mice
4.1. Introduction ...... 83 4.2. Materials and methods ...... 85 4.2.1. Generation of PyMT/Adamts1+/+, PyMT/Adamts1+/- and PyMT/Adamts1-/- breeding colony ...... 85 4.2.2. Microarray ...... 85 4.2.3. Genotyping ...... 86 4.2.3.1. BCO18473 ...... 86 4.2.3.2. Rnf160 ...... 86
Tan IA XI 4.2.4. Quantitative RT-PCR ...... 87 4.2.5. Statistics ...... 88 4.3. Results ...... 88 4.3.1. Microarray ...... 88 4.3.2. PyMT/Adamts1-/- mammary tumours expresses a non-coding transcript of Adamts1 ...... 93 4.3.3. Validation of Rnf160 upregulation in PyMT/Adamts1-/- ...... 96 4.3.4. Long non-coding RNA BC018473 is downregulated in PyMT/Adamts1-/- mammary tumours ...... 98 4.3.5. Strain imbalance between the two Adamts1 cohorts ...... 101 4.3.6. Cell-based experiments and published mouse cohorts possess homogenous strain mixing in Adamts1+/+ and Adamts1-/- mice ...... 101 4.4. Discussion ...... 105
CHAPTER 5 – Conclusion and future directions
5.1. Promoted matrix adhesion is a novel prometastatic behaviour associated with the ADAMTS1 metalloprotease ...... 110 5.2. Potential mechanisms underlying the role of ADAMTS1 in cell-ECM adhesion ...... 112 5.3. Clinical significance ...... 115 5.4. Summary and future directions ...... 116
CHAPTER 6 – Bibliography ...... 118
Tan IA XII List of figures
Figure 1.1 – ADAMTS1 protein synthesis and function ...... 6 Figure 1.2 – ADAMTS1 expression in normal and corresponding cancer tissues ...... 8 Figure 1.3 – Aggravation of inflammatory response in liver fibrosis by ADAMTS1 to promote hepatocellular carcinoma ...... 14 Figure 1.4 – ADAMTS1 mediated pathways to promote proliferation and cell survival of breast cancer cells ...... 22 Figure 2.1 – Schematic representation of annealing sites of Adamts1 genotyping primers ...... 33 Figure 2.2 – Real-time cell proliferation and adhesion assays with the xCelligence system ...... 40 Figure 2.3 – PyMT/Adamts1-/- mice have smaller mammary tumours than Adamts1+/+ and Adamts1+/- littermates, and isolated 1omMCC were predominantly of mammary epithelial cancer cell type ...... 42 Figure 2.4 – Cell adhesion is impaired in PyMT/Adamts1-/- mammary cancer cells ...... 46 Figure 2.5 – Cell adhesion of #2756 Adamts1 null 1omMCC transduced to overexpress Adamts1 ...... 49 Figure 2.6 – Adamts1 overexpression in MCF10A cells ...... 52 Figure 2.7 –Cell adhesion of MCF10 breast cancer cell line overexpressing Adamts1 ...... 54 Figure 2.8 - Schematic representation of ECD-integrin motif in ADAMTS1 ...... 57 Figure 2.9 - Potential pathways facilitated by ADAMTS1 to promote mammary cancer cell adhesion to the ECM ...... 58 Figure 3.1 – Real-time invasion and migration assessment using the xCelligence system ...... 65 Figure 3.2 – Loss of Adamts1 impedes primary mammary cancer cell migration ...... 69 Figure 3.3 – Adamts1-/- 1oMCC exhibited accelerated cell invasion than Adamts1+/+ cells ...... 72 Figure 3.4 – Migration of wild type MF10A, MCF10A-Adamts1 and MCF10A-GFP cells over 45 hours 74 Figure 3.5 – Invasion of wild type MCF10A, MCF10A-Adamts1 and MCF10A-GFP cells over 45 hours 77 Figure 4.1 – PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumour samples assessed in microarray90 Figure 4.2 – PCA plot of sample distribution based on global gene expression patterns ...... 92 Figure 4.3 – Confirmation of Adamts1 genotypes ...... 94 Figure 4.4 – Rnf160 transcript expression ...... 97 Figure 4.5 – Downregulation of BC018473 transcript in PyMT/Adamts1-/- mammary tumours ...... 99
Tan IA XIII Figure 4.6 – Unequal representation of BC018473 is unique to the microarray cohort ...... 103 Figure 5.1 – The role of ADAMTS1 in mammary cancer cell adhesion and how it may influence the migration and invasion of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary carcinoma cells ...... 114
Tan IA XIV List of tables
Table 1.1 – ADAMTS1 expression with cancer progression ...... 11 Table 1.2 – ADAMTS1-mediated pathways in cancer progression and metastasis ...... 24 Table 2.1 – Genotyping PCR primers: MMTV-PyMT and Adamts1 ...... 33 Table 2.2 – RT-PCR primers: Adamts1 and ADAMTS1 ...... 37 Table 4.1 – Genotyping PCR primers: BC018473 and Rnf160 ...... 87 Table 4.2 – Gene expression analysis of PyMT/Adamts1+/+ vs PyMT/Adamts1-/- breast tumours ...... 89 Table 4.3 – Individual microarray probe sets against specific exons of Rnf160 ...... 96 Table 4.4 – BC018473 microarray expression across different mouse strains detected in other studies107
Tan IA XV Abbreviations
1omMCC primary mouse mammary cancer cell ADAMTS1 a disintegrin and metalloproteinase with thrombospondin motifs 1 Adamts1-/- Adamts1 knockout Adamts1+/- Adamts1 heterozygous Adamts1+/+ Adamts1 wild type ADPC androgen dependent prostate cancer ANOVA analysis of variance AR androgen receptor bp base pair C-terminal carboxyl terminal cDNA complemtary deoxyribonucleic acid CHO chinese hamster ovary cm centimetre CRC colorectal carcinoma CRPC castrate resistant prostate cancer DMEM dulbecco’s minimum essential medium DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dNTP deoxyribonucleic triphosphate ECD glutamic acid-cysteine-aspartic acid ECM extracellular matrix EDTA ethlyenediaminetetraacetic acid EGF epidermal growth factor ER estrogen receptor FBS fetal bovine serum GFP green fluorescent protein GSE genomic spatial event h hour
Tan IA XVI HA hyaluronan HB-EGF heparin binding epidermal growth factor HCC hepatocellular carcinoma HCC hepatocellular carcinoma HS heparan sulphate HSC hepatic stellate cells HSPG heparan sulphate proteoglycans IRES internal ribosomal entry site LSD least significant difference mg milligram min minute ml millilitre mm millimetre mM millimolar MMP matrix metalloproteinase MMTV mouse mammary tumour virus MTV mammary tumour virus N-terminal amino terminal
Na2HPO4 disodium hydrogen phosphate NaCl sodium chloride
NaH2PO4 sodium dihydrogen phosphate ng nanogram No. number NSCLC non-small cell lung cancer oC degrees Celsius OPG osteoprotegrin PBS phosphate buffer saline PCR polymerase chain reaction PSF penicillin/streptomycin/fungizone PPAR-γ peroxisome proliferator-activated receptor gamma PyMT polyoma middle-T qRT-PCR quantitative reverse transcription polymerase chain reaction R2 coefficient of determination
Tan IA XVII RANKL nuclear kappa B RGD arginine-glycine-asparagine RNA ribonucleic acid rpm revolutions per minute sec second SEM standard error of mean shRNA short hairpin ribonucleic acid TGF-β transforming growth factor beta TNFα tumour necrosis factor alpha TSP thrombospondin U/ml units per ml v/v volume per volume percentage solution VEGF vascular endothelial growth factor w/v weight per volume percentage solution wt wild type μg microgram μl microlitre
Tan IA XVIII
CHAPTER 1
Introduction
CHAPTER 1 Introduction
STATEMENT OF AUTHORSHIP
The metalloproteinase ADAMTS1: A comprehensive review of its role in tumorigenic and metastatic pathways
Izza de Arao Tan (Candidate) Development and writer of manuscript Certification that the statement of contribution is accurate
Carmela Ricciardelli (co-author) Supervised development of work and manuscript preparation Certification that the statement of contribution is accurate and permission is given for the inclusion of the paper in the thesis
Darryl L Russell (co-author) Supervised development of work and manuscript preparation Certification that the statement of contribution is accurate and permission is given for the inclusion of the paper in the thesis
Tan IA 2 CHAPTER 1 Introduction
1.1. INTRODUCTION
In cancer, the remodeling of the host tissue environment is fundamental for the dismantling of normal tissue structural constraints, establishment of vascular supply, and invasion of metastatic cells. For this aberrant remodeling to occur, cancers cells rely on the proteolytic activity of extracellular matrix (ECM) proteases to facilitate the degradation of structural proteins that make up the ECM (Koblinski et al., 2000; Rucci et al., 2011). Metalloproteases belonging to the ADAMTS family have been widely implicated in tissue remodeling events manifested in cancer development, growth and progression (Cross et al., 2005; Demircan et al., 2009; El Hour et al., 2010; Kumar et al., 2012; Llamazares et al., 2007; Lo et al., 2010; Molokwu et al., 2010; Okada, 2000; Porter et al., 2006; Ricciardelli et al., 2011; Viloria et al., 2009; Wang et al., 2011).
There are 19 ADAMTS proteases identified in humans. They share homology in the catalytic metalloprotease and disintegrin domains, but differ in the variable numbers of thrombospondin-like motifs and other carboxyl-terminal domains associated with ECM interaction (Kuno et al., 1997a; Porter et al., 2005). Unlike the transmembrane ADAM family, ADAMTS proteases are secreted and bind to the ECM through their C-terminal regions (Kuno and Matsushima, 1998). Some family members facilitate the polymerisation of ECM proteins (ADAMTS2 (Colige et al., 2005; Colige et al., 2002), ADAMTS3 (Fernandes et al., 2001), ADAMTS10 (Kutz et al., 2011), ADAMTS17 (Morales et al., 2009)) while others catalyse proteoglycan degradation (ADAMTS1 (Kuno et al., 2000; Ricciardelli et al., 2011; Russell et al., 2003), ADAMTS4 (Horber et al., 2000; Sugimoto et al., 1999), ADAMTS5 (Abbaszade et al., 1999), ADAMTS7 (Liu et al., 2006a; Luan et al., 2008), ADAMTS8 (Porter et al., 2005), ADAMTS9 (Somerville et al., 2003), ADAMTS12 (Liu et al., 2006b; Luan et al., 2008), ADAMTS15 (Peluffo et al., 2011), ADAMTS16 (Gao et al., 2007), ADAMTS18 (Li et al., 2010), ADAMTS20 (Llamazares et al., 2003; Silver et al., 2008). There is considerable functional redundancy among the proteoglycan-degrading proteases and many cancers exhibit dysregulated activity of several ADAMTS enzymes (Wagstaff et al., 2011). Among them, the modulated expression of ADAMTS1 has been the most characterised in cancer and is the focus of this review.
ADAMTS1 was initially described as a mediator of inflammation (Kuno et al., 1997b) but its activity has since become appreciated in organogenesis (Gunther et al., 2005; Mittaz et al., 2004; Shindo et al., 2000; Thai and Iruela-Arispe, 2002), blood/lymph vessel formation (Brown et al., 2006; Brown et al., 2010c; Iruela-Arispe et al., 2003; Krampert et al., 2005; Lee et al., 2006; Luque et al., 2003;
Tan IA 3 CHAPTER 1 Introduction
Misra et al., 2008), ovarian folliculogenesis (Brown et al., 2006) and ovulation (Brown et al., 2010b; Russell et al., 2003). In these physiological events, ADAMTS1 remodels the ECM through the proteolytic degradation of key substrates such as chondroitin sulphated proteoglycans (Kuno et al., 2000; Nakamura et al., 2005; Rodriguez-Manzaneque et al., 2009; Sandy et al., 2001) and collagen (Hu et al., 2012; Lind et al., 2006b; Rehn et al., 2007). ADAMTS1 also acts as an inhibitor of angiogenesis by sequestering pro-angioigenic stimuli, vascular epithelial growth factor (VEGF), and preventing its interaction with its receptor (Iruela-Arispe et al., 2003; Luque et al., 2003). The dysregulation of ADAMTS1 often leads to pathological manifestations of altered ECM (Stankunas et al., 2008; Wachsmuth et al., 2004; Zreiqat et al., 2010) and/or vascular density (Fu et al., 2011; Misra et al., 2010; Reynolds et al., 2010; Yatabe et al., 2009) and many studies have highlighted its functional activity during tumourigenic transformation (Gustavsson et al., 2008; Gustavsson et al., 2009; Kuno et al., 2004; Lind et al., 2006a; Liu et al., 2006c; Masui et al., 2001; Ricciardelli et al., 2011; Rocks et al., 2008)
ADAMTS1 dysregulation is linked to four of the most commonly diagnosed cancers. But conflicting reports currently surround its expression in cancer, as different studies have shown both up- (Bonuccelli et al., 2009; Hirano et al., 2011; Ricciardelli et al., 2011; Tyan et al., 2012) and downregulated (Choi et al., 2008; Gustavsson et al., 2008; Ifon et al., 2005; Kohno et al., 2010; Lind et al., 2006a; Yegnasubramanian et al., 2011) expression of ADAMTS1 in primary tumours compared with healthy tissue controls. For this reason, ADAMTS1 has been ascribed both pro- and anti-tumourigenic activities but with poor understanding of the specific mechanisms it mediates to promote or inhibit tumourigenesis. However, irrespective of the direction of regulation, perturbations in ADAMTS1 expression are commonly associated with the transition to malignancy and changes in the peritumoural environment (Ricciardelli et al., 2011), tumour vascularity (Gustavsson et al., 2008; Gustavsson et al., 2010; Liu et al., 2006c; Luque et al., 2003) and tumour cell behaviour (Kuno et al., 2004; Liu et al., 2006c; Ricciardelli et al., 2011). This comprehensive review explores the regulation of ADAMTS1 gene expression and its consequences during cancer development and progression. It will also review current literature, which supports an anti-tumourigenic role for ADAMTS1 since expression is most commonly reduced in primary cancers versus matched normal organs. Conversely, pro-metastatic abilities appear to be conferred in tumours that gain high ADAMTS1 expression during subsequent tumour progression.
Tan IA 4 CHAPTER 1 Introduction
1.2. THE SYNTHESIS, STRUCTURE AND PROTEIN INTERACTIONS OF ADAMTS1
Key distinctions in the structure of each ADAMTS protease lie at the carboxyl-end (Porter et al., 2005). For ADAMTS1, this region consists of 3 thrombospondin1 (TSP1) motifs separated by a cysteine-rich domain and spacer region (Kuno et al., 1997a; Kuno et al., 1997b) (Fig. 1). ADAMTS1 is synthesised as a zymogen and undergoes N-linked glycosylation following protein translation (Kuno and Matsushima, 1998). The secretion of ADAMTS1 to the ECM requires the excision of its pro-domain from the 87kD mature protein by furin-related endopeptidases (Kuno and Matsushima, 1998). In the ECM, the C-terminal region of the mature protease directly binds to the ECM (Kuno and Matsushima, 1998; Kuno et al., 1999) and associates with other proteins such fibulin-1 (Lee et al., 2005), latent TGF-β (Bourd- Boittin et al., 2011) and sulphated proteoglycans (Kuno and Matsushima, 1998; Kuno et al., 1999; Rodriguez-Manzaneque et al., 2009; Rodriguez-Manzaneque et al., 2002) (Fig.1). The protease component of ADAMTS1 catalyses the breakdown of collagen type 1 (Rehn et al., 2007), tissue-specific stromal proteoglycans such as versican (Ricciardelli et al., 2011; Russell et al., 2003; Sandy et al., 2001), aggrecans (Rodriguez-Manzaneque et al., 2002), syndecan-4 (Rodriguez-Manzaneque et al., 2009) and basement membrane proteins like nidogen 1 and 2 (Canals et al., 2006) (Figure 1.1). Thus, the complex structure of ADAMTS1 can therefore influence the cancer environment by a range of means.
Tan IA 5 CHAPTER 1 Introduction
ites for latent n Ib and TSP1 and n Ib
1/2 (nidogen teins r region. ADAMTS1 is is ADAMTS1 r region. catalytic metalloprotease metalloprotease catalytic TSP1 - associates with TSP1 receptors (CD36, CD47) and glycoprotein lb TSP1 TSP1 TSP1 TSP1 motifs and spacer region - ECM binding region TSP1 motifs associate with - sulfated glycosaminoglycans in ECM TSP1-like motifs directly interacts - with EGF-like repeats of fibulin-1 TSP1 spacer
β spacer space y cys rich s yy cys rich -glycosylation. Furin endopeptidases cleave the pro-domain of ADAMTS1, after after ADAMTS1, of pro-domain the cleave endopeptidases Furin -glycosylation. Ng N-glycosylates N
TSP1 TSP1 e s DIS DIS Proximal TSP1 -contains a WGPW and KTFR amino acid sequence that directly interact peptides, with RKPK and LKSL of latent-TGF- respectively, . The amino-half of ADAMTS1 is composed of a pro-domain (PRO), metalloproteinase (MP) and and (MP) metalloproteinase (PRO), a pro-domain of composed is ADAMTS1 of The amino-half . Disintegrin domain - unknown functions and/or interactions Furin endopeptidase MP MP O MP MP M PRO Immature ADAMTS1 CYTOPLASM CYTOPLASM Metalloproteinase domain - cleaves ECM proteolycans Aggrecan, (Versican, Syndecan-4) - cleaves basement membrane proteins (Nidogen 1/2, Collagen I IV) TSP1/2 at LRRPPL - cleaves peptide region Mature ADAMTS1 ECM ADAMTS1 protein synthesis and function protein ADAMTS1 synthesis
. The TSP1 motifs of ADAMTS1 associates with sulfated glycosaminoglycan and fibulin-1. TSP1 directly interacts with glycoprotei with interacts directly TSP1 and fibulin-1. glycosaminoglycan sulfated with associates ADAMTS1 of motifs . The TSP1 β disintegrin (DIS) domain, while the carboxyl region of ADAMTS1 consists of 3 TSP1-like motifs, cysteine-rich domain and a space and domain cysteine-rich motifs, TSP1-like of 3 consists of ADAMTS1 region carboxyl the while domain, (DIS) disintegrin Figure 1.1. Figure initially synthesised as a pro-zymogen and undergoes post translation translation post undergoes and a pro-zymogen as synthesised initially the ECM, the In region. spacer its at bound becomes it where matrix extracellular the into secreted is enzyme mature the which pro membrane basement and syndecan-4), aggrecans (versican, proteoglycans of stromal the cleavage ADAMTS1 facilitates of domain s docking as act which peptides, and KTFR a WGPW has of ADAMTS1 motif TSP1 The proximal proteins. and TSP1 I) type and collagen TGF and CD47. as CD36 such receptors
Tan IA 6 CHAPTER 1 Introduction
1.3. EPIGENETIC DOWNREGULATION OF ADAMTS1 IN PRIMARY CANCERS
Low ADAMTS1 expression is often reported in primary tumours compared to their tissue of origin (Choi et al., 2008; Gustavsson et al., 2008; Gustavsson et al., 2010; Gustavsson et al., 2009; Kohno et al., 2010; Lind et al., 2006a). Metadata we extracted from the Genesapiens repository of global gene expression studies support the consistent downregulation of ADAMTS1 in primary tumours compared with healthy tissue controls (Figure 1.2) (Kilpinen et al., 2008; Kilpinen et al., 2011; Masui et al., 2001). Further investigations in lung (Choi et al., 2008; Kohno et al., 2010; Rocks et al., 2008), prostate (Gustavsson et al., 2008; Gustavsson et al., 2010; Gustavsson et al., 2009) and colorectal cancers (Ahlquist et al., 2008; Lind et al., 2006a) revealed epigenetic silencing through promoter hypermethylation to be the key mechanism underlying ADAMTS1 repression during tumour development (Ahlquist et al., 2008; Choi et al., 2008; Lind et al., 2006a; Yegnasubramanian et al., 2011). In prostate and non-small cell lung cancer (NSCLC), ADAMTS1 promoter methylation was present in more than 75% and 30% of tumour biopsies, respectively, and only 5-7% methylation in normal tissue counterparts (Choi et al., 2008; Yegnasubramanian et al., 2011). Cell lines from prostate and NSCLC cancers also possessed methylated-rich ADAMTS1 promoters and low ADAMTS1 expression, which was restored when treated with demethylating agents (Choi et al., 2008; Yegnasubramanian et al., 2011). The pathological consequence/s of diminished ADAMTS1 levels in NSCLC is unknown. However, for prostate cancer, the decrease in ADAMTS1 mRNA follows disease progression (see Section 1.4.1)
Hypermethylation of ADAMTS1 was also exhibited in more than 80% of colorectal cancer cell lines (Ahlquist et al., 2008; Lind et al., 2006a) and approximately 70% and 40% of colorectal carcinoma and adenoma biopsies, respectively (Lind et al., 2006a). On the other hand, distant healthy colon tissues from CRC patients only presented 9% methylated ADAMTS1, while healthy colon samples from patients without cancer exclusively presented its unmethylated form (Ahlquist et al., 2008). These findings implicate the methylation state of ADAMTS1 as a potential early biomarker for predicting CRC development. Furthermore, the increase in ADAMTS1 silencing from adenomas to carcinomas parallels epidemiological and histological evidence that strongly imply colorectal carcinomas evolving from benign adenomas (Leslie et al., 2002). The negative correlation between ADAMTS1 and CRC progression suggests a potential suppressive activity of ADAMTS1 during adenoma-carcinoma transformation. How the loss of ADAMTS1 results in or aids this transition in CRC as well as in NSCLC and prostate cancer remains unknown and warrants further investigation.
Tan IA 7 CHAPTER 1 Introduction
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600
400
200 ADAMTS1 expression value 0 b. Healthy Cancer
ADAMTS1 expression values Tissue Type N Min Median Max Mean SD SEM P-value 0 1000 2000 3000 4000 5000 Healthy 16 277.7 1684.0 3895.0 1907.0 1339.0 334.6 adipose * 0.0287 Y Cancer 10 226.0 526.6 3002.0 849.8 861.2 272.3 R Healthy 10 577.2 1388.0 3215.0 1616.0 937.6 296.5 bladder ** 0.0026 Y Cancer 77 15.5 419.1 4077.0 902.4 1132.0 129.0 R blood lymphoid Healthy 131 13.2 59.4 1404.0 99.0 151.5 13.2 **** < 0.0001Y cell Cancer 348 14.8 106.7 476.9 117.1 80.2 4.3 R bone marrow Healthy 2 41.8 61.0 80.2 61.0 27.2 19.2 n/a Y lymphoid cell Cancer 572 14.7 64.1 1847.0 111.1 168.5 7.0 R bone marrow Healthy 9 33.2 80.8 189.5 95.8 48.5 16.2 * 0.0174 Y myeloid cell Cancer 294 14.5 48.1 1566.0 78.2 126.7 7.4 R Healthy 8 162.5 327.3 913.1 390.7 236.8 83.7 breast ns 0.325 Y Cancer 1312 19.5 247.6 3355.0 415.6 459.6 12.7 R central nervous Healthy 290 15.4 169.2 2866.0 266.8 328.0 19.3 ns 0.075 Y system Cancer 412 14.7 175.9 2160.0 318.4 376.3 18.5 R Healthy 4 404.0 660.5 1100.0 706.3 289.2 144.6 cervix ** 0.003 Y Cancer 55 49.2 146.6 761.2 199.0 161.4 21.8 R Healthy 23 72.1 737.5 3336.0 967.8 863.4 180.0 colorectal **** < 0.0001Y Cancer 504 15.9 189.9 3086.0 255.6 238.5 10.6 R endocrine Healthy 33 15.3 310.1 3326.0 600.2 843.8 146.9 ns 0.0771 Y system Cancer 51 18.2 201.8 1984.0 282.0 313.4 43.9 R Healthy 4 67.3 166.1 245.0 161.1 73.9 36.9 eye n/a Y Cancer 1 255.2 255.2 255.2 255.2 0.0 0.0 R Healthy 68 32.8 333.3 3018.0 730.8 884.4 107.3 kidney ** 0.0087 Y Cancer 232 26.6 532.5 3324.0 666.4 499.3 32.8 R Healthy 13 21.8 1207.0 2238.0 1024.0 771.9 214.1 liver * 0.0476 Y Cancer 7 72.9 213.8 794.2 295.3 268.4 101.4 R liver and biliary Healthy 7 31.8 119.2 1043.0 262.6 356.8 134.9 n/a Y system Cancer 2 99.3 170.1 241.0 170.1 100.2 70.9 R lymphatic Healthy 90 14.5 171.4 1873.0 242.0 296.8 31.3 ns 0.2718 Y system Cancer 52 15.6 123.8 804.4 157.0 149.0 20.7 R Healthy 4 357.8 1084.0 1727.0 1063.0 570.6 285.3 mesothelium ns 0.1844 Y Cancer 14 178.4 592.7 1781.0 646.9 427.2 114.2 R Healthy 48 69.2 527.6 3242.0 826.1 779.8 112.6 muscle ns 0.1336 Y Cancer 47 101.1 734.1 3043.0 883.3 625.0 91.2 R Healthy 33 71.9 264.7 2602.0 537.3 602.9 104.9 other GI system ns 0.9922 Y Cancer 47 30.6 313.3 2050.0 413.1 354.7 51.7 R other urogenital Healthy 11 102.2 530.8 967.9 529.7 284.5 85.8 ns 0.1511 Y system Cancer 23 92.0 256.2 2602.0 470.1 553.2 115.3 R Healthy 5 1347.0 3008.0 3322.0 2519.0 913.6 408.6 ovary *** 0.0003 Y Cancer 243 15.4 398.3 2464.0 548.1 504.1 32.3 R Healthy 25 38.4 456.1 3568.0 1189.0 1304.0 260.8 pancreas ns 0.2062 Y Cancer 42 39.9 209.4 3490.0 477.6 663.7 102.4 R peripheral Healthy 18 81.1 1053.0 3434.0 1255.0 1083.0 255.3 ns 0.58 Y nervous system Cancer 4 229.9 704.0 1188.0 706.4 416.3 208.1 R Healthy 12 513.0 1187.0 3230.0 1351.0 699.1 201.8 prostate **** < 0.0001Y Cancer 86 28.0 406.7 2866.0 575.5 535.3 57.7 R respiratory Healthy 102 13.8 75.5 3648.0 445.8 847.5 83.9 **** < 0.0001Y system Cancer 183 15.7 324.1 3242.0 484.5 488.1 36.1 R Healthy 7 28.4 121.0 267.3 140.3 99.5 37.6 salivary gland ns 0.202 Y Cancer 5 145.7 227.0 461.0 252.3 126.0 56.3 R Healthy 3 648.3 814.9 1176.0 879.9 270.0 155.9 skin * 0.0364 Y Cancer 12 110.0 312.0 798.5 403.5 253.4 73.2 R Healthy 26 36.5 77.7 1840.0 279.2 457.8 89.8 testis **** < 0.0001Y Cancer 207 25.3 273.4 4054.0 788.9 970.7 67.5 R Healthy 11 17.8 314.6 1057.0 406.3 323.9 97.7 tongue ns 0.9671 Y Cancer 8 63.5 446.9 1137.0 442.5 355.8 125.8 R Healthy 28 66.8 1462.0 3613.0 1643.0 1105.0 208.9 uterus **** < 0.0001Y Cancer 187 26.3 348.8 3192.0 452.9 386.4 28.3 R 0 1000 2000 3000 4000 5000
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Figure 1.2. ADAMTS1 expression in normal and corresponding cancer tissues. a) Total ADAMTS1 expression in combined normal and cancer tissue samples. Data represented as mean±SEM, Student’s t-test ****P<0.0001. b) ADAMTS1 expression in 30 normal and cancer tissues obtained from a publicly available database (www.genesapiens.org). ADAMTS1 is downregulated in cancer tissues found in the adipose, bladder, bone marrow myeloid cell, cervix, colorectal, liver, ovary, prostate, skin and uterine cancer, upregulated in blood lymphoid cell, kidney, respiratory and testis, and not differentially regulated in breast, central nervous system, endocrine, lymphatic, mesothelium, muscle, other G1/urogenital, pancreas, peripheral nervous system, salivary and tongue. Vertical lines on scatter plot indicate median value for each tissue type. Mann Whitney U
t-test to determine statistical significance.
1.4. ADAMTS1 EXPRESSION IN CANCER
Numerous studies have documented changes in ADAMTS1 mRNA and protein levels during tumour advancement in the prostate, liver and mammary gland (Table 1.1). The subsequent sections discuss the implications of altered ADAMTS1 levels in tumour development and how this relates to the current knowledge surrounding prostate, hepatocellular and breast cancer development.
1.4.1. ADAMTS1 is downregulated as prostate cancer becomes castrate resistant
Castrate resistant prostate cancer (CRPC) is an aggressive form of prostate malignancy. Its development is typically associated with pre-existing androgen dependent prostate cancer (ADPC) that acquire resistance to androgen ablation therapy (Culig and Bartsch, 2006; Devlin and Mudryj, 2009; Mellado et al., 2009; Navarro et al., 2002). Resistance to androgen deprivation therefore permits prostate cancer cell longevity and disease advancement that is independent of androgen control (Shore et al., 2012).
Many groups have illustrated in vitro a decrease in ADAMTS1 expression with transition of prostate cancer cells towards castrate resistance (Best et al., 2005; Gustavsson et al., 2008; Gustavsson et al., 2010; Gustavsson et al., 2009; Ifon et al., 2005; Jennbacken et al., 2009; Varambally et al., 2005) (Table 1.1). For instance LNCaP19, a castrate resistant derivative of the ADPC cell line, displayed significantly lower ADAMTS1 than wild-type LNCaP cells (Gustavsson et al., 2008; Gustavsson et al., 2010; Gustavsson et al., 2009). Conversely, introduction of tumour suppressor gene,
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U94 in advanced castrate resistant prostate PC3 cells not only reverted tumour development to an androgen dependent phenotype, but also instigated a 7-fold induction in ADAMTS1 expression (Ifon et al., 2005). This same pattern of ADAMTS1 downregulation was also identified in prostate tumours removed from hormone refractory patients (Best et al., 2005; Varambally et al., 2005). The concomitant decrease in ADAMTS1 may suggest an androgen-dependent mechanism driving ADAMTS1 transcription. However, this notion is refuted by the lack of effect dihydrotestosterone treatment have on basal ADAMTS1 expression in prostate cancer cells (Cross et al., 2005). Alternatively, signaling pathways downstream of androgen receptors (AR) and/or TGF-β, which become disrupted and over- stimulated in CRPC (Danielpour, 2005; Decker et al., 2012; Edwards et al., 2003; Marques et al., 2005; Russell and Bennett…, 1998; Visakorpi et al., 1995), have been implicated in eliciting gene methylation changes in prostate cancer (Aitchison et al., 2008; Friedlander et al., 2012; Tewari et al., 2012). As ADAMTS1 promoter hypermethylation occur during prostate cancer development, epigenetic gene expression changes induced by TGF-β and/or AR may underpin the downregulation of ADAMTS1 with increasing castrate resistance.
The reduction in ADAMTS1 with CRPC progression implicates a relevant function for this enzyme that may impede the detrimental phenotype exhibited in CRPC. Based on previous studies, ADAMTS1 modifies the prostate tumour vasculature integral in CRPC and is further discussed in the angiogenesis section of this review.
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Table 1.1 ADAMTS1 expression with cancer progression CANCER ADAMTS1 EXPRESSION WITH CANCER PROGRESSION TYPE
Type of Disease model Samples ADAMTS1 levels analysis
Benign prostate (n=6) 2746 ±342.6 Prostate cancer Localised primary biopsies 942.1 ±191.5 tumour (n=8) Microarray (Varambally et al., Hormone refractory 2005) 265.8 ±87.8 tissue (n=5) Androgen-dependent 603 ±157.4 (n=10) Prostate cancer Prostate Prostate cancer biopsies Microarray cancer patients with (Best et al., 2005) 269.5 ±32.9 metastatic disease (n=10) PC3+U94 tumour 7 ±2 RT-PCR CRPC PC3 cell line suppressor (n=2) (fold- (Ifon et al., 2005) PC3 + empty vector 1 change) (n=1) Subcutaneous Androgen sensitive 1 RT-PCR xenografts LNCaP cells (fold- (Gustavsson et al., Castrate resistant -12.7 change) 2008) LNCaP-19 cells Paired HCC patients Cirrhotic liver (n=16) 1.5 ±0.3 samples Western HCC (n=16) 0.5 ±0.2 (Masui et al., 2001) Resected tissues Normal (n=10) 682.8 ±161.6 from patients with Cirrhosis (n=13) 1339.2 ±277.3 cirrhosis and HCC Cancer-early (n=18) 233.6 ±37.2 Microarray (Wurmbach et al., Cancer-advanced 164.9 ±31.8 2007) ((n=17) HCC Normal (n=19) 881.8 ±177.1 Liver biopsies from Cirrhosis (n=41) 2279 ±168.3 human patients Microarray Cirrhosis/HCC (n=17) 1554.9 ±146.7 (Mas et al., 2009) HCC (n=41) 771.9 ±122.4 Matched patients Control (n=10) Low RT-PCR samples and (Bourd-Boittin et al., Cirrhotic (n=10) High immunoblot 2011)
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Human breast Non-neoplastic (n=10) 2.1 (0.86-9.4) RT-PCR tissues (Porter et al., Invasive breast (median & 0.6 (0.2-2.7) 2004) carcinoma (n=48) range) Mammary xenografts Caveolin-1 mutation 1.5 RT-PCR (Bonuccelli et al., (fold No mutation 1 2009) change) MMTV-PyMT Large tumour burden High ADAMTS1 transgenic mice Intermediate tumour IHC Intermediate ADAMTS1 Breast cancer (Ricciardelli et al., burden 2011) Low tumour burden No ADAMTS1
No anti-ADAMTS1 Ab 4T1 mammary cells treatment; large High ADAMTS1 xenografted in tumours Western BALB/c mice 100ug anti-ADAMTS1 Reduced ADAMTS1 (Hirano et al., 2011) Ab; repressed tumour activity growth
1.4.2. ADAMTS1 promotes hepatocellular carcinoma by aggravating liver fibrosis
ADAMTS1 is poorly expressed in hepatocellular carcinoma (HCC) compared to a healthy liver (Braconi et al., 2009; Chen et al., 2002) (Table 1.1 and Figure 1.2). In spite of this, ADAMTS1 is thought to play a key role in hepatic tumourigenesis indirectly through the aggravation of hepatic fibrogenesis (Bourd-Boittin et al., 2011; Schwettmann et al., 2008), where it is produced in abundance (Masui et al., 2001; Schwettmann et al., 2008).
Among many genetic risk factors and viral susceptibility to HCC, cirrhosis or liver fibrosis, contributes to more than 80% of HCC cases (Desjardins, 2002; Srivatanakul et al., 2004). The pathogenesis of HCC from cirrhosis is initiated by the inflammatory response to hepatocyte cell damage (Hayashi and Di Bisceglie, 2005; Starr and Raines, 2011). The influx of active inflammatory cytokines, particularly TGF-β in the liver, stimulates the substantial remodeling events required for tissue regeneration (Neubauer et al., 2001). This regenerative event leads to two major pathological consequences that ultimately result in hepatic tumour growth. First is that it promotes an increase in myofibroblast population to generate a fibrin/fibronectin-rich ECM leading to liver fibrogenesis (Neubauer et al., 2001; Starr and Raines, 2011). Secondly, as HSC proliferation and differentiation occur in such a
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pernicious environment where there is high oxidative stress from inflammatory cytokines and apoptotic hepatocytes, the likelihood of mutations during cell division becomes more pronounced. Indeed, this fibrogenic regeneration process in liver disease provides the most ideal opportunity for the development of HCC (Hayashi and Di Bisceglie, 2005).
The key function of ADAMTS1 in liver fibrosis, and indirectly, liver tumourigenesis lies with its ability to activate latent TGF-β normally bound to ECM proteins such as fibrilins in the hepatic extracellular environment (Hayashi and Sakai, 2012) (Figure 1.3). In HSC cultures, latent TGF-β was found to dock with the KRKF peptide sequence in the TSP1 motifs of ADAMTS1 (Bourd-Boittin et al., 2011). This interaction allowed the cleavage of the latent binding protein from TGF-β releasing the active form in the extracellular environment (Bourd-Boittin et al., 2011; Hayashi and Di Bisceglie, 2005). TGF-β is the main cytokine that activates quiescent HSC to undergo cell proliferation and differentiation to matrix-producing myofibroblasts (Hayashi and Di Bisceglie, 2005; Neubauer et al., 2001). The activation of HSC also promotes the production of ADAMTS1 (Bourd-Boittin et al., 2011), which facilitates further latent TGF-β cleavage and activation. The cyclic increase in ADAMTS1 and TGF-β activation leads to an exacerbation of the inflammatory response in chronically diseased livers. Hallmark characteristics such as elevated smooth muscle actin and increased matrix deposition have been consistently shown to positively correlate with ADAMTS1 abundance in chronic liver disease (Neubauer et al., 2001; Starr and Raines, 2011) and progressive grades of liver fibrosis (Bourd-Boittin et al., 2011; Schwettmann et al., 2008). In fact, by inhibiting this interaction between ADAMTS1 and latent TGF-β in liver damage, the inflammatory response that follows is also prevented (Bourd-Boittin et al., 2011) abrogating the hostile environment that encourages HSC to acquire mutations.
