DETERMINING THE ROLE OF MUC1 AND BETA-CATENIN ON THE EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING AND LOCALIZATION IN BREAST CANCER
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Authors Bitler, Benjamin Guy
Publisher The University of Arizona.
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Link to Item http://hdl.handle.net/10150/194738 1
DETERMINING THE ROLE OF MUC1 AND BETA-CATENIN ON THE EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING AND LOCALIZATION IN BREAST CANCER
By
Benjamin Guy Bitler
A Dissertation Submitted to the Faculty of the
GRADUATE INTERDISCIPLINARY PROGRAM IN CANCER BIOLOGY
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2010 2
THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE
As members of the Dissertation Committee, we certify that we have read the dissertation
prepared by Benjamin Guy Bitler
entitled Determining the role of MUC1 and beta-catenin on the epidermal growth factor receptor signaling and localization in breast cancer.
and recommend that it be accepted as fulfilling the dissertation requirement for the
Degree of Doctor of Philosophy
Dr. Joyce Schroeder ______Date: 05/07/2010
Dr. Eugene Gerner ______Date: 05/07/2010
Dr. Ron Heimark ______Date: 05/07/2010
Dr. Jesse Martinez ______Date: 05/07/2010
Dr. Raymond Nagle ______Date: 05/07/2010
Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.
______Date: 05/07/2010 Dissertation Director: Dr. Joyce Schroeder
3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department of the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
SIGNED: Benjamin Guy Bitler
4
ACKNOWLEDGMENTS
First and foremost I would like to acknowledge my wife, Jamie Bitler. Her support in my pursuit of a PhD has been unwavering and constant. She is my best-friend and has been by my side every step of the way. We have experienced a lot in our short lives and I cannot imagine anyone else I would rather be with.
I would like to acknowledge my mentor, Dr. Joyce Schroeder, for her direction, encouragement and friendship. I can never begin to describe my appreciation for Joyce and all of the things she has done for me. I admire her appreciation and understanding of science and the constant drive to make me a better scientist.
I would like to acknowledge my family who have been my distractions in times experimental successes and failures. My dad and step-mother, my brothers, Brandon, John and Jack, and sisters, Stephanie and Savannah, have all supported me and given me the motivation to persevere. I am extremely proud of all of them.
I would like to acknowledge my lab family past and present for making my lab experiences enjoyable. I can honestly say for the past five years I have not been to “work” once, because I do not consider doing experiments, scientific discussions, and writing about my passions with people that share my same passions “work.” All of the friends I have made are lifelong friends and colleagues.
I would like to acknowledge the Cancer Biology program. More specifically I would like to thank Dr. Tim Bowden and Anne Cione for their guidance. I feel honored to have been a part of Dr. Bowden’s life and I feel as if I am a better person because of our relationship. There are not enough words to describe how much I appreciate all of the administrative assistance I have received from Anne Cione. She is an amazing person with a love and dedication to the Cancer Biology students that is unmatched.
I would like to acknowledge my committee members for their experimental suggestions and guidance. 5
DEDICATION
I am dedicating this work to my mother Lynne D. Bitler whose fight against breast
cancer was courageous and brave. I remember one incidence during a short period of
remission when my mom completed the 5K Susan G. Komen Race for the Cure. She was still bald from the chemotherapy, in agony from the cancer and was extremely exhausted and yet by her determination to defeat breast cancer she finished the race. Most people who have cancer and are undergoing treatment will sometimes have trouble just getting out bed or sitting up in a chair, but not my mom. I imagine when she completed the race she was telling the cancer that “I will not be defeated.” She was a warrior who wanted live to see her kids grow up, get married, graduate college, have kids of their own and be