Whether ADAMTS1 also facilitates TGF-β activation in other organs has yet to be investigated. The complexity surrounding TGF-β signaling and the different signaling pathways it facilitates in various tissues (Zi et al., 2012), suggests that this interaction may be tissue specific and dependent on the ECM environment. Inhibiting the proteolytic processing of latent TGF-β mediated by ADAMTS1 in the liver is a potential preventative treatment for HCC development in patients with chronic liver disease.
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CHRONIC LIVER DISEASE
Hepatocyte cell damage
Inflammatory response
Recruitment of inflammatory cytokines Oxidative stress Active TGF-β Latent TGF-β TGF-β TGF-β TGF-βTGF β TGF-βT β TGF-β TGF-ββ
Quiescent Activated ADAMTS1 Mutated HSCs HSCs Oxidative mature HSCs stress
TGF-β ADAMTS1
ADAMTS1 Myofibroblasts
Matrix deposition and fibrogenesis
CIRRHOSIS
HEPATOCELLULAR CARCINOMA
Figure 1.3. Aggravation of inflammatory response in liver fibrosis by ADAMTS1 to promote hepatocellular carcinoma. Inflammation induced by hepatocyte cell damage in chronic liver disease promotes the production of inflammatory cytokine, TGF-. TGF- activates quiescent hepatic stellate cells (HSC) and stimulates their differentiation to matrix producing myofibroblasts. Active HSCs also produce ADAMTS1, which facilitates the cleavage of latent TGF- to its active form. This cyclic activation of TGF- and ADAMTS1 causes further activation of HSCs, matrix deposition and liver cirrhosis-the most prevalent risk factor in HCC development. Persisted HSC activation and differentiation in a hostile extracellular environment with high in oxidative stress, encourages HSC mutation to occur.
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1.4.3. Upregulated expression of ADAMTS1 promotes breast cancer progression
Expression profiling of ADAMTS enzymes in breast cancer initially revealed significant down regulation of ADAMTS1 in malignant mammary tumours compared with non-neoplastic tissues (Porter et al., 2004). While this inferred a negative role for ADAMTS1 in mammary tumourigenesis (Porter et al., 2004; Porter et al., 2006), subsequent studies, have strongly shown the importance of ADAMTS1 activity in aiding breast cancer development and progression (Bonuccelli et al., 2009; Hirano et al., 2011; Liu et al., 2006c; Ricciardelli et al., 2011).
Induced overexpression of ADAMTS1 results in poor survival and accelerated tumour growth in mouse models of breast cancer (Bonuccelli et al., 2009; Liu et al., 2006c; Ricciardelli et al., 2011) (Table 1). This was evident in mice inoculated with TA3 mammary cancer cells transfected with ADAMTS1, which grew rapid subcutaneous tumours and lived significantly shorter lifespan than mice bearing wild- type TA3 grafts (Liu et al., 2006c). These detrimental effects observed in TA3-ADAMTS1 xenografted mice were suppressed when TA3 transfectants expressed catalytic inactive mutants of ADAMTS1 (Liu et al., 2006c). More recently, treatment of ADAMTS1-neutralising antibodies in mice significantly inhibited and delayed tumour formation of aggressive 4T1 breast cancer cells (Hirano et al., 2011). A caveolin-1 mutation, which is a common genetic abnormality present in a third of ER-α-positive breast cancers (Li et al., 2006), is accompanied by a substantial increase in Adamts1 expression and the formation of larger mammary xenografts (Bonuccelli et al., 2009).
A pro-tumourigenic role for ADAMTS1 is also supported by in vivo evidence from our group (Ricciardelli et al., 2011). Through ablation of Adamts1 from the genetic repertoire of MMTV-PyMT transgenic mice, we observed reduced tumour growth and prolonged survival compared with Adamts1+/+/PyMT and Adamts1+/-/PyMT littermates (Ricciardelli et al., 2011). More importantly, loss of Adamts1 also impaired mammary cancer advancement to invasive disease as pathological grading identified predominantly non-invasive lesions (DCIS) and less invasive cancer in the Adamts1 null tumour cohort (~25% DCIS, ~65% Grade II), while mainly highly invasive tumours in Adamts1+/+/PyMT (~30% Grade II and ~70% Grade III) (Ricciardelli et al., 2011). Our results, together with previously mentioned findings strongly support the tumourigenic importance of ADAMTS1 in breast tumourigenesis.
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1.5. ADAMTS1 IN METASTATIC CANCER
Although diminished ADAMTS1 expression frequently occurs with tumour initiation of many primary cancers, progression towards metastatic disease is often associated with increased ADAMTS1 (Casimiro et al., 2012; Chen et al., 2012; Kang et al., 2003; Liu et al., 2006c; Lu et al., 2009; Masui et al., 2001; Minn et al., 2005; Ricciardelli et al., 2011). The reduced levels of ADAMTS1 during tumour development but an increase with metastatic progression implicate a binary role of ADAMTS1 in cancer. Elevated ADAMTS1 expression may confer changes in cancer cell properties/behaviour that aid metastatic spread. The duality in ADAMTS1 regulation is evident between primary gastric tumours and corresponding metastatic growths (Chen et al., 2012). Primary tumours derived from the gastrointestinal tract expressed significantly lower levels of ADAMTS1 mRNA and protein than normal tissues (Chen et al., 2012). In contrast, matched lymph node metastases expressed significantly higher ADAMTS1 than the primary tumour origin (Chen et al., 2012). In a pancreatic cancer cohort, which collectively expressed lower ADAMTS1 than healthy pancreatic tissues, tumours with relatively high ADAMTS1, correlated with more lymph node metastatic incidents and retroperitoneal invasion (Masui et al., 2001).
For breast cancer, the increase in ADAMTS1 during tumourigenesis continues with metastatic progression. ADAMTS1 was the third most overexpressed gene in highly metastatic MDA-MB-231 clones, with an 18-fold upregulation of ADAMTS1 over weakly metastatic cells (Kang et al., 2003). Subsequent knock down of ADAMTS1 in these highly metastatic cells not only regressed disease spread but also reduced secondary tumour burden (Lu et al., 2009). Elevated ADAMTS1 was also identified in primary breast tumours complicated with distant metastases in the lung and bone, (Casimiro et al., 2012; Minn et al., 2005). Pulmonary and breast cancer cells overexpressing ADAMTS1 established more frequent metastases in mice compared with wild-type controls or cells expressing truncated ADAMTS1 mutants (Liu et al., 2006c). In cancer cells that possessed high metastatic affinity to bone, ADAMTS1 facilitated the paracrine/autocrine release of EGF-like ligands (Lu et al., 2009). This in turn stimulated the production of osteoclast stimulating factor, receptor activator of nuclear-κB ligand (RANKL), while downregulating RANKL antagonist, osteoprotegrin (OPG) (Lu et al., 2009). The altered levels of RANKL and OPG in the bone created an encouraging environment for circulating cancer cells to colonise and establish secondary growths. We further confirm the pro-metastatic property of Adamts1 in our MMTV-PyMT transgenic breast cancer model, as Adamts1 null mice developed smaller and fewer pulmonary metastases than wild type or heterozygous littermates (Ricciardelli et al., 2011).
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In conflict with the pro-metastatic activity of ADAMTS1, Chinese hamster ovary (CHO) cells overexpressing full-length ADAMTS1 showed increased tumour growth while C-terminal fragments of
ADAMTS1 displayed significantly lower pulmonary metastatic incidence compared with wild-type CHO cells (Kuno et al., 2004) and this activity required the spacer domain. Thus the complexity of ADAMTS1 effects may arise through post-translational cleavage (Liu et al., 2006c). While increased metastatic incidence was observed with expression of full-length ADAMTS1 in mammary xenografts, the expression of C- and N-terminal truncated fragments of ADAMTS1 significantly reduced metastases of TA3 mammary cancer cells (Liu et al., 2006c). Cleavage in the C-terminal linker domain region of recombinant ADAMTS1 in vitro has been reported in a number of cell lines (Kuno et al., 2004; Liu et al., 2006c; Rodriguez-Manzaneque et al., 2000). The extent and the type of processing in vivo may also depend on the tumour microenvironment, indeed, heparin (Rodriguez-Manzaneque et al., 2000) and heparin sulphate (Liu et al., 2006c), can prevent ADAMTS1 cleavage in vitro. Thus differential processing potentially explains the opposing effects of ADAMTS1 between different experimental systems.
1.6. ADAMTS1-MEDIATED PATHWAYS IN CANCER DEVELOPMENT AND METASTASIS
ADAMTS1 has been implicated in a wide range of mechanistic pathways involved in cancer progression and metastasis including, but most likely not limited to angiogenesis, cell proliferation, survival and invasive capacity (Table 1.2).
1.6.1. Angiogenesis
Angiogenic inhibition was one of the first activities ascribed to ADAMTS1 (Iruela-Arispe et al., 2003; Iruela-Arispe et al., 1999; Lee et al., 2006; Luque et al., 2003; Vazquez et al., 1999; Wagstaff et al., 2011). It sequesters potent angiogenic stimulant, vascular endothelial growth factor (VEGF), away from interacting with its receptors and preventing an angiogenic effect (Iruela-Arispe et al., 2003; Luque et al., 2003; Rodriguez-Manzaneque et al., 2000). ADAMTS1 also cleaves TSP1 homotrimers, to
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release active TSP1 monomers (Lee et al., 2006) that possess inherit anti-angiogenic properties (Dawson et al., 1997). As a result, ADAMTS1 activity impairs endothelial cell proliferation (Luque et al., 2003; Vazquez et al., 1999) and capillary formation (Krampert et al., 2005; Lee et al., 2006) of normal vascular endothelial cells. This inhibition underlies the attenuated capillary formation found in non- neoplastic pathologies such as Down’s syndrome (Reynolds et al., 2010; Ryeom and Folkman, 2009) and renal ischemia (Basile et al., 2008).
However, the putative role of ADAMTS1 in suppressing vascularisation is not consistently observed in cancer (Esselens et al., 2010; Masui et al., 2001; Ricciardelli et al., 2011; Rocks et al., 2006). Loss of Adamts1 for instance, did not affect tumour vascularity in Adamts-/-/PyMT mice (Ricciardelli et al., 2011) nor did it alter the vascular phenotype in pancreatic, lung and hepatic tumours (Masui et al., 2001; Rocks et al., 2008). Meanwhile, other studies have reported pro-angiogenic effects with promoted microvascularisation in an experimental model of tumour angiogenesis (Fu et al., 2011) and increased blood vessel density in mammary xenografts that overexpressed ADAMTS1 (Kuno et al., 2004). Though by and large, reports surrounding ADAMTS1 and tumour angiogenesis concur with its ability to inhibit blood vessel formation (Chen et al., 2002; Gustavsson et al., 2008; Gustavsson et al., 2010; Gustavsson et al., 2009; Keightley et al., 2010; Kuno et al., 2004; Obika et al., 2012). CHO xenografts overexpressing ADAMTS1 presented with reduced CD31 positivity than empty vector controls (Kuno et al., 2004). Increased ADAMTS1 also inhibited endothelial cell proliferation in endometrial carcinomas (Keightley et al., 2010), and a significant reduction in tube formation was observed when endothelial cells were supplemented with conditioned media abundant in ADAMTS1 (Obika et al., 2012). In this latter study, the angio-inhibitory effects elicited by this protease repressed the growth of DU145, CHO and HT1080 xenografts (Obika et al., 2012).
In prostate cancer, where ADAMTS1 downregulation occurs with ADPC-CRPC progression, ADAMTS1 inversely correlated with tumour microvasculature (Gustavsson et al., 2008; Gustavsson et al., 2009). Microvessel density (MVD) is a prognostic indicator of prostate cancer severity (Borre et al., 1998; Gustavsson et al., 2008; Gustavsson et al., 2010; Gustavsson et al., 2009; Mydlo et al., 1998; Offersen et al., 1998; Offersen et al., 2003; Siegal et al., 1995; Steiner et al., 2012; Strohmeyer et al., 2000). Interestingly, rather than impairing tumour vascularisation indiscriminately, ADAMTS1 selectively inhibited the formation of small blood vessels and instigated an inverse shift in tumour vasculature from one predominantly comprised of large blood vessels, to one rich in small blood vessels (Gustavsson et al., 2010). This specific modulation of tumour vascular phenotype may implicate the involvement of
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angiogenic factors, such as angiopoietins, which can mediate the formation of specific blood vessels (Lind et al., 2005; Morrissey et al., 2008; Tomic et al., 2012; Winter et al., 2007). However, no study has yet been conducted demonstrating any associations between ADAMTS1, angiopoietins and formation of specific vascular phenotype.
With relation to ADAMTS1 interaction/s with other modulators of angiogenesis to inhibit tumour vascularisation, supplementation of exogenous ADAMTS1 resulted in the formation of ADAMTS1-VEGF complexes in mammary cancer cells (Luque et al., 2003). The direct interaction between ADAMTS1 and VEGF suggested a parallel angio-inhibitory mechanism instigated by ADAMTS1 in physiology. However, tumour vascularity in these tumours was not assessed and thus whether tumour vascularity reflected the diminished VEGF bioavailability was not determined. Alternatively, in pulmonary (Kuno et al., 2004; Lee et al., 2010) and hepatic (Lee et al., 2010) metastatic tumours treated with exogenous ADAMTS1 increased proteolytic processing of TSP1 by ADAMTS1 was postulated to be the causal event that impaired vascularisation.
1.6.2. Cell proliferation
The effect of ADAMTS1 in cell proliferation varies between different cancer types. In renal carcinomas (Grigo et al., 2008) and mammary xenografts (Liu et al., 2006c), loss of functional ADAMTS1 coincided with inhibited cell proliferation. However, in the majority of studies, ADAMTS1 expression exerted no effect on cancer cell proliferation (Gustavsson et al., 2010; Liu et al., 2006c; Lu et al., 2009; Ricciardelli et al., 2011; Rocks et al., 2008). This was evident in in vivo (Liu et al., 2006c; Ricciardelli et al., 2011) and in vitro (Lu et al., 2009) models of breast cancer, whereby breast cancer cells that lacked ADAMTS1 shared similar cell proliferation index as cancer cells expressing ADAMTS1. Likewise, no difference in proliferative index was found between androgen dependent LNCaP wild type and LNCaP19 cells (Gustavsson et al., 2010) as well as in pulmonary BZR cancer cells (Rocks et al., 2008), which displayed the same proliferative phenotype as cells in which ADAMTS1 was silenced.
As most studies have reported unchanged proliferation in cancer cells, there is limited knowledge on the mitogenic pathways where ADAMTS1 participates. Evidence has shown ADAMTS1 promotes proliferation by activating latent growth factors into their bioactive forms through ectodomain cleavage (Figure 1.4). At the molecular level, the proteolytic activity of ADAMTS1 facilitated ectodomain
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shedding of mitogen precursors, EGF, heparin-binding EGF (HB-EGF) and amphiregulin, which promote their activation (Liu et al., 2006c). The release of EGF-ligands by ADAMTS1 was later corroborated in a metastatic breast cancer study, which showed that the loss of ADAMTS1 in breast cancer cells diminished bioavailable EGF-like ligands such as amphiregulin, HB-EGF and TGFα in the ECM (Lu et al., 2009).
ADAMTS1 also facilitates the proteolytic processing of structural ECM proteins that inherently possess mitogenic abilities. The most notable of these substrates is the chondroitin sulphate proteoglycan versican (Russell et al., 2003; Sandy et al., 2001), which is highly abundant in Adamts1+/+/PyMT tumours compared with Adamts1-/-PyMT mammary tumours (Ricciardelli et al., 2011). Versican cleavage by ADAMTS1 liberates G1- and G3- fragments, both of which have been shown to promote tumour cell proliferation via two pathways (Lebaron, 1996; LeBaron et al., 1992; Ricciardelli et al., 2009; Yang et al., 1999; Zhang et al., 1999). G3-versican mediates proliferation through its terminal EGF-like motifs (Wu et al., 2004; Zhang et al., 1998; Zheng et al., 2004), which can activate EGF receptors while G1, which lack the EGF motifs, promotes proliferation by destabilising cell adhesion to its substratum and interactions with hyaluronan (HA) (Yang et al., 1999; Zhang et al., 1999).
1.6.3. Cell survival
An alternative pathway in which ADAMTS1 exacerbates tumour growth is by promoting cancer cell survival (Liu et al., 2006c; Ricciardelli et al., 2011). In PyMT transgenic mice, we identified a greater proportion of apoptotic cells in tumours from Adamts1-/- mice compared with Adamts1+/+ littermates (Ricciardelli et al., 2011). A significant decrease in apoptosis was also evident in TA3 tumours with full length ADAMTS1 versus those without full-length protein (Liu et al., 2006c). Together these findings suggest, that elevated ADAMTS1 in some contexts elicits a greater propensity to escape cell death.
In the PyMT mouse mammary cancer model, the apoptotic inhibition mediated by ADAMTS1 was potentially attributable to an enhanced cytotoxic environment in Adamts1-/-/PyMT tumours (Ricciardelli et al., 2011) (Figure 1.4). Adamts1-/-/PyMT mammary cancers have significantly higher positivity for Th1-cytotoxic cell markers and more CD45+ leukocytes than in wild-type tumours (Ricciardelli et al., 2011). Th1-immune cytotoxicity is associated with tumour rejection, and this type of response is often suppressed in cancer (Yu et al., 2006; Zhang et al., 1997). The interaction of
Tan IA 20 CHAPTER 1 Introduction
ADAMTS1 with peritumoural environmental factors may modulate changes that promote a more tolerant or Th2 type immune response. Interestingly, versican, a known substrate of ADAMTS1 is emerging as a modulator of immune responses (Kim et al., 2009; Wang et al., 2009). The G1 fragment of versican also has its inherent anti-apoptotic effects by downregulating the pro-apoptotic molecule Bax (Cattaruzza et al., 2004; Sheng et al., 2005), thus inhibiting the formation of the apoptotic mitochondria machinery (De Giorgi et al., 2002). Increased versican processing by ADAMTS1 may therefore be an indirect pathway enabling tumour cells to escape programmed death. Given that Adamts1-/-/PyMT tumours showed reduced levels of cleaved versican (Ricciardelli et al., 2011), modulation of versican may be an alternative but indirect pathway that ADAMTS1 allow cancer cells to survive.
Tan IA 21 CHAPTER 1 Introduction
une cytotoxicity, une cytotoxicity, and promote cell cell promote and ndent studies have have studies ndent . These domains are domains . These e response inhibition inhibition response e g cells and promotes and promotes g cells ith ECM binding affinity. affinity. binding ECM ith
NK cells γ IFN- ADAMTS1 facilitates ectodomain ectodomain facilitates ADAMTS1 PTO O , ultimately committing to a Th1-immune Th1-immune a to committing ultimately , Th1 cells γ IL-12 APOPTOSIS APOPTOSIS T-helper cells T-helper T Th1 immune response
DPEEAE DPE apoptotic machinery Suppresses mitochondrial Suppre G3 G1 Versican Versican A R
duce β E Reduced contact inhibition Re F GAG- Destabilises ECM-cell adhesion ion and cell survival of breast cancer cells. cancer breast of survival and cell ion ROLIFERATION G1 PROLIFERATION PROLIFERATION P G3 ADAMTS1 A EGF receptors
Active EGF/EGF- like ligands ADAMTS1 mediated pathways to promote proliferat to promote pathways mediated ADAMTS1
shedding of transmembrane bound EGF and EGF-like ligands, liberating activate mitogens to bind to their corresponding receptor receptor corresponding their to bind to mitogens activate liberating ligands, EGF-like and EGF bound transmembrane of shedding domains with EGF-like a G3 fragment releasing thereby of versican cleavage the proteolytic mediates also ADAMTS1 proliferation. w a G1 domain releases also of versican cleavage Proteolytic proliferation. cell and promote EGF receptors to bind to able then This domain destabilises ECM-cell adherence, reduces contact inhibition promotesand proliferation.cell ADAMTS1 blocks Th1-imm presentin antigen by produced are cytokines IL-12 cancer. in suppressed often is and rejection, tumour with associated is which IFN- of expression the induce to Tbet factor transcription activates also IL-12 activation. cell killer natural Figure1.4. response. Meanwhile, CD40-ligand is an essential stimuli that activates CD40 receptors and promote a cytotoxic response. Indepe response. cytotoxic a promote and receptors CD40 activates that stimuli essential an is CD40-ligand Meanwhile, response. shown these ligands to have pro-apoptotic effects of in cancer. ADAMTS1 may therefore have an important role in cytotoxic immun cytotoxic in role an important have therefore may ADAMTS1 cancer. in of effects pro-apoptotic have to ligands these shown apoptosis escape to cells allowing
Tan IA 22 CHAPTER 1 Introduction
1.6.4. Cell migration and invasion
Investigations so far surrounding ADAMTS1 and pro-metastatic cell behaviour have consistently associated increased cell migration and invasion with elevated ADAMTS1 (Bonuccelli et al., 2009; Braconi et al., 2009; Esselens et al., 2010; Keightley et al., 2010; Tyan et al., 2012). Supplementation of exogenous ADAMTS1 to MDA-MB-468 and MDA-MB-231 breast cancer cells, promoted a significant increase in cancer cell invasion not observed in the absence of ADAMTS1 (Esselens et al., 2010; Tyan et al., 2012). shRNA treatment verified the specificity of this altered behaviour to ADAMTS1 as ADAMTS1-silencing significantly attenuated the invasive phenotype (Tyan et al., 2012). Prostaglandin treatment, which enhanced Adamts1 expression, also promoted the invasiveness of endometrial cancer cells (Keightley et al., 2010). Likewise, invasive HCC cells treated with an anti-cancer drug, 17-AAG repressed ADAMTS1 expression and inhibited cell invasiveness (Braconi et al., 2009). Caveolin-1 mutant breast cancer cells, with upregulated Adamts1, become highly invasive and migratory (Bonuccelli et al., 2009). Though these changes may be primarily attributed to caveolin-1 mutation, the corresponding shift in gene profile that included high Adamts1 implies a functional role for this protease.
As an ECM degrading protease, ADAMTS1 imposes its pro-metastatic effects indirectly by cleaving its substrates in the ECM and liberating cancer cells from the structural barriers they adhere to. In 293T kidney cells, ADAMTS1 facilitates the cleavage of syndecan-4 to impair cell adhesion and subsequently promote migration (Rodriguez-Manzaneque et al., 2009). Syndecan-4 interacts with focal adhesion complexes integral in cell-cell and cell-matrix interactions (Altemeier et al., 2012a; Altemeier et al., 2012b; Okina et al., 2012; Zong et al., 2011). Furthermore, versican, the most characterised ADAMTS1 substrate, has repeatedly been shown to independently promote highly invasive and motile phenotypes in prostate (Ricciardelli et al., 2007) and ovarian cancer cells (Ween et al., 2011). In PyMT/Adamts1+/+ mice, which developed frequent pulmonary metastases, a concurrent increase in cleaved versican abundance was observed. (Ricciardelli et al., 2011). Versican cleavage liberates a G1 fragment that possesses HA binding affinity (Yang et al., 1999). Stromal accumulation of G1-versican can destabilise cell adhesion (Yang et al., 1999) and may underlie the increase in metastatic incidents in PyMT/Adamts1+/+ transgenic mice.
Tan IA 23 CHAPTER 1 Introduction
Table 1.2. ADAMTS1 mediated pathways in cancer progression and metastasis PATHWAYS EFFECTS OF ADAMTS1 • ADAMTS1 promoted angiogenesis in breast xenografts (Fu et al., 2011; Gustavsson et al., 2008; Gustavsson et al., 2010; Promotes Gustavsson et al., 2009; Kuno et al., 2004; Obika et al., 2012)
• Pulmonary and hepatic metastases with exogenous ADAMTS1 have reduced CD31 positivity (Lee et al., 2010) • Endothelial tube formation was inhibited in the presence of ADAMTS1 (Obika et al., 2012) • ADAMTS1 inhibited endothelial cell proliferation in endometrial Inhibits carcinomas (Keightley et al., 2010) Angiogenesis • ADAMTS1 in CRPC promoted formation of small blood vessel and inhibited formation of large blood vessels (Gustavsson et al., 2008; Gustavsson et al., 2010; Gustavsson et al., 2009) • ADAMTS1 in DU145, HT1080 and CHO cell inhibited tumour angiogenesis (Kuno et al., 2004; Obika et al., 2012)
• Vascular density in mammary, pancreatic, lung and hepatic tumours were not different between high and low ADAMTS1 No effect expressing tumour cohorts (Masui et al., 2001; Ricciardelli et al., 2011; Rocks et al., 2008),
• Proliferation of TA3 transfectants expressing ADAMTS1 was significantly reduced after inhibiting ADAMTS1 activity (Liu et al., 2006c) Promotes • HNF-4α-inhibited renal cancer cell proliferation by downregulating ADAMTS1 (Grigo et al., 2008) Proliferation • ADAMTS1 promoted no difference in proliferation in pancreatic, breast, pulmonary (Gustavsson et al., 2010; Liu et al., 2006c; No effect Lu et al., 2009; Masui et al., 2001; Ricciardelli et al., 2011; Rocks et al., 2008)
• Adamts1-/-/PyMT mammary tumours have more apoptotic cells than wild-type tumours (Ricciardelli et al., 2011) Cell survival Promotes • TA3-ADAMTS1 transfectants were less apoptotic than wild-type TA3 (Liu et al., 2006c)
Tan IA 24 CHAPTER 1 Introduction
• Increased invasion of MDA-MB-468, MDA-MB-231and MCF7 breast cancer cells supplemented with exogenous ADAMTS1 (Esselens et al., 2010; Tyan et al., 2012) • ADAMTS1 transfection in TA3 cells promoted cell invasion (Liu et al., 2006c) Invasion • Highly invasive and motile caveolin-1 breast cancer mutants expressed high Adamts1 (Bonuccelli et al., 2009) and Promotes • Treatment of invasive HCC cells with 17-AAG inhibited invasion Migration and downregulated ADAMTS1 (Braconi et al., 2009) • ADAMTS1 promoted epithelial cell invasion of endometrial cancer cells (Keightley et al., 2010) • ADAMTS1 overexpression increased migratory potential of 293T cells (Rodriguez-Manzaneque et al., 2009)
1.7. CONCLUSION
Extracellular degrading enzymes play an essential role in the aberrant tissue remodeling of the peritumoural environment. Perturbations in ADAMTS1 evoke significant changes that ultimately promote cancer development and metastatic progression. As dysregulation of ADAMTS1 is present in most commonly reported cancers, further understanding of the mechanism(s) by which it may help promote metastasis in prostate, breast, lung, colon, liver and other cancers will enrich our insight into tumourigenesis in these organs and reveal novel targets to delay cancer development and progression.
Tan IA 25 CHAPTER 1 Introduction
1.8. HYPOTHESES AND AIMS
ADAMTS1 overexpression is strongly associated with metastatic disease in breast cancer. This has been identified through microarray analyses of breast cancer cell lines with various metastatic capacities and in experimental metastatic models where Adamts1 has been exogenously supplemented. Consistent with these reports, in vivo evidence in the MMTV-PyMT model further strengthened evidence for the pro-metastatic role of ADAMTS1 in breast cancer, with a reduction in incidence and pulmonary metastatic growth upon ablation of functional Adamts1.
The aim of my project is to elucidate the signalling pathways and mechanistic events through which ADAMTS1 mediates its pro-metastatic effects, focusing on its influence on cell- matrix adhesion, migration and invasion. Matrix adhesion, migration and invasion are interdependent cell processes, integral to the cascade of events leading to metastasis. The ability of cancer cells to adhere to the surrounding environment is essential for the determination of front-rear cell polarity driving cancer cell migration, which is in turn necessary for cell invasion. Attachment of cells to the basement membrane is also required for the activation of proteolytic enzymes that breakdown the structural constraints restricting cell movement and thereby allowing cancer cells to traverse through the surrounding extracellular environment or penetrate tissue boundaries.
I hypothesise that the prometastatic effect of ADAMTS1 is underpinned by an enhanced capacity of cells to adhere to the ECM, which in turn promotes cell motility and increases tumour invasiveness.
To address these hypotheses, I aim to:
1. Determine the consequence of Adamts1 ablation on the population and rate of breast carcinoma cell adhesion to a matrigel substratum, migration and invasion through a matrigel barrier. 2. Determine whether introducing Adamts1 into non-transformed mammary epithelial cells enhances their capacity to exhibit cell-matrix adherence, migration and invasion.
Furthermore, I also hypothesise that the loss of ADAMTS1 has significant effects on metastatic phenotype of mammary tumours and alters the tumour gene expression profile in favour of a more benign breast cancer sub-types.
Tan IA 26 CHAPTER 1 Introduction
Hence, to elucidate the transcriptional aberrations and molecular pathways resulting from ADAMTS1, this study also aimed to conduct comparative microarray analysis between PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours. Performing this analysis can potentially translate how the loss of Adamts1 in the MMTV-PyMT breast cancer model parallel to the clinical prognostic outcomes of human breast cancers.
Tan IA 27
Chapter 2 ADAMTS1 enhances breast cancer cell adhesion to the extracellular matrix
CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
2.1. INTRODUCTION
Metastasis is a detrimental disease stage in cancer that often leads to patient mortality due to the lack of effective treatment to prevent progression and the eventual spread of malignancy to vital organs. Metastatic disease is a complex process that requires the invasion of cancer cells into the surrounding non-malignant tissue, followed by the sequential escape of cancer cells from the primary tumour site, intravasation into vessel portals and contact independent survival of cancer cells in circulation. Finally, cells extravasate out of blood/lymph vessels and migrate into a new tissue where they eventually establish secondary tumour growth/s. It is with this complexity and dynamic evolution that make the mechanistic pathways underlying the metastatic cascade difficult to understand.
The acquisition of a dynamic adhesion capacity is a hallmark of metastatic cells and plays an integral role in the critical steps involved throughout the metastatic cascade (Balzer and Konstantopoulos, 2012; Li and Feng, 2011; Schneider et al., 2007). Interactions between a cancer cell and the peritumoral matrix for instance, is essential in determining front-rear cell polarity driving cancer cell migration and invasion (Muthuswamy and Xue, 2012; Yamana et al., 2006). Cancer cell attachment to vessels walls is required in the intravasation and extravasation of cancer cells in and out of the blood/lymph circulation during metastasis (Dua et al., 2005; Wong et al., 2002). The adherence of cancer cells to the basement membrane also stimulates the activity of proteolytic enzymes to degrade the structural proteins making up the tissue barrier, surrounding premalignant mammary glands and allowing cancer cells to traverse and consequently escape the primary tumour site (Guiet et al., 2011; Haberern and Kupchik, 1985; Kirmse et al., 2011; Kramer et al., 1989; Sahai and Marshall, 2003).
Metalloproteinase enzymes play a key role in facilitating and modulating cell-ECM interactions necessary for cell adherence to ECM proteins. Hepatic stellate cells for instance, rely on ADAM12 activity to adhere to homogeneous collagen I, laminin and fibronectin matrices (Leyme et al., 2012), while MMP-9 activity promotes the adhesion of endometrial stromal cells to ECM proteins such as collagen I, collagen IV, fibrinogen and fibronectin (Kim et al., 2012; Mei et al., 2012). In cancer, studies have shown an interdependent relationship between cancer cell adhesion, elevated production of metalloproteases and metastases. This has been reported in human melanoma cells which exhibited elevated MT1-MMP expression and a corresponding increase in adhesion ability (Kirmse et al., 2011). Similarly, ovarian and prostate cancer cell adhesion to matrix proteins induced the production of MMP-2 and MMP-9 proteases, which was concomitant to high incidence of metastasis (Cai et al., 2012; Van
Tan IA 29 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
Slambrouck et al., 2009; Wang et al., 2012a; Wang et al., 2012b).
The ADAMTS family is the most recent addition to the metalloprotease class of enzymes and of its 19 members, ADAMTS1 has been the most studied in cancer. Prevailing in vivo and in vitro evidence in breast cancer points to a role for ADAMTS1 in the metastatic progression of breast cancers. In the MMTV-PyMT transgenic breast cancer model, the development of aggressive mammary tumours, which frequently disseminate to the lung, was significantly inhibited in mice null for ADAMTS1 (Ricciardelli et al., 2011).
The action/s mediated by ADAMTS1 to promote breast cancer metastasis are currently unclear. To understand the underlying events influenced by ADAMTS1 and provide a mechanism by which tumours expressing this protease exhibit elevated metastasis, this study explored mammary cancer cell adhesion to basement membrane related ECM. Using primary mammary carcinoma cells derived from PyMT mammary tumours and a human mammary epithelial cell line, I assessed the effect of Adamts gene ablation and Adamts1 overexpression, respectively, on cell adhesiveness to a biological membrane substratum. It is my hypothesis that the loss of Adamts1 impairs the ability of mammary cancer cells to adhere to the matrix surface, while conversely, introduction of ADAMTS1 to breast epithelial cells lacking this protease will promote mammary cell interaction with the ECM-like membrane.
2.2. MATERIALS AND METHODS
2.2.1. Animals
PyMT/Adamts1+/+, Adamts1+/- and Adamts1-/- transgenic C57/Bl6/129SvxFvBN mice were housed at the Laboratory Animal Services (University of Adelaide, Australia) and maintained on a 12 h: 12 h day/night cycle with rodent chow and water ad libitum. Experiments using mice were approved by the University of Adelaide ethics committee and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. All mice were anaesthetised then killed by cervical dislocation, in compliance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
Tan IA 30 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
2.2.2. DNA extraction
Ear and tail biopsies collected from mice were digested in 250 μl digestion buffer [20 mM EDTA, 50 mM Tris, 120 mM NaCl and 1% (w/v) SDS, pH 8.0] and 200μg/ml Proteinase K (Promega Corporation, Alexandria, NSW, Australia) at 55 oC for 2 h with shaking. After 2 h, 250 μl 4 M Ammonium was added to precipitate proteins and cellular debris. Solution was vortexed for 10 secs and incubated at room temperature for 15 mins with shaking. Precipitate was allowed to settle for 10 mins and pelleted by centrifugation at 14,0000 rpm for 10 mins. 400 μl of supernatant was transferred to a clean tube, to which 800 μl of 100% ethanol was added, and vortexed for 10 secs. DNA precipitate was pelleted by centrifugation at 14,000 rpm for 8 mins. After discarding the supernatant, DNA pellet was washed with 1 ml 70% ethanol and spun at 14,000 rpm for 5 mins. Washed DNA was air dried, and was dissolved in 500 μl water for injection (AstraZeneca, North Ryde, NSW, Australia) at 55 oC for 30 mins. Resuspended DNA was stored at -20 oC.
2.2.3. PCR genotyping and gel electrophoresis
PCR genotyping was performed using DNA material extracted from tail biopsies or 5 ng/μl complementary DNA synthesised from breast tumour RNA extracts. PCR amplified products were visualised by gel electrophoresis in 1% (w/v) agarose (Promega Corporation, Annandale, NSW, Australia) gel made up in 0.5X TBE [44.5 mM Tris, 44.5 mM Boric Acid, 1 mM EDTA pH 8.0] and 2 μg/ml Ethidium Bromide. Amplicon band size was referenced against a DNA 100 bp ladder (Promega Corporation, Annandale, NSW, Australia). Gels were visualised using UV transilluminator and gel documentation system (Kodak DC120).
2.2.3.1. MMTV-PyMT
PyMT genotyping using previously designed primer sets to generate a 195 bp amplicon (Table 2.1). Each PCR reaction contained 5 μl 5x Green GoTaq® Flexi buffer (Promega Corporation, Annandale, NSW, Australia), 2.5 mM Magnesium Chloride, 0.5 mM dNTP mix, 1.25 μl 10 mM forward and reverse primers, 1.25 μl GoTaq® polymerase (Promega Corporation, Annandale, NSW, Australia)
o and made up to a total volume of 25 μl with H2O. Cycling conditions were 94 C for 5 mins, 35 cycles of 94 oC for 1 min, 60 oC for 1 min and 72 oC for 30 secs, and lastly, 72 oC for 5 mins.