successful. This was just one of example of her strength and perseverance, which
allowed her to battle cancer. The thought of my mom struggling with breast cancer has
motivated and pushed me to do research that will make a difference. When experiments
did not work or I had setbacks, I would refocus by reminding myself of my mom and all
the women that are dealing with this awful disease. I hope and pray that one day I will be
able to demonstrate the same courage as my mom in dealing with adversity. My mom is
my inspiration for my work and I hope that I have made her proud by continuing the fight
against breast cancer. 6
TABLE OF CONTENTS
LIST OF FIGURES ...... 10
LIST OF TABLES ...... 12
ABSTRACT ...... 13
I. INTRODUCTION...... 15
BREAST CANCER ...... 16 ERBB RECEPTORS...... 18 ERBB RECEPTOR TRAFFICKING ...... 24 ERBB RECEPTORS IN BREAST CANCER ...... 30 ERBB RECEPTOR THERAPEUTICS ...... 31 CELLULAR POLARITY...... 34 MUC1 ...... 37 MUC1 STRUCTURE ...... 38 MUC1 TRANSCRIPTIONAL REGULATION AND TRAFFICKING ...... 41 MUC1 CYTOPLASMIC TAIL PROTIEN INTERACTIONS ...... 43 BETA-CATENIN STRUCTURE FUNCTION ...... 46 BETA-CATENIN/CANONICAL WNT SIGNALING ...... 47 BETA-CATENIN IN BREAST CANCER ...... 50 PEPTIDE THERAPY ...... 51 STATEMENT OF THE PROBLEM...... 53
II. MATERIALS AND METHODS ...... 55
CELL LINES ...... 55 TRANSFECTION WITH SIRNA ...... 55 ANTIBODIES AND GROWTH FACTORS ...... 56 IMMUNOPRECIPITATION AND IMMUNOBLOTTING ...... 57 IMMUNOFLUORESCENCE ...... 58 GROWTH FACTOR TREATMENT ...... 58 DIFFERENTIAL DETERGENT FRACTIONATION ...... 58 CHROMATIN-BOUND PROTIEN ISOLATION ...... 60 HIGH-SALT PROTEIN EXTRACTION ...... 60 CHROMATIN IMMUNOPRECIPITATION ...... 61 INVASION ASSAY ...... 62 MTT ASSAY ...... 63
7
TABLE OF CONTENTS (Contd.)
PEPTIDE SYNTHESIS ...... 63 MICE ...... 64 MOUSE GENOTYPING ...... 64 MOUSE PROTEIN LYSATES ...... 64 MAMMARY GLAND WHOLE MOUNTS ...... 65 XENOGRAFT TUMOR STUDIES ...... 65 GENETICALLY-DRIVEN TUMOR STUDIES ...... 66 MMTV-WNT-1 TRANSGENIC MOUSE STUDY ...... 66 STATISTICAL ANALYSIS ...... 67
III. MUC1 REGULATES NUCLEAR LOCALIZATION AND FUNCTION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR ...... 68 INTRODUCTION ...... 68 RESULTS ...... 70 MUC1 PROMOTES ACCUMULATION OF NUCLEAR EGFR ...... 71 INHIBITING MUC1/EGFR INTERACTIONS DECREASES NUCLEAR EGFR ...... 77 MUC1 AND EGFR INTERACT IN THE NUCLEUS ...... 79 EGFR COLOCALIZES WITH PHOSPHORYLATED-RNA POLYMERASE II ...... 82 MUC1 PROMOTES EGFR-MEDIATED CYCLIN D1 EXPRESSION ...... 85 DISCUSSION ...... 88
IV. EGFR ACTIVITY PROMOTES TUMORIGENESIS IN MMTV-WNT-1 MOUSE MODEL OF BREAST CANCER ...... 93 INTRODUCTION ...... 93 RESULTS ...... 97 EGFR KINASE ACTIVITY PROMOTES WNT-1 DEPENDENT TUMORIGENESIS ...... 97 BETA-CATENIN AND E-CADHERIN PROTEIN EXPRESSION IS EGFR KINASE INDEPENDENT ...... 98 BETA-CATENIN LOCALIZATION IN MMTV-WNT-1 TUMORS IS EGFR KINASE INDEPENDENT ...... 98 EGFR KINASE ACTIVITY ALTERS MAMMARY GLAND DEVELOPMENT ...... 101 DISCUSSION ...... 102
8
TABLE OF CONTENTS (Contd.) V. INTRACELLULAR MUC1 PEPTIDES INHIBIT CANCER PROGRESSION ...... 105 INTRODUCTION ...... 105 RESULTS ...... 108 PMIP EFFICIENTLY ENTERS THE CELLS, REDUCES EGFR PROTEIN LEVELS AND INHIBITS MUC1 COLOCALIZATION WITH BETA- CATENIN ...... 108 PMIP INHIBITS CELLULAR INVASION AND PROLIFERATION IN VITRO ...... 113 PMIP INHIBITS TUMOR GROWTH AND RECURRENCE IN A XENOGRAFT BREAST CANCER MODEL ...... 115 PMIP INHIBITS TUMOR GROWTH AND INDUCES REGRESSION IN A GENETICALLY-DRIVEN BREAST CANCER ...... 118 MSPMIP TREATMENT RESULTS IN A REDUCTION OF EGFR AND MUC1 LEVELS ...... 123 PMIP DOWNREGULATES MET EXPRESSION...... 125 DISCUSSION ...... 128