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2.2.3.2. Adamts1
Adamts1 genotyping was performed using two primer sets; one targeted against the wild type Adamts1 allele and another for the Adamts1 null allele (Mittaz et al., 2004) (Figure 2.1). Adamts1 wild type specific primers amplified the region spanning Exon 2-Intron 2-Exon 3 to generate a 577 bp amplicon, while Adamts1 knockout primers targeted the region between Intron 1 to Exon 3 to generate a 278 bp (knockout allele) or a 1323 bp (wild-type allele) product (Table 2.1). PCR reactions for both primer sets each contained 5 μl 5x Green GoTaq® Flexi buffer, 3 mM Magnesium Chloride, 0.5 mM dNTP mix, 0.125 μl 10 mM forward and reverse primers, 1.25 μl GoTaq® polymerase and made up to a
o o total volume of 25 μl with H2O. Cycling conditions were 94 C for 5 mins, 35 cycles of 94 C for 1 min 60 oC for 1 min and 72 oC for 30 secs, and lastly, 72 oC for 5 mins.
Tan IA 32 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
a. Intron 1 to Exon 3 ampliconn (1323bp)(1323 bp) Exon 2 to Exon 3 ampliconn (577(577bp)(577bp bp)) Adamts1 wild-type allele Ex 1 Ex 2 Ex 3 Ex 4 Ex 5 Ex 6 Ex 7 Ex 8 Ex 9
Exon 2 deletion
Intron 1 to Exon 3 ampliconn ((278bp)(278278bp bp)) b. No target sequence for Exon 2 forward primer (no amplicon) Adamts1 knockout allele Ex 1 Ex 3 Ex 4 Ex 5 Ex 6 Ex 7 Ex 8 Ex 9
Figure 2.1. Schematic representation of annealing sites for Adamts1 genotyping primers. Adamts1 genotypes were identified using two primer sets designed specifically against the a) Adamts1 wild type or b) knockout alleles. Forward and reverse ‘wild type primers’ (red arrows) were designed to anneal to exon 2 and exon 3, respectively of Adamts1. These primers generate a 577 bp amplicon in the Adamts1 wild type allele but no amplicon can be generated with the Adamts1 knockout allele due to the deletion of exon 2. Forward and reverse ‘knockout primers’ (blue arrows) were designed to anneal to intron 1 and exon 3, respectively of Adamts1. These primers generate a
1323 bp and a 278 bp amplicons, which correspond to the Adamts1 wild type and knockout alleles, respectively. The elongation period during the amplification stage of the PCR reaction is insufficient
for complete synthesis of the 1323 bp amplicon, and thus only the 278 bp PCR product can be
visualise following gel electrophoresis.
Table 2.1 Genotyping PCR primers Gene name (Accession Primer Sequence (5’-3’) Amplicon size No.) Forward CGTCCAGAAAACCACAGTCA MMTV-PyMT 195 bp Reverse CCGCTCGTCACTTATCCTTC Wild type AGTTACCTCCAATGCAGCTCTCAC wild type allele – 577 bp Forward knockout allele - no Wild type ATCCCGAGAGTGTCACACGTGTGG amplicon Adamts1 Reverse (NM_009621) Knockout TCCTCAAGCCCCACCCCTTGG Forward wild type allele – 1323 bp Knockout knockout allele – 278 bp TCCTGCTGGGGTCACATACAG Reverse *amplicon not amplified due to short amplification period during PCR
Tan IA 33 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
2.2.4. Isolation and propagation of primary mammary cancer cells
Mammary tumours were resected from Adamts1+/+, Adamts1+/- and Adamts1-/- MMTV-PyMT transgenic mice at 16-20 weeks of age or when ethical euthanasia was necessary due to tumours reaching 3 cm3. Mammary tumours from all glands were weighed, collected and cut into 1mm3 pieces. Tumour tissues were incubated in digestion buffer [2 mg/ml collagenase A, 5 U/ml hyaluronidase, 0.1% w/v deoxyribonuclease 1] (10 mls) for 1 h at 37 °C with agitation. Tissue suspension was filtered through a sterilised 40 μm nylon net filter (Steriflip® Filter unit, Merck Millipore, Billerica, MA, USA) to separate undigested material from isolated primary mouse mammary cancer cells (1°mMCC). The cell suspension was spun at 1500 rpm for 5 mins to separate the digestion buffer solution from the isolated cell fraction. The top digestion buffer layer was removed and the cell pellet was resuspended in 3ml of growth media [DMEM + 10% FBS + 1% PSF]. The 1°mMCC suspension (1 ml) was then transferred into T75 flasks containing 10 ml growth media and grown for approximately 3-4 days or until reaching 90% confluence. After reaching 90% confluence, 1°mMCC were cryopreserved with 15% DMSO cryopreserving agent (Sigma-Aldrich Pty Ltd., Sydney, Australia). All primary cell lines were used at passage 2 in subsequent experiments. Briefly, cells were revived from liquid nitrogen into a T25 flask containing 5mls of growth media. After 48 h, primary cells were transferred into three T25 flasks and allowed to reach 70-80% confluence. This took approximately 48-72 h post-passage, after which cells were trypsinised and used in experiments described below. Growth media replacement was performed daily.
2.2.5. Viral transduction of #2756 knockout primary mouse mammary cancer cells
To further investigate the influence of ADAMTS1 on mammary cancer cell adhesion, the Adamts1 mRNA sequence was introduced into an Adamts1-/- 1°mMCC by viral transduction. Lentivirus constructs were engineered to express the full-length mouse ADAMTS1 protein by the method previously described (Barry et al., 2001; Brown et al., 2010a). Adamts1 and the GFP control sequence were inserted into the pLenti-III-mir-GFP vector under the control of the EF-1 promoter. Primary cell sample #2756 was chosen as the preferred knockout sample to introduce ADAMTS1 into as it exhibited good survival in culture. To maintain primary cells undergoing two passages prior to an experiment, viral transduction was performed as follows. Primary cell lines #2756 and #11 cells were revived from liquid nitrogen storage into a T25 flask containing 5mls growth media. After reaching 70-80%, 1x105 cells were
Tan IA 34 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
seeded in a 6-well plate and allowed to grow overnight to reach 70% confluence. After washing the cells
5 with PBS [80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl], 0.5x10 pfu lentivirus containing the Adamts1-GFP construct or GFP vector control was diluted into 25 0μl growth medium (DMEM + 10% FBS) and immediately added to cells. Cells were incubated with lentivirus for 10 mins at room temperature and then in a 37 oC incubator for 1 h. Additional growth media (1.75 ml) was then added and left overnight. After 24 h, the virus mixture was replaced with 2 ml growth media. Adamts1-/- 1°mMCC cells were allowed to grow for 24 h prior to being used in an adhesion assay. Non-transduced #2756 and an Adamts1+/+ 1omMCC sample (#11) underwent the same process of incubation but received only the vehicle (growth media).
2.2.6. Generation of MCF10A-Adamts1 and MCF10A-GFP clones
MCF10A is a human breast cell line of epithelial origin, which we have previously demonstrated to express negligible transcript levels of ADAMTS1. Because of its low level of ADAMTS1 expression, it was the ideal cell line to introduce ADAMTS1 into and examine the direct effects of ADAMTS1 on metastatic cell behaviour. MCF10A cells were maintained in Dulbecco’s Modified Eagle’s Medium/Ham F-12 (DMEM/F12) Nutrient Mixture (Sigma-Aldrich Pty Ltd, Sydney, Australia) supplemented with 5% (v/v) horse serum (Sigma-Aldrich Pty Ltd, Australia), 20 ng/ml EGF (Peprotech, Rocky Hill, New Jersey, USA), 0.5 μg/ml hydrocortisone (Sigma-Aldrich Pty Ltd, Sydney, Australia), 100 ng/ml cholera toxin and 10 μg/ml insulin (Sigma-Aldrich Pty Ltd, Sydney, Australia) and 1% (v/v) Pen/strep antibiotic (Sigma- Aldrich Pty Ltd, Sydney, Australia).
The Adamts1 mRNA sequence conjugated with a GFP reporter construct was introduced in MCF10A cells by viral transduction as described in Section 2.2.5. Transduction of a GFP only expression vector was also performed in parallel in separate cells and was used as an empty vector control in subsequent experiments. The initial transduction procedure achieved 70% transduction efficiency. In order to increase the proportion of cells expressing ADAMTS1, MCF10A cells were seeded at 1 cell/well in a 96-well plate and allowed to grow until reaching 80-90% confluence. Cells were monitored daily and upon reaching the desired confluence, MCF10A cells expressing ADAMTS1 were progressively transferred into 1 well of a 48-well, 12-well, 6-well plates and T75 flask to amplify the desired clones. MCF10A cells expressing the GFP vector control underwent the same selection and
Tan IA 35 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
growth conditions. Upon reaching 70-80% confluence in a T75 flask, MCF10A cells were cryopreserved and stored in liquid nitrogen.
2.2.7. Quantitative real-time RT-PCR
Total RNA was isolated using TRIzol® reagent (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia) according to the manufacturer’s instructions. Briefly, cells at 70-80% confluence in a T25 flask were lysed with 3 mls of TRIzol® reagent and mechanically agitated using a pipette. Homogenised cell lysate was incubated for 5 mins at room temperature after which 600 μl Chloroform was added. The cell lysate solution was mixed vigorously by hand, incubated for 2-3 mins to allow phase separation and spun at 12,000 rcf for 10 mins at 4 oC. The upper phase was transferred into a new tube and mixed with 1.5 ml isopropanol alcohol. To precipitate the RNA material, the solution was incubated at room temperature and spun at 12,000 rcf at 4 oC to pellet the RNA. The RNA pellet was then washed with 3 mls 70% ethanol, air dried for 30 mins and dissolved in 200 μl water. Any remaining genomic DNA contaminants were removed using DNase 1 (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia) according to manufacturer’s instructions. 1-10 μg/μl DNase-treated RNA was used as template to synthesise cDNA from using SuperScript III Reverse Transcriptase (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia) as per manufacturer’s instructions. Quantitative real-time RT-PCR (qRT-PCR) was performed in triplicate. Custom made primers against the Adamts1 and ADAMTS1 mRNA primers have been previously designed in the group (Dunning et al., 2007) using Primer Express (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia) and manufactured by Sigma-Aldrich (Table 2.2). Primers against housekeeper, L19 were commercially available (Qiagen (Mm_Rpl19_1_SG QuantiTect Primer Assay QT00166145, Qiagen Australia Pty Ltd.). cDNA amplification occurred in a 20 μl reaction volume, comprised of 10 μl SYBR Green master mix (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia), 0.2 μl 50mM forward and reverse primers (Adamts1 and ADAMTS1) or 1.25 μl (L19) and nuclease free water, using 7900HT Fast Real-Time PCR system (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia). PCR cycling conditions were as follows 50 °C for 2 mins, 95 °C for 10 mins and 40 cycles of amplification at 95 °C for 15 secs and 60 °C for 60 secs. Relative expression levels were calculated using 2-ΔΔCT and calibrated against high-ADAMTS1 expressing Hs578T cell line or #11, which is a PyMT/Adamts1+/+ 1°mMCC. Each qRT-PCR run also had a negative control, which lacked a cDNA template.
Tan IA 36 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
Table 2.2 Primer sequences used in RT-PCR Gene name Primer Sequence (5’-3’) (Accession No) Adamts1 Forward TGATAAGTGTGGCGTTTGTGG (NM_009621) Reverse CCCCCTTTGATTCCGATGT
ADAMTS1 Forward GCACTGCAAGGCGTAGGAC (NM_006988) Reverse AAGCATGGTTTCCACATAGCG
2.2.8. Immunocytochemistry
In order to quantify the percentage of mammary epithelial cancer cells comprising our isolated 1omMCC culture, 5x103 primary cells were grown in 1 well of an 8-well chamber slide (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia) with 500 μl growth media for 24 h in a 37 oC humidified incubator. After 24 h, media was removed and cells were fixed with -20 oC methanol (1 ml/well) (ChemSupply, Gillman, SA, Australia) for 10 mins followed by 1 min incubation in -20 oC acetone (1 ml/well) (Merck Millipore, Billerica, MA, USA). Each well was washed with PBS twice (1 ml/well) and incubated in 1 ml PBS with 3% (v/v) H2O2 (Sigma-Aldrich Pty Ltd, Sydney, Australia) to inhibit endogenous peroxidase activity. Non-specific antibody binding was subsequently blocked with 5% rabbit serum (Sigma-Aldrich Pty Ltd, Sydney Australia) for 20 mins and incubated overnight at 4 oC with 1:100 anti-cytokeratin endo-A primary antibody (DSHB, University of Iowa, USA). A negative control well retained the 5% rabbit serum blocking solution during the overnight incubation. All wells were washed with 1 ml PBS, incubated for 1 h with 1:400 rabbit anti-rat secondary antibody (Millipore Corporation, Billerica, MA, USA) and then with 1:500 Streptavidin (Sigma-Aldrich Pty Ltd, Sydney, Australia). Visualisation of positive staining was achieved after a 6 min reaction with Diaminobenzidene (Sigma-Aldrich Pty Ltd, Sydney, Australia). Chamber wells were then detached from the slide and the slide was subsequently washed with Ethanol and Xylene solution and mounted using Pertex mounting medium (HD Scientific Supplies Pty Ltd, NSW, Australia).
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2.2.9. Quantification of cytokeratin-positive mammary epithelial cancer cells
High-resolution images of 8-well chamber slides were captured using the Nanozoomer digital slide scanner (Hamamatsu Photonics, Hamamatsu, Japan). 10 images of each well were taken at 20x magnification using the NDP viewing software (Hamamatsu Photonics, Hamamatsu, Japan). Positive and negative cells were quantified by manual counting and percentage of positive cells was calculated. Image analysis was performed blinded to the mouse genotypes.
2.2.10. Real time cell-based assay
Cell proliferation and ECM adhesion was assessed using the xCelligence system (ACEA Biosciences, San Diego, CA, USA) (Figure 2.2).
2.2.10.1. In vitro proliferation assay
At approximately 70-80% confluence, cells were trypsinised and spun at 1500 rpm for 5 mins. The cell pellet was then resuspended in 1ml growth media and counted using the TC10 automated cell counter (Bio-Rad Laboratories Pty Ltd, NSW, Australia) at a 1:1 cell suspension and trypan blue (Sigma-Aldrich Pty Ltd, Sydney, Australia). Cells were then aliquoted at 5x104, 1x105 and 2x105 cells/ml. 25 μl of normal growth media was added into each well of an E-plate (ACEA Biosciences Inc., San Diego, CA, USA) to obtain background reading followed by the addition of 100 μl of cell suspension into quadruplicate wells. The E-plate was then loaded into the xCelligence and cell proliferation was measured for 40 h (Figure 2.2 b).
2.2.10.2. In vitro adhesion assay
For the adhesion assay, an E-plate (ACEA, Roche Diagnostics, Australia) was coated with 20 μl of 5% v/v matrigel (BD Biosciences, North Ryde, NSW, Australia) and left to solidify for 4 h in a 37 oC humidified incubator. Cells at approximately 70-80% confluence were trypsinised and spun at 1500 rpm for 5 mins. After the supernatant was removed, the cell pellet was resuspended in 1 ml growth media and counted using a TC10 automated cell counter at a 1:1 cell suspension and trypan blue. Cells were aliquoted at 5x104 cells/ml. Normal growth media (25 μl) was added into each well to obtain a
Tan IA 38 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
background reading and the cell suspensions (100 μl) were then added into each well accordingly prior to loading into xCelligence for measurement of cell impedance (Figure 2.2 b). Cell impedance for each cell type was assessed in triplicate or quadruplicate and recorded over a 2 h-time period. Linear regression analyses of adhesion impedance between 0-2 h were performed to all samples using GraphPad Prism® version 6 (GraphPad Software, La Jolla, California, USA). This analysis identified the slope of the line, which indicated the rate of cell adhesion to matrigel during the period of the assay
2.2.11. Statistics
Statistical significance was determined by One-Way ANOVA and Fisher’s LSD post hoc test in GraphPad Prism® version 6 (GraphPad Software, La Jolla, California, USA). Non-normally distributed data were log-transformed prior to statistical analysis. Statistical significance was defined at p≤0.05.
Tan IA 39 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
a.
No cells on electrodes Some cells on electrodes More cells on electrodes = = = 0 cell impedance cell impedance increases cell impedance increases further
Cell Impedance
Time
b. E-plate Growth media w/ 10% FCS) 1o MCC
1:20 Matrigel coat Microelectrode arrays
Components Proliferation Adhesion Cell density 5x104/mL 5x104/mL 1:20 Matrigel none 20μL
Media 100μL 100μL
Figure 2.2. Real-time cell proliferation and adhesion assays using the xCelligence system.
The xCeliigence system can measure the real-time proliferative and adherent behaviour of cells
using specialised 16-well plates known as the E-plate. E-plate wells consist of an underlying sheet of microelectrodes that can detect cell interaction upon contact. When cells interact with the underlying electrode surface, it impedes the ability of the electrodes to sense the surrounding conductive culture media. Interaction of cells to the microelectrodes is therefore reported as cell impedance, and is
corresponding to cell number and strength of the interaction. a) Prior to any cell contact, microelectrodes are unimpeded and thus no increase in cell impedance is recorded. As cells begin to
attach to the bottom of the well, cell impedance increases progressively. After adhering to the surface within the first 2 h after seeding, cells undergo proliferation, which also promotes an increase in cell impedance recorded in the period after 2 h until confluence is reached. b) Assessment of PyMT/Adamts1+/+, Adamts1+/- and Adamts1-/- cell proliferation and adhesion was performed at a density of 5x104 cells/mL in a 100 μL volume. Adhesion assays were performed with a matrigel substratum, which is a commercially available analogue of the basement membrane, to mimic the adhesive interaction between cells and extracellular environment.
Tan IA 40 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
2.3. RESULTS
2.3.1. Isolated cells from Adamts1+/+, Adamts+/- and Adamts1-/- mammary tumours were predominantly mammary epithelial cancer cells.
Previously published data from our group identified significantly smaller mammary tumour mass as well as less frequent and smaller pulmonary metastases in 16-20 week old PyMT/Adamts1-/- mice compared with PyMT/Adamts1+/+/ and PyMT/Adamts1+/- littermates (Ricciardelli et al., 2011). In order to understand the cellular behaviour underlying the decreased growth and metastatic incidence in PyMT/Adamts1-/- transgenic mice, mammary tumours from 16-20 week old PyMT mice of different Adamts1 genotypes were collected. Tumours collected from any of the ten mouse mammary glands were pooled and mammary cancer cells were isolated (Figure 2.3 b). Consistent with our previous finding (Ricciardelli et al., 2011), total tumour mass as a proportion of body weight was significantly lower in PyMT/Adamts1-/- (6.88% ± 0.77) mice than in PyMT/Adamts1+/- (12.94% ± 1.31) and PyMT/Adamts1+/+ (10.66% ± 1.55) littermates (p=0.0019, One-way ANOVA; Figure 2.3 c).
As there are heterogeneous cell populations that comprise mammary tumours, it was essential to determine the percentage of mammary epithelial cancer cells present in the cell dispersates. 1omMCC grown under in vitro cell culture conditions maintained a healthy cell morphology following revival from liquid nitrogen (Figure 2.3 d-f). To quantify the proportion of mammary epithelial cancer cells, immunocytochemistry was performed against the epithelial cell marker, cytokeratin endo-A (Figure 2.3 g-i). This identified a predominant epithelial cancer cell populations in all isolates from mammary tumours, which did not differ between PyMT/Adamts1+/+ (61.76% ± 13.45), Adamts1+/- (61.70% ± 6.30) and Adamts1-/- (74.66% ± 4.41) 1omMCC (Figure 2.3 j). The additional, cytokeratin negative cells are expected to be stromal fibroblasts and endothelial cells.
Tan IA 41 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
a. PyMT Adamts1 wild Adamts1 primers type primers knockout primers (198 bp) (577 bp) (278 bp)
PyMT/Adamts1+/+
PyMT/Adamts1+/-
PyMT/Adamts1-/-
b.
R1 L1 R2 L2 R3 L3
R4 L4 R5 L5
* 15 *** PyMT/Adamts1+/+
c. +/- PyMT/Adamts1 PyMT/Adamts1-/-
10
5
tumour buden (% body weight) Total 0
Tan IA 42 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
Brightfield Cytokeratin d. g. +/+
Adamts1
PyMT/
e. h. +/- +/-
Adamts1
PyMT/
f. i.
-/-
Adamts1
PyMT/
Tan IA 43 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
+/+ j. 90 ns PyMT/Adamts1 +/- 80 PyMT/Adamts1 PyMT/Adamts1-/- 70
60 50 40 30 20
10 (% positive cells) Cytokeratin endo-A 0 Figure 2.3. PyMT/Adamts1-/- mice have smaller mammary tumours than Adamts1+/+ and Adamts1+/- littermates, and isolated 1omMCC were predominantly of mammary epithelial cancer cell type. a) Genotyping amplicons generated with primers specific for either the PyMT transgene, Adamts1 wild type or knockout alleles using DNA templates obtained from PyMT/Adamts1+/+, PyMT/Adamts1+/- and PyMT/Adamts1-/- mice. b) Mammary tumours were excised from any of the 10 mammary glands where tumour growth was found and digested to isolate mammary epithelial cancer cells. c) Total tumour weight of Adamts1+/+ (n=10), Adamts1+/- (n=13) and Adamts1-/- (n=15) mammary tumours in proportion to body weight. d, e, f) Brightfield images of Adamts1+/+, Adamts1+/- and Adamts1-/- 1omMCC, revived from liquid nitrogen and grown under in vitro cell culture conditions. g, h, i) 1omMCC immunocytostained against epithelial cell marker, cytokeratin endo-A. Images were taken at 20x magnification. j) Proportion of mammary epithelial cancer cells isolated from PyMT/Adamts1+/+, Adamts1+/- and Adamts1-/- mammary. Data is represented as mean ±SEM. Statistical analysis was performed using log-transformed data and One-way ANOVA with Fisher’s LSD post hoc test. Significance was determined if p≤0.05 (*p≤0.05, ***p≤0.0005).
Tan IA 44 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
2.3.2. PyMT/Adamts1-/- primary cells exhibited reduced adhesion to matrigel compared with Adamts1+/+ and Adamts1+/- primary mouse mammary cancer cells.
The optimal cell density used in cell adhesion assays was determined by performing an assay with titrated cell densities over 40 h. This preliminary assay allowed for the determination of the cell adhesion over the initial period after seeding, as described previously (Atienza et al., 2006) as well as assessing the sensitivity of detection of cell proliferation in the later stages of culture. Cells plated at 2x104, 1x104 and 0.5x104 cells/ml all showed a rapid increase in cell impedance that plateaued after 2 h (Figure 2.4 a). This 2 h-period represented cell adherence to the underlying well surface and was therefore used as reference to evaluate cell adhesion. Cell proliferation during the period from 2 h to 40 h in culture was not different between the three Adamts1 cohorts (Figure 2.4 b).
Figure 2.4 c depicts the adhesion profiles comparing the MCF10A mammary epithelial cell line, as well as PyMT/Adamts1+/+, PyMT/Adamts1+/- and PyMT/Adamts1-/- primary cells over 2 h. MCF10A was included as a homogeneous inter-assay experimental control and the results indicate the small variability between replicate experiments. (Figure 2.3 c, grey plot). 1omMCC derived from PyMT/Adamts1+/+, PyMT/Adamts1+/- and PyMT/Adamts1-/- adhered to the matrigel substratum progressively throughout the 2 h time period (Figure 2.3 c). When comparing the adhesion cell index reached by Adamts1+/+, Adamts1+/- and Adamts1-/- at 30 mins intervals, no significant differences in adhesion were observed between the three Adamts1 genotypes at 30 mins after the commencement of the assay. However, after 1 h in culture, Adamts1-/- cells (0.088 ± 0.013) had significantly lower adhesion cell index than Adamts1+/+ (0.129 ± 0.006) and Adamts1+/- (0.140 ± 0.025) (p=0.015, One-way ANOVA; Figure 2.4 d). The differences in adhesion between Adamts1 deficient and Adamts1 expressing cells became more pronounced at 1.5 h (p=0.005, One-way ANOVA) and 2 h (p=0.003, One-way ANOVA) (Figure 2.4 d) in culture.
Consistent with the progressive deviation in adhesion cell index at fixed times over the course of the adhesion experiments, comparing the rate of adhesion demonstrated a lower adhesive rate in PyMT/Adamts-/- cells compared Adamts1+/+ and Adamts1+/- cells (p=0.0056, One-way ANOVA, Figure 2.4 d). The rate of adhesion was determined from the slope of the regression line fitted between 0-2 h (Adamts1+/+ (R2=0.992, p=<0.0001); Adamts1+/- (R2=0.977, p=<0.0001); Adamts1-/- (R2=0.9370, p=<0.0001); data not shown).
Tan IA 45 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
a.6 2x104 cells/well 5 1x104 cells/well 4 0.5x10 cells/well 4
3
2
Proliferation cell index
1
0 0123 45 6789101112 13 14 15 16 17 18 19 20 Time(hours)
b. 3.0 MCF10A MCF10A +/+ 2.5 PyMT/Adamts1+/+ Adamts1 +/- PyMT/Adamts1+/- Adamts1 -/- PyMT/Adamts1-/- 2.0 Adamts1
1.5
1.0
Proliferation cell index 0.5
0.0 123456 789 10 11 12 13 14 15 16 17 18 19 20 -0.5 Time(hours)
0.4 c. MCF10AMCF10A +/+ Adamts1PyMT/Adamts1+/+ 0.3 Adamts1+/- +/- PyMT/Adamts1-/- Adamts1 -/- PyMT/Adamts1
0.2
Adhesion cell index 0.1
0.0 0 102030405060708090100110120 Time(minutes)
Tan IA 46 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
d. 0.5 +/+ PyMT/Adamts1 ** PyMT/Adamts1+/- ** 0.4 PyMT/Adamts1-/- ** 0.3 ** * 0.2 * Adhesion cell index
0.1
0.0 0.5 h 1.0 h 1.5 h 2.0 h
0.0035 ** e. **
0.0030 PyMT/Adamts1+/+
PyMT/Adamts1+/- 0.0025 PyMT/Adamts1-/- 0.0020
0.0015
0.0010
0.0005 Rate of adhesion (cell index/minute)
0.0000
Figure 2.4. Cell adhesion is impaired in Adamts1-/- breast cancer cells. a) Cell adhesion and proliferation of Adamts1+/- primary cells at a density of 0.5x104, 1x104 and 2x104 cells/mL (n=1, quadruplicate) over 40 h. Area within dotted line (2 h) indicates immediate period after seeding when rapid impedance changes correspond to cell adhesion. b) Adhesion cell index profiles of individual Adamts1+/+ (n=8), Adamts1+/- (n=7) and Adamts1-/- (n=9) 1o mMCC over 2 h. Each replicate was a unique primary cell line and adhesion of each sample was assayed in quadruplicate. The MCF10A cell line (grey circle) was included in all adhesion assays as an inter-assay control to assess the consistency of the method. c) Cell adhesion profiles of 1omMCC over 2 h. d) Comparison of adhesion cell index of PyMT/Adamts1+/+, Adamts1+/- and Adamts1-/- 1omMCC at 0.5, 1, 1.5 and 2 h. e) Rate of cell adhesion over 2 h as determined by slope of the line of the linear regression model. Data is represented as mean ± SEM. Statistical analysis was performed using log-transformed data and One-way ANOVA with Fisher’s LSD post hoc test. Significance was determined if p≤0.05 (*p≤0.05, ** p≤0.005, ***p≤0.0005).
Tan IA 47 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
2.3.3. Induced Adamts1 expression in knockout primary mammary carcinoma cells did not affect cell adhesion due to poor transduction efficiency.
To further validate the suggestion that ADAMTS1 promotes cell adhesion, an Adamts1 lentivirus construct was developed to restore Adamts1 expression into an Adamts1 null 1omMCC. Cell line #2756 was chosen as the preferred knockout sample to introduce Adamts1 into as it exhibited good survival in culture, and low cell adhesion index among the Adamts1-/- cohort. A PyMT/Adamts1+/+ 1omMCC (#11) was also included in the assay to represent the expected adherent behaviour of wild type cells. Transduction of the cells after 24 h treatment with lentivirus was verified by imaging of GFP expression in fluorescence microscopy.
Surprisingly, 2756-Adamts1 cells displayed cell adhesion over 2 h that was comparable to control 2756-GFP transduced and non-transduced 2756 cells, while #11 mammary cancer cells exhibited a more progressive adherent behaviour, reflective of Adamts1+/+ mammary cancer cells (Figure 2.5 a). Comparison of the adhesion cell index at 0.5 h, 1h, 1.5 h and 2 h further illustrated the similarities between #2756 cells and the higher cell adhesion index reached by wild type #11 cells (Figure 2.5 b). A similar pattern in cell adhesion rates between non-transduced 2756, 2756-GFP, 2756- Adamts1 and #11 primary cells were also observed, with 2756, 2756-GFP and 2756-Adamts1 cells exhibited comparable rates in adhesion while #11 wild type cells adhered relatively faster (Figure 2.5 c).
Since restoration of Adamts1 expression in #2756 knockout cells exerted no apparent changes in adherence of mammary cancer cells to a matrigel layer, confirmation of Adamts1 expression in 2756- Adamts1 by quantitative RT-PCR was performed and this surprisingly showed no increase in Adamts1 expression in 2756-Adamts1 cells (Figure 2.5 d). Thus, the influence of ADAMTS1 on cell adhesion could not be examined in this experiment due to unexplained lack of success in inducing Adamts1 expression in #2756 knockout cells. In an effort to rectify this shortcoming I next investigated Adamts1 transduction into MCF10A cell line.
Tan IA 48 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
a. 0.20 2756 (non-transduced) 2756-GFP 0.15 2756 -Adamts1 11 (non-transduced) 0.10
Adhesion cell index 0.05
0.00 0.5 1 1.5 2 Time (hours) b. 0.20
2756 (non-transduced) 0.15 2756-GFP 2756 -Adamts1 11 (non-transduced) 0.10
Adhesion cell index 0.05
0.00 0.5 1 1.5 2 Time (hours) c. 0.0018 2756 (non-transduced) 2756-GFP 2756 -Adamts1 11 (non-transduced) 0.0012
0.0006
Rate of adhesion (cell index/minute) 0.0000
Tan IA 49 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
2756 (non-transduced) d. ) 1.0 CT 2756-GFP ΔΔ
- 2756-Adamts1 0.8 11 (non-transduced) 0.6
0.4 expression (2 0.2
Adamts1 0.0 Figure 2.5. Cell adhesion of #2756 Adamts1-/- cells induced to overexpress Adamts1. a) Cell adhesion index profiles of non-transduced 2756, 2756-GFP, 2756-Adamts1 and Adamts1+/+ 1omMCC over 2 h. b) Comparison of adhesion cell index between non-transduced 2756, 2756-GFP, 2756-Adamts1 and Adamts1+/+ 1omMCC at 0.5, 1, 1.5 and 2 h. Data is represented as mean ± SEM. Due to shortage of available cryopreserved #2756 and #11 stocks, only two experimental replicates were performed and thus statistical analysis could not be conducted. c) Rates of cell adhesion over 2 h as determined by the slope of a linear regression model. Data is represented as mean ± SEM. d) Adamts1 mRNA expression in #2756 (non-transduced, GFP and Adamts1) and #11 primary cells. Adamts1 expression was normalised to a mouse L19 internal control and calibrated against sample #11. Data represent 2-ΔΔCT (n=1). Note the low detection of Adamts1 mRNA in Adamts1-/- cells was expected, as the knockout strategy is known to produce some residual non-sense transcript.
Tan IA 50 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
2.3.4. Lentiviral transduction of Adamts1 in MCF10A mammary epithelial cells promoted cell adhesion to matrigel.
Previous characterisation of ADAMTS1 expression in various human breast cancer cell lines including MDA-MB-468, MDA-MB-231, MCF10A, MCF7 and Hs578T (MCF10A vs Hs578T, Figure 2.6 a) demonstrated MCF10A express little to no ADAMTS1 mRNA and was therefore the ideal cell line to introduce Adamts1 and assess the consequences.
The MCF10A cell line is an immortalised non-transformed human breast epithelial cell line (Soule et al., 1990) which recapitulates normal mammary gland development in three-dimensional in vitro culture (Debnath et al., 2003). Introduction of Adamts1 into MCF10A cells was achieved through transduction with a lentiviral construct comprising GFP with Adamts1 separated by an internal ribosomal entry site (IRES), or a GFP only control lentivirus. Adamts1 expression was confirmed by RT-PCR and this revealed greater expression of Adamts1 in MCF10A-Adamts1 than in an Adamts1+/+ 1omMCC sample (Figure 2.6 b). MCF10A-GFP transduced cells expressed no detectable Adamts1. As the Adamts1 transcript was conjugated with a GFP reporter gene, the success and efficiency of Adamts1 induction was also confirmed by fluorescent imaging (Figure 2.6 c-e). Likewise, high transduction efficiency was also achieved in MCF10A with the GFP-only control vector (Figure 2.6 f-h).
MCF10A-Adamts1 and MCF10A-GFP displayed more progressive adherence to matrigel than wild type MCF10A cells (Figure 2.7 a), which was significant across all time points analysed (0.5 h, p=0.0004; 1 h, p=0.0006; 1.5 h, p=0.0053; 2 h, p=0.014, One-way ANOVA; Figure 2.7 b). The promoted adhesion index in both overexpressing clones suggested that the transduction procedure alone had a positive effect on the adhesive behaviour of this cell line. Despite this effect, when compared to each other, MCF10A-Adamts1 cell adherence over the 2 h period was consistently greater than GFP expressing MCF10A clones (Figure 2.7 a). This was significant at 0.5 h (p=0.036, Fisher’s LSD post hoc) and trending at 1 h (p=0.06, Fisher’s LSD post hoc; Figure 2.7 b). This demonstrated that Adamts1 overexpression in MCF10A-Adamts1 cells further promoted an increase in higher adhesion index.
The same trends were found when considering the rate of adhesion between wild type MCF10A, MCF10A-Adamts1 and MCF10-GFP cells over the whole 2 h adhesion period. MCF10A- Adamts1 and MCF10A-GFP adhered to the matrigel layer at a faster rate than wild type cells (p=0.0009,
Tan IA 51 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
One-way ANOVA; Figure 2.7 c), while Adamts1 expression trended to exhibit a faster rate of cell adhesion than MCF10A-GFP (P=0.056, Fisher’s LSD post hoc; Figure 2.7 c).
1.0
a. )
CT 0.8 ΔΔ - 0.6
0.4 expression (2 0.2 0.01
ADAMTS1 0.00 HS578t MCF10A wild type
b. 3×106
)
-ddCT 2×106
expression (2 1×106
Adamts1
0 PyMT/Adamts1+/+ MCF10A-Adamts1 MCF10A-GFP
Tan IA 52 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
MCF10A-Adamts1 MCF10A-GFP
c. f.
Brightfield
d. g.
FITC
e. h.
Merged
Figure 2.6. Adamts1 overexpression in MCF10A cells. a) ADAMTS1 expression in MCF10A breast cancer cell line relative to Hs578T. b) Adamts1 expression of MCF10A-Adamts1 and MCF10A-GFP stables clones used in adhesion assays. Expression was normalised to a mouse L19 internal control and calibrated against a PyMT/Adamts1+/+ primary cell line. Data represents 2-ΔΔCT of triplicate readings (n=1). Brightfield and fluorescent images of (c, d, e) MCF10A-Adamts1 and (f, g, h) MCF10A- GFP cells growing in monolayer at 20x magnification. Scale bar=200µm.
Tan IA 53 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
a. 0.25 MCF10A wt 0.20 MCF10A Adamts1 MCF10A GFP 0.15 0.10
Adhesion cell index 0.05
0.00 0.5 1 1.5 2 Time (hours) 0.25 * b. MCF10A wt MCF10A-Adamts1 0.20 MCF10A-GFP ** *** 0.15 p=0.06 p=0.06 * *** 0.10 ** * Adhesion cell index ** 0.05 0.00 0.5 1 1.5 2 Time (hours) *** c. 0.0025 p=0.056 MCF10A wt MCF10A GFP 0.0020 MCF10A Adamts1 ** 0.0015
0.0010
0.0005
Rate of adhesion (cell index/minute) 0.0000 Figure 2.7. Cell adhesion of MCF10A breast cancer cell line overexpressing Adamts1. a) Adhesion of wild type MCF10A, MCF10A-GFP and MCF10A-Adamts1 over 2 h. b) Comparison of adhesion cell index of wild-type, GFP- and Adamts1-overexpressing MCF10A cells at 0.5, 1, 1.5 and
2 h. c) Rate of MCF10A, MCF10A-GFP and MCF10A-Adamts1 cell adhesion to matrigel (cell index/minute) over 2 h. Data is represented as mean ± SEM (n=3 independent experiments). Statistical analysis was performed using log-transformed data and One-way ANOVA with Fisher’s LSD post hoc test. Significance was determined if p≤0.05 (*p≤0.05, ** p≤0.005, ***p≤0.0005).