VI. ANTI-CANCER THERAPIES THAT UTILIZE CELL PENETRATING PEPTIDES ...... 131 INTRODUCTION ...... 131 CELL PENETRATING PEPTIDES ...... 132 MECHANISM OF CELL PENETRATING PEPTIDES ...... 135 NON-IMMUNOGENIC ...... 138 ANTI-CANCER CELL PENETRATING PEPTIDES ...... 138 NUCLEAR TRANSLOCATION ...... 141 ONCOGENIC SIGNALING MODULATION ...... 145 MODULATION OF ECM INTERACTIONS ...... 150 NOVEL THERAPEUTIC TARGETS ...... 152 CURRENT & FUTURE DEVELOPMENTS ...... 152 ENDOSOMAL ESCAPE ...... 153 CPP STABILITY AND MODIFICATIONS ...... 153
VII. CONCLUDING STATEMENTS ...... 156 MUC1 AND EGFR’S ROLE IN AUTOPHAGY ...... 158 TARGETING MUC1/EGFR INTERACTION AND EGFR ACTIVITY IN COMBINATION ...... 159 EGFR AS A BIOMARKER ...... 161 PREVENTING LOSS OF CELL POLARITY ...... 161
9
TABLE OF CONTENTS (Contd.) ROLE OF HEAT SHOCK PROTIENS IN EGFR AND MUC1-DEPENDENT TUMORIGENESIS ...... 162
MUC1’S ROLE IN EGFR CYTOSOLIC RELEASE ...... 163 NUCLEAR EGFR TRAFFICKING: ANTI-CANCER TARGET ...... 164
IIX. REFERENCES ...... 168
10
LIST OF FIGURES
Figure 1.1: Cellular Origin and Subtypes of Breast Cancer ...... 17
Figure 1.2: ErbB Dimers and Adaptor Protiens ...... 20
Figure 1.3: EGFR Signaling Pathways and Biological Outputs ...... 22
Figure 1.4: EGFR Trafficking ...... 27
Figure 1.5: EGFR Oncogenic Function ...... 29
Figure 1.6: Maintenance of Cell Polarity ...... 35
Figure 1.7: Structure of MUC1 ...... 40
Figure 1.8: MUC1’s Cytoplasmic Tail and it Protein Interaction ...... 43
Figure 1.9: Canonical Wnt Signaling ...... 49
Figure 1.10: Working Model ...... 54
Figure 3.1: MUC1 Promotes Nuclear EGFR Localization ...... 74
Figure 3.2: MUC1 Promotes Nuclear EGFR Accumulation ...... 76
Figure 3.3: Inhibiting MUC1/EGFR Interaction Inhibitt Nuclear EGFR Accumulation . 78
Figure 3.4: MUC1/EGFR Interaction in the Nucleus Promotes EGFR/Chromatin
Interaction ...... 81
Figure 3.5: EGFR Colocalizes with Phosphorylated RNA Polymerase II ...... 84
Figure 3.6: EGFR Interaction with the cyclin D1 promoter is MUC1-dependent ...... 87
Figure 3.7: MUC1 Promotes Cyclin D1 Expression ...... 87
Figure 3.8: Model ...... 92
Figure 4.1: Loss of EGFR kinase activity significantly delays Wnt-1 dependent
tumorigenesis ...... 99 11
Figure 4.2: MMTV-Wnt-1 Egfr waved-2 tumors grow significantly slower ...... 99
Figure 4.3: Total β-catenin protein is independent of EGFR tyrosine kinase activity ... 100
Figure 4.4: β-catenin localization is independent of EGFR tyrosine kinase activity ...... 100
Figure 4.5: EGFR kinase activity is required for normal branching morphogenesis ...... 101
Figure 5.1: MUC1Peptide can effeciently enter cells ...... 111
Figure 5.2: PMIP blocks MUC1/beta-catenin and MUC1/EGFR Interactions ...... 112
Figure 5.3: PMIP inhibits cell proliferation and invasion of breast cancer cells ...... 114
Figure 5.4: PMIP significantly inhibits tumor growth and recurrence in vivo ...... 117
Figure 5.5: PMIP significantly slows progression of MMTV-pyV mT induced mammary
glands tumors ...... 121
Figure 5.6: MMTV-pyV mT tumors have differential response to msPMIP ...... 122
Figure 5.7: PMIP is associated with reduced Muc1 Expression ...... 124
Figure 5.8: hPMIP treatment results in reduced levels of phosphorylated EGFR and
dpERK ...... 125
Figure 5.9: MET is significantly downregulated in response to PMIP ...... 127
Figure 6.1: Mechanism of Peptide Membrane Transduction ...... 137
Figure 6.2: Mechanism of CTT Anti-Cancer Therapy ...... 140
Figure 7.1: EGFR Tyrosine kinase inhibitor (TKI) and PMIP combination significantly
inhibit proliferation ...... 160
Figure 7.2: Blocking nuclear EGFR translocation inhibits cell proliferation ...... 165
Figure 7.3: Working Model with PMIP Treatment ...... 167
12
LIST OF TABLES
Table 1.1: Anti-Erb Receptor Therapies ...... 33
Table 1.2: Role of MUC1 in mouse models of breast cancer ...... 37
Table 2.1: Antibodies ...... 56
Table 6.1: Commonly Utilized Cationic and Amphipathic Cell Penetrating Peptides ...... 134
13
ABSTRACT
The epidermal growth factor family of receptors is important in the development
and progression of many types of cancers including, breast, lung, and glioblastoma. The
family consists of 4 members (EGFR/erbB1, Her2/erbB2, erbB3, and erbB4). In all