Tan IA 54 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
2.4. DISCUSSION
MMTV-PyMT transgenic mice develop breast cancers that eventually spread and form pulmonary metastases with high frequency, making them an important model for investigation of spontaneous metastatic progression (Sahai and Marshall, 2003). Ablation of the Adamts1 gene significantly reduced primary breast tumour growth in these mice and more importantly, the fatal spread of metastases to the lungs (Ricciardelli et al., 2011). These findings demonstrated a key pro-metastatic role for ADAMTS1 in breast cancer, but how ADAMTS1 bestows pro-metastatic cell behaviour/s to mammary cancer cells is unknown.
As described above, dynamically changing cell adhesion to the ECM is a key determinant of all stages of metastasis (Haberern and Kupchik, 1985; Kirmse et al., 2011; Kramer et al., 1989; Palecek et al., 1997; Schneider et al., 2007; Wolf et al., 2003; Yamana et al., 2006) making it necessary to focus on the influence of ADAMTS1 on mammary cancer cell adhesion to the ECM. To determine whether ADAMTS1 elicits a change in adhesive capacity in mammary cancer cells, a series of primary cell isolates were established from Adamts1+/+, Adamts1+/- and Adamts1-/- PyMT mammary tumours and allowed to adhere to matrigel. Matrigel is a heterogeneous mix of laminin, collagen IV, heparin sulphate proteoglycan, entactin and nidogen (Kleinman et al., 1986). These proteins are the major constituents of the ECM and basement membrane in vivo (Palecek et al., 1997) and thus, matrigel is a widely accepted biological analogue of the extracellular environment and basement membrane in vitro.
Finding from this study strongly demonstrated impaired adherence of Adamts1-deficient mouse mammary carcinoma cells to matrigel. While attempts to restore Adamts1 expression in PyMT/Adamts1- /- primary breast cancer cells was unable to consequently revert the adherent phenotype that of PyMT/Adamts1+/+ cells, this was due to an unexplained inability to induce Adamts1 expression in these primary cells. Poor success of the lentiviral transduction to induce gene expression is not an uncommon event as other groups have also reported poor efficiency of viral-mediated gene delivery in primary cells (Chandrashekran et al., 2004; Ellis et al., 2013; Farina et al., 1998; Finer et al., 1994). This limitation with primary cells is not observed with established cell lines (Chandrashekran et al., 2004; Ellis et al., 2013; Farina et al., 1998; Finer et al., 1994). Thus, Adamts1 expression was transduced into immortalised MCF10A breast cell line, which lack ADAMTS1. Although transduction alone had a positive influence on MCF10A cell adhesion, overexpression of Adamts1 accelerated the adhesion rate and increased total adherent MCF10A cell population. This finding, together with the impaired adhesion
Tan IA 55 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
exhibited by Adamts1-/- cells implicates ADAMTS1 as promoter of cell adhesion in mammary cancer cells. This is a novel characteristic, which has yet been associated with ADAMTS1 in breast cancer and is an important cell behaviour that could underlie the metastatic phenotype associated with Adamts1+/+ and Adamts1+/- mammary tumours.
Interactions between a cancer cell and the peritumoural matrix is integral for the determination of front-rear polarity driving cancer cell migration and invasion (Palecek et al., 1997; Wolf et al., 2003; Yamana et al., 2006). It also instigates the activation of metalloproteolytic activity to degrade ECM proteins and allow cancer cells to traverse from the primary tumour site and/or breach the basement membrane barrier to disseminate to distant tissues (Haberern and Kupchik, 1985; Kirmse et al., 2011; Kramer et al., 1989). Cell-ECM interactions are mediated mainly by the integrin adhesion receptors. Binding of cell surface-bound integrin receptors with protein ligands in the extracellular milieu, transduces a signalling cascade that alters focal adhesion formation as well as actin polymerisation via GTPase proteins normally activated in migrating metastatic cells (Clark et al., 1998; Hotchin and Hall, 1995; Price et al., 1998; Ren et al., 1999; Ridley and Hall, 1992). Historically, ligand partners for integrins were first defined by the presence of an RGD (arginine-glycine-asparagine) tripeptide recognition motif (Pierschbacher and Ruoslahti, 1984). Over the years however, new integrin- recognition sites continue to emerge (Humphries et al., 1988; McCarthy et al., 1990; Soteriadou et al., 1992) and ligand partners have diversified from ECM structural proteins, to other adhesion molecules, thrombospondin, disintegrins and metalloproteases (reviewed in (Humphries et al., 2006; Plow et al., 2000). As a protease with a cysteine-rich disintegrin-like domain and thrombospondin motifs, ADAMTS1 may associate with integrins through a non-RGD motif in its disintegrin (McLane et al., 2004) and/or thrombospondin domains (Wolf et al., 2003). Indeed, a novel integrin recognition site, ECD (glutamic acid-cysteine-aspartic acid), present in the ADAM metalloprotease family (Kirmse et al., 2011) is also present in the first thrombospondin motif of ADAMTS1 (Figure 2.8) This tripeptide region is known to interact with αvβ3 (Yokotsuka et al., 2011), αvβ5 (Yu and Machesky, 2012) and α6β1 (Tester et al., 2000) integrin receptors and poses as a non-RGD binding motif to which integrins such as αvβ3, αvβ5 and α6β1 can recognise and bind to ADAMTS1. Verification of this interaction through protein binding assessment has yet been performed and is warranted in future studies.
How ADAMTS1-integrin interaction relates to the increased capacity of cancer cells to adhere to the ECM is also associated with its ability to directly bind to soluble heparan sulphate (HS) residues of HS proteoglycans (HSPG) (Kuno and Matsushima, 1998). The potential ligand binding of ADAMTS1
Tan IA 56 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
to cell-surface integrin receptor/s and its known interaction with HS and HSPG in the ECM, presents ADAMTS1 as an intermediate factor bridging cancer cell interaction with the ECM (Figure 2.9 a).
Tsp1 protein sequence WGPWGPWGDCSRTCGGGVQYTMRECDNPVPKNGGKYCEGKRVRYRSCNIEDCP
ADAMTS1 protease MP DIS TSP1 TSP1 TSP1
Figure 2.8. Schematic representation of ECD-integrin motif in ADAMTS1. The N-terminal thrombospondin motif of ADAMTS1 (protein sequence accession number NP_033751) contains an ECD tripeptide sequence (underlined), which is a known integrin binding recognition motif.
Alternatively, ADAMTS1 may enhance mammary cancer cell adhesion to the ECM, through the upregulation of integrin receptors (Figure 2.9 b); a role which has been postulated for ADAMTS12 in human trophoblastic cells (Beristain et al., 2011). In this context and through an unknown control mechanism, ADAMTS12 induced the upregulation αvβ3 and αv integrins in trophoblasts, enhancing their binding affinity to ECM proteins, collagen II and collagen IV (Beristain et al., 2011). This reported activity attributable to ADAMTS12 proposes a mechanistic alternative with which ECM-secreted enzymes such as ADAMTS1, may promote cell adherence with the ECM. For this reason, identifying any gene expression changes induced by ADAMTS1, as undertaken in Chapter 4, is considered valuable to understanding the total mechanism of ADAMTS1 action.
Tan IA 57 CHAPTER 2 ADAMTS1 enhances breast cancer cell adhesion to the ECM
s) ll b) rotein
l p noma ce to form form to i G ECM carc y n i r
g otifs (TSP1). (TSP1). otifs -ECM adhesion, adhesion, -ECM nte i
mammary carcinoma cell mammar /+ of various structura +/+ + nd n Adamts1 Adamts1 s1 ou b Findings in this study study this in Findings mt ade up Adamts1 Ada rane / b (m T/ TSP1 em Heparan sulfate proteoglycans (HSPG) PyMT/ PyM Membrane bound integrin M ECM ECM (made up of various structural proteins) TSP1 T
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on receptors integrin linking factor intermediate be an may ADAMTS1 a)
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Adamts1 Adamts1 demonstrate a functional role for ADAMTS1 in matrix adhesion of mammary cancer cells. Cell surface-bound integrins mediate cell mediate integrins surface-bound Cell cells. cancer mammary of adhesion matrix in for ADAMTS1 role a functional demonstrate pathways. potential these through ADAMTS1 by enhanced be may which HSP of residues HS to binds motifs, thrombospondin C-terminal its via ADAMTS1, ECM, the In matrix. ECM the and surface cell the 1 m type and thrombospondin (DIS) disintegrin (MP), metalloprotease its via receptors integrin with interacting while proteins, able receptors of adhesion the number increases turn in which receptors, of integrin expression the the may upregulate ADAMTS1 contacts with the ECM. Figure 2.9. Potential pathways facilitated by ADAMTS1 facilitated pathways Potential Figure 2.9.
Tan IA 58
Chapter 3
ADAMTS1 accelerates mammary cancer cell migration but does not alter mammary cancer cell invasion
CHAPTER 3 ADAMTS1 accelerates mammary cancer cell invasion
3.1. INTRODUCTION
As discussed in Chapter 2, increased cell adherence to the extracellular matrix and basement membrane promotes a series of events in metastasis. Contacts to the ECM by cancer cells is important for the determination of cell polarity (Muthuswamy and Xue, 2012; Yamana et al., 2006) and activation of ECM-degrading metalloproteases (Guiet et al., 2011; Haberern and Kupchik, 1985; Kirmse et al., 2011; Kramer et al., 1989; Sahai and Marshall, 2003). These support the migratory and invasive behaviours, which cancer cells must acquire to escape the primary tumour, penetrate tissue boundaries and disseminate into new organs to develop secondary tumours (reviewed in (Bravo-Cordero et al., 2012; Palm et al., 2005; van Zijl et al., 2011; Yilmaz and Christofori, 2010)).
Cancer cell migration and invasion are complex processes that require multiple events to occur, and tumour cells often undergo epithelial-mesenchymal transition (EMT) to promote these pro- metastatic behaviours (Thompson et al., 2005). One of the main characteristic of EMT is the modification of cell shape from an epithelial cell body with a well-defined rigid structure and tight cell-cell junctions, to an amoeboid or mesenchymal-shaped cell with polymerised-actin projections (Raviraj et al., 2012a; Raviraj et al., 2012b) and focal contacts with the ECM (Chaturvedi et al., 2012; Friedl et al., 1997; Friedl et al., 1998; Golembieski et al., 2008; Hall et al., 2002; Niggemann et al., 1997; Odenwald et al., 2013; Sales et al., 2008). This drastic change in cell morphology is accompanied by the abrogation of physical cell-cell connections and the acquisition of front-rear cell polarity, for which cell-ECM adhesion is integral (Li et al., 2005; Muthuswamy and Xue, 2012; Yamana et al., 2006). Front-rear polarisation is essential in the directional assembly of adherent junctions between a cancer cell and the ECM at its leading edge (front) and the coordinated retraction of cell-ECM interactions at its trailing edge (rear) (Hegerfeldt et al., 2002; Pignatelli et al., 2012; Ridley et al., 2003). These focal points of contacts at the tumour cell’s leading edge act as traction sites, that with the traction force generated by the concomitant loss of ECM contacts at the trailing edge, translocates the cancer cell forward in the ECM (Hegerfeldt et al., 2002; Pignatelli et al., 2012; Ridley et al., 2003). Cell invasion during metastasis requires migration and the degradation of ECM proteins, particularly those comprising the tissue basement membrane. The breakdown and subsequent penetration of the basement membrane are hallmark histological characteristics found in metastatic cancer tissues, for which, the proteolytic activity of metalloproteases is essential (Canals et al., 2006; Saiki et al., 1993).
Tan IA 60 CHAPTER 3 ADAMTS1 accelerates mammary cancer cell invasion
Augmenting metalloprotease activity in cancer cells through up regulation of its expression or exogenous supplementation can alone induce and even enhance the invasiveness and motility of an otherwise weakly or non-metastatic cancer cell (Foley et al., 2012; Gaida et al., 2010; Li et al., 2013; Nakada et al., 2001; Saiki et al., 1993). In colon cancer cells, the upregulation of ADAM9 promoted cell invasion (Li et al., 2013), while MMP1a overexpression increased the invasion and subsequent metastatic occurrence of lung carcinoma and skin melanoma cells (Foley et al., 2012). Increased expression of other MMP’s such as MMP2 and MMP9 also result in the dissemination and invasion of astrocytic (Nakada et al., 2001) and, renal, fibrosarcoma and melanoma cancer cells (Saiki et al., 1993), respectively.
Studies surrounding ADAMTS1 and cancer metastasis have consistently correlated promoted cancer cell invasion and motility with elevated ADAMTS1 expression. This effect has been reported in endometrial cancer induced to express Adamts1 (Keightley et al., 2010) and invasive HCC cells (Braconi et al., 2009). In breast cancer, exogenous ADAMTS1 have also induced a significant increase in cell invasion and migration (Esselens et al., 2010; Liu et al., 2006c; Tyan et al., 2012). The question of whether endogenous ADAMTS1 abundance in highly metastatic breast cancer in vivo directly induces a parallel enhancement of mammary cancer cell invasion and migration as observed with exogenous Adamts1 supplementation, remain to be investigated.
In addressing this question, our group has looked at the effects of endogenous Adamts1 using the MMTV-PyMT transgenic breast cancer model. The MMTV-PyMT model recapitulates the typical progression of pulmonary metastases from primary breast tumours and possesses a similar gene expression signature as HER2 positive/ER negative human breast cancers (Herschkowitz et al., 2007). In this model, the deletion of a functional Adamts1 gene significantly attenuated the growth and reduced the incidence of pulmonary metastases arising from primary breast tumours (Ricciardelli et al., 2011). This current study endeavoured to determine whether ADAMTS1 enhances the ability of mammary cancer cells to metastasise by directly mediating cell behavioural changes in particular cell migration and invasion. Since showing in Chapter 2 that ADAMTS1 promoted mammary cancer cell-ECM adhesion, and that cancer cell-ECM interaction aid migration and cell invasion, I hypothesised that the loss of Adamts1 in PyMT transgenic mice, demotes their ability to migrate and invade through a biological barrier.
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3.2. MATERIALS AND METHODS
3.2.1. Cell culture
Isolated 1omMCC were generated as described in Section 2.1.2. Prior to an experiment, cells were revived from liquid nitrogen and grown in a T25 flask containing 5ml of growth media. Cells were
o incubated in a sterile 37 C humidified incubator with 5% CO2. After 48 h, primary cells were transferred into three T25 flasks and allowed to reach 70-80% confluence, which approximately took 48-72 h post- passage. Replacement of growth media was performed daily. Wild type MCF10A, MCF10A-GFP and MCF10A-Adamts1 breast cancer cell lines were maintained in DMEM/F12 (Sigma-Aldrich Pty Ltd, Sydney, Australia) supplemented with 5% (v/v) horse serum, 20 ng/ml EGF, 0.5 μg/ml hydrocortisone, 100 ng/ml cholera toxin and 10 μg/ml insulin and 1% (v/v) Pen/strep antibiotic (Sigma-Aldrich Pty Ltd, Sydney, Australia). Cells were passaged at 1:2 split upon reaching 70%-80% confluence and allowed to
o grow in a sterile 37 C humidified incubator with 5% CO2. Wild type MCF10A and MCF10A overexpressing clones were passaged twice prior to an experiment. Adamts1 stably overexpressing MDA-MB-231 clone, previously established in the group, was maintained in RPMI-1640 culture media (Sigma-Aldrich Pty Ltd, Sydney, Australia), 10% FBS, 1% (v/v) Pen/strep antibiotic and 0.4 mg/ml Geneticin® selective antibiotic (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia).
3.2.2. Quantitative real-time RT-PCR
Total RNA was isolated using TRIzol reagent (Life Technologies Australia Pty Ltd.) according to the manufacturer’s instructions and as described in Section 2.2.7. Quantitative real-time PCR against the Adamts1 transcript was also performed as described in Section 2.2.7.
3.2.3. Real time cell-based assay
Assessment of cancer cell migration and invasion were performed using the xCelligence system (ACEA Biosciences, California, USA) and a CIM plate (ACEA Biosciences, California, USA) (Figure 3.1).
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3.2.3.1. In vitro migration assay
Cells at approximately 70-80% confluence were trypsinised and spun at 1500 rpm for 5 mins. After the supernatant was removed, the cell pellet was resuspended in 1 ml serum-free media and counted using the TC10 automated cell counter (BioRad Laboratories Pty Ltd, NSW, Australia) at a 1:1 cell suspension and trypan blue (Sigma-Aldrich Pty Ltd., Australia) dilution. Cells were aliquoted at 2x105 cells/ml in serum-free media. 160 μl of normal growth media was added to the bottom chamber wells and 25 μl of serum free media was added to the top chamber wells of the CIM plate. A background reading was obtained with the xCelligence, after which the cells (100 μl/well) were aliquoted into the top wells and the migration assay commenced. The change in electrical impedance recorded in the electrodes placed on the underside of the 8 μm porous membrane indicated the migration cell index. The rate of migration for each sample was identified by interpolating of a sigmoidal curve over the 10 h migration profile using GraphPad Prism® version 6 (GraphPad Software, La Jolla, California, USA) (Figure 3.1 c). The sigmoid regression model identified the slope of the curve, which represented the rate when cells were migrating exponentially, as well as an upper plateau phase where little or no further cell migration occurred. The point at which the upper plateau phase began determined the maximum migration index reached by each samples and the time that this was reached.
3.2.3.2. In vitro invasion assay
For invasion assays, each well of the top CIM plate chamber (ACEA, Roche Diagnostics, Australia) was coated with 20 μl 5% v/v matrigel. Matrigel was left to polymerise for 4 h in a 37 °C humidified incubator. Cells at approximately 70-80% confluence were trypsinised and spun at 1500 rpm for 5 mins. After the supernatant was removed, the cell pellet was resuspended in 1 ml serum-free media and counted using the TC10 automated cell counter at a 1:1 cell suspension and trypan blue dilution. Cells were aliquoted at 4x105 cells/ml. After a 4 h incubation, 160μl of normal growth media was added to the bottom chamber wells and 25 μl of serum free media was added to the top chamber wells. A background reading was obtained in the xCelligence, and the cell suspension (100 μl/well) was added to top chamber and the invasion experiment commenced. Cell invasion was assessed in triplicate or quadruplicate over 40 h. The change in electrical impedance recorded in the electrodes placed on the underside of the 8 μm porous membrane indicated the invasion cell index. The 40 h invasion profile of each sample was interpolated with a sigmoid regression curve using GraphPad Prism® version 6 (GraphPad Software, La Jolla, California, USA) (Figure 3.1 c). The sigmoid regression
Tan IA 63 CHAPTER 3 ADAMTS1 accelerates mammary cancer cell invasion
model identified the slope of the curve, which represented the rate when cells were migrating exponentially, as well as an upper plateau phase where little or no further cell migration occurred. The point at which the upper plateau phase began determined the maximum migration index reached by each samples and the time that this was reached.
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a. CIM-plate
Growth media w/ 10% FCS)
Serum free media
1o MCC
1:20 Matrigel coat Components Migration Invasion PET membrane with 8μm pore Top Cell density 2x105/mL 4x105/mL Gold electrode arrays well 1:20 none 20μL Matrigel Media 100μL 100μL
Bottom Cell density No cells No cells well Media 160μL 160μL b.
Cells adhere to filter Cells actively migrate Migrated cells cover the membrane with some and adhere to the microelectrode surface beginning to migrate electrode underside = through = No further increase in cell = Rapid increase in cell impedance Slow increase in cell impedance impedance Migration
Invasion
Cell Impedance
Time
Tan IA 65 CHAPTER 3 ADAMTS1 accelerates mammary cancer cell invasion
c. START OF PLATEAU Peak PLATEAU PHASE cell index X reached
Cell Index SLOPE OF THE CURVE (Rate of migration/invasion)
Time(hours) Time at peak invasion/migration
Figure 3.1. Real-time invasion and migration assessment using the xCelligence system. Cell migration and invasion assays were performed using specialised plates known as a CIM-plate. a) Specific components added to a CIM plate well for a migration and invasion assay. b) Each well of a CIM-plate mimics the basic design of a transwell chamber in that each well is comprised of two units:
a top well where cells are seeded in a chemoattractant-free media and a bottom well with
chemoattractant-rich media. Both wells are separated by an 8μm porous filter membrane interface, which cells pass through in order to migrate towards the chemoattractant. For assessing invasion, a matrigel barrier was added to the top well, which cells need to breakdown in order to migrate towards the chemoattractant rich media. The underside of each filter membrane has a sheath of microelectrode arrays, which cells adhere to once they migrate towards the chemoattractant-rich media and penetrate the filter membrane. Cell adherence to the microelectrode surface causes a change in electrical impedance, and is indicative of the cell population that have successfully penetrated through the porous membrane and the strength of adherence between the cells and the electrode surface. Once cells cover the entire electrode surface, cell impedance can no longer be detected and the migration/invasion profile plateaus. c) Invasion and migration profiles were interpolated with a sigmoid regression curve using GraphPad Prism® version 6. Sigmoid analysis determined the slope of the logarithmic increase in cell index, which represented the rate at which cells progressively migrated/invaded through the microporous membrane. It also identified the point at which cell index plateaued and this defined the maximum cell index and the time taken for this to be reached.
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3.2.4. Statistics
Statistical significance was determined by One-Way ANOVA and Fisher’s LSD post-hoc test in GraphPad Prism® version 6 (GraphPad Software, La Jolla, California, USA). Non-normally distributed data were log-transformed prior to statistical analysis. Statistical significance was defined at p≤0.05.
3.3. RESULTS
3.3.1. Loss of Adamts1 impedes mammary carcinoma cell migration.
To investigate whether loss of ADAMTS1 in mouse mammary carcinoma cells affected cancer cell motility, cell migration assays were performed using primary mammary cancer cells isolated from PyMT/Adamts1+/+, Adamts1+/- and Adamts1-/- transgenic mice (Figure 3.2 a). MCF10A breast cancer cells were also included in each experiment as an inter-assay quality control of assay reproducibility and indicated a high degree of consistency between all migration assays (Figure 3.2 a, grey plot). Migratory profiles of all 1oMCC displayed an initial phase of increasing migration index that peaked at different times for each ADAMTS1 genotype and plateaued thereafter (Figure 3.2 a). Peak migration was reached when cells saturate the underlying electrode surface, preventing further detection of cell impedance (for schematic illustration see Figure 3.1 b).
Sigmoid regression analysis was performed to mathematically identify the time and maximum cell index corresponding to peak migration. It also identified the slope of the exponential increase in cell migration, indicative of the migration rate. This model fitted the profiles of each 1omMCC almost
2 +/+ 2 +/- 2 perfectly with an R coefficient close to 1 (Adamts1 (R =0.990, p<0.0001), Adamts1 (R =0.987, p<0.0001), Adamts1-/- (R2=0.991, p<0.0001)). All primary cell lines reached similar maximum cell indices (Figure 3.2 b), though for PyMT/Adamts1-/- (7.78 h ± 0.086) and Adamts1+/- (7.48 h ± 0.60) maximum migration was reached at a significantly later time point than Adamts+/+ mammary carcinoma cells (5.13 h ± 0.251) (p=0.05, One-way ANOVA; Figure 3.2 c). The rate of migration as determined by the slope of the sigmoidal curve, depicted a similar pattern, in that PyMT/Adamts1+/- (0.3124 cell index/hour ± 0.042) and PyMT/Adamts1-/- (0.3012 cell index/hour ± 0.053) migrated at a slower rate relatively to wild type cells (0.562 cell index/hour ± 0.132) (Figure 3.2 d). Although the latter finding did not reach statistical significance (p=0.15, One-way ANOVA), the corresponding differences in migration
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rate and period that maximum migration index was reached in PyMT/Adamts1+/- and PyMT/Adamts1-/- against PyMT/Adamts1+/+ 1omMCC implied that the partial or complete loss of Adamts1 expression decelerates the migratory capacity of mammary carcinoma cells.
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a. 2.5
MCF10AMCF 10A PyMT/Adamts1+/+ +/+ 2.0 Adamts1 PyMT/Adamts1Adamts1+/- +/- PyMT/Adamts1Adamts1-/- -/- 1.5
1.0 Migration cell index
0.5
0.0 0 1 2 3 4 5 6 7 8 9 10 Time(hours) PyMT/Adamts1+/+ PyMT/Adamts1+/- b. PyMT/Adamts1-/- c. d. * 2.5 10 0.8
* 2.0 8 0.6
1.5 6 0.4
1.0 (hours) 4 (cell index/hour) Rate of migration 0.2 2 0.5 Maximum migration cell index 0.0 to maximum migration Time 0 0.0
Figure 3.2. Loss of Adamts1 impedes primary mammary cancer cell migration. a) Migration profiles of MCF10A (n=6) and 1omMCC isolated from PyMT/Adamts1+/+ (n=5), Adamts1+/- (n=4) and Adamts1-/- (n=6) mice over 10 h. The b) maximum migration cell index, c) time at which maximum migration was reached and d) rate of cell migration were determined using the sigmoid regression model. Statistical analysis was performed using log-transformed data and One-way ANOVA with Fisher’s LSD post-hoc test. Significance was determined if p≤0.05 (*).
Tan IA 69 CHAPTER 3 ADAMTS1 accelerates mammary cancer cell invasion
3.3.2. The loss of ADAMTS1 did not alter the invasive capacity of Adamts1-/- 1omMCC.
Primary cancer cell invasion through a matrigel barrier was assessed over 40 h. Figure 3.3 a presents the average invasion profiles for PyMT/Adamts1+/+, Adamts1+/- and Adamts1-/- cells and MCF10A. MCF10A was again used an inter-assay experimental control and revealed a high level of experimental consistency (Figure 3.5 a, grey plot). No significant differences in invasion cell index were found between the three Adamts1 1oMCC at any time points recorded (Figure 3.3 a).
The invasion profile of the three PyMT mammary carcinoma cell genotypes featured an initial lag period, logarithmic phase of cell invasion and an upper plateau. The lag phase of invasion represented the delay in 1oMCC transition towards a serum-rich media. This delay was attributed to the matrigel barrier, which cells must initially degrade in order to translocate to the chemoattractive environment. Over time and upon acquiring the ability to degrade the ECM-like substratum, cells progressively invade and this period is represented by the logarithmic increase in invasion profile. Invaded cells eventually saturate the underlying microelectrode surface thereby preventing further detection of cell impedance and causing a plateau in invasion cell index (for schematic illustration see Figure 3.1 b). The 40 h invasion profile was interpreted with a sigmoid regression curve to identify the time and cell index corresponding to peak invasion, and the rate of invasion. This model represented cell invasion of each sample over 40 h to a high degree (Adamts1+/+ (R2=0.997, p<0.0001), Adamts1+/- (R2=0.990, p<0.0001), Adamts1-/- (R2=0.994, p<0.0001)).
Although it did not reach statistical significance, PyMT/Adamts1-/- 1omMCC (1.983 ± 0.226) reached a relatively lower maximum invasion index than wild type counterparts (2.762 ± 0.05) (Figure 3.3 b). The total time taken for this to be reached was significantly shorter in the PyMT/Adamts1-/- (25.6 h ± 2.14) cohort than in the PyMT/Adamts1+/+ 1omMCC (40.6 h ± 2.84) (p=0.0064, One-way ANOVA with Fisher’s LSD hoc test; Figure 3.3 c). However, the peak at an earlier time-point in Adamts1-/- cells was in part due to a lower in relative maximum invasion index. In the PyMT/Adamts1+/+ cohort for instance, the longer invasion period corresponded to a relatively high peak invasion index while similarly, the shorter invasion period in PyMT/Adamts1-/- corresponded to a relatively lower cell index. The lower peak invasion may be attributable to the reduced adhesion capacity of the Adamts1-/- cells in the presence of matrigel, demonstrated in Chapter 2. Considering the rate of invasion between the two Adamts1 genotypes calculated from the sigmoidal curve fitting removes the influence of the height of the peak index in each genotype and was in fact similar between the genotypes. These findings
Tan IA 70 CHAPTER 3 ADAMTS1 accelerates mammary cancer cell invasion
therefore implicate a minimal role for ADAMTS1 in mammary cancer cell invasion. As PyMT/Adamts1-/- cells retain the capacity to invade, these findings suggest an alternative mechanism for cell invasion that may be independent of ADAMTS1 proteolytic activity.
Tan IA 71 CHAPTER 3 ADAMTS1 accelerates mammary cancer cell invasion
a. 3.0 MCF10AMCF10A +/+ +/+ PyMT/Adamts1Adamts1 2.5 PyMT/Adamts1Adamts1+/- +/- PyMT/Adamts1Adamts1-/- -/- 2.0
1.5
1.0 Invasion cell index
0.5
0.0 5 10 15 20 25 30 35 40 Time (hours) PyMT/Adamts1+/+ PyMT/Adamts1+/-
b. 3 PyMT/Adamts1-/- c. d. 50 ** 0.15
40
2 0.10 30
(hours) 20 1 0.05 (cell index/hour) 10 Rate of invasion
to maximum invasion Time Maximum invasion cell index 0 0 0.00
Figure 3.3. Adamts1-/- 1omMCC exhibited accelerated cell invasion than Adamts1+/+ cells. a) Invasion profiles of MCF10A (n=7) and 1omMCC isolated from Adamts1+/+ (n=5), Adamts1+/- (n=5) and Adamts1-/-/PyMT (n=8) mice over 40 h. b) Maximum invasion cell index, c) time at which maximum invasion was reached and d) rate of cell invasion of PyMT/Adamts1+/+, Adamts1+/- and Adamts1-/- 1omMCC as determined by sigmoidal regression model. Statistical analysis was performed using log- transformed data and One-way ANOVA with Fisher’s LSD post hoc test. Significance was determined if p≤0.05 (**p≤0.005).
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3.3.3. Adamts1 overexpression in mammary epithelial MCF10A cells promotes cell migration over 45 h.
Cell migration and invasion assays were performed using four different MCF10A clones stably overexpressing either Adamts1 or the GFP vector control. Adamts1 expression in these clones was quantified by real-time transcript detection and verified upregulation of Adamts1 in all MCF10A-Adamts1 clones but not in any MCF10A-GFP (Figure 3.4 a).
Cell migration of MCF10A cells occurred at a slower rate than 1omMCC and was therefore assessed for 45 h. Wild type and GFP-overexpressing MCF10A cells displayed similar sigmoidal migration profiles as both migrated slowly for approximately the first 15 h and increased considerably for the next 30-35 h (Figure 3.4 b, grey plot). For MCF10A-Adamts1, cell migration was more rapid than wild type and MCF10A-GFP clones (Figure 3.4 b, purple and green plot, respectively). Assessed at 5 h intervals following the commencement of the assay, MCF10A-Adamts1 cell migration index was significantly higher than parental and MCF10A-GFP at each interval within the first 15 h of the assay (5 h, p=0.036; 10 h, p=0.047; 15 h, p=0.046; One-way ANOVA; Figure 3.4 c). At 20 h and thereafter MCF10A-Adamts1 migration index remained higher than both wild type and GFP-overexpressing MCF10A, and this trended close to reaching statistical significance. No difference in cell migration index was identified between wild type and MCF10A-GFP cells at any time points analysed (Figure 3.4 c). The similarities in cell migration between wild type and MCF10A-GFP, but a different migration of MCF10A- Adamts1 cells imply that neither the transduction nor subsequent selection of transduced cells, affected the migratory behaviour of MCF10A cells and that the difference in migratory behaviour displayed by MCF10A-Adamts1 cells is likely attributed to Adamts1 expression induction.
2 The sigmoid regression model fitted all migration profiles very well with an R coefficient close to 1 (MCF10A wild type, R2=0.999, p<0.0001; MCF10A-GFP, R2=0.999, p<0.0001; MCF10A-Adamts1, R2=0.999, p<0.0001). After applying this model to each sample, we found no significant difference in peak migration cell index reached when comparing the three MCF10A cell types (Figure 3.4 d). Although not significant, in terms of time and rate to peak migration, Adamts1 overexpressing cells had a relatively shorter time to reach peak migration index (Figure 3.4 e) and a corresponding faster migration rate (Figure 3.4 f) than MCF10A-GFP and wild type cells. Together, these suggests an enhancement of migration capacity in MCF10A when expressing Adamts1 and supported the potential pro-migratory role for ADAMTS1 as observed in primary mammary carcinoma isolates.
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a. 0.20
) CT 0.15 ΔΔ -
0.10 expression (2
0.05 Adamts1
0.00 #2 #4 #6 #7 #1 #4 #6 #7 MCF10A-Adamts1 MCF10A-GFP
b. 7
6 MCF10AMCF10A wild wt type MCF10A-GFPMCF10A Adamts1 MCF10A-Adamts1MCF10A GFP 5
4
3
Migration cell index 2
1
0 5 10 15 20 25 30 35 40 45 Time (hours)
Tan IA 74 CHAPTER 3 ADAMTS1 accelerates mammary cancer cell invasion
c. 8 MCF10AMCF10A wildwt type 7 MCF10A-Adamts1MCF10A-GFP MCF10A-GFPMCF10A-Adamts1 6
5 4 * 3 * Migration cell index 2 * * * 1 *
0 5 10 15 20 25 30 35 40 45 Time (hours) MCF10A wt MCF10A GFP d. MCF10A Adamts1 e. f. 0.12 8 80
0.09 6 60
4 40 0.06 (hours) (cell index/hour) 2 20 Rate of migration 0.03
at maximum migration Time Maximum migration cell index 0 0 0.00
Figure 3.4. Migration of wild type MCF10A, MCF10A-Adamts1 and MCF10A-GFP cells over 45 hours. a) Adamts1 expression of four stable MCF10A-Adamts1 and MCF10A-GFP clones used in migration and invasion assays. Expression was normalised to a high Adamts1-overexpressing MDA- ΔΔ MB-231 stable cell line. Data represents 2- CT of triplicate readings (n=1). b) Migration profiles of MCF10A wild type, MCF10A-Adamts1 and MCF10A-GFP cells over 45 h. c) Comparison of
migration cell index of MCF10A cell lines every 5 h until experimental endpoint. Data represents mean ± SEM (Independent experiments, n=4). d) Maximum migration cell index, e) time at which maximum migration was reached and f) rate of migration of MCF10A wild type, MCF10-GFP and MCF10A-Adamts1 as determined by sigmoidal regression model. Statistical analysis was performed using log-transformed data and One-way ANOVA with Fisher’s LSD post hoc test. Significance was determined if p≤0.05 (*).
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3.3.4. Adamts1 overexpression in MCF10A cells had no effect on cell invasion.
Cell invasion through a matrigel barrier was assessed for 45 h and cell lines invaded consistently throughout this period (Figure 3.5 a). Cell invasion ceased at an earlier time point in MCF10A-Adamts1 cohort than MCF10A-GFP or wild type counterparts. However, these differences in cell invasion were not significantly different (Figure 3.5 b). In these experiments, invasion was inherently variable, and this may be caused by small inconsistencies in the matrigel coating or other experimental factors.
To determine the rate and peak invasion of wild type, GFP-overexpressing and Adamts1- overexpressing cells, the sigmoid regression model was once again applied (MCF10A wild type, R2=0.997, p<0.0001; MCF10A-GFP, R2=0.998, p<0.0001; MCF10A-Adamts1, R2=0.995, p<0.0001). Peak invasion impedance (Figure 3.5 c), and the time taken to reach peak invasion (Figure 3.5 d) were not significantly different between the three MCF10A cell lines. Likewise, rates of invasion were also comparable between the different MCF10A cell lines (Figure 3.5 e). These findings therefore indicate that the expression of Adamts1 does not alter the invasive behaviour of MCF10A mammary epithelial cells.
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a. 6
MCF10AMCF10A wild wt type 5 MCF10A-GFPMCF10A Adamts1 MCF10A-Adamts1MCF10A GFP 4
3
Invasion cell index 2
1
0 5 10 15 20 25 30 35 40 45 Time (hours)
5 b. MCF10AMCF10A wild wt type MCF10A-GFPMCF10A-Adamts1 4 MCF10A-Adamts1MCF10A-GFP
3
2
Invasion cell index 1
0 5 10 15 20 25 30 35 40 45 Time (hours)
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MCF10A wt MCF10A GFP c. MCF10A Adamts1 d. e. 8 80 0.15
6 60 0.10 4 40 (hours) 0.05 (cell index/hour) 2 20 Rate of invasion
Time at maximum invasion Time
Maximum invasion cell index 0 0 0.00
Figure 3.5. Invasion of wild type MCF10A, MCF10A-Adamts1 and MCF10A-GFP cells over 45 h. a) Invasion profiles of wild type MCF10A, MCF10A-Adamts1 and MCF10A-GFP cells over 45 h. b) Comparison of invasion cell index of MCF10A cell lines every 5 h until experimental endpoint. Data represents mean ± SEM (Independent experiments, n=4). c) Maximum invasion cell index, d) time at which maximum invasion was reached and e) rate of cell invasion of MCF10A-wild type, MCF10- GFP and MCF10A-Adamts1 as determined by sigmoidal regression analysis.
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3.4. DISCUSSION
That ADAMTS1 is a promoter of cell migration and invasion concurs with its reported activity in non-neoplastic events such as wound healing (Krampert et al., 2005; Su et al., 2008), organ development (Capehart, 2010; Ducros et al., 2007; Mittaz et al., 2004; Rehn et al., 2007) and angiogenesis (Dunlap et al., 2010; Garcia-Conesa et al., 2009; Hatipoglu et al., 2009; Jönsson-Rylander et al., 2005). Similar associations have also been reported in cancer whereby ADAMTS1 stimulates motile and invasive behaviours in tumour cells (Aravindan et al., 2013; Carver et al., 2009; Esselens et al., 2010; Masui et al., 2001; Tyan et al., 2012; Zhang et al., 2010) and in stromal cells supporting tumourigenesis (Ricciardelli et al., 2011; Rocks et al., 2008). In determining whether ADAMTS1 elicits a similar influence on breast cancer cells, to consequently promote metastasis, mammary carcinoma cells and an immortalised human breast cell line were assessed in cell migration and invasion assays following the ablation and induction of Adamts1, respectively.
For many years, in vitro cell migration and invasion have been defined solely through endpoint quantification of migratory and invasive competent cells. However, a heightened capacity to migrate/invade cannot only be defined by an increase in cell number at a fixed time. The rate and period at which these characteristics are manifested are also important measures that more fully define migratory and invasive cell capacity (De Wever et al., 2010; Huang et al., 2005). Assessments of these behaviours in real-time provides a unique opportunity to identify not only the population of cancer cells that have invaded or migrated, respectively, but also the rate and period at which cells displayed these behaviours. A real time method of assessment was applied in our current study, and through this approach we revealed that ADAMTS1 enhanced the migratory capacity of breast cancer cells not by increasing the population of migratory-competent cells but by enabling cell migration to occur faster.
The ability to promote breast cancer cell migration is consistent with our observed role for ADAMTS1 in matrix adhesion and suggests the reduced capacity to adhere to the ECM substratum is likely to be at least a partial mechanistic explanation for the reduced migratory capacity in Adamts1 null breast cancer cells. Matrix adhesion is a major cue for the acquisition of front-rear cell polarity, which dictates the opposing formation of cell-ECM contacts at the leading edge and the dissociation of cell- ECM contacts at the trailing edge (Muthuswamy and Xue, 2012; Yamana et al., 2006). The unidirectional formation of focal ECM contacts and a corresponding build up of actin-cytoskeletal
Tan IA 79 CHAPTER 3 ADAMTS1 accelerates mammary cancer cell invasion
structure at the cell’s leading edge, generate protrusion (Bugyi and Carlier, 2010; Le Clainche and Carlier, 2004; Pollard and Borisy, 2003) and actomyosin contractile forces (Iwadate and Yumura, 2008) that together, mechanically propel a cell forward. In enhancing the capacity of breast cancer cells to adhere to the extracellular matrix and accelerating the rate at which it occurs, ADAMTS1 may effectively initiate a cascade of events in cell migration more immediately, thereby allowing cell migration to advance sooner. A study in osteosarcoma cells supports this notion by its demonstration of increased abundance of ADAMTS1 at the leading edge protrusion interfacing migrating cells and the adjacent matrix (Rehn et al., 2007). A similar localisation of ADAMTS1 orthologue, GON-1, was also reported in nematode Caenorhabditis elegans (C. elegans). In this organism, gonadal morphogenesis, which required the elongation of gonadal structures is initiated by the migration of specialised cells known as leader cells (Blelloch and Kimble, 1999). During active gonadogenesis, GON-1 expression was found specifically at the tip of the leading edge of motile leader cells (Blelloch and Kimble, 1999). Although cell adherence was not assessed in these studies, the locality of ADAMTS1 at the leading edge corresponded to the specific site where focal matrix contacts form.
Changes in migration could reasonably be expected to correspondingly underpin changes in invasion; however, invasion through matrigel was not demonstrably altered by either ablation or overexpression of Adamts1. It has been noted in other studies that cells can switch invasive modality, between utilising either ECM remodelling or adapt amoeboid mechanisms (Farina et al., 1998; Rösel et al., 2008; Sabeh et al., 2009; Sahai and Marshall, 2003; Wolf et al., 2003). The latter mechanism occurs independent of metalloproteolytic enzymes (Grise et al., 2012; Guiet et al., 2011; Hancox et al., 2009; Yokotsuka et al., 2011) and is not governed by cell-matrix interactions and integrin-driven migration (Sahai and Marshall, 2003; Terry et al., 2012; Wolf et al., 2003) like mesenchymal invasion, which necessitates ECM remodelling through proteolytic degradation (Tester et al., 2000; Yu and Machesky, 2012) and integrin-ECM contact (Paulus et al., 1996). Perhaps the invasion exhibited by Adamts1 null cells involved the amoeboid modality. Studies have found that depletion of metalloprotease activity in cancer cells (Meierjohann et al., 2010) including breast carcinoma cells (Wolf et al., 2003) instigate the assumption of amoeboid-like invasive phenotype as a compensatory mechanism to infiltrate the ECM and breach the basement membrane. While exhibiting similar capacity to invade through matrigel, the method of invasion to which Adamts1-expressing and Adamts1 null breast carcinoma cells commit may be different, and perhaps loss of ADAMTS1, instead of hindering cell invasion, altered the mode of invasion from a mesenchymal, protease-dependent pathway to an amoeboid, protease-independent one. This hypothesis is indeed in keeping with the reduced capacity of Adamts1 null cells to adhere to
Tan IA 80 CHAPTER 3 ADAMTS1 accelerates mammary cancer cell invasion
matrix proteins, as inhibition of cell-ECM adhesion can also trigger the assumption of amoeboid invasion by mammary cancer cells (Hancox et al., 2009).
It may also be likely that Adamts does not directly elicit any change in cell invasion, but as an ECM degrading protease, ADAMTS1 may impose its effects on cell invasion indirectly by the processing of protein substrates in the peritumoural stroma. Versican is a chondroitin sulphate proteoglycan and is the most characterised proteolytic substrates of ADAMTS1 (Brown et al., 2010b; Cooley et al., 2012; Fu et al., 2011; Russell et al., 2003). It has repeatedly been shown to positively associate with metastatic disease and/or relapse in breast (Ricciardelli et al., 2002; Suwiwat et al., 2004), prostate (Ricciardelli et al., 2007; Sakko et al., 2001) and ovarian cancer (Voutilainen et al., 2003). The cleavage of versican occurs at its glycosaminoglycan-beta (GAG-β) domain and this liberates a G1 fragment, which has been shown to promote cancer cell invasion (Cattaruzza et al. 2004). In Adamts1-deficient PyMT mammary tumours, reduced metastatic incidence corresponded to a significant reduction of versican cleavage (Ricciardelli et al., 2011). The abrogation of versican cleavage may present as an indirect pathway by which ADAMTS1 alters cancer cell invasion and thus in its absence, as in our in vitro invasion experiments, ADAMTS1 elicits no effect on cell invasion. To elucidate whether this indirect mechanism participates in the metastatic transition of breast cancer cells, further studies on mammary cancer cell invasion are required recapitulating the proteolytic interaction between ADAMTS1 and ECM substrates including versican.
Tan IA 81
Chapter 4
Differential gene expression analysis of mammary tumours derived from PyMT/ Adamts1+/+ and PyMT/Adamts1-/- mice
CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
4.1. INTRODUCTION
The Polyomavirus middle-T (PyMT) antigen is an oncogene that potently induces hyperproliferation leading to cancer development in any tissues where it is introduced (Dawe et al., 1987). Its expression under the Mouse Mammary Tumour Virus (MMTV) promoter specifically induces sporadic and aggressive tumours to develop in the mammary gland, which subsequently spread in the lungs (Fluck and Haslam, 1996; Guy et al., 1992).
Using the MMTV-PyMT transgenic mouse model, our group demonstrated the functional relevance of the metalloprotease ADAMTS1 in breast cancer and in the eventual development of pulmonary metastases. Adamts1 is endogenously expressed in tumours of MMTV-PyMT transgenic mice, where palpable mammary tumours develop at the median age of 14.3 weeks and exceed 3cm3 in size (Ricciardelli et al., 2011). PyMT/Adamts1+/+ mice also grew frequent and numerous pulmonary metastatic lesions, which imposed a high morbidity within this cohort (Ricciardelli et al., 2011). However, in Adamts1 null mice, breast cancer development was substantially alleviated and the advancement to a more adverse tumour grade was delayed. Breast tumours that grew in Adamts1 null transgenic mice were also smaller and mammary tumour cells underwent more programmed cell death and displayed a more cytotoxic immune environment than PyMT breast tumours that expressed the ADAMTS1 protease (Ricciardelli et al., 2011). Of more significance, the lack of ADAMTS1 also impeded the spread and growth of pulmonary metastatic cancers (Ricciardelli et al., 2011), which based on evidence illustrated in preceding chapters, may be attributed to poor cell-matrix contacts and reduced cell motility.
With knowledge of the influence of ADAMTS1 on breast cancer development, growth and metastatic progression, and its involvement in cancer cell apoptosis (Ricciardelli et al., 2011), matrix adhesion (see Chapter 2) and motility (see Chapter 3), what remain unknown are the molecular mechanisms underlying these changes in tumour phenotype. The aim of this current study was to identify perturbations in mammary cancer cell gene expression associated with ADAMTS1 deficiency through comparative microarray analysis of Adamts1+/+ and Adamts1-/- breast tumours derived from MMTV-PyMT transgenic mice.
Microarray gene analysis is an invaluable tool used extensively to differentiate genetic disparities between healthy and diseased tissues. Its application in cancer research has endowed significant revelations towards the genetic signatures that mediate benign vs malignant or, metastatic
Tan IA 83 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
disease. This technology has also been integral in the discovery of prognostic markers and novel targets for cancer prevention. At present, there has not yet been any studies conducted that identified in breast or other types of cancers, the consequences for global gene expression that is primarily attributed to Adamts1. Attaining this new knowledge, will not only unravel some of the molecular mechanisms behind the pro-tumourigenic and pro-metastatic role of ADAMTS1 in breast cancer, but will also provide new insights into the potential molecular mechanisms it influences in other cancers such as prostate (Gustavsson et al., 2009; Jennbacken et al., 2009), endometrial (Keightley et al., 2010), hepatic (Braconi et al., 2009) and pancreatic cancers (Masui et al., 2001) where it is also differentially regulated.
Breast cancers induced by the MMTV-PyMT transgene exhibit close genetic resemblance to luminal-B subtype human breast tumours (Brenton et al., 2005; Herschkowitz et al., 2007; Sørlie et al., 2001; Sorlie et al., 2003), which is associated to poor prognosis due to metastasis complication occurring less than 5 years of diagnosis as well as resistance to endocrine and chemotherapy (Tran and Bedard, 2011). Through analysis of the gene expression profiles, the similarity of Adamts1 null tumours to human breast cancer subtypes can be inferred based on the known gene signatures. Performing this analysis can potentially translate how the loss of Adamts1 in the MMTV-PyMT breast cancer model may be relevant to the clinical prognostic outcomes of human breast cancers. As PyMT/Adamts1-/- mice manifested with less advanced and aggressive breast cancer disease, we predicted that loss of ADAMTS1 reverts the gene signature of PyMT mammary tumours from one resembling luminal B human breast cancer to a sub type that has a better clinical outcome.
Tan IA 84 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
4.2. MATERIALS AND METHODS
4.2.1. Generation of PyMT/Adamts1+/+, PyMT/Adamts1+/- and PyMT/Adamts1-/- breeding colony
An FvBN male mouse carrying the PyMT transgene was mated with a female C57/Bl6/129Sv mix mouse, heterozygote for Adamts1. This parental breeder pair generated an FvBN:C57/Bl6/129Sv F1 progeny that were either positive or negative for the PyMT transgene and were either Adamts1+/+ or
+/- Adamts1 . From this F1 colony, a PyMT+ male mouse and PyMT- female mouse, both heterozygous
- for Adamts1, were mated to generate an F2 progeny positive or negative for PyMT transgene and were either heterozygous, homozygous wild-type or homozygous null for Adamts1. Successive breeding between PyMT+ males and PyMT- Adamts1+/- females, were performed to produce later generations.
4.2.2. Microarray
Mammary tumours were dissected from the left 5th (L5) mouse mammary gland of PyMT/Adamts1+/+ (n=6) and PyMT/Adamts1-/- (n=6) mice and weighed. Only the L5 glands of consistent size were used in order to exclude the possibility of confounding differences in gene expression between the different glands or different tumour sizes. RNA was extracted from tumour tissue using TRIzol® reagent (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia) according to the manufacturer’s instructions then stored at -80 ºC (Figure 4.1 a). Thawed RNA samples were subjected to spectrophotometer analysis using Nanodrop ND-1000 (Thermo Scientific, Scoresby, Victoria, Australia) as previously described (Desjardins and Conklin, 2010), to determine RNA quality. RNA quality is indicated by the A260/A280 absorbance ratio and pure RNA has an A260/A280 ratio between 1.8-2 (Gallagher and Desjardins, 2007). 10 μg/μl RNA was DNAse treated using DNA-free™ Kit (Life Technologies Australia Pty ltd., Mulgrave, Victoria, Australia) as per the manufacturer’s procedure. DNase-treated RNA was again assessed for RNA quality. 3 μl DNase-treated RNA (100 ng/μl) from each PyMT/Adamts1 genotypes were sent to the Adelaide Microarray Facility (Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia) for microarray analysis using the Affymetrix Mouse Gene 1.0ST platform (Affymetrix® Microarray Solutions, Santa Clara, California, USA). A 2 μl
Tan IA 85 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
aliquot of DNAse-treated RNA from each sample was also sent for their independent assessment of RNA quality.
4.2.3. Genotyping
PCR genotyping was performed using DNA extracted from tail biopsies (described in Section 2.2.2) or from 5 ng/μl complementary DNA synthesised from breast tumour RNA extracts. PCR amplified products were visualised by gel electrophoresis in 1% (w/v) agarose (Promega Corporation, Annandale, NSW, Australia) gel made up in 0.5X TBE and 2 μg/ml Ethidium Bromide. PyMT and Adamts1 genotyping was performed as described in Section 2.2.3.
4.2.3.1. BC018473
BC018473 gene detection was determined using tail biopsy DNA and genotyping primers that amplified the region spanning Exon3-Intron3-Exon4 to generate a 1008 bp amplicon. Primer design against published BC018473 genomic sequence (Accession No. NC_000077) was performed using Primer Express Software (Applied Biosystems, Scoresby, Victoria, Australia) and synthesised by Sigma-Aldrich (Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia) (Table 4.1). Each PCR reaction comprised of 5 μl 5x Green GoTaq® Flexi buffer (Promega Corporation, Annandale, NSW, Australia), 4 mM Magnesium Chloride (Promega Corporation, Annandale, NSW, Australia), 0.5 mM dNTP (Promega Corporation, Annandale, NSW, Australia), 0.125 μl 10 mM forward and reverse primers, 1.25 μl
o GoTaq® polymerase and made up to a total volume of 25 μl H2O. Cycling conditions were 95 C for 3 mins, 35 cycles of 95 oC for 30 secs, 56 oC for 0.30 secs and 72 oC for 1.30 min, and lastly, 72 oC for 5 mins.
4.2.3.2. Rnf160 Rnf160 genotyping primers were designed using published genomic sequence (Accession No. NM_001081068) against the region spanning Exon23 to Exon25 to generate a 3185 bp amplicon. Primer designed used Primer Express Software (Applied Biosystems, Scoresby, Victoria, Australia) and synthesised by Sigma-Aldrich (Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia) (Table 4.1). Each PCR reaction comprised of 5 μl 5x Green GoTaq® Flexi buffer, 4 mM Magnesium Chloride, 0.5 mM dNTP, 0.125 μl 10 mM forward and reverse primers, 1.25 μl GoTaq® polymerase and made up to a
Tan IA 86 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
o o total volume of 25 μl H2O. Cycling conditions were 95 C for 3 mins, 35 cycles of 95 C for 30 secs, 56 oC for 0.30 secs and 72 oC for 3.30 mins, and lastly, 72 oC for 5 mins.
Table 4.1 Genotyping PCR primers Gene name Amplicon Primer Sequence (5’-3’) (Gene ID) size Forward GACTCCTAGCCACGAGATGC 1008 bp BC018473 (DNA (Gene ID: 193217) Reverse TTGGTGCACATCCATCAAGT template) Forward CCTGTGCAAATTGCTGCTTA 182 bp Rnf160 (cDNA (Gene ID: 78913) Reverse ACACAGCCCAGGACATTCTC template)
4.2.4. Quantitative RT-PCR
Quantitative real time RT-PCR was performed in triplicate. Custom-made primers against the Adamts1 mRNA (RefSeq# NM_009621) designed using Primer Express (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia) and manufactured by Sigma-Aldrich have been previously described (Dunning et al., 2007). Primers against L19 were commercially available (Qiagen (Mm_Rpl19_1_SG QuantiTect Primer Assay QT00166145, Qiagen Australia Pty Ltd.). cDNA amplification occurred in a 20 μl reaction volume, comprised of 10 μl SYBR Green master mix (Life Technologies Australia Pty Ltd.), 0.2 μl 50 mM Adamts1 forward and reverse primers, or 1.25 μl (L19) and nuclease free water, using 7900HT Fast Real-Time PCR system (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia).
Quantitation of BC018473 transcripts (RefSeq# NR_003364) was performed using Taqman® Gene expression assay (Life Technologies Australia Pty Ltd, Mulgrave, Victoria, Australia) as per manufacturer’s instructions.
PCR cycling conditions were as follows 50 °C for 2 mins, 95 °C for 10 mins and 40 cycles of amplification at 95 °C for 15 secs and 60 °C for 60 secs. Relative expression levels were calculated
Tan IA 87 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
using 2-ΔΔCT and compared against an Adamts1-overexpressing MDA-MB231 cell line or #11, an Adamts1+/+ 1°mMCC. Each qRT-PCR run had a negative control, which lacked a cDNA template.
4.2.5. Statistics
Statistical significance was determined by Student T-test in GraphPad Prism® version 6 (GraphPad Software, La Jolla, California, USA). Non-normally distributed data were log-transformed prior to statistical analysis. Statistical significance was defined at p≤0.05.
4.3. RESULTS
4.3.1. Microarray
Microarray gene expression analysis was performed on six Adamts1+/+ and Adamts1-/- L5 mammary tumours dissected from 16-20 week old MMTV-PyMT transgenic mice (Figure 4.1 a). Samples were matched for total tumour mass as a proportion of body weight (PyMT/Adamts1+/+ (median, 15.44%; range, 9.90-29.57); PyMT/Adamts1-/- (median,12.94%; range 4.77-17.69); Figure 4.1 b) and L5 tumour burden (PyMT/Adamts1+/+ (median, 0.395g; range 0.288-1.025); PyMT/Adamts1-/- (median, 0.398g; range, 0.248-1.245); Figure 4.1 c) to eliminate possible gene transcription changes that may be attributable to differences in pathological state or rate of tumour growth. The Adamts1 genotype of each sample was also verified using corresponding DNA extracts of tail biopsies taken at the time tumours were excised (Figure 4.1 d).
Prior to sending DNase-treated RNA for microarray analysis, RNA quality was determined.
Spectrophotometer analysis demonstrated high purity of all RNA samples with A260-A280 ratios close to 2 (PyMT/Adamts1+/+ (median, 1.985; range, 1.93-2.01); PyMT/Adamts1-/- (median, 2.005; range, 1.87- 2.06); Figure 4.1 e). An additional independent assessment of RNA quality with an Agilent bioanalyser also reported all DNAse-treated RNA to be of high quality (data not shown).
Microarray analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours surprisingly revealed only two differentially expressed genes as determined by the False Discovery Rate (FDR). The
Tan IA 88 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
most highly differentially expressed gene is a non-coding transcript, 129SvJ strain specific marker (Stegalkina et al., 1999) known as BC108473. This transcript was found to be down regulated by almost 160-fold in Adamts1 null compared with Adamts1+/+ tumours (FDR=0.0000557; Table 4.2). The second differentially expressed gene is zinc finger protein, Rnf160 (also referred to as Ltn1; Listerin E3 ubiquitin ligase), which was upregulated by ~3-fold in Adamts1-/- tumours (FDR=0.00507; Table 4.2). Surprisingly, no difference in Adamts1 transcript levels was identified between the two tumour cohorts. The huge similarity in global gene expression between these two cohorts is reflected in the principal component analysis (PCA) plot, which showed considerable overlap between individual Adamts1+/+ and Adamts1-/- samples and no discernible clustering of samples according to their Adamts1 genotype (Figure 4.2).
Table 4.2. Gene expression analysis of PyMT/Adamts1-/- vs PyMT/Adamts1+/+ breast tumours Transcript Fold Rank Gene symbol RefSeq FDR ID change 1 10382846 BC018473 NR_003364 -158.495 5.57E-05 2 10440566 Rnf160 NM_001081068 2.796 0.00506583 3 10393373 St6galnac1 NM_011371 -2.589 0.118442 4 10466521 Gcnt1 NM_173442 -1.720 0.152212 5 10470950 Endog NM_007931 1.229 0.387337 6 10442954 Axin1 NM_009733 1.230 0.452962 7 10541426 Cpamd8 NM_008646 -1.224 0.554741 8 10544252 E330009J07Rik NM_175528 1.454 0.558096 9 10584758 Fam103a1 BC096399 1.254 0.558096 10 10430447 Micall1 NM_177461 1.234 0.558096
818 10440522 Adamts1 NM_009621 -2.427 0.651136
Tan IA 89 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
a.
L1
L2 L3
L4 L5
b. 40
30
20
(% body weight) 10
tumour burden Total
0 Adamts1+/+ Adamts1-/-
c. 1.5
1.0
0.5
L5 gland tumour burden (grams) 0.0 Adamts1+/+ Adamts1-/-
Tan IA 90 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
PyMT/Adamts1+/+ PyMT/Adamts1-/- d. 1497 1499 1507 1702 1988 1702 1680 1888 1914 2018 2026 2070 PRIMERS ko wt ko wt ko wt ko wt ko wt ko wt ko wt ko wt ko wt ko wt ko wt ko wt
e. ) 2.2 280
/A 260 2.0
Good RNA quality 1.8 Poor RNA quality
RNA quality (A RNA
Figure 4.1. Mammary tumour weights and RNA quality of PyMT/Adamts1+/+ and PyMT/Adamts1-/- microarray samples. a) Mammary tumour samples were derived from the left 5th mammary gland of 16-20 week old PyMT/Adamts1+/+ (n=6) and PyMT/Adamts1-/- (n=6) mice. Samples in each cohort were matched for b) total tumour burden as proportion of body weight and c) L5 tumour weight to eliminate possible gene expression changes attributable to difference/s in tumour grade or size. d) Adamts1 genotypes were re-confirmed using DNA-extracted tail biopsies and Adamts1 genotyping primers. e) Spectrophotometer analysis determined all DNAse-treated RNA extracted from L5 mammary tumours to be of high quality, with A260/A280 absorbance ratios of ≈2.
Tan IA 91 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
Figure 4.2. PCA plot of sample distribution based on global expression patterns. The PCA plot is a visual representation of the variation in global gene expression between each sample. PyMT/Adamts1+/+ (blue) and PyMT/Adamts1-/- (red) samples overlapped considerably and did not form distinct groups.
Tan IA 92 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
4.3.2. PyMT/Adamts1-/- mammary tumours express a non-coding transcript of Adamts1
The Adamts1 transcript was detected by microarray using a probe set (Transcript cluster ID 10440522) comprised of multiple oligonucleotides that can hybridise with the 9 different exon regions of full-length Adamts1 (Figure 4.3 a). According to our microarray data, Adamts1 expression was surprisingly not differentially expressed between Adamts1 null and wild type samples (Table 4.1, rank# 818). Of the six PyMT/Adamts1-/- tumour samples, two (#1680 and #1914) displayed expression values as high as Adamts1 wild type tumours (Figure 4.3 b). To verify this finding, Adamts1 expression was validated by qRT-PCR using primers spanning the Exon8-Exon9 region of the Adamts1 mRNA (Figure 4.3 a) and cDNA synthesised from the same stock of DNAse-treated RNA as the microarray samples. Transcript expression levels presented relatively high Adamts1 in all PyMT/Adamts1+/+ samples, which was expected in this cohort (Figure 4.3 c). However, similar to our microarray results, PyMT/Adamts-/- samples #1680 and #1914 once again showed comparable Adamts1 transcript levels as PyMT/Adamts1+/+ (Figure 4.3 c).
The Adamts1 null gene was generated through exon 2 deletion (Mittaz et al., 2004) and was the determining factor that differentiated Adamts1 homozygous wild type, heterozygous and homozygous null animals (Figure 2.1). No ADAMTS1 protein can be synthesised from the Adamts1 null gene and this has been confirmed repeatedly in previous studies (Brown et al., 2006; Mittaz et al., 2004; Ricciardelli et al., 2011; Russell et al., 2003). The absence of exon 2 in the genome of all PyMT/Adamts1 null mice, from which our microarray samples were acquired, was confirmed repeatedly using DNA material extracted from tail and ear biopsies (Figure 4.1 d). For this reason, any transcript transcribed from the Adamts1 null gene is a non-coding form lacking exon 2. To confirm this, Adamts1 genotyping primers targeting the exon 2-exon 3 region (Figure 4.3 d) was performed using cDNA as template and this verified the presence of exon 2 in the Adamts1 transcribed by all PyMT/Adamts1+/+, but its absence in all PyMT/Adamts1-/- cDNA (Figure 4.3 e). Hence, despite evidence of Adamts1 transcription in some Adamts1-/- tumours, the mRNA produce could only be a redundant Adamts1 transcript that lacks a second exon.
Tan IA 93 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
a. qRT-PCR primers ExonExonEx o8n to 8 toExon ExExono 9n amplicon9 aampliconmplicon (148 (148bp)(148bp bp)) Adamts1 transcript Ex 1 Ex 2 Ex 3 Ex 4 Ex 5 Ex 6 Ex 7Ex 8 Ex 9
Microarray probes
b. 8 )
2 #1988
#1993 #1702 #1680 #1499 6 #1914 #1507 #1497
#1888
4 #2018 #2026 #2070
expression value (Log 2
Adamts1
0 Adamts1+/+ Adamts1-/-
c. ) 1.5
-ddCT #1499 #1702 #1680
#1507 1.0 #1497 #1914
expression (2
#1988
Adamts1 0.5 #1993 #1888
#2018
Normalised #2070 0.0 #2026 Adamts1+/+ Adamts1-/-
Tan IA 94 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
Genotyping primers d. Exon 2 to Exon 9 amplicon (156 bp) Adamts1 mRNA Ex 1 Ex 2 Ex 3 Ex 4 Ex 5 Ex 6 Ex 7Ex 8 Ex 9
e. PyMT/Adamts1+/+ PyMT/Adamts1-/-
1497 1499 1507 1702 1988 1993 1914 2070 2026 1888 2018 1680
Figureigure 44.3.3 ConfirmationConfirmation of of Adamts1Adamts1 genotypes.genotypes a) ExonExon regregionsions targetetargetedd by RRT-PCRT-PCR primersprimers and microarray probes. RT-PCR primers anneal to exon 8 (forward primer) and exon 9 (reverse primer) of Adamts1 (green annotations). Microarray identification of Adamts1 used 10 different probe sets, with at least one probe set for each of the 9 exons comprising Adamts1 (orange annotation). Adamts1 transcript expression value identified by b) microarray and c) quantitative RT-PCR. d) Adamts1 wild type genotyping primers anneals at exon 2 (forward primer) and exon 3 (reverse primer) to generate a 156 bp amplicon. This region was deleted in the Adamts1 knockout allele. e) Using a cDNA template synthesised from the same RNA stock, from which microarray samples were derived, the 156 bp was only found in all PyMT/Adamts1+/+ but not in any of the six PyMT/Adamts1-/-. Gel lanes were spliced to arrange samples according to Adamts1 genotype.
Tan IA 95 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
4.3.3. Validation of Rnf160 upregulation in PyMT/Adamts1-/-
Prior to validating the differential expression of Rnf160 between the two Adamts1 cohorts, the target sequence of the microarray probe/s targeted against this transcript was mapped to the reference mRNA sequence of Rnf160 (Accession No. NM_001081068). The Mouse Gene 1.0ST microarray platform has sixteen different probe sets against Rnf160, with each targeting different exons of Rnf160 (Figure 4.4 a and Table 4.3). Of these, only one probe set (Probe set ID 10440566) against exon 17, was found to be upregulated in PyMT/Adamts1-/-. As the microarray expression values for the 29 alternative exons of Rnf160 were highly expressed to a similar extent in both PyMT/Adamst1+/+ and PyMT/Adamts1-/- breast tumour cohorts, the disparity in exon 17 may be consistent with a different splicing event in which exon 17 is missing in the wild type samples. However, no splicing event/s surrounding Rnf160 have been reported that correspond to the particular excision of the exon 17.
Amplification of Rnf160 using cDNA from the same DNAse-treated RNA samples used for microarray analysis and Rnf160-specific primers (Figure 4.4 b), showed all samples expressed similar levels of Rnf160 (Figure 4.4 c). For this reason, further verification assays was not performed.
Table 4.3. Individual microarray probe sets against specific exons of Rnf160 Probe set Target Adamts1+/+ Adamts1-/- FDR ID exon/s 10440578 1-11 7.486±0.312 7.423±0.280 ns 10440576 12 8.267±0.246 8.228±0.300 ns 10440574 13 8.565±0.213 8.421±0.257 ns 10440572 14 8.805±0.214 8.669±0.331 ns 10440570 15 8.813±0.215 9.065±0.254 ns 10440568 16 7.952±0.190 8.544±0.263 ns 10440566 17 6.770±0.121 8.254±0.277 0.00507 10440564 18 8.098±0.232 8.442±0.245 ns 10440562 19 7.497±0.245 7.454±0.354 ns 10440560 20 8.076±0.179 8.079±0.187 ns 10440558 21 8.630±0.219 8.519±0.244 ns 10440556 22 8.364±0.259 8.216±0.257 ns 10440554 23 8.573±0.216 8.411±0.205 ns 10440552 24 8.241±0.311 7.997±0.262 ns 10440550 25 7.554±0.267 7.325±0.232 ns 10440543 26-30 7.036±0.265 7.308±0.261 ns
Tan IA 96 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
a. Rnf160 transcript
Microarray probes Exon 23 to Exon 25 ExonExon 2233 to ExonExon 2525 Genotyping primers Exon amplicon (183 (182bp)(182bp bp) ) b. Rnf160 transcript
PyMT/Adamts1+/+ PyMT/Adamts1-/- c.
1497 1499 1507 1702 1988 1933 1914 2070 2026 1888 2018 1680
Figure 4.4. Rnf160 transcript expression. a) The Rnf160 transcript is comprised of 30 exons and each of these have a corresponding microarray probe set. Of these, a single probe set (dark orange) identified a difference in Rnf160 transcript levels between the two Adamts1 tumour cohorts. b) Genotyping primers for Rnf160 anneal at exon 23 (forward primer) and exon 24 (reverse primer) to generate a 183 bp amplicon. c) Rnf160-specific amplicons were identified in cDNA synthesised from DNAse-treated RNA of both PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours.
Tan IA 97 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
4.3.4. Long non-coding RNA BC018473 is downregulated in PyMT/Adamts1-/- mammary tumours
BC108473 is a non-coding transcript derived from a gene found to be specifically abundant in 129/Sv-strain (Stegalkina et al., 1999). Microarray oligoprobes against this transcript were targeted against exon 4 (Figure 4.5 a). Thus, to validate the differential expression of this transcript between the two Adamts1 mammary tumour cohorts, qRT-PCR was performed using primers designed to amplify exon 3-exon 4 (Figure 4.5 a). Microarray analysis revealed BC108473 was significantly downregulated by ~158-fold in PyMT/Adamts1-/- when compared to PyMT/Adamts1+/+ breast carcinomas (Figure 4.4 b). Confirmation through RT-PCR concurred with the differential expression of BC018473 with variable levels in PyMT/Adamts1+/+ (median, 1.06; range, 0.34-3.250) but was undetectable in all PyMT/Adamts1-/- samples (median, 0; range, 0-0.01; Student t-test, p=0.0003). Importantly, the same pattern in BC018473 disparity between Adamts1+/+ and Adamts1-/- was reflected at the genomic level, with PCR identification of an amplicon, corresponding to the exon 3-intron 3- exon 4 region of BC108473 gene (Figure 4.5 d) found in Adamts+/+ but not in Adamts1-/- samples (Figure 4.5 e).
Tan IA 98 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
qRT-PCR primers a. ExonExon 3 to ExonExon 44 ampliconampampliconlicon (130(130bp) bp) BC018473 mRNA Ex 1 Ex 2 Ex 3 Ex 4
Microarray probes b. 15 )
2 ****
10
expression value (Log 5
BC018473
0 Adamts1+/+ Adamts1-/- c. 4 ***
)
CT ΔΔ - 3
2
expression (2
1
BC018473
0 +/+ -/- Adamts1 Adamts1
Tan IA 99 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
d. Genotyping primers ExonExon 3 toto Exon Exon 4 4ampamplicon ampliconlicon (685bp)(686 8(10085b5bp)p) bp) BC018473 gene Ex 1 Ex 2 Ex 3 Ex 4
PyMT/Adamts1+/+ PyMT/Adamts1-/- e. 2026 2070 1497 1499 1507 1702 1988 1993 1680 1888 2018 1914
Figure 4.5. Downregulation of BC018473 transcript in PyMT/Adamts1-/- mammary tumours. a) Schematic representation of regions targeted by Taqman primers and microarray probe set. Taqman primers (green annotations) were designed against the exon 3 and exon 4 region of the BC018473 transcript. BC018473 was identified by microarray by a single probe set designed against exon 4 (orange annotations). PyMT/Adamts1-/- mammary tumours express low BC018473 transcript levels than PyMT/Adamts1-/- as determined by b) microarray and confirmed by c) RT-PCR. d) Schematic representation of target region of BC018473-specific PCR primers. These were designed to anneal to exon 3 (forward primer) and exon 4 (reverse primer) of BC018473 to generate a 1008 bp amplicon. e. BC018473 PCR amplicon was identified only in PyMT/Adamts1+/+ but not in PyMT/Adamts1-/- mammary tumours. Gel lanes were spliced to arrange samples according to Adamts1 genotype.
Tan IA 100 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
4.3.5. Strain imbalance between Adamts1 null and Adamts1+/+ transgenic mice
The discovery of BC018473 downregulation in the Adamts1+/+ PyMT tumours revealed an intrinsic genetic variation between Adamts1+/+ and Adamts1-/- populations in this study. The known strain specific presence of the BC018473 gene suggests a mouse strain distinction between Adamts1 null and Adamts1+/+ genotype mice. This was unexpected as both cohorts were derived from the same inbred mouse colony. To further investigate this finding and delineate a possible cause of this strain imbalance, we traced back the strain lineage of the parental breeders in this mouse colony.
The breeding strategy applied to produce PyMT transgenic, Adamts1+/+, Adamts1+/- and Adamts1-/- female mice is depicted in Figure 4.6 a and as described in Section 4.2.1. All microarray samples of each Adamts1 genotype were drawn from F2 progeny from this mixed strain breeding program. As a 129 strain marker, the expression of the BC018473 mRNA and the presence of this gene in the genomic DNA of only the Adamts1+/+ cohort indicated an imbalance in the distribution of the background strain between our two genotype groups and that Adamts1+/+ animals inherited a more 129/Sv mouse strain than Adamts1-/- littermates. This finding is of critical importance to the interpretation of the microarray experiment as the comparison of global gene expression patterns between two genotypes must assume that all background genes apart from that being investigated are equally or randomly inherited between groups. The lack of strain homogeneity identified confounds the attribution of any differential gene expression to an effect of ADAMTS1. Importantly the discovery that the background genetics are non-uniformly distributed between Adamts1 genotypes suggests that the genetic background in the F2 generation of mice was highly variable between individual mice most likely introduced variation into the expression analysis. This finding may explain the surprising paucity of differentially expressed genes identified between the two genotypes.
4.3.6. Cell-based experiments and published mouse cohorts possess homogenous strain mixing in Adamts1+/+ and Adamts1-/- mice.