breast cancer cases, EGFR expression is deregulated 20 to 30% of the time; however in
the most aggressive form of breast cancer (basal-like) EGFR expression is upregulated in
60% of cases. EGFR’s expression and activity can be altered in transformed cells
through a variety of mechanisms, such as novel protein-protein interactions, gene amplification, mutations, and loss of regulatory proteins. In this work we have examined the role of cancer specific protein interactions of EGFR with MUC1 and beta-catenin in the progression of breast cancer.
Herein I report that the interaction of MUC1 and EGFR in breast cancer cells alters EGFR localization by promoting EGFR nuclear translocation. Importantly, I discovered that the presence of MUC1 mediates EGFR’s interaction with chromatin.
More specifically, I found that EGFR interacts with the cyclin D1 promoter region in a
MUC1-dependent fashion which resulted in a significant increase in cyclin D1 protein expression. Nuclear EGFR localization has been shown to correlate with resistance to anti-EGFR therapies, which indicates that MUC1’s interaction with EGFR could be a mechanism of resistance.
MUC1’s interaction with both EGFR and beta-catenin can promote transformation therefore a peptide therapy was developed, PMIP, which mimics the 14
hypothesized interaction domains of MUC1’s cytoplasmic tail. PMIP was designed to
inhibit the interaction of MUC1/EGFR and MUC1/beta-catenin thereby regulating EGFR
expression and promoting beta-catenin localization to adherens junctions. PMIP
effectively enters the cytosol of cells and inhibits the target interactions. Importantly,
PMIP inhibited invasion and proliferation of breast cancer cells and in mice significantly reduced the growth rate of breast cancer xenograft and genetically-driven tumors. This study demonstrated that the use of peptides to inhibit intracellular protein interactions is a viable option that would have limited toxic side-effects. Overall, this work reveals a new regulatory role of EGFR localization and activity by MUC1 and that this mechanism is
viable therapeutic breast cancer target.
Lastly, in a mouse model of breast cancer I examined the role of EGFR
tyrosine kinase activity in beta-catenin dependent tumorigenesis. A transgenic mouse
model of breast cancer, MMTV-Wnt-1, was bred onto an EGFR kinase deficient
background. I discovered that the loss of EGFR kinase activity in this model resulted in a
significant delay in tumor onset and inhibited tumor growth. These findings indicate a
cooperation of EGFR and beta-catenin dependent signaling pathways, which promote
transformation of glandular epithelial cells.
15
I. INTRODUCTION
Aberrant cellular localization and activity of proteins is often observed in human
cancers. The mislocalization of these proteins is frequently the result of other mutations
or cellular events, but the novel role for mislocalized proteins in promoting disease
progression and therapeutic resistance is starting to be appreciated. In cancer, abnormal
trafficking of proteins will result in atypical protein-protein interactions that will support
cell growth, metastases, and inhibit chemotherapy efficacy. Specifically, I was interested
in the how aberrant localization and interactions of the oncoproteins, MUC1, EGFR, and
β-catenin contribute to the progression of breast cancer.
MUC1, EGFR, and β-catenin have breast cancer specific interactions, which have
been determined to promote cancer progression. I discovered that EGFR/MUC1 interactions enhance atypical EGFR nuclear localization and transcriptional activation.
In addition, by reducing MUC1 expression or EGFR activity I could significantly inhibit
β-catenin-dependent tumorigenesis. These findings prompted the development of a mimetic peptide that could inhibit these cancer-specific protein-protein interactions.
Importantly, inhibition of MUC1/EGFR and MUC1/β-catenin interactions resulted in a significant anti-tumor effect both in in vitro and in vivo models of breast cancer.