The finding that Adamts1 genotype was in linkage disequilibrium with BC018473 and thus potentially linked to a wider strain specific gene set led us to investigate whether the same disparity between Adamts1 genotypes existed in our other studies with similar mixed strain breeding colonies. The 1omMCC described in Chapters 2 and 3 of this study (collectively referred to henceforth as the
Tan IA 101 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
Xcelligence cohort) were extracted from female mice of the same breeding colony as the microarray cohort. A similar investigation of BC018473 transcript expression between wild type and knockout mice, determined that BC018473 abundance was not significantly different between PyMT/Adamts1+/+ and PyMT/Adamts1-/- samples. Likewise, both Adamts1 genotype cohorts used in our published study (referred to henceforth as the AmJPathol, 2011 cohort) of in vivo tumour development in Adamts1-/- (Ricciardelli et al., 2011) mice did not exhibit this disparity in BC018473 mRNA. It is important to note that the AmJPathol, 2011 cohort originated from a different F0 parental pair than the microarray and xCelligence cohorts.
It was in fact the PyMT/Adamts1+/+ microarray tumour cohort that was distinctly different, as BC018473 expression was not elevated in other PyMT/Adamts1+/+ cohorts (Figure 4.5 b), while Adamts1 null expression of this strain marker was found consistently negligible across the three cohorts (Figure 4.5 b).
Tan IA 102 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
COHORT COHORT TUMOUR MICROARRAY MICROARRAY
+/-
+/+
Adamts1 -/- / / + +
FvBN:C57/ Bl6/129sv PyMT FvBN PyMT Adamts1 +/- -
- or Adamts1 or +/- Adamts1 or
+/+ Adamts1 PyMT+ or PyMT or
PyMT+ or PyMT FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv +/+ /
- Adamts1 +/- +/-
1 PyMT F Bl6/129sv Bl6/129sv Adamts1 Adamts1 Adamts1 FvBN:C57/ / - C57/Bl6/129sv C57/Bl6/129sv
2 PyMT
0 F F
COHORT COHORT TUMOUR xCELLIGENCE
+/-
+/-
+/-
+/+
Adamts1 -/- -/- / /
Adamts1 + + -/- / - Adamts1 / -
FvBN:C57/ Bl6/129sv PyMT Adamts1 FvBN PyMT +/- FvBN:C57/ Bl6/129sv PyMT FvBN:C57/ Bl6/129sv PyMT -
- - - or Adamts1 or or Adamts1 or or Adamts1 or +/- +/- +/- Adamts1 or -
+/+ Adamts1 Adamts1 Adamts1 PyMT+ or PyMT or or or
PyMT+ or PyMT PyMT+ or PyMT
PyMT+ or PyMT FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv +/+ +/+ FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv +/+
/
- Adamts1 +/- +/- +/- +/-
1 PyMT F Bl6/129sv Bl6/129sv Bl6/129sv Bl6/129sv Bl6/129sv Bl6/129sv Adamts1 FvBN:C57/ Adamts1 Adamts1 Adamts1 Adamts1 Adamts1 / FvBN:C57/ Adamts1 FvBN:C57/ - / / - - C57/Bl6/129sv C57/Bl6/129sv
4 2 3 PyMT PyMT PyMT
F 0 F F
F
,2011 ,2011 , 2011 , 2011 cohort #2: xCelligence assay Tumour cohort #3: Microarray Tumour COHORT COHORT COHORT TUMOUR TUMOUR
Am J Path Am J Path
+/-
+/-
+/- +/-
+/+
Adamts1 -/- -/- / /
Adamts1 + + -/- -/- / Adamts1 - / Adamts1 + / -
FvBN:C57/ Bl6/129sv PyMT Adamts1 FvBN PyMT +/- FvBN:C57/ Bl6/129sv PyMT FvBN:C57/ Bl6/129sv PyMT FvBN:C57/ Bl6/129sv PyMT Am J Pathol -
- - - - or Adamts1 or or Adamts1 or or Adamts1 or Adamts1 or +/- +/- +/- +/- Adamts1 or -
+/+ Adamts1 Adamts1 Adamts1 Adamts1 PyMT+ or PyMT or or or or
PyMT+ or PyMT PyMT+ or PyMT
PyMT+ or PyMT PyMT+ or PyMT FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv +/+ +/+ FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv FvBN:C57/Bl6/129sv +/+ +/+
/
- Adamts1 +/- +/- +/- +/- +/-
1 PyMT F Bl6/129sv Bl6/129sv Bl6/129sv Bl6/129sv Bl6/129sv Bl6/129sv Bl6/129sv Bl6/129sv Adamts1 FvBN:C57/ Adamts1 Adamts1 Adamts1 Adamts1 FvBN:C57/ Adamts1 Adamts1 Adamts1 FvBN:C57/ / Adamts1 FvBN:C57/ / - / / - - - C57/Bl6/129sv C57/Bl6/129sv
4 5 2 3 PyMT PyMT PyMT PyMT
0 F F F F Tumour cohort #1: Tumour F
a.
Tan IA 103 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
b. ** 4 ) **
-ddCT ns 3
2
1 1.0
BC018473 expression (2 0.5 0.0 AmJPathol, 2011 cohort Xceligence cohort Microarray cohort
Figure 4.6 Unequal representation of BC018473 is unique to the microarray cohort. BC018473 is a marker for the 129Sv mouse strain. a) Schematic diagram of breeding strategy performed to generate PyMT/Adamts1+/+, PyMT/Adamts1+/- and PyMT/Adamts1-/- mice. The PyMT transgene originated from an FvBN male mouse wild type for Adamts1. To generate Adamts1 heterozygous and null mice, PyMT +ve Adamts1+/+ FvBN male mouse was mated with a PyMT –ve Adamts1+/- C57/Bl6/129SvJ female. Progenies of the parental breeder pair were mated to generate successive generations of PyMT/Adamts1+/+, PyMT/Adamts1+/- and PyMT/Adamts1-/- on a C57/Bl6129SvJ/FvBN background. This breeding strategy was employed to generate PyMT mammary tumour samples used for experimental studies. Tumour cohort analysed in a previously published data were derived from F and F progenies (Tumour cohort#1: Am J Pathol, 2011). Tumour cohort used in real-time 4 5 cell based experiments (Tumour cohort#2: xCelligence assay) belonged to the F4 generation, while samples used in microarray analysis (Tumour cohort#3: Microarray) were all F2 progenies. b) bc018473 expression in Adamts1 wild type (blue) and null mammary tumours (red) belonging to the three different tumour cohorts. Expression was normalised to a mouse L19 internal control and calibrated against a PyMT/Adamts1 +/+ primary cell line (#1497). Bar represents mean. Statistical analysis was performed using One-way ANOVA and Fisher’s LSD post-hoc test and significance is defined if p≤0.05 (**p≤0.005).
Tan IA 104 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
4.4. DISCUSSION
Comparative gene analyses through DNA microarray have been advantageous in revealing unknown molecular mechanisms that underpin pathologies such as cancer development and metastasis. This technology has yet been utilised to understand the molecular changes attributed solely to ADAMTS1 in breast cancer metastasis, with which it is strongly associated with (Kang et al., 2003; Kuno et al., 2004; Liu et al., 2006c; Minn et al., 2005; Ricciardelli et al., 2011). Hence, through microarray gene expression analyses of mammary cancers derived from PyMT/Adamts1+/+ and PyMT/Adamts1-/- female mice, this study aimed to reveal the molecular pathways altered by the deletion of Adamts1 in breast tumours. As the global gene transcriptome of MMTV-PyMT mammary tumours parallel those of human luminal-B breast cancer (Brenton et al., 2005; Herschkowitz et al., 2007; Sørlie et al., 2001; Sorlie et al., 2003), which is clinically associated with poor patient outcome (Tran and Bedard, 2011), we also sought to identify whether the loss of Adamts1 imposed a change in global gene signature from an aggressive human breast cancer sub-type to that resembling a subtype with a better prognosis (e.g. luminal-A).
Surprisingly, over the 21,041 gene transcripts targeted in our microarray platform, only two were differentially regulated. The foremost differentially regulated transcript was BC018473, which was downregulated in PyMT/Adamts1-/- tumours by ~156-fold, followed by Rnf160, which is a zinc finger ubiquitin ligase upregulated in the knockout cohort by ~2.7-fold. The finding that Adamts1 was not differentially regulated was consistent with residual expression of a missense transcript in Adamts1 null mice with the absence of exon 2 in the PyMT/Adamts1-/- samples as expected. The loss of exon 2 deletes a part of the protease domain of the protein and alters the reading frame sequence of the Adamts1 and hence complete loss of the protein has been confirmed (Mittaz et al., 2004; Russell et al., 2003).
BC018473 is a long non-coding transcript found in Chromosome 11, and has currently no known functional role in physiology or pathology. It was initially described as a set of unique exons in the 5’ long terminal repeat upstream of the coding sequence of mammary tumour virus 3 (MTV3), with transcripts only existing in 129/SvJ mice but not in the C57Bl/6 cohort (Stegalkina et al., 1999). The unique expression of this non-coding transcript in 129SvJ provided the initial evidence demonstrating the 129-strain specificity of the BC018473 transcript. Subsequent findings followed in microarray analyses conducted by other groups in which high BC018473 transcript levels were found exclusively in
Tan IA 105 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
substrains of 129 (129S2, 129SV and 129B6/FVB) but not in C57, A/J, AKR/J, C3He/FeJ or DBA2 mice (Table 4.3) (Henrichsen et al., 2009; Kõks et al., 2009; Zhu et al., 2011).
As a 129-strain specific gene, the disequilibrium of BC018473 found in Adamts1+/+ and Adamts1-/- mammary tumours indicated a strain imbalance between the two mouse cohorts analysed. With BC018473 highly transcribed, and the gene only detected in PyMT/Adamts1+/+ mice, this indicated our wild-type progeny preferentially inherited this aspect of the 129 strain background over their knockout littermates. The lack of background homogeneity across the two Adamts1 genotypes was a significant confounding factor that may influence gene expression between PyMT/Adamts1+/+ and PyMT/Adamts1-/- tumours in an unpredictable fashion. Potential change/s in gene expression that could perhaps be attributed to Adamts1 were therefore undermined by the fact that the genetic backgrounds were not equal in the two experimental groups of animals.
The lack of strain homogeneity between wild type and knockout animals was unique to the microarray cohort. PyMT/Adamts1+/+ and PyMT/Adamts1-/- mice analysed in other experiments, such as the ones referred to in this project as well as in previous work do not present with a difference in BC018473 transcript expression between the genotypes. More specifically, it was the PyMT/Adamts1+/+ microarray cohort that stood apart from all other cohorts, as Adamts1 wild type and knockout mice from other studies have no or negligible levels of BC018473, similar to the PyMT/Adamts1-/- microarray cohort. How the co-inheritance of the Adamts1 gene and the 129 strain occurs is fascinating considering that the genes are on different chromosomes, but identifying the mechanism for genetic linkage was beyond the scope of my project.
For this microarray study, matched tumour bearing mice were selected from F2 progeny after a single backcross of the offspring from FVBn;PyMT+/Adamts1-/- male and C57Bl/6;PyMT-/Adamts1+/- female progenitors. The cohorts utilised in other studies presented far more uniform BC018473 expression. It is possibly important that these cohorts were either F4 or F5 generations; however, disequilibrium in the inheritance of any gene linked to the genotype of the Adamts1 locus could not have been predicted.
Tan IA 106 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
Table 4.4. BC108473 microarray expression across different mouse strains detected in other studies ) 2 129B6/ 9.5 4.5 15 FVB 129S2
129SV
4.0
7.5 10
BC018473
expression 3.5 C57
C3He/ C57BL/
5.5 A/J AKR/J FeJ 6J DBA2/J 5 FVB 3.0 Balb/C
3.5 2.5 BC018473 microarray expression value (Log 0 Affymetrix Mouse Affymetrix Mouse Microarray Affymetrix Mouse Genome 430 2.0 Array Genome 430 2.0 Genome 430A 2.0 platform Array Array GEO accession GSE10744 GSE15293 GSE23938 number (Kõks et al., Reference (Henrichsen et al., 2009) (Zhu et al., 2011) 2009) • Female mice • 15-18 week old • Tissue: primary male wfs1 wild- mouse mammary • 11-14 week old male mice type and tumours derived from • Tissues (pooled): lung, kidneys, brain, deficient mice Samples 8 transgenic models heart, testis and liver • Tissue: • 129B6/FVB (1 model; • n=18 for each mouse strain temporal lobe n=5) • 129SV (n=19) Balb/c (1 model; n=7) C57 (n=18) FVB (6 models; n=29)
Tan IA 107 CHAPTER 4 Expression analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- mammary tumours
The second differentially regulated gene in PyMT/Adamts-/- mice, Rnf160 is a listerin E3 ubiquitin ligase and a component of the ubiquitin protein degradation machinery (Bengtson and Joazeiro, 2010). The upregulation of Rnf160 in PyMT/Adamts1-/- mammary tumours was not consistently observed in different probe sets that targeted this transcript and suggests that a splice variant lacking exon 17 may have been underrepresented in the Adamts1+/+ mice. Since a background strain imbalance was also evident within our microarray samples, attribution of the upregulation of Rnf160 to the difference in strain background rather than to the loss of ADAMTS1 cannot be ruled out.
In this study, the effect of genetic variations between different strains unfortunately corrupted the capacity to identify gene expression changes that may be attributable to ADAMTS1. It is therefore warranted that our microarray study be replicated using mice from later generations, such as an F4 or F5 progeny which the present study showed eliminates any strain-specific bias in the two Adamts1 genotype populations allowing isolation of the effects of ADAMTS1 on the global gene signature and molecular pathways underlying its role in mammary tumourigenesis and metastasis.
Tan IA 108
Chapter 5
Conclusion and Future Directions
CHAPTER 5 Conclusion and future directions
Cancer cells acquire essential molecular changes that promote the atypical remodelling of the peritumoural ECM environment, which ultimately permit metastatic disease progression. The upregulation of metalloproteases, enzymes involved in extracellular protein degradation, is a vital molecular event that accompany and support the transition to metastasis. Metalloproteases facilitate the significant remodelling of the peritumoural stroma by breaking down the structural components of the ECM and disintegrating the tissue framework that would otherwise inhibit cancer cell escape and restrict cell motility. In breast cancer, increased abundance of the metalloprotease ADAMTS1 strongly correlated with metastasis (Bonuccelli et al., 2009; Casimiro et al., 2012; Kang et al., 2003; Kuno et al., 2004; Liu et al., 2006c; Lu et al., 2009; Minn et al., 2005; Ricciardelli et al., 2011). This had been demonstrated through comparative gene expression analysis between breast cancers with either no, weak or strong metastatic capacity (Casimiro et al., 2012; Kang et al., 2003; Minn et al., 2005) and in various experimentally-induced metastasis models (i.e. intravenous injection of breast cancer cells) (Kuno et al., 2004; Liu et al., 2006c). The pro-metastatic influence of ADAMTS1 was also observed in an in vivo model of breast tumourigenesis whereby the loss of Adamts1 significantly impeded the aggressive advancement and eventual metastasis of MMTV-PyMT breast tumours (Ricciardelli et al., 2011).
This project undertook both targeted and holistic approaches to understand the underlying events imposed by ADAMTS1 in breast cancer metastasis; with much focus on its influence on the integral characteristics acquired by mammary tumour cells to facilitate metastasis. Results from this project associated a novel characteristic with ADAMTS1 that in turn may potentially underlie its role in metastasis.
5.1. Promoted cell matrix adhesion is a novel prometastatic behaviour associated with the ADAMTS1 metalloprotease
Through real time assessment of cell adhesion, migration and invasion, this study demonstrated that ADAMTS1 enhances mammary cancer cell adhesion to structural proteins that make up the ECM and basement membrane. This enhanced ability to adhere to the ECM underpinned the promoted migratory behaviour observed in mammary cancer cells expressing this protease by providing points of contact that instigate the determination of front-rear polarity (Hegerfeldt et al., 2002; Pignatelli et al.,
Tan IA 110 CHAPTER 5 Conclusion and future directions
2012; Ridley et al., 2003) and traction sites necessary for the translocation of cancer cell in the ECM (Hegerfeldt et al., 2002; Pignatelli et al., 2012; Ridley et al., 2003). As cell-ECM adhesion rely mainly on membrane-bound integrin receptors, ADAMTS1 may potentially promote this metastatic cell characteristic by augmenting integrin binding to extracellular protein ligands. Potentially, ADAMTS1 may conduct its role in cell-ECM adhesion by acting as a linker protein between membrane-bound integrins and structural matrix proteins (Figure 5.1 a) or through upregulation of integrins (Figure 5.1 b) (discussed in detail in Section 5.2).
Unlike the observed effects on matrix adhesion and migration, the capacity of breast carcinoma cells to invade through matrigel seemed unaffected by ADAMTS1 ablation. This finding was not surprising as in the absence of matrix interaction and metalloprotease activity, metastatic cells often retain the capacity to invade by assuming an amoeboid-mode of invasion (Grise et al., 2012; Guiet et al., 2011; Hancox et al., 2009; Yokotsuka et al., 2011). The use of a strong and chemically complex chemoattractant in the in vitro invasion assays may have been able to activate more than one invasive mechanism that the cells used. The presence of ADAMTS1, promoted cell adherence to the ECM matrix and a mesenchymal mode of invasion, which requires metalloprotease activity (Figure 5.1 c). This is consistent with the observed degradation of ECM structure surrounding invasive tumours in the mammary gland. In contrast, in the absence of ADAMTS1, adhesion to the ECM is impaired (Figure 5.1 d) and may switch cancer cell invasion to an amoeboid-like mechanism (Hancox et al., 2009) (Figure 5.1 e).
In attempting to gain mechanistic understanding into how ADAMTS1 mediated its effect in cancer cell adhesion as well as elucidate the key differences in gene expression and molecular pathways attributed to ADAMTS1 in breast cancer, microarray gene analysis was performed. Unfortunately, this comparative and comprehensive analysis revealed a biased inheritance of a strain specific gene between the PyMT/Adamts1+/+ and PyMT/Adamts1-/- cohorts, which indicated non- homogeneity in strain between our two tumour cohorts. The disparity in strain corrupted the underlying genetic effects attributable to Adamts1 and thus warrants repetition using mouse progenies in which strain inheritance is uniformly distributed. Due to the unforeseen and unavoidable strain difference found between our two Adamts1 microarray tumour cohorts, future studies are still required to identify the mechanisms, which underpin the functional role of ADAMTS1 in mammary cancer cell-ECM adhesion and more importantly, its effect in mammary tumorigenesis and metastatic disease as a whole.
Tan IA 111 CHAPTER 5 Conclusion and future directions
5.2. Potential mechanisms underlying the role of ADAMTS1 in cell-ECM adhesion
Interactions between cells and the ECM are carried out by membrane-bound integrin receptors. Integrin-ligand binding stimulates actin polymerisation around focal adhesions, which mediate the formation of cellular protrusion (or lamepodia) and produces an intracellular cytoskeleton required for generating motile forces. It also transduces a signalling cascade that activate gene expression for cell motility (Clark et al., 1998; Hotchin and Hall, 1995; Price et al., 1998; Ren et al., 1999; Ridley and Hall, 1992). Integrin binding was initially defined exclusively by the presence of an RGD tripeptide motif in its ligands ((Pierschbacher and Ruoslahti, 1984). However, subsequent studies have later found an array of non-RGD binding regions, which many integrins have affinity to (reviewed in (Humphries et al., 2006; Plow et al., 2000; Ruoslahti, 1996). These non-RGD integrin binding motifs are found in other proteins including adhesion molecules, thrombospondin, disintegrins and metalloproteases (reviewed in (Humphries et al., 2006; Kamiguti et al., 1998; Lu et al., 2007; Plow et al., 2000; Ruoslahti, 1996).
Many studies have implied a cooperative relationship between metalloproteases and integrins in cell-ECM adhesion and migration. For instance, mammary cancer cells exhibited co-localised expression of MMP13 with α1, α2 and β1 integrins when attached to collagen 1 (Ibaragi et al., 2011), while migration of metastatic breast cancer cells required the co-expression αvβ3 and MMP9 (Rolli et al.,
2003). The association of MMP2 with β1 integrins also promoted astrocyte motility by instigating the reorganisation of the actin cytoskeleton and formation of lamellipodia and filopodia structures (Ogier et al., 2006). Furthermore, loss of function in other members of the ADAMTS metalloprotease family such as ADAMTS18 (Wei et al., 2010) and ADAMTS12 (Beristain et al., 2011) resulted in diminished cell adherence to structural proteins that make up the ECM as well as reduced integrin activity. The mechanism by which ADAMTS1 promotes cell-ECM adhesion is not known. However, as integrin binding is essential to ECM adhesion, ADAMTS1 may likely promote cell adhesion by increasing integrin-ligand interaction between mammary cancer cells and the surrounding ECM.
ADAM12, a closely related metalloproteinase to ADAMTS1, was previously reported to support
cell-matrix contacts by directly interacting to HSPG proteoglycans in the ECM and αvβ3 integrins on
mammary cancer cells (Iba et al., 1999). αvβ3 integrins specifically bound to the cysteine-rich region of ADAM12 and led to the polymerisation of actin-containing projections that enabled cell migration (Iba et al., 1999). . Integrin binding, which occur canonically with ligands that possess an RGD tripeptide motif
Tan IA 112 CHAPTER 5 Conclusion and future directions
can also occur with proteins with non-RGD motifs (reviewed in (Humphries et al., 2006; Plow et al., 2000; Ruoslahti, 1996). Within the third thrombospondin motif of ADAMTS1 is an ECD tripeptide region, which has been characterised as an integrin recognition motif to many ADAM metalloproteases (Lu et al., 2007). The ECD tripeptide sequence is a motif which αvβ3 (Yokotsuka et al., 2011), αvβ5 (Yu and
Machesky, 2012) and α6β1 (Tester et al., 2000) integrins have affinity for. Since ADAMTS1 is anchored to the HS side chains of HSPG in the ECM (Kuno and Matsushima, 1998), the presence of this known integrin recognition site within ADAMTS1 could suggest a direct interaction between ADAMTS1 in ECM and integrins in mammary cancer cells, thereby presenting a parallel mechanism to ADAM12.
Alternatively, ADAMTS1 may promote cell-ECM adherence by upregulating integrin expression (Figure 5.1 b). This mechanism is akin to the observed integrin upregulation induced by another ADAMTS metalloprotease, ADAMTS12 (Beristain et al., 2011). ADAMTS12 promoted the expression of
αvβ3 and αv integrins in trophoblasts to consequently enhance trophoblast adhesion to ECM proteins, collagen II and collagen IV (Beristain et al., 2011). In glioblastoma cells, the downregulation of secreted protease, MMP9 resulted in a concomitant inhibited expression of several integrins including α6, α10, αv,
β1 and β3, and impaired cell adhesion to collagen I, vitronectin, fibronectin and laminin matrices (Veeravalli et al., 2010). Consequently, inhibited MMP9 expression also impaired the migratory potential of glioblastoma cells (Veeravalli et al., 2010). Elucidating whether ADAMTS1 facilitate its role in cell- ECM adhesion by inducing integrin overexpression can be determined by surveying the expression of α and β integrin sub-unit in ADAMTS1 expressing and deficient mammary tumours.
Whether ADAMTS1 facilitates its effect on cell-ECM adhesion by directly interacting to integrins or inducing integrin overexpression necessitate further studies. Nevertheless, the role of ADAMTS1 in promoting cell adherence to the surround extracellular environment dictates its potential role in cell migration and invasion. In contributing new understanding towards this metastatic cell characteristic, the underlying function of ADAMTS1 in metastasis can be extended, not only in breast cancer malignancy but also in other cancer types where ADAMTS1 mediates metastasis..
Tan IA 113 CHAPTER 5 Conclusion and future directions )
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mammary carcinoma cells. cells. carcinoma mammary acting as a linker protein that binds to integrin receptors on the cell surface and the ECM proteins, in particular HS HS residues particular in proteins, ECM the and surface cell the on receptors integrin to binds that protein a as linker acting
-/- a) upregulating the expression of integrin receptors, which in turn increases the number of adhesion receptors able to form conta form to able receptors adhesion of number the increases turn in which receptors, integrin of expression the upregulating
th With impaired. is ECM the to adhesion cell carcinoma mammary b) With an enhanced capacity and possibly increased affinity to adhere to the ECM, cell migration begins, and with the secretion secretion the with and begins, migration cell ECM, the to adhere to affinity increased possibly and capacity enhanced an With Adamts1 d)
c)
and PyMT/ the ECM either by by either ECM the the ECM. ECM. the the in abs contrary, In invasion. of mesenchymal-mode a assume and ECM the surrounding degrade cells carcinoma mammary ADAMTS1, ADAMTS1, diminished. also is cells, carcinoma mammary deficient cells and thus cancer of still mode abilit invasion, the retain amoeboid HSPG, or by Figure Figure 5.1. The role in cancer and how cell of mammary adhesion ADAMTS1 influence it the and invasionmigration of PyMT/ d) d) ADAMTS1-independent adhesion
Tan IA 114 CHAPTER 5 Conclusion and future directions
5.3. Clinical significance
The most pernicious stage in cancer is metastasis. Breast cancer mortality due to metastatic disease accounts for more than 80% deaths in breast cancer patients (Greenberg et al., 1996; Jung et al., 2012; Tai et al., 2004). This high mortality has been a major motivation for many studies including ours to further the knowledge behind the mechanistic events leading to this advanced disease stage in breast cancer malignancy.
Basal-like, HER2+, luminal A and luminal B are four different subtypes of human breast cancers defined through their intrinsic genetic profile (Fan et al., 2006; Sørlie et al., 2001; Sorlie et al., 2003). Of these four subtypes, basal-like, HER2+ and luminal B breast cancers have the poorest prognosis, with luminal-B breast cancers possessing the most resistance to chemotherapy (Bhargava et al., 2010; Carey et al., 2007; Esserman et al., 2012; Rouzier et al., 2005) and associated with the highest recurrence rate of metastatic disease occurring within 5 years since becoming disease-free (Fan et al., 2006). The MMTV-PyMT transgenic model for breast cancer produces aggressive and highly metastatic murine breast tumours that share considerable genetic resemblance to human luminal-B breast cancers (Herschkowitz et al., 2007). Through this model cancer research can further our understanding of the aberrant molecular pathways that promote oncogenesis and metastasis of luminal B tumours.
Findings presented in this project elucidate the potential mechanisms that are directly influenced by ADAMTS1 to promote breast cancer metastasis. As the abrogation of ADAMTS1 leads to the attenuation of tumourigenesis, delay in tumour advancement and inhibition of metastatic occurrence in MMTV-PyMT transgenic mice (Ricciardelli et al., 2011), this new knowledge implicates the functional role of ADAMTS1 in promoting the metastatic cell behaviour that could be targeted in advanced luminal- B breast cancers. By targeting the inhibition of ADAMTS1, mammary carcinoma cells dissemination could potentially be delayed, thereby impeding the progression of metastasis and augmenting the efficacy of existing therapeutic agents designed to kill cancer cells about to enter the metastatic cascade.
Tan IA 115 CHAPTER 5 Conclusion and future directions
5.4. Limitations, future directions and summary
Metastatic disease stage requires a series of complex events beginning with the escape of cancer cells from the primary tumour mass and subsequent dissemination into the vascular supply. In order for the cascade of events involved in metastasis to occur, cancer cells require phases of increased adherence to the ECM and enhanced abilities to move within and invade through the ECM.
Cell adhesion to the peritumoural matrix is an integral component of many events in metastasis including the migratory and invasive behaviour of cells. The ability of cancer cells to adhere to the surrounding environment is essential for the determination of front-rear cell polarity driving cancer cell migration (Cao et al., 2007; Mi et al., 2007; Muthuswamy and Xue, 2012; Yamana et al., 2006), which is in turn necessary for cell invasion. Attachment of cells at the leading migratory edge to ECM barriers such as the basement membrane also promotes the activation of proteolytic enzymes (Kirmse et al., 2011) that breakdown the structural constraints restricting cell movement and allowing cancer cells to traverse through its surrounding extracellular environment or penetrate the tissue boundary (Haberern and Kupchik, 1985; Kirmse et al., 2011; Kramer et al., 1989). Understanding this behaviour concomitantly as cancer cell migration and invasion will provide a more complete picture on the metastatic potential acquired by cancer cells.
The relevance of understanding the adhesiveness of cancer cells to its surrounding extracellular environment is one aspect of cancer research that has been well investigated but has been relatively unexplored in the context of ADAMTS1. With new knowledge implicating ADAMTS1 as a promoter of adherent interactions between mammary cancer cells and surrounding matrix proteins, it is therefore essential to determine how ADAMTS1 mediates this process and the specific matrix proteins ADAMTS1- expressing tumour cells can bind. In particular, it will be insightful to investigate the integrin expression in Adamts1-expressing and -deficient mammary cancer cells, as these transmembrane receptors are essential to the attachment of cancer cells to the ECM and may somehow be altered by ADAMTS1. The functionality of the ECD tripeptide sequence, within the third thrombospondin domain of ADAMTS1 should also be further investigated through protein-bindings assays between ADAMTS1 and integrins such as αvβ3, αvβ5 and α6β1 known to recognise this motif. As secreted ADAMTS1 is anchored to HSPG in the ECM, interacting directly to integrin presents as a possible mechanism of how ADAMTS1 encourages mammary cancer cell adherence to the ECM.
Tan IA 116 CHAPTER 5 Conclusion and future directions
Providing a mechanistic explanation behind the observed effects of ADAMTS1 in breast cancer was the primary intention behind the microarray analysis of PyMT/Adamts1+/+ and PyMT/Adamts1-/- breast tumours. In identifying the disparity in global gene expression in breast cancers can greatly unravel the molecular mechanisms behind the pro-tumourigenic and pro-metastatic role of ADAMTS1 in breast. Furthermore, as PyMT/Adamts1-/- mice manifested with less advanced and aggressive breast cancer disease, we can compare the gene signature resulting from ADAMTS1 loss and identify whether the absence of this particular protease reverts the clinical tumour phenotype from a luminal B subtype human breast cancer to a more benign breast cancer sub type with a better prognostic patient outcome. However, an unpredictable artefact impeded any conclusions to be drawn from our results and thus, the mechanisms underlying ADAMTS1 in breast cancer metastasis remain unknown. It is therefore warranted that a comparative gene expression analysis between PyMT/Adamts1+/+ and PyMT/Adamts1- /- mammary tumours be repeated using mouse cohorts that possess balanced strain mixing. Similarly, alternative parallel experiments such as proteomic analysis or a comprehensive gene expression analysis by real-time quantitative PCR or protein expression by western analysis can also effectively address this aim. Lastly, despite validating the overexpression of Adamts1 in both PyMT breast carcinoma cells and in MCF10A-Adamts1 cell line, confirmation that the ADAMTS1 protein by western analysis will also be ideal.
Tan IA 117
CHAPTER 6
Bibliography
CHAPTER 6 Bibliography
Abbaszade, I., Liu, R. Q., Yang, F., Rosenfeld, S. A., Ross, O. H., Link, J. R., Ellis, D. M., Tortorella, M. D., Pratta, M. A., Hollis, J. M., et al. (1999). Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family. The Journal of biological chemistry 274, 23443-23450.
Ahlquist, T., Lind, G. E., Costa, V. L., Meling, G. I., Vatn, M., Hoff, G. S., Rognum, T. O., Skotheim, R. I., Thiis-Evensen, E., and Lothe, R. A. (2008). Gene methylation profiles of normal mucosa, and benign and malignant colorectal tumors identify early onset markers. Molecular cancer 7, 94.
Aitchison, A., Veerakumarasivam, A., Vias, M., Kumar, R., Hamdy, F., Neal, D., and Mills, I. (2008). Promoter methylation correlates with reduced Smad4 expression in advanced prostate cancer. Prostate 68, 661-674.
Altemeier, W. A., Schlesinger, S. Y., Buell, C. A., Brauer, R., Rapraeger, A. C., Parks, W. C., and Chen, P. (2012a). Transmembrane and extracellular domains of syndecan-1 have distinct functions in regulating lung epithelial migration and adhesion. The Journal of biological chemistry 287, 34927- 34935.
Altemeier, W. A., Schlesinger, S. Y., Buell, C. A., Parks, W. C., and Chen, P. (2012b). Syndecan-1 controls cell migration by activating Rap1 to regulate focal adhesion disassembly. Journal of cell science.
Aravindan, S., Natarajan, M., Herman, T., and Aravindan, N. (2013). Radiation-induced TNFα cross signaling-dependent nuclear import of NFκB favors metastasis in neuroblastoma. Clinical & experimental metastasis.
Atienza, J., Yu, N., Kirstein, S., Xi, B., Wang, X., Xu, X., and Abassi, Y. (2006). Dynamic and label-free cell-based assays using the real-time cell electronic sensing system. Assay and drug development technologies 4, 597-607.
Balzer, E. M., and Konstantopoulos, K. (2012). Intercellular adhesion: mechanisms for growth and metastasis of epithelial cancers. Wiley interdisciplinary reviews Systems biology and medicine 4, 171- 181.
Tan IA 119 CHAPTER 6 Bibliography
Barry, S., Harder, B., Brzezinski, M., Flint, L., Seppen, J., and Osborne, W. (2001). Lentivirus vectors encoding both central polypurine tract and posttranscriptional regulatory element provide enhanced transduction and transgene expression. Human gene therapy 12, 1103-1108.
Basile, D. P., Fredrich, K., Chelladurai, B., Leonard, E. C., and Parrish, A. R. (2008). Renal ischemia reperfusion inhibits VEGF expression and induces ADAMTS-1, a novel VEGF inhibitor. American journal of physiology Renal physiology 294, F928-936.
Bengtson, M., and Joazeiro, C. (2010). Role of a ribosome-associated E3 ubiquitin ligase in protein quality control. Nature 467, 470-473.
Beristain, A. G., Zhu, H., and Leung, P. C. (2011). Regulated expression of ADAMTS-12 in human trophoblastic cells: a role for ADAMTS-12 in epithelial cell invasion? PloS one 6, e18473.
Best, C., Gillespie, J., Yi, Y., Chandramouli, G., Perlmutter, M., Gathright, Y., Erickson, H., Georgevich, L., Tangrea, M., Duray, P., et al. (2005). Molecular alterations in primary prostate cancer after androgen ablation therapy. Clinical cancer research : an official journal of the American Association for Cancer Research 11, 6823-6834.
Bhargava, R., Beriwal, S., Dabbs, D., Ozbek, U., Soran, A., Johnson, R., Brufsky, A., Lembersky, B., and Ahrendt, G. (2010). Immunohistochemical surrogate markers of breast cancer molecular classes predicts response to neoadjuvant chemotherapy: a single institutional experience with 359 cases. Cancer 116, 1431-1439.
Blelloch, R., and Kimble, J. (1999). Control of organ shape by a secreted metalloprotease in the nematode Caenorhabditis elegans. Nature 399, 586-590.
Bonuccelli, G., Casimiro, M. C., Sotgia, F., Wang, C., Liu, M., Katiyar, S., Zhou, J., Dew, E., Capozza, F., Daumer, K. M., et al. (2009). Caveolin-1 (P132L), a common breast cancer mutation, confers mammary cell invasiveness and defines a novel stem cell/metastasis-associated gene signature. The American journal of pathology 174, 1650-1662.
Tan IA 120 CHAPTER 6 Bibliography
Borre, M., Offersen, B. V., Nerstrom, B., and Overgaard, J. (1998). Microvessel density predicts survival in prostate cancer patients subjected to watchful waiting. Br J Cancer 78, 940-944.
Bourd-Boittin, K., Bonnier, D., Leyme, A., Mari, B., Tuffery, P., Samson, M., Ezan, F., Baffet, G., and Theret, N. (2011). Protease profiling of liver fibrosis reveals the ADAM metallopeptidase with thrombospondin type 1 motif, 1 as a central activator of transforming growth factor beta. Hepatology 54, 2173-2184.
Braconi, C., Meng, F., Swenson, E., Khrapenko, L., Huang, N., and Patel, T. (2009). Candidate therapeutic agents for hepatocellular cancer can be identified from phenotype-associated gene expression signatures. Cancer 115, 3738-3748.
Bravo-Cordero, J., Hodgson, L., and Condeelis, J. (2012). Directed cell invasion and migration during metastasis. Current opinion in cell biology 24, 277-283.
Brenton, J., Carey, L., Ahmed, A., and Caldas, C. (2005). Molecular classification and molecular forecasting of breast cancer: ready for clinical application? Journal of clinical oncology : official journal of the American Society of Clinical Oncology 23, 7350-7360.
Brown, C., Sadlon, T., Gargett, T., Melville, E., Zhang, R., Drabsch, Y., Ling, M., Strathdee, C., Gonda, T., and Barry, S. (2010a). Robust, reversible gene knockdown using a single lentiviral short hairpin RNA vector. Human gene therapy 21, 1005-1017.
Brown, H. M., Dunning, K. R., Robker, R. L., Boerboom, D., Pritchard, M., Lane, M., and Russell, D. L. (2010b). ADAMTS1 cleavage of versican mediates essential structural remodeling of the ovarian follicle and cumulus-oocyte matrix during ovulation in mice. Biology of reproduction 83, 549-557.
Brown, H. M., Dunning, K. R., Robker, R. L., Pritchard, M., and Russell, D. L. (2006). Requirement for ADAMTS-1 in extracellular matrix remodeling during ovarian folliculogenesis and lymphangiogenesis. Developmental biology 300, 699-709.
Brown, H. M., Robker, R. L., and Russell, D. L. (2010c). Development and hormonal regulation of the ovarian lymphatic vasculature. Endocrinology 151, 5446-5455.
Tan IA 121 CHAPTER 6 Bibliography
Bugyi, B., and Carlier, M.-F. (2010). Control of actin filament treadmilling in cell motility. Annual review of biophysics 39, 449-470.
Cai, J., Tang, H., Xu, L., Wang, X., Yang, C., Ruan, S., Guo, J., Hu, S., and Wang, Z. (2012). Fibroblasts in omentum activated by tumor cells promote ovarian cancer growth, adhesion and invasiveness. Carcinogenesis 33, 20-29.
Canals, F., Colome, N., Ferrer, C., Plaza-Calonge Mdel, C., and Rodriguez-Manzaneque, J. C. (2006). Identification of substrates of the extracellular protease ADAMTS1 by DIGE proteomic analysis. Proteomics 6 Suppl 1, S28-35.
Cao, Y., Chang, H., Li, L., Cheng, R. C., and Fan, X. N. (2007). Alteration of adhesion molecule expression and cellular polarity in hepatocellular carcinoma. Histopathology 51, 528-538.
Capehart, A. A. (2010). Proteolytic cleavage of versican during limb joint development. Anatomical record (Hoboken, NJ : 2007) 293, 208-214.
Carey, L., Dees, E., Sawyer, L., Gatti, L., Moore, D., Collichio, F., Ollila, D., Sartor, C., Graham, M., and Perou, C. (2007). The triple negative paradox: primary tumor chemosensitivity of breast cancer subtypes. Clinical cancer research : an official journal of the American Association for Cancer Research 13, 2329-2334.
Carver, B. S., Tran, J., Gopalan, A., Chen, Z., Shaikh, S., Carracedo, A., Alimonti, A., Nardella, C., Varmeh, S., Scardino, P. T., et al. (2009). Aberrant ERG expression cooperates with loss of PTEN to promote cancer progression in the prostate. Nature genetics 41, 619-624.
Casimiro, S., Luis, I., Fernandes, A., Pires, R., Pinto, A., Gouveia, A. G., Francisco, A. F., Portela, J., Correia, L., and Costa, L. (2012). Analysis of a bone metastasis gene expression signature in patients with bone metastasis from solid tumors. Clinical & experimental metastasis 29, 155-164.
Cattaruzza, S., Schiappacassi, M., Kimata, K., Colombatti, A., and Perris, R. (2004). The globular domains of PG-M/versican modulate the proliferation-apoptosis equilibrium and invasive capabilities of
Tan IA 122 CHAPTER 6 Bibliography
tumor cells. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 18, 779-781.
Chandrashekran, A., Gordon, M., and Casimir, C. (2004). Targeted retroviral transduction of c-kit+ hematopoietic cells using novel ligand display technology. Blood 104, 2697-2703.
Chaturvedi, A., Hoffman, L., Welm, A., Lessnick, S., and Beckerle, M. (2012). The EWS/FLI Oncogene Drives Changes in Cellular Morphology, Adhesion, and Migration in Ewing Sarcoma. Genes & cancer 3, 102-116.
Chen, J., Zhi, Y., Chang, X., Zhang, S., and Dai, D. (2012). Expression of ADAMTS1 and Its Correlation with Angiogenesis in Primary Gastric Cancer and Lymph Node Metastasis. Digestive diseases and sciences.
Chen, X., Cheung, S. T., So, S., Fan, S. T., Barry, C., Higgins, J., Lai, K. M., Ji, J., Dudoit, S., Ng, I. O., et al. (2002). Gene expression patterns in human liver cancers. Mol Biol Cell 13, 1929-1939.
Choi, J. E., Kim, D. S., Kim, E. J., Chae, M. H., Cha, S. I., Kim, C. H., Jheon, S., Jung, T. H., and Park, J. Y. (2008). Aberrant methylation of ADAMTS1 in non-small cell lung cancer. Cancer genetics and cytogenetics 187, 80-84.
Clark, E., King, W., Brugge, J., Symons, M., and Hynes, R. (1998). Integrin-mediated signals regulated by members of the rho family of GTPases. The Journal of cell biology 142, 573-586.
Colige, A., Ruggiero, F., Vandenberghe, I., Dubail, J., Kesteloot, F., Van Beeumen, J., Beschin, A., Brys, L., Lapière, C., and Nusgens, B. (2005). Domains and maturation processes that regulate the activity of ADAMTS-2, a metalloproteinase cleaving the aminopropeptide of fibrillar procollagens types I- III and V. The Journal of biological chemistry 280, 34397-34408.
Colige, A., Vandenberghe, I., Thiry, M., Lambert, C. A., Van Beeumen, J., Li, S. W., Prockop, D. J., Lapiere, C. M., and Nusgens, B. V. (2002). Cloning and characterization of ADAMTS-14, a novel ADAMTS displaying high homology with ADAMTS-2 and ADAMTS-3. The Journal of biological chemistry 277, 5756-5766.
Tan IA 123 CHAPTER 6 Bibliography
Cooley, M. A., Fresco, V. M., Dorlon, M. E., Twal, W. O., Lee, N. V., Barth, J. L., Kern, C. B., Iruela- Arispe, M. L., and Argraves, W. S. (2012). Fibulin-1 is required during cardiac ventricular morphogenesis for versican cleavage, suppression of ErbB2 and Erk1/2 activation, and to attenuate trabecular cardiomyocyte proliferation. Developmental dynamics : an official publication of the American Association of Anatomists 241, 303-314.
Cross, N. A., Chandrasekharan, S., Jokonya, N., Fowles, A., Hamdy, F. C., Buttle, D. J., and Eaton, C. L. (2005). The expression and regulation of ADAMTS-1, -4, -5, -9, and -15, and TIMP-3 by TGFbeta1 in prostate cells: relevance to the accumulation of versican. Prostate 63, 269-275.
Culig, Z., and Bartsch, G. (2006). Androgen axis in prostate cancer. J Cell Biochem 99, 373-381.
Danielpour, D. (2005). Functions and regulation of transforming growth factor-beta (TGF-beta) in the prostate. Eur J Cancer 41, 846-857.
Dawe, C., Freund, R., Mandel, G., Ballmer-Hofer, K., Talmage, D., and Benjamin, T. (1987). Variations in polyoma virus genotype in relation to tumor induction in mice. Characterization of wild type strains with widely differing tumor profiles. The American journal of pathology 127, 243-261.
Dawson, D., Pearce, S., Zhong, R., Silverstein, R., Frazier, W., and Bouck, N. (1997). CD36 mediates the In vitro inhibitory effects of thrombospondin-1 on endothelial cells. The Journal of cell biology 138, 707-717.
De Giorgi, F., Lartigue, L., Bauer, M., Schubert, A., Grimm, S., Hanson, G., Remington, S., Youle, R., and Ichas, F. (2002). The permeability transition pore signals apoptosis by directing Bax translocation and multimerization. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 16, 607-609.
De Wever, O., Hendrix, A., De Boeck, A., Westbroek, W., Braems, G., Emami, S., Sabbah, M., Gespach, C., and Bracke, M. (2010). Modeling and quantification of cancer cell invasion through collagen type I matrices. The International journal of developmental biology 54, 887-896.
Tan IA 124 CHAPTER 6 Bibliography
Debnath, J., Muthuswamy, S., and Brugge, J. (2003). Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods (San Diego, Calif) 30, 256-268.
Decker, K. F., Zheng, D., He, Y., Bowman, T., Edwards, J. R., and Jia, L. (2012). Persistent androgen receptor-mediated transcription in castration-resistant prostate cancer under androgen-deprived conditions. Nucleic acids research.
Demircan, K., Gunduz, E., Gunduz, M., Beder, L. B., Hirohata, S., Nagatsuka, H., Cengiz, B., Cilek, M. Z., Yamanaka, N., Shimizu, K., and Ninomiya, Y. (2009). Increased mRNA expression of ADAMTS metalloproteinases in metastatic foci of head and neck cancer. Head & neck 31, 793-801.
Desjardins, L. A. (2002). Hepatocellular carcinoma. Clin J Oncol Nurs 6, 107-108.
Desjardins, P., and Conklin, D. (2010). NanoDrop microvolume quantitation of nucleic acids. Journal of visualized experiments : JoVE.
Devlin, H. L., and Mudryj, M. (2009). Progression of prostate cancer: multiple pathways to androgen independence. Cancer Lett 274, 177-186.
Dua, R., Gui, G., and Isacke, C. (2005). Endothelial adhesion molecules in breast cancer invasion into the vascular and lymphatic systems. European journal of surgical oncology : the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 31, 824-832.
Ducros, E., Berthaut, A., Mirshahi, P., Lemarchand, S., Soria, J., Legeais, J. M., and Mirshahi, M. (2007). Expression of extracellular matrix proteins fibulin-1 and fibulin-2 by human corneal fibroblasts. Current eye research 32, 481-490.
Dunlap, K. A., Kwak, H. I., Burghardt, R. C., Bazer, F. W., Magness, R. R., Johnson, G. A., and Bayless, K. J. (2010). The sphingosine 1-phosphate (S1P) signaling pathway is regulated during pregnancy in sheep. Biology of reproduction 82, 876-887.
Tan IA 125 CHAPTER 6 Bibliography
Dunning, K. R., Lane, M., Brown, H. M., Yeo, C., Robker, R. L., and Russell, D. L. (2007). Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes. Human reproduction (Oxford, England) 22, 2842-2850.
Edwards, J., Krishna, N., Grigor, K., and Bartlett, J. (2003). Androgen receptor gene amplification and protein expression in hormone refractory prostate cancer. Br J Cancer 89, 552-556.
El Hour, M., Moncada-Pazos, A., Blacher, S., Masset, A., Cal, S., Berndt, S., Detilleux, J., Host, L., Obaya, A. J., Maillard, C., et al. (2010). Higher sensitivity of Adamts12-deficient mice to tumor growth and angiogenesis. Oncogene 29, 3025-3032.
Ellis, B., Hirsch, M., Barker, J., Connelly, J., Steininger, R., and Porteus, M. (2013). A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno- associated virus (AAV1-9) and one engineered adeno-associated virus serotype. Virology journal 10, 74.
Esselens, C., Malapeira, J., Colome, N., Casal, C., Rodriguez-Manzaneque, J. C., Canals, F., and Arribas, J. (2010). The cleavage of semaphorin 3C induced by ADAMTS1 promotes cell migration. The Journal of biological chemistry 285, 2463-2473.
Esserman, L., Berry, D., Cheang, M., Yau, C., Perou, C., Carey, L., DeMichele, A., Gray, J., Conway- Dorsey, K., Lenburg, M., et al. (2012). Chemotherapy response and recurrence-free survival in neoadjuvant breast cancer depends on biomarker profiles: results from the I-SPY 1 TRIAL (CALGB 150007/150012; ACRIN 6657). Breast cancer research and treatment 132, 1049-1062.
Fan, C., Oh, D., Wessels, L., Weigelt, B., Nuyten, D., Nobel, A., van't Veer, L., and Perou, C. (2006). Concordance among gene-expression-based predictors for breast cancer. The New England journal of medicine 355, 560-569.
Farina, K., Wyckoff, J., Rivera, J., Lee, H., Segall, J., Condeelis, J., and Jones, J. (1998). Cell motility of tumor cells visualized in living intact primary tumors using green fluorescent protein. Cancer research 58, 2528-2532.
Tan IA 126 CHAPTER 6 Bibliography
Fernandes, R., Hirohata, S., Engle, J., Colige, A., Cohn, D., Eyre, D., and Apte, S. (2001). Procollagen II amino propeptide processing by ADAMTS-3. Insights on dermatosparaxis. The Journal of biological chemistry 276, 31502-31509.
Finer, M., Dull, T., Qin, L., Farson, D., and Roberts, M. (1994). kat: a high-efficiency retroviral transduction system for primary human T lymphocytes. Blood 83, 43-50.
Fluck, M., and Haslam, S. (1996). Mammary tumors induced by polyomavirus. Breast cancer research and treatment 39, 45-56.
Foley, C., Luo, C., O'Callaghan, K., Hinds, P., Covic, L., and Kuliopulos, A. (2012). Matrix metalloprotease-1a promotes tumorigenesis and metastasis. The Journal of biological chemistry 287, 24330-24338.
Friedl, P., Maaser, K., Klein, C., Niggemann, B., Krohne, G., and Zänker, K. (1997). Migration of highly aggressive MV3 melanoma cells in 3-dimensional collagen lattices results in local matrix reorganization and shedding of alpha2 and beta1 integrins and CD44. Cancer research 57, 2061-2070.
Friedl, P., Zänker, K., and Bröcker, E. (1998). Cell migration strategies in 3-D extracellular matrix: differences in morphology, cell matrix interactions, and integrin function. Microscopy research and technique 43, 369-378.
Friedlander, T., Roy, R., Tomlins, S., Ngo, V., Kobayashi, Y., Azameera, A., Rubin, M., Pienta, K., Chinnaiyan, A., Ittmann, M., et al. (2012). Common structural and epigenetic changes in the genome of castration-resistant prostate cancer. Cancer research 72, 616-625.
Fu, Y., Nagy, J. A., Brown, L. F., Shih, S. C., Johnson, P. Y., Chan, C. K., Dvorak, H. F., and Wight, T. N. (2011). Proteolytic cleavage of versican and involvement of ADAMTS-1 in VEGF-A/VPF-induced pathological angiogenesis. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 59, 463-473.
Tan IA 127 CHAPTER 6 Bibliography
Gaida, M., Haag, N., Günther, F., Tschaharganeh, D., Schirmacher, P., Friess, H., Giese, N., Schmidt, J., and Wente, M. (2010). Expression of A disintegrin and metalloprotease 10 in pancreatic carcinoma. International journal of molecular medicine 26, 281-288.
Gallagher, S., and Desjardins, P. (2007). Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Current protocols in human genetics / editorial board, Jonathan L Haines [et al] Appendix 3.
Gao, S., De Geyter, C., Kossowska, K., and Zhang, H. (2007). FSH stimulates the expression of the ADAMTS-16 protease in mature human ovarian follicles. Molecular human reproduction 13, 465-471.
Garcia-Conesa, M. T., Tribolo, S., Guyot, S., Tomas-Barberan, F. A., and Kroon, P. A. (2009). Oligomeric procyanidins inhibit cell migration and modulate the expression of migration and proliferation associated genes in human umbilical vascular endothelial cells. Molecular nutrition & food research 53, 266-276.
Golembieski, W., Thomas, S., Schultz, C., Yunker, C., McClung, H., Lemke, N., Cazacu, S., Barker, T., Sage, E., Brodie, C., and Rempel, S. (2008). HSP27 mediates SPARC-induced changes in glioma morphology, migration, and invasion. Glia 56, 1061-1075.
Greenberg, P., Hortobagyi, G., Smith, T., Ziegler, L., Frye, D., and Buzdar, A. (1996). Long-term follow- up of patients with complete remission following combination chemotherapy for metastatic breast cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 14, 2197-2205.
Grigo, K., Wirsing, A., Lucas, B., Klein-Hitpass, L., and Ryffel, G. U. (2008). HNF4 alpha orchestrates a set of 14 genes to down-regulate cell proliferation in kidney cells. Biological chemistry 389, 179-187.
Grise, F., Sena, S., Bidaud-Meynard, A., Baud, J., Hiriart, J.-B., Makki, K., Dugot-Senant, N., Staedel, C., Bioulac-Sage, P., Zucman-Rossi, J., et al. (2012). Rnd3/RhoE Is down-regulated in hepatocellular carcinoma and controls cellular invasion. Hepatology (Baltimore, Md) 55, 1766-1775.
Tan IA 128 CHAPTER 6 Bibliography
Guiet, R., Van Goethem, E., Cougoule, C., Balor, S., Valette, A., Al Saati, T., Lowell, C., Le Cabec, V., and Maridonneau-Parini, I. (2011). The process of macrophage migration promotes matrix metalloproteinase-independent invasion by tumor cells. Journal of immunology (Baltimore, Md : 1950) 187, 3806-3814.
Gunther, W., Skaftnesmo, K. O., Arnold, H., Bjerkvig, R., and Terzis, A. J. (2005). Distribution patterns of the anti-angiogenic protein ADAMTS-1 during rat development. Acta histochemica 107, 121-131.
Gustavsson, H., Jennbacken, K., Welen, K., and Damber, J. E. (2008). Altered expression of genes regulating angiogenesis in experimental androgen-independent prostate cancer. Prostate 68, 161-170.
Gustavsson, H., Tesan, T., Jennbacken, K., Kuno, K., Damber, J. E., and Welen, K. (2010). ADAMTS1 alters blood vessel morphology and TSP1 levels in LNCaP and LNCaP-19 prostate tumors. BMC cancer 10, 288.
Gustavsson, H., Wang, W., Jennbacken, K., Welen, K., and Damber, J. E. (2009). ADAMTS1, a putative anti-angiogenic factor, is decreased in human prostate cancer. BJU international 104, 1786- 1790.
Guy, C. T., Cardiff, R. D., and Muller, W. J. (1992). Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Molecular and cellular biology 12, 954-961.
Haberern, C., and Kupchik, H. (1985). Diversity of adhesion to basement membrane components of human pancreatic adenocarcinomas. Cancer research 45, 5246-5251.
Hall, J., Maitland, N., Stower, M., and Lang, S. (2002). Primary prostate stromal cells modulate the morphology and migration of primary prostate epithelial cells in type 1 collagen gels. Cancer research 62, 58-62.
Hancox, R., Allen, M., Holliday, D., Edwards, D., Pennington, C., Guttery, D., Shaw, J., Walker, R., Pringle, J., and Jones, J. (2009). Tumour-associated tenascin-C isoforms promote breast cancer cell
Tan IA 129 CHAPTER 6 Bibliography
invasion and growth by matrix metalloproteinase-dependent and independent mechanisms. Breast cancer research : BCR 11.
Hatipoglu, O., Hirohata, S., Cilek, M., Ogawa, H., Miyoshi, T., Obika, M., Demircan, K., Shinohata, R., Kusachi, S., and Ninomiya, Y. (2009). ADAMTS1 is a unique hypoxic early response gene expressed by endothelial cells. The Journal of biological chemistry 284, 16325-16333.
Hayashi, H., and Sakai, T. (2012). Biological Significance of Local TGF-beta Activation in Liver Diseases. Front Physiol 3, 12.
Hayashi, P. H., and Di Bisceglie, A. M. (2005). The progression of hepatitis B- and C-infections to chronic liver disease and hepatocellular carcinoma: epidemiology and pathogenesis. Med Clin North Am 89, 371-389.
Hegerfeldt, Y., Tusch, M., Bröcker, E.-B., and Friedl, P. (2002). Collective cell movement in primary melanoma explants: plasticity of cell-cell interaction, beta1-integrin function, and migration strategies. Cancer research 62, 2125-2130.
Henrichsen, C., Vinckenbosch, N., Zöllner, S., Chaignat, E., Pradervand, S., Schütz, F., Ruedi, M., Kaessmann, H., and Reymond, A. (2009). Segmental copy number variation shapes tissue transcriptomes. Nature genetics 41, 424-429.
Herschkowitz, J., Simin, K., Weigman, V., Mikaelian, I., Usary, J., Hu, Z., Rasmussen, K., Jones, L., Assefnia, S., Chandrasekharan, S., et al. (2007). Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome biology 8.
Hirano, T., Hirose, K., Sakurai, K., Makishima, M., Sasaki, K., and Amano, S. (2011). Inhibition of tumor growth by antibody to ADAMTS1 in mouse xenografts of breast cancer. Anticancer research 31, 3839- 3842.
Horber, C., Buttner, F. H., Kern, C., Schmiedeknecht, G., and Bartnik, E. (2000). Truncation of the amino-terminus of the recombinant aggrecan rAgg1mut leads to reduced cleavage at the aggrecanase
Tan IA 130 CHAPTER 6 Bibliography
site. Efficient aggrecanase catabolism may depend on multiple substrate interactions. Matrix Biol 19, 533-543.
Hotchin, N., and Hall, A. (1995). The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases. The Journal of cell biology 131, 1857-1865.
Hu, L., Jonsson, K. B., Andersen, H., Edenro, A., Bohlooly, Y. M., Melhus, H., and Lind, T. (2012). Over- expression of Adamts1 in mice alters bone mineral density. Journal of bone and mineral metabolism 30, 304-311.
Huang, J., Bridges, L., and White, J. (2005). Selective modulation of integrin-mediated cell migration by distinct ADAM family members. Molecular biology of the cell 16, 4982-4991.
Humphries, J., Byron, A., and Humphries, M. (2006). Integrin ligands at a glance. Journal of cell science 119, 3901-3903.
Humphries, M., Yasuda, Y., Olden, K., and Yamada, K. (1988). The cell interaction sites of fibronectin in tumour metastasis. Ciba Foundation symposium 141, 75-93.
Iba, K., Albrechtsen, R., Gilpin, B. J., Loechel, F., and Wewer, U. M. (1999). Cysteine-rich domain of human ADAM 12 (meltrin alpha) supports tumor cell adhesion. The American journal of pathology 154, 1489-1501.
Ibaragi, S., Shimo, T., Hassan, N. M., Isowa, S., Kurio, N., Mandai, H., Kodama, S., and Sasaki, A. (2011). Induction of MMP-13 expression in bone-metastasizing cancer cells by type I collagen through integrin α1β1 and α2β1-p38 MAPK signaling. Anticancer research 31, 1307-1313.
Ifon, E. T., Pang, A. L., Johnson, W., Cashman, K., Zimmerman, S., Muralidhar, S., Chan, W. Y., Casey, J., and Rosenthal, L. J. (2005). U94 alters FN1 and ANGPTL4 gene expression and inhibits tumorigenesis of prostate cancer cell line PC3. Cancer Cell Int 5, 19.
Iruela-Arispe, M. L., Carpizo, D., and Luque, A. (2003). ADAMTS1: a matrix metalloprotease with angioinhibitory properties. Ann N Y Acad Sci 995, 183-190.
Tan IA 131 CHAPTER 6 Bibliography
Iruela-Arispe, M. L., Lombardo, M., Krutzsch, H. C., Lawler, J., and Roberts, D. D. (1999). Inhibition of angiogenesis by thrombospondin-1 is mediated by 2 independent regions within the type 1 repeats. Circulation 100, 1423-1431.
Iwadate, Y., and Yumura, S. (2008). Actin-based propulsive forces and myosin-II-based contractile forces in migrating Dictyostelium cells. Journal of cell science 121, 1314-1324.
Jennbacken, K., Gustavsson, H., Tesan, T., Horn, M., Vallbo, C., Welen, K., and Damber, J. E. (2009). The prostatic environment suppresses growth of androgen-independent prostate cancer xenografts: an effect influenced by testosterone. Prostate 69, 1164-1175.
Jönsson-Rylander, A.-C., Nilsson, T., Fritsche-Danielson, R., Hammarström, A., Behrendt, M., Andersson, J.-O., Lindgren, K., Andersson, A.-K., Wallbrandt, P., Rosengren, B., et al. (2005). Role of ADAMTS-1 in atherosclerosis: remodeling of carotid artery, immunohistochemistry, and proteolysis of versican. Arteriosclerosis, thrombosis, and vascular biology 25, 180-185.
Jung, S., Rosenzweig, M., Sereika, S., Linkov, F., Brufsky, A., and Weissfeld, J. (2012). Factors associated with mortality after breast cancer metastasis. Cancer causes & control : CCC 23, 103-112.
Kamiguti, A., Zuzel, M., and Theakston, R. (1998). Snake venom metalloproteinases and disintegrins: interactions with cells. Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica [et al] 31, 853-862.
Kang, Y., Siegel, P., Shu, W., Drobnjak, M., Kakonen, S., Cordón-Cardo, C., Guise, T., and Massagué, J. (2003). A multigenic program mediating breast cancer metastasis to bone. Cancer cell 3, 537-549.
Keightley, M. C., Sales, K. J., and Jabbour, H. N. (2010). PGF2alpha-F-prostanoid receptor signalling via ADAMTS1 modulates epithelial cell invasion and endothelial cell function in endometrial cancer. BMC cancer 10, 488.
Kilpinen, S., Autio, R., Ojala, K., Iljin, K., Bucher, E., Sara, H., Pisto, T., Saarela, M., Skotheim, R., Björkman, M., et al. (2008). Systematic bioinformatic analysis of expression levels of 17,330 human genes across 9,783 samples from 175 types of healthy and pathological tissues. Genome biology 9.
Tan IA 132 CHAPTER 6 Bibliography
Kilpinen, S., Ojala, K., and Kallioniemi, O. (2011). Alignment of gene expression profiles from test samples against a reference database: New method for context-specific interpretation of microarray data. BioData mining 4, 5.
Kim, J. H., Yang, Y. I., Ahn, J. H., Lee, J. G., Lee, K. T., and Choi, J. H. (2012). Deer (Cervus elaphus) antler extract suppresses adhesion and migration of endometriotic cells and regulates MMP-2 and MMP-9 expression. Journal of ethnopharmacology 140, 391-397.
Kim, S., Takahashi, H., Lin, W. W., Descargues, P., Grivennikov, S., Kim, Y., Luo, J. L., and Karin, M. (2009). Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate metastasis. Nature 457, 102-106.
Kirmse, R., Otto, H., and Ludwig, T. (2011). Interdependency of cell adhesion, force generation and extracellular proteolysis in matrix remodeling. Journal of cell science 124, 1857-1866.
Kleinman, H., McGarvey, M., Hassell, J., Star, V., Cannon, F., Laurie, G., and Martin, G. (1986). Basement membrane complexes with biological activity. Biochemistry 25, 312-318.
Koblinski, J., Ahram, M., and Sloane, B. (2000). Unraveling the role of proteases in cancer. Clinica chimica acta; international journal of clinical chemistry 291, 113-135.
Kohno, T., Otsuka, A., Girard, L., Sato, M., Iwakawa, R., Ogiwara, H., Sanchez-Cespedes, M., Minna, J. D., and Yokota, J. (2010). A catalog of genes homozygously deleted in human lung cancer and the candidacy of PTPRD as a tumor suppressor gene. Genes, chromosomes & cancer 49, 342-352.
Kõks, S., Soomets, U., Paya-Cano, J., Fernandes, C., Luuk, H., Plaas, M., Terasmaa, A., Tillmann, V., Noormets, K., Vasar, E., and Schalkwyk, L. (2009). Wfs1 gene deletion causes growth retardation in mice and interferes with the growth hormone pathway. Physiological genomics 37, 249-259.
Kramer, R., McDonald, K., Crowley, E., Ramos, D., and Damsky, C. (1989). Melanoma cell adhesion to basement membrane mediated by integrin-related complexes. Cancer research 49, 393-402.
Tan IA 133 CHAPTER 6 Bibliography
Krampert, M., Kuenzle, S., Thai, S. N., Lee, N., Iruela-Arispe, M. L., and Werner, S. (2005). ADAMTS1 proteinase is up-regulated in wounded skin and regulates migration of fibroblasts and endothelial cells. The Journal of biological chemistry 280, 23844-23852.
Kumar, S., Sharghi-Namini, S., Rao, N., and Ge, R. (2012). ADAMTS5 Functions as an Anti-Angiogenic and Anti-Tumorigenic Protein Independent of Its Proteoglycanase Activity. The American journal of pathology 181, 1056-1068.
Kuno, K., Bannai, K., Hakozaki, M., Matsushima, K., and Hirose, K. (2004). The carboxyl-terminal half region of ADAMTS-1 suppresses both tumorigenicity and experimental tumor metastatic potential. Biochemical and biophysical research communications 319, 1327-1333.
Kuno, K., Iizasa, H., Ohno, S., and Matsushima, K. (1997a). The exon/intron organization and chromosomal mapping of the mouse ADAMTS-1 gene encoding an ADAM family protein with TSP motifs. Genomics 46, 466-471.
Kuno, K., Kanada, N., Nakashima, E., Fujiki, F., Ichimura, F., and Matsushima, K. (1997b). Molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motifs as an inflammation associated gene. The Journal of biological chemistry 272, 556-562.
Kuno, K., and Matsushima, K. (1998). ADAMTS-1 protein anchors at the extracellular matrix through the thrombospondin type I motifs and its spacing region. The Journal of biological chemistry 273, 13912- 13917.
Kuno, K., Okada, Y., Kawashima, H., Nakamura, H., Miyasaka, M., Ohno, H., and Matsushima, K. (2000). ADAMTS-1 cleaves a cartilage proteoglycan, aggrecan. FEBS letters 478, 241-245.
Kuno, K., Terashima, Y., and Matsushima, K. (1999). ADAMTS-1 is an active metalloproteinase associated with the extracellular matrix. The Journal of biological chemistry 274, 18821-18826.
Tan IA 134 CHAPTER 6 Bibliography
Kutz, W. E., Wang, L. W., Bader, H. L., Majors, A. K., Iwata, K., Traboulsi, E. I., Sakai, L. Y., Keene, D. R., and Apte, S. S. (2011). ADAMTS10 protein interacts with fibrillin-1 and promotes its deposition in extracellular matrix of cultured fibroblasts. The Journal of biological chemistry 286, 17156-17167.
Le Clainche, C., and Carlier, M.-F. (2004). Actin-based motility assay. Current protocols in cell biology / editorial board, Juan S Bonifacino [et al] Chapter 12.
Lebaron, R. G. (1996). Versican. Perspectives on developmental neurobiology 3, 261-271.
LeBaron, R. G., Zimmermann, D. R., and Ruoslahti, E. (1992). Hyaluronate binding properties of versican. The Journal of biological chemistry 267, 10003-10010.
Lee, N. V., Rodriguez-Manzaneque, J. C., Thai, S. N., Twal, W. O., Luque, A., Lyons, K. M., Argraves, W. S., and Iruela-Arispe, M. L. (2005). Fibulin-1 acts as a cofactor for the matrix metalloprotease ADAMTS-1. The Journal of biological chemistry 280, 34796-34804.
Lee, N. V., Sato, M., Annis, D. S., Loo, J. A., Wu, L., Mosher, D. F., and Iruela-Arispe, M. L. (2006). ADAMTS1 mediates the release of antiangiogenic polypeptides from TSP1 and 2. The EMBO journal 25, 5270-5283.
Lee, Y. J., Koch, M., Karl, D., Torres-Collado, A. X., Fernando, N. T., Rothrock, C., Kuruppu, D., Ryeom, S., Iruela-Arispe, M. L., and Yoon, S. S. (2010). Variable inhibition of thrombospondin 1 against liver and lung metastases through differential activation of metalloproteinase ADAMTS1. Cancer research 70, 948-956.
Leslie, A., Carey, F. A., Pratt, N. R., and Steele, R. J. (2002). The colorectal adenoma-carcinoma sequence. Br J Surg 89, 845-860.
Leyme, A., Bourd-Boittin, K., Bonnier, D., Falconer, A., Arlot-Bonnemains, Y., and Theret, N. (2012). Identification of ILK as a new partner of the ADAM12 disintegrin and metalloprotease in cell adhesion and survival. Mol Biol Cell 23, 3461-3472.
Li, D. M., and Feng, Y. M. (2011). Signaling mechanism of cell adhesion molecules in breast cancer metastasis: potential therapeutic targets. Breast cancer research and treatment 128, 7-21.
Tan IA 135 CHAPTER 6 Bibliography
Li, J., Ji, Z., Qiao, C., Qi, Y., and Shi, W. (2013). Overexpression of ADAM9 Promotes Colon Cancer Cells Invasion. Journal of investigative surgery : the official journal of the Academy of Surgical Research.
Li, S., Guan, J.-L., and Chien, S. (2005). Biochemistry and biomechanics of cell motility. Annual review of biomedical engineering 7, 105-150.
Li, T., Sotgia, F., Vuolo, M., Li, M., and Yang…, W. (2006). Caveolin-1 mutations in human breast cancer: functional association with estrogen receptor α-positive status. The American journal of ….
Li, Z., Zhang, W., Shao, Y., Zhang, C., Wu, Q., Yang, H., Wan, X., Zhang, J., Guan, M., Wan, J., and Yu, B. (2010). High-resolution melting analysis of ADAMTS18 methylation levels in gastric, colorectal and pancreatic cancers. Medical oncology (Northwood, London, England) 27, 998-1004.
Lind, A. J., Wikstrom, P., Granfors, T., Egevad, L., Stattin, P., and Bergh, A. (2005). Angiopoietin 2 expression is related to histological grade, vascular density, metastases, and outcome in prostate cancer. Prostate 62, 394-399.
Lind, G. E., Kleivi, K., Meling, G. I., Teixeira, M. R., Thiis-Evensen, E., Rognum, T. O., and Lothe, R. A. (2006a). ADAMTS1, CRABP1, and NR3C1 identified as epigenetically deregulated genes in colorectal tumorigenesis. Cellular oncology : the official journal of the International Society for Cellular Oncology 28, 259-272.
Lind, T., Birch, M. A., and McKie, N. (2006b). Purification of an insect derived recombinant human ADAMTS-1 reveals novel gelatin (type I collagen) degrading activities. Molecular and cellular biochemistry 281, 95-102.
Liu, C.-J., Kong, W., Ilalov, K., Yu, S., Xu, K., Prazak, L., Fajardo, M., Sehgal, B., and Di Cesare, P. (2006a). ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20, 988-990.
Tan IA 136 CHAPTER 6 Bibliography
Liu, C.-j., Kong, W., Xu, K., Luan, Y., Ilalov, K., Sehgal, B., Yu, S., Howell, R., and Di Cesare, P. (2006b). ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein. The Journal of biological chemistry 281, 15800-15808.
Liu, Y. J., Xu, Y., and Yu, Q. (2006c). Full-length ADAMTS-1 and the ADAMTS-1 fragments display pro- and antimetastatic activity, respectively. Oncogene 25, 2452-2467.
Llamazares, M., Cal, S., Quesada, V., and López-Otín, C. (2003). Identification and characterization of ADAMTS-20 defines a novel subfamily of metalloproteinases-disintegrins with multiple thrombospondin- 1 repeats and a unique GON domain. The Journal of biological chemistry 278, 13382-13389.
Llamazares, M., Obaya, A. J., Moncada-Pazos, A., Heljasvaara, R., Espada, J., Lopez-Otin, C., and Cal, S. (2007). The ADAMTS12 metalloproteinase exhibits anti-tumorigenic properties through modulation of the Ras-dependent ERK signalling pathway. Journal of cell science 120, 3544-3552.
Lo, P. H., Lung, H. L., Cheung, A. K., Apte, S. S., Chan, K. W., Kwong, F. M., Ko, J. M., Cheng, Y., Law, S., Srivastava, G., et al. (2010). Extracellular protease ADAMTS9 suppresses esophageal and nasopharyngeal carcinoma tumor formation by inhibiting angiogenesis. Cancer research 70, 5567-5576.
Lu, X., Lu, D., Scully, M., and Kakkar, V. (2007). Structure-activity relationship studies on ADAM protein-integrin interactions. Cardiovascular & hematological agents in medicinal chemistry 5, 29-42.
Lu, X., Wang, Q., Hu, G., Van Poznak, C., Fleisher, M., Reiss, M., Massague, J., and Kang, Y. (2009). ADAMTS1 and MMP1 proteolytically engage EGF-like ligands in an osteolytic signaling cascade for bone metastasis. Genes & development 23, 1882-1894.
Luan, Y., Kong, L., Howell, D., Ilalov, K., Fajardo, M., Bai, X. H., Di Cesare, P., Goldring, M., Abramson, S., and Liu, C. J. (2008). Inhibition of ADAMTS-7 and ADAMTS-12 degradation of cartilage oligomeric matrix protein by alpha-2-macroglobulin. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society 16, 1413-1420.
Tan IA 137 CHAPTER 6 Bibliography
Luque, A., Carpizo, D. R., and Iruela-Arispe, M. L. (2003). ADAMTS1/METH1 inhibits endothelial cell proliferation by direct binding and sequestration of VEGF165. The Journal of biological chemistry 278, 23656-23665.
Marques, R., Erkens-Schulze, S., de Ridder, C., Hermans, K., Waltering, K., Visakorpi, T., Trapman, J., Romijn, J., van Weerden, W., and Jenster, G. (2005). Androgen receptor modifications in prostate cancer cells upon long-termandrogen ablation and antiandrogen treatment. International journal of cancer Journal international du cancer 117, 221-229.
Mas, V., Maluf, D., Archer, K., Yanek, K., Kong, X., Kulik, L., Freise, C., Olthoff, K., Ghobrial, R., McIver, P., and Fisher, R. (2009). Genes involved in viral carcinogenesis and tumor initiation in hepatitis C virus- induced hepatocellular carcinoma. Molecular medicine (Cambridge, Mass) 15, 85-94.
Masui, T., Hosotani, R., Tsuji, S., Miyamoto, Y., Yasuda, S., Ida, J., Nakajima, S., Kawaguchi, M., Kobayashi, H., Koizumi, M., et al. (2001). Expression of METH-1 and METH-2 in pancreatic cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 7, 3437- 3443.
McCarthy, J., Skubitz, A., Qi, Z., Yi, X., Mickelson, D., Klein, D., and Furcht, L. (1990). RGD- independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities. The Journal of cell biology 110, 777-787.
McLane, M., Sanchez, E., Wong, A., Paquette-Straub, C., and Perez, J. (2004). Disintegrins. Current drug targets Cardiovascular & haematological disorders 4, 327-355.
Mei, J., Jin, L. P., Ding, D., Li, M. Q., Li, D. J., and Zhu, X. Y. (2012). Inhibition of IDO1 suppresses cyclooxygenase-2 and matrix metalloproteinase-9 expression and decreases proliferation, adhesion and invasion of endometrial stromal cells. Molecular human reproduction 18, 467-476.
Meierjohann, S., Hufnagel, A., Wende, E., Kleinschmidt, M., Wolf, K., Friedl, P., Gaubatz, S., and Schartl, M. (2010). MMP13 mediates cell cycle progression in melanocytes and melanoma cells: in vitro studies of migration and proliferation. Molecular cancer 9, 201.
Tan IA 138 CHAPTER 6 Bibliography
Mellado, B., Codony, J., Ribal, M. J., Visa, L., and Gascon, P. (2009). Molecular biology of androgen- independent prostate cancer: the role of the androgen receptor pathway. Clin Transl Oncol 11, 5-10.
Mi, Z., Oliver, T., Guo, H., Gao, C., and Kuo, P. (2007). Thrombin-cleaved COOH(-) terminal osteopontin peptide binds with cyclophilin C to CD147 in murine breast cancer cells. Cancer research 67, 4088-4097.
Minn, A. J., Kang, Y., Serganova, I., Gupta, G. P., Giri, D. D., Doubrovin, M., Ponomarev, V., Gerald, W. L., Blasberg, R., and Massague, J. (2005). Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. J Clin Invest 115, 44-55.
Misra, S., Lee, N., Fu, A. A., Raghavakaimal, S., Mandrekar, J., Bjarnason, H., McKusick, M. A., Iruela- Arispe, L., and Mukhopadhyay, D. (2008). Increased expression of a disintegrin and metalloproteinase thrombospondin 1 in thrombosed hemodialysis grafts. Journal of vascular and interventional radiology : JVIR 19, 111-119.
Misra, S., Shergill, U., Yang, B., Janardhanan, R., and Misra, K. D. (2010). Increased expression of HIF- 1alpha, VEGF-A and its receptors, MMP-2, TIMP-1, and ADAMTS-1 at the venous stenosis of arteriovenous fistula in a mouse model with renal insufficiency. Journal of vascular and interventional radiology : JVIR 21, 1255-1261.
Mittaz, L., Russell, D. L., Wilson, T., Brasted, M., Tkalcevic, J., Salamonsen, L. A., Hertzog, P. J., and Pritchard, M. A. (2004). Adamts-1 is essential for the development and function of the urogenital system. Biology of reproduction 70, 1096-1105.
Molokwu, C. N., Adeniji, O. O., Chandrasekharan, S., Hamdy, F. C., and Buttle, D. J. (2010). Androgen regulates ADAMTS15 gene expression in prostate cancer cells. Cancer investigation 28, 698-710.
Morales, J., Al-Sharif, L., Khalil, D., Shinwari, J., Bavi, P., Al-Mahrouqi, R., Al-Rajhi, A., Alkuraya, F., Meyer, B., and Al Tassan, N. (2009). Homozygous mutations in ADAMTS10 and ADAMTS17 cause lenticular myopia, ectopia lentis, glaucoma, spherophakia, and short stature. American journal of human genetics 85, 558-568.
Tan IA 139 CHAPTER 6 Bibliography
Morrissey, C., True, L. D., Roudier, M. P., Coleman, I. M., Hawley, S., Nelson, P. S., Coleman, R., Wang, Y. C., Corey, E., Lange, P. H., et al. (2008). Differential expression of angiogenesis associated genes in prostate cancer bone, liver and lymph node metastases. Clinical & experimental metastasis 25, 377-388.
Muthuswamy, S., and Xue, B. (2012). Cell polarity as a regulator of cancer cell behavior plasticity. Annual review of cell and developmental biology 28, 599-625.
Mydlo, J. H., Kral, J. G., Volpe, M., Axotis, C., Macchia, R. J., and Pertschuk, L. P. (1998). An analysis of microvessel density, androgen receptor, p53 and HER-2/neu expression and Gleason score in prostate cancer . preliminary results and therapeutic implications. Eur Urol 34, 426-432.
Nakada, M., Kita, D., Futami, K., Yamashita, J., Fujimoto, N., Sato, H., and Okada, Y. (2001). Roles of membrane type 1 matrix metalloproteinase and tissue inhibitor of metalloproteinases 2 in invasion and dissemination of human malignant glioma. Journal of neurosurgery 94, 464-473.
Nakamura, M., Sone, S., Takahashi, I., Mizoguchi, I., Echigo, S., and Sasano, Y. (2005). Expression of versican and ADAMTS1, 4, and 5 during bone development in the rat mandible and hind limb. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 53, 1553- 1562.
Navarro, D., Luzardo, O. P., Fernandez, L., Chesa, N., and Diaz-Chico, B. N. (2002). Transition to androgen-independence in prostate cancer. J Steroid Biochem Mol Biol 81, 191-201.
Neubauer, K., Saile, B., and Ramadori, G. (2001). Liver fibrosis and altered matrix synthesis. Can J Gastroenterol 15, 187-193.
Niggemann, B., Maaser, K., Lü, H., Kroczek, R., Zänker, K., and Friedl, P. (1997). Locomotory phenotypes of human tumor cell lines and T lymphocytes in a three-dimensional collagen lattice. Cancer letters 118, 173-180.
Tan IA 140 CHAPTER 6 Bibliography
Obika, M., Ogawa, H., Takahashi, K., Li, J., Hatipoglu, O., Cilek, M., Miyoshi, T., Inagaki, J., Ohtsuki, T., Kusachi, S., et al. (2012). Tumor growth inhibitory effect of ADAMTS1 is accompanied by the inhibition of tumor angiogenesis. Cancer science.
Odenwald, M., Prosperi, J., and Goss, K. (2013). APC/β-catenin-rich complexes at membrane protrusions regulate mammary tumor cell migration and mesenchymal morphology. BMC cancer 13, 12.
Offersen, B. V., Borre, M., and Overgaard, J. (1998). Immunohistochemical determination of tumor angiogenesis measured by the maximal microvessel density in human prostate cancer. APMIS 106, 463-469.
Offersen, B. V., Borre, M., and Overgaard, J. (2003). Quantification of angiogenesis as a prognostic marker in human carcinomas: a critical evaluation of histopathological methods for estimation of vascular density. Eur J Cancer 39, 881-890.
Ogier, C., Bernard, A., Chollet, A.-M., Le Diguardher, T., Hanessian, S., Charton, G., Khrestchatisky, M., and Rivera, S. (2006). Matrix metalloproteinase-2 (MMP-2) regulates astrocyte motility in connection with the actin cytoskeleton and integrins. Glia 54, 272-284.
Okada, Y. (2000). Tumor cell-matrix interaction: pericellular matrix degradation and metastasis. Verhandlungen der Deutschen Gesellschaft fur Pathologie 84, 33-75.
Okina, E., Grossi, A., Gopal, S., Multhaupt, H. A., and Couchman, J. R. (2012). Alpha-actinin interactions with syndecan-4 are integral to fibroblast-matrix adhesion and regulate cytoskeletal architecture. The international journal of biochemistry & cell biology 44, 2161-2174.
Palecek, S., Loftus, J., Ginsberg, M., Lauffenburger, D., and Horwitz, A. (1997). Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 385, 537-540.
Palm, D., Lang, K., Brandt, B., Zaenker, K., and Entschladen, F. (2005). In vitro and in vivo imaging of cell migration: two interdepending methods to unravel metastasis formation. Seminars in cancer biology 15, 396-404.
Tan IA 141 CHAPTER 6 Bibliography
Paulus, W., Baur, I., Beutler, A., and Reeves, S. (1996). Diffuse brain invasion of glioma cells requires beta 1 integrins. Laboratory investigation; a journal of technical methods and pathology 75, 819-826.
Peluffo, M. C., Murphy, M. J., Baughman, S. T., Stouffer, R. L., and Hennebold, J. D. (2011). Systematic analysis of protease gene expression in the rhesus macaque ovulatory follicle: metalloproteinase involvement in follicle rupture. Endocrinology 152, 3963-3974.
Pierschbacher, M., and Ruoslahti, E. (1984). Variants of the cell recognition site of fibronectin that retain attachment-promoting activity. Proceedings of the National Academy of Sciences of the United States of America 81, 5985-5988.
Pignatelli, J., Jones, M., LaLonde, D., and Turner, C. (2012). Beta2-adaptin binds actopaxin and regulates cell spreading, migration and matrix degradation. PloS one 7.
Plow, E., Haas, T., Zhang, L., Loftus, J., and Smith, J. (2000). Ligand binding to integrins. The Journal of biological chemistry 275, 21785-21788.
Pollard, T., and Borisy, G. (2003). Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453-465.
Porter, S., Clark, I. M., Kevorkian, L., and Edwards, D. R. (2005). The ADAMTS metalloproteinases. The Biochemical journal 386, 15-27.
Porter, S., Scott, S., Sassoon, E., Williams, M., Jones, J., Girling, A., Ball, R., and Edwards, D. (2004). Dysregulated expression of adamalysin-thrombospondin genes in human breast carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research 10, 2429-2469.
Porter, S., Span, P. N., Sweep, F. C., Tjan-Heijnen, V. C., Pennington, C. J., Pedersen, T. X., Johnsen, M., Lund, L. R., Romer, J., and Edwards, D. R. (2006). ADAMTS8 and ADAMTS15 expression predicts survival in human breast carcinoma. International journal of cancer Journal international du cancer 118, 1241-1247.
Price, L., Leng, J., Schwartz, M., and Bokoch, G. (1998). Activation of Rac and Cdc42 by integrins mediates cell spreading. Molecular biology of the cell 9, 1863-1871.
Tan IA 142 CHAPTER 6 Bibliography
Raviraj, V., Fok, S., Zhao, J., Chien, H.-Y., Lyons, J., Thompson, E., and Soon, L. (2012a). Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices. BMC cell biology 13, 12.
Raviraj, V., Zhang, H., Chien, H.-y., Cole, L., Thompson, E., and Soon, L. (2012b). Dormant but migratory tumour cells in desmoplastic stroma of invasive ductal carcinomas. Clinical & experimental metastasis 29, 273-292.
Rehn, A. P., Birch, M. A., Karlstrom, E., Wendel, M., and Lind, T. (2007). ADAMTS-1 increases the three-dimensional growth of osteoblasts through type I collagen processing. Bone 41, 231-238.
Ren, X., Kiosses, W., and Schwartz, M. (1999). Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. The EMBO journal 18, 578-585.
Reynolds, L. E., Watson, A. R., Baker, M., Jones, T. A., D'Amico, G., Robinson, S. D., Joffre, C., Garrido-Urbani, S., Rodriguez-Manzaneque, J. C., Martino-Echarri, E., et al. (2010). Tumour angiogenesis is reduced in the Tc1 mouse model of Down's syndrome. Nature 465, 813-817.
Ricciardelli, C., Brooks, J. H., Suwiwat, S., Sakko, A. J., Mayne, K., Raymond, W. A., Seshadri, R., LeBaron, R. G., and Horsfall, D. J. (2002). Regulation of stromal versican expression by breast cancer cells and importance to relapse-free survival in patients with node-negative primary breast cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 8, 1054- 1060.
Ricciardelli, C., Frewin, K. M., Tan Ide, A., Williams, E. D., Opeskin, K., Pritchard, M. A., Ingman, W. V., and Russell, D. L. (2011). The ADAMTS1 protease gene is required for mammary tumor growth and metastasis. The American journal of pathology 179, 3075-3085.
Ricciardelli, C., Russell, D. L., Ween, M. P., Mayne, K., Suwiwat, S., Byers, S., Marshall, V. R., Tilley, W. D., and Horsfall, D. J. (2007). Formation of hyaluronan- and versican-rich pericellular matrix by prostate cancer cells promotes cell motility. The Journal of biological chemistry 282, 10814-10825.
Ricciardelli, C., Sakko, A. J., Ween, M. P., Russell, D. L., and Horsfall, D. J. (2009). The biological role and regulation of versican levels in cancer. Cancer metastasis reviews 28, 233-245.
Tan IA 143 CHAPTER 6 Bibliography
Ridley, A., and Hall, A. (1992). The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70, 389-399.
Ridley, A., Schwartz, M., Burridge, K., Firtel, R., Ginsberg, M., Borisy, G., Parsons, J., and Horwitz, A. (2003). Cell migration: integrating signals from front to back. Science (New York, NY) 302, 1704-1709.
Rocks, N., Paulissen, G., Quesada Calvo, F., Polette, M., Gueders, M., Munaut, C., Foidart, J. M., Noel, A., Birembaut, P., and Cataldo, D. (2006). Expression of a disintegrin and metalloprotease (ADAM and ADAMTS) enzymes in human non-small-cell lung carcinomas (NSCLC). Br J Cancer 94, 724-730.
Rocks, N., Paulissen, G., Quesada-Calvo, F., Munaut, C., Gonzalez, M. L., Gueders, M., Hacha, J., Gilles, C., Foidart, J. M., Noel, A., and Cataldo, D. D. (2008). ADAMTS-1 metalloproteinase promotes tumor development through the induction of a stromal reaction in vivo. Cancer research 68, 9541-9550.
Rodriguez-Manzaneque, J. C., Carpizo, D., Plaza-Calonge Mdel, C., Torres-Collado, A. X., Thai, S. N., Simons, M., Horowitz, A., and Iruela-Arispe, M. L. (2009). Cleavage of syndecan-4 by ADAMTS1 provokes defects in adhesion. The international journal of biochemistry & cell biology 41, 800-810.
Rodriguez-Manzaneque, J. C., Milchanowski, A. B., Dufour, E. K., Leduc, R., and Iruela-Arispe, M. L. (2000). Characterization of METH-1/ADAMTS1 processing reveals two distinct active forms. The Journal of biological chemistry 275, 33471-33479.
Rodriguez-Manzaneque, J. C., Westling, J., Thai, S. N., Luque, A., Knauper, V., Murphy, G., Sandy, J. D., and Iruela-Arispe, M. L. (2002). ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors. Biochemical and biophysical research communications 293, 501-508.
Rolli, M., Fransvea, E., Pilch, J., Saven, A., and Felding-Habermann, B. (2003). Activated integrin alphavbeta3 cooperates with metalloproteinase MMP-9 in regulating migration of metastatic breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America 100, 9482-9487.
Tan IA 144 CHAPTER 6 Bibliography
Rösel, D., Brábek, J., Tolde, O., Mierke, C., Zitterbart, D., Raupach, C., Bicanová, K., Kollmannsberger, P., Panková, D., Vesely, P., et al. (2008). Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces. Molecular cancer research : MCR 6, 1410-1420.
Rouzier, R., Perou, C., Symmans, W., Ibrahim, N., Cristofanilli, M., Anderson, K., Hess, K., Stec, J., Ayers, M., Wagner, P., et al. (2005). Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clinical cancer research : an official journal of the American Association for Cancer Research 11, 5678-5685.
Rucci, N., Sanità, P., and Angelucci, A. (2011). Roles of metalloproteases in metastatic niche. Current molecular medicine 11, 609-622.
Ruoslahti, E. (1996). RGD and other recognition sequences for integrins. Annual review of cell and developmental biology 12, 697-715.
Russell, D. L., Doyle, K. M., Ochsner, S. A., Sandy, J. D., and Richards, J. S. (2003). Processing and localization of ADAMTS-1 and proteolytic cleavage of versican during cumulus matrix expansion and ovulation. The Journal of biological chemistry 278, 42330-42339.
Russell, P., and Bennett…, S. (1998). Growth factor involvement in progression of prostate cancer. Clinical chemistry.
Ryeom, S., and Folkman, J. (2009). Role of endogenous angiogenesis inhibitors in Down syndrome. The Journal of craniofacial surgery 20 Suppl 1, 595-596.
Sabeh, F., Shimizu-Hirota, R., and Weiss, S. (2009). Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited. The Journal of cell biology 185, 11-19.
Sahai, E., and Marshall, C. (2003). Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nature cell biology 5, 711-719.
Tan IA 145 CHAPTER 6 Bibliography
Saiki, I., Fujii, H., Yoneda, J., Abe, F., Nakajima, M., Tsuruo, T., and Azuma, I. (1993). Role of aminopeptidase N (CD13) in tumor-cell invasion and extracellular matrix degradation. International journal of cancer Journal international du cancer 54, 137-143.
Sakko, A. J., Ricciardelli, C., Mayne, K., Tilley, W. D., Lebaron, R. G., and Horsfall, D. J. (2001). Versican accumulation in human prostatic fibroblast cultures is enhanced by prostate cancer cell- derived transforming growth factor beta1. Cancer research 61, 926-930.
Sales, K., Boddy, S., and Jabbour, H. (2008). F-prostanoid receptor alters adhesion, morphology and migration of endometrial adenocarcinoma cells. Oncogene 27, 2466-2477.
Sandy, J. D., Westling, J., Kenagy, R. D., Iruela-Arispe, M. L., Verscharen, C., Rodriguez-Mazaneque, J. C., Zimmermann, D. R., Lemire, J. M., Fischer, J. W., Wight, T. N., and Clowes, A. W. (2001). Versican V1 proteolysis in human aorta in vivo occurs at the Glu441-Ala442 bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4. The Journal of biological chemistry 276, 13372-13378.
Schneider, D., Liaw, L., Daniel, C., Athanasopoulos, A., Herrmann, M., Preissner, K., Nawroth, P., and Chavakis, T. (2007). Inhibition of breast cancer cell adhesion and bone metastasis by the extracellular adherence protein of Staphylococcus aureus. Biochemical and biophysical research communications 357, 282-288.
Schwettmann, L., Wehmeier, M., Jokovic, D., Aleksandrova, K., Brand, K., Manns, M. P., Lichtinghagen, R., and Bahr, M. J. (2008). Hepatic expression of A disintegrin and metalloproteinase (ADAM) and ADAMs with thrombospondin motives (ADAM-TS) enzymes in patients with chronic liver diseases. J Hepatol 49, 243-250.
Sheng, W., Wang, G., Wang, Y., Liang, J., Wen, J., Zheng, P. S., Wu, Y., Lee, V., Slingerland, J., Dumont, D., and Yang, B. B. (2005). The roles of versican V1 and V2 isoforms in cell proliferation and apoptosis. Mol Biol Cell 16, 1330-1340.
Shindo, T., Kurihara, H., Kuno, K., Yokoyama, H., Wada, T., Kurihara, Y., Imai, T., Wang, Y., Ogata, M., Nishimatsu, H., et al. (2000). ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function. J Clin Invest 105, 1345-1352.
Tan IA 146 CHAPTER 6 Bibliography
Shore, N., Mason, M., and de Reijke, T. (2012). New developments in castrate-resistant prostate cancer. BJU international 109 Suppl 6, 22-32.
Siegal, J. A., Yu, E., and Brawer, M. K. (1995). Topography of neovascularity in human prostate carcinoma. Cancer 75, 2545-2551.
Silver, D. L., Hou, L., Somerville, R., Young, M. E., Apte, S. S., and Pavan, W. J. (2008). The secreted metalloprotease ADAMTS20 is required for melanoblast survival. PLoS genetics 4, e1000003.
Somerville, R., Longpre, J.-M., Jungers, K., Engle, J., Ross, M., Evanko, S., Wight, T., Leduc, R., and Apte, S. (2003). Characterization of ADAMTS-9 and ADAMTS-20 as a distinct ADAMTS subfamily related to Caenorhabditis elegans GON-1. The Journal of biological chemistry 278, 9503-9513.
Sørlie, T., Perou, C., Tibshirani, R., Aas, T., Geisler, S., Johnsen, H., Hastie, T., Eisen, M., van de Rijn, M., Jeffrey, S., et al. (2001). Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proceedings of the National Academy of Sciences of the United States of America 98, 10869-10874.
Sorlie, T., Tibshirani, R., Parker, J., Hastie, T., Marron, J., Nobel, A., Deng, S., Johnsen, H., Pesich, R., Geisler, S., et al. (2003). Repeated observation of breast tumor subtypes in independent gene expression data sets. Proceedings of the National Academy of Sciences of the United States of America 100, 8418-8423.
Soteriadou, K., Remoundos, M., Katsikas, M., Tzinia, A., Tsikaris, V., Sakarellos, C., and Tzartos, S. (1992). The Ser-Arg-Tyr-Asp region of the major surface glycoprotein of Leishmania mimics the Arg- Gly-Asp-Ser cell attachment region of fibronectin. The Journal of biological chemistry 267, 13980- 13985.
Soule, H., Maloney, T., Wolman, S., Peterson, W., Brenz, R., McGrath, C., Russo, J., Pauley, R., Jones, R., and Brooks, S. (1990). Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer research 50, 6075-6086.
Tan IA 147 CHAPTER 6 Bibliography
Srivatanakul, P., Sriplung, H., and Deerasamee, S. (2004). Epidemiology of liver cancer: an overview. Asian Pac J Cancer Prev 5, 118-125.
Stankunas, K., Hang, C., Tsun, Z.-Y., Chen, H., Lee, N., Wu, J., Shang, C., Bayle, J., Shou, W., Iruela- Arispe, M., and Chang, C.-P. (2008). Endocardial Brg1 represses ADAMTS1 to maintain the microenvironment for myocardial morphogenesis. Developmental cell 14, 298-609.
Starr, S. P., and Raines, D. (2011). Cirrhosis: diagnosis, management, and prevention. Am Fam Physician 84, 1353-1359.
Stegalkina, S., Guerrero, A., Walton, K., Liu, X., Robinson, G., and Hennighausen, L. (1999). Transcription originating in the long terminal repeats of the endogenous mouse mammary tumor virus MTV-3 is activated in Stat5a-null mice and picks Up hitchhiking exons. Journal of virology 73, 8669- 8676.
Steiner, I., Jung, K., Miller, K., Stephan, C., and Erbersdobler, A. (2012). Expression of endothelial factors in prostate cancer: a possible role of caveolin-1 for tumour progression. Oncol Rep 27, 389-395.
Strohmeyer, D., Rossing, C., Bauerfeind, A., Kaufmann, O., Schlechte, H., Bartsch, G., and Loening, S. (2000). Vascular endothelial growth factor and its correlation with angiogenesis and p53 expression in prostate cancer. Prostate 45, 216-224.
Su, S. C., Mendoza, E. A., Kwak, H. I., and Bayless, K. J. (2008). Molecular profile of endothelial invasion of three-dimensional collagen matrices: insights into angiogenic sprout induction in wound healing. American journal of physiology Cell physiology 295, C1215-1229.
Sugimoto, K., Takahashi, M., Yamamoto, Y., Shimada, K., and Tanzawa, K. (1999). Identification of aggrecanase activity in medium of cartilage culture. Journal of biochemistry 126, 449-455.
Suwiwat, S., Ricciardelli, C., Tammi, R., Tammi, M., Auvinen, P., Kosma, V. M., LeBaron, R. G., Raymond, W. A., Tilley, W. D., and Horsfall, D. J. (2004). Expression of extracellular matrix components versican, chondroitin sulfate, tenascin, and hyaluronan, and their association with disease outcome in
Tan IA 148 CHAPTER 6 Bibliography
node-negative breast cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 10, 2491-2498.
Tai, P., Yu, E., Vinh-Hung, V., Cserni, G., and Vlastos, G. (2004). Survival of patients with metastatic breast cancer: twenty-year data from two SEER registries. BMC cancer 4, 60.
Terry, S., Elbediwy, A., Zihni, C., Harris, A., Bailly, M., Charras, G., Balda, M., and Matter, K. (2012). Stimulation of cortical myosin phosphorylation by p114RhoGEF drives cell migration and tumor cell invasion. PloS one 7.
Tester, A., Ruangpanit, N., Anderson, R., and Thompson, E. (2000). MMP-9 secretion and MMP-2 activation distinguish invasive and metastatic sublines of a mouse mammary carcinoma system showing epithelial-mesenchymal transition traits. Clinical & experimental metastasis 18, 553-560.
Tewari, A., Yardimci, G., Shibata, Y., Sheffield, N., Song, L., Taylor, B., Georgiev, S., Coetzee, G., Ohler, U., Furey, T., et al. (2012). Chromatin accessibility reveals insights into androgen receptor activation and transcriptional specificity. Genome biology 13.
Thai, S. N., and Iruela-Arispe, M. L. (2002). Expression of ADAMTS1 during murine development. Mechanisms of development 115, 181-185.
Thompson, E., Newgreen, D., and Tarin, D. (2005). Carcinoma invasion and metastasis: a role for epithelial-mesenchymal transition? Cancer research 65, 5991.
Tomic, T. T., Gustavsson, H., Wang, W., Jennbacken, K., Welen, K., and Damber, J. E. (2012). Castration resistant prostate cancer is associated with increased blood vessel stabilization and elevated levels of VEGF and Ang-2. Prostate 72, 705-712.
Tran, B., and Bedard, P. (2011). Luminal-B breast cancer and novel therapeutic targets. Breast cancer research : BCR 13, 221.
Tyan, S. W., Hsu, C. H., Peng, K. L., Chen, C. C., Kuo, W. H., Lee, E. Y., Shew, J. Y., Chang, K. J., Juan, L. J., and Lee, W. H. (2012). Breast cancer cells induce stromal fibroblasts to secrete ADAMTS1 for cancer invasion through an epigenetic change. PloS one 7, e35128.
Tan IA 149 CHAPTER 6 Bibliography
Van Slambrouck, S., Jenkins, A. R., Romero, A. E., and Steelant, W. F. (2009). Reorganization of the integrin alpha2 subunit controls cell adhesion and cancer cell invasion in prostate cancer. International journal of oncology 34, 1717-1726. van Zijl, F., Krupitza, G., and Mikulits, W. (2011). Initial steps of metastasis: cell invasion and endothelial transmigration. Mutation research 728, 23-34.
Varambally, S., Yu, J., Laxman, B., Rhodes, D., Mehra, R., Tomlins, S., Shah, R., Chandran, U., Monzon, F., Becich, M., et al. (2005). Integrative genomic and proteomic analysis of prostate cancer reveals signatures of metastatic progression. Cancer cell 8, 393-406.
Vazquez, F., Hastings, G., Ortega, M. A., Lane, T. F., Oikemus, S., Lombardo, M., and Iruela-Arispe, M. L. (1999). METH-1, a human ortholog of ADAMTS-1, and METH-2 are members of a new family of proteins with angio-inhibitory activity. The Journal of biological chemistry 274, 23349-23357.
Veeravalli, K., Chetty, C., Ponnala, S., Gondi, C., Lakka, S., Fassett, D., Klopfenstein, J., Dinh, D., Gujrati, M., and Rao, J. (2010). MMP-9, uPAR and cathepsin B silencing downregulate integrins in human glioma xenograft cells in vitro and in vivo in nude mice. PloS one 5.
Viloria, C. G., Obaya, A. J., Moncada-Pazos, A., Llamazares, M., Astudillo, A., Capella, G., Cal, S., and Lopez-Otin, C. (2009). Genetic inactivation of ADAMTS15 metalloprotease in human colorectal cancer. Cancer research 69, 4926-4934.
Visakorpi, T., Hyytinen, E., Koivisto, P., Tanner, M., Keinänen, R., Palmberg, C., Palotie, A., Tammela, T., Isola, J., and Kallioniemi, O. (1995). In vivo amplification of the androgen receptor gene and progression of human prostate cancer. Nature genetics 9, 401-406.
Voutilainen, K., Anttila, M., Sillanpaa, S., Tammi, R., Tammi, M., Saarikoski, S., and Kosma, V. M. (2003). Versican in epithelial ovarian cancer: relation to hyaluronan, clinicopathologic factors and prognosis. International journal of cancer Journal international du cancer 107, 359-364.
Tan IA 150 CHAPTER 6 Bibliography
Wachsmuth, L., Bau, B., Fan, Z., Pecht, A., Gerwin, N., and Aigner, T. (2004). ADAMTS-1, a gene product of articular chondrocytes in vivo and in vitro, is downregulated by interleukin 1beta. The Journal of rheumatology 31, 315-320.
Wagstaff, L., Kelwick, R., Decock, J., and Edwards, D. R. (2011). The roles of ADAMTS metalloproteinases in tumorigenesis and metastasis. Frontiers in bioscience : a journal and virtual library 16, 1861-1872.
Wang, D., Zhu, T., Zhang, F. B., and He, C. (2011). Expression of ADAMTS12 in colorectal cancer- associated stroma prevents cancer development and is a good prognostic indicator of colorectal cancer. Digestive diseases and sciences 56, 3281-3287.
Wang, W., Xu, G. L., Jia, W. D., Ma, J. L., Li, J. S., Ge, Y. S., Ren, W. H., Yu, J. H., and Liu, W. B. (2009). Ligation of TLR2 by versican: a link between inflammation and metastasis. Archives of medical research 40, 321-323.
Wang, Y., Li, L., Guo, X., Jin, X., Sun, W., Zhang, X., and Xu, R. C. (2012a). Interleukin-6 signaling regulates anchorage-independent growth, proliferation, adhesion and invasion in human ovarian cancer cells. Cytokine 59, 228-236.
Wang, Y., Xu, R., Zhang, X., Niu, X., Qu, Y., Li, L., and Meng, X. (2012b). Interleukin-8 secretion by ovarian cancer cells increases anchorage-independent growth, proliferation, angiogenic potential, adhesion and invasion. Cytokine 59, 145-155.
Ween, M. P., Hummitzsch, K., Rodgers, R. J., Oehler, M. K., and Ricciardelli, C. (2011). Versican induces a pro-metastatic ovarian cancer cell behavior which can be inhibited by small hyaluronan oligosaccharides. Clinical & experimental metastasis 28, 113-125.
Wei, X., Prickett, T., Viloria, C., Molinolo, A., Lin, J., Cardenas-Navia, I., Cruz, P., Program, N. C. S., Rosenberg, S., Davies, M., et al. (2010). Mutational and functional analysis reveals ADAMTS18 metalloproteinase as a novel driver in melanoma. Molecular cancer research : MCR 8, 1513-1525.
Tan IA 151 CHAPTER 6 Bibliography
Winter, S. F., Acevedo, V. D., Gangula, R. D., Freeman, K. W., Spencer, D. M., and Greenberg, N. M. (2007). Conditional activation of FGFR1 in the prostate epithelium induces angiogenesis with concomitant differential regulation of Ang-1 and Ang-2. Oncogene 26, 4897-4907.
Wolf, K., Mazo, I., Leung, H., Engelke, K., von Andrian, U., Deryugina, E., Strongin, A., Bröcker, E.-B., and Friedl, P. (2003). Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. The Journal of cell biology 160, 267-277.
Wong, C., Song, C., Grimes, M., Fu, W., Dewhirst, M., Muschel, R., and Al-Mehdi, A.-B. (2002). Intravascular location of breast cancer cells after spontaneous metastasis to the lung. The American journal of pathology 161, 749-753.
Wu, Y., Chen, L., Cao, L., Sheng, W., and Yang, B. B. (2004). Overexpression of the C-terminal PG- M/versican domain impairs growth of tumor cells by intervening in the interaction between epidermal growth factor receptor and beta1-integrin. Journal of cell science 117, 2227-2237.
Wurmbach, E., Chen, Y.-b., Khitrov, G., Zhang, W., Roayaie, S., Schwartz, M., Fiel, I., Thung, S., Mazzaferro, V., Bruix, J., et al. (2007). Genome-wide molecular profiles of HCV-induced dysplasia and hepatocellular carcinoma. Hepatology (Baltimore, Md) 45, 938-947.
Yamana, N., Arakawa, Y., Nishino, T., Kurokawa, K., Tanji, M., Itoh, R., Monypenny, J., Ishizaki, T., Bito, H., Nozaki, K., et al. (2006). The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src. Molecular and cellular biology 26, 6844- 6858.
Yang, B. L., Zhang, Y., Cao, L., and Yang, B. B. (1999). Cell adhesion and proliferation mediated through the G1 domain of versican. J Cell Biochem 72, 210-220.
Yatabe, T., Mochizuki, S., Takizawa, M., Chijiiwa, M., Okada, A., Kimura, T., Fujita, Y., Matsumoto, H., Toyama, Y., and Okada, Y. (2009). Hyaluronan inhibits expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic chondrocytes. Annals of the rheumatic diseases 68, 1051-1058.
Tan IA 152 CHAPTER 6 Bibliography
Yegnasubramanian, S., Wu, Z., Haffner, M. C., Esopi, D., Aryee, M. J., Badrinath, R., He, T. L., Morgan, J. D., Carvalho, B., Zheng, Q., et al. (2011). Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences. BMC genomics 12, 313.
Yilmaz, M., and Christofori, G. (2010). Mechanisms of motility in metastasizing cells. Molecular cancer research : MCR 8, 629-642.
Yokotsuka, M., Iwaya, K., Saito, T., Pandiella, A., Tsuboi, R., Kohno, N., Matsubara, O., and Mukai, K. (2011). Overexpression of HER2 signaling to WAVE2-Arp2/3 complex activates MMP-independent migration in breast cancer. Breast cancer research and treatment 126, 311-318.
Yu, J., Ren, X., Cao, S., Zhang, W., and Hao, X. (2006). Th1 polarization and apoptosis-inducing activity of CD4+ T -cells in cytokine-induced killers might favor the antitumor cytotoxicity of cytokine- induced killers in vivo. Cancer biotherapy & radiopharmaceuticals 21, 276-284.
Yu, X., and Machesky, L. (2012). Cells assemble invadopodia-like structures and invade into matrigel in a matrix metalloprotease dependent manner in the circular invasion assay. PloS one 7.
Zhang, J., Niu, X.-M., Liao, M.-L., Liu, Y., Sha, H.-F., Zhao, Y., Yu, Y.-F., Tan, Q., Xiang, J.-Q., Fang, J., et al. (2010). Discrepancy of biologic behavior influenced by bone marrow derived cells in lung cancer. Oncology reports 24, 1185-1192.
Zhang, X., Brunner, T., Carter, L., Dutton, R. W., Rogers, P., Bradley, L., Sato, T., Reed, J. C., Green, D., and Swain, S. L. (1997). Unequal death in T helper cell (Th)1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis. The Journal of experimental medicine 185, 1837- 1849.
Zhang, Y., Cao, L., Kiani, C., Yang, B. L., Hu, W., and Yang, B. B. (1999). Promotion of chondrocyte proliferation by versican mediated by G1 domain and EGF-like motifs. J Cell Biochem 73, 445-457.
Tan IA 153 CHAPTER 6 Bibliography
Zhang, Y., Cao, L., Kiani, C. G., Yang, B. L., and Yang, B. B. (1998). The G3 domain of versican inhibits mesenchymal chondrogenesis via the epidermal growth factor-like motifs. The Journal of biological chemistry 273, 33054-33063.
Zheng, P. S., Wen, J., Ang, L. C., Sheng, W., Viloria-Petit, A., Wang, Y., Wu, Y., Kerbel, R. S., and Yang, B. B. (2004). Versican/PG-M G3 domain promotes tumor growth and angiogenesis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 18, 754- 756.
Zhu, M., Yi, M., Kim, C., Deng, C., Li, Y., Medina, D., Stephens, R., and Green, J. (2011). Integrated miRNA and mRNA expression profiling of mouse mammary tumor models identifies miRNA signatures associated with mammary tumor lineage. Genome biology 12.
Zi, Z., Chapnick, D., and Liu, X. (2012). Dynamics of TGF-β/Smad signaling. FEBS letters 586, 1921- 1928.
Zong, F., Fthenou, E., Mundt, F., Szatmari, T., Kovalszky, I., Szilak, L., Brodin, D., Tzanakakis, G., Hjerpe, A., and Dobra, K. (2011). Specific syndecan-1 domains regulate mesenchymal tumor cell adhesion, motility and migration. PloS one 6, e14816.
Zreiqat, H., Belluoccio, D., Smith, M. M., Wilson, R., Rowley, L. A., Jones, K., Ramaswamy, Y., Vogl, T., Roth, J., Bateman, J. F., and Little, C. B. (2010). S100A8 and S100A9 in experimental osteoarthritis. Arthritis research & therapy 12, R16.
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