Breast Cancer 16
Breast cancer is the second most common cancer in women and accounts for 27%
of all cancers cases diagnosed in American women. In 2009, there were approximately
41,000 deaths due to breast cancer, which makes breast the second leading cause of cancer deaths in women. Additionally, women that do respond to treatment have a 30 to
50% chance of recurring within the first ten years. Recurrence of the cancer is highly dependent on the stage and subtype of the primary cancer [9]. There are several subtypes of breast cancer that are important in the determining the appropriate clinical approach.
These subtypes are determined by their genetic and proteomic signatures. The subtypes include luminal A and B, Her-2/erbB2 overexpressing, claudin-low, basal-like, and normal-breast-like [10]. Figure 1.1 depicts the hypothesized cellular linage of the subtypes of breast cancer [Reviewed in [7]] (Figure 1.1). Subtypes are most often distinguished by their estrogen/progesterone receptor and Her-2/erbB2 expression.
Approximatley 65% of breast cancers diagnosed are part of luminal A or B subtypes,
which are normally characterized by smaller less invasive tumors that are estrogen and progesterone receptor positive and erbB2 negative [10-12]. In contrast, basal-like breast cancers account for only 13-28% of all breast cancer cases, but the 5-year survival rate
for this subtype is significantly shorter [11]. This a result of the increase incidence of
metastases observed in the basal-like subtype, which is frequently the cause of cancer
mortalities. 17
Figure 1.1. Cellular Origin and Subtypes (blue) of Breast Cancer. Percentage incidence of subtype (red). Modified from [8].
Basal-like breast cancer is the most aggressive and metastatic subtype, mainly due
to the limited therapeutic options and advanced nature of the disease. Basal-like breast
cancers are often characterized by being triple negative (ER-, PR-, and HER2/erbB2-)
however it is important to note that not all basal-like breast cancers are triple negatives.
In addition to the hormone receptors and erbB2 other molecular markers have been positively correlated such as epidermal growth factor receptor (EGFR), β-catenin, p63 and high molecular weight keratins [11-13]. Interestingly, in the basal-subtype there is a
strong correlation of aberrant EGFR expression and mutated breast cancer 1 gene
(BRCA1) [14, 15]. BRCA1 is a tumor suppressor protein that is critical in the repair of double-stranded DNA breaks and limits genetic aberrations [16-19]. The correlation of
BRCA1 mutations and EGFR overexpression is hypothesized to be linked, because of the 18
high frequency of EGFR amplifications seen in the basal-like subtype [Reviewed in
[20]]. Expression of EGFR in basal-like breast cancers has made it a significant therapeutic target, however efficacy of anti-EGFR drugs has not translated from the lab bench to the clinical setting [Reviewed in [21]]. This demonstrates the need for more understanding on how EGFR expression and therapeutic efficacy are linked.
ErbB Receptors
The erbB family (EGFR/erbB1, Her2/Neu/erbB2, erbB3, and erbB4) of receptor tyrosine kinases (RTKs) is a well studied group of tyrosine kinases mainly because of their role in development and cancer progression. Loss of EGFR in the mouse results in embryonic lethality because of severe developmental defects [22]. In contrast, EGFR was also the first receptor tyrosine kinase associated with cancer and has since become a
major focus in anti-cancer therapeutics [23].
The 170 kDa type I receptor tyrosine kinase ErbB1/EGFR was first described by
Carpenter et al. in 1978 when treatment with EGF resulted in an increase in plasma membrane associated phosphorylation [24]. In 1980 the egfr gene was mapped to chromosome 7, which allowed for cloning and ultimately genetic manipulation of the gene [25]. Cloning and sequencing of egfr led to its description as the first receptor tyrosine kinase to play a significant role in cancer [23]. Transcriptional regulation of
EGFR occurs in a non-classical fashion, the lack of a TATA and CAAT box in the 5’
promoter region results in multiple transcriptional initiation sites [26]. However, several 19
transcription factors, such as Sp1 and c-Jun, have been discovered to promote egfr
mRNA production [27, 28]. Following transcription the mRNA is translated and the
resulting EGFR protein is inserted into the endoplasmic reticulum membrane. Mediated
by a signal peptide, EGFR is then trafficked through the golgi to the basolateral
membrane [29]. Trafficking of EGFR and other erbB receptors to the plasma membrane
allows the interaction of receptor and extracellular ligand.
Normally, these membrane bound receptors function by binding ligand and there
are three different groups of ligands: 1) EGFR-specific (Epidermal Growth Factor, EGF;
Transforming Growth Factor α, TGFα; and Amphiregulin, AR) 2) EGFR and ErbB4-
specfic (beta-cellulin, BTC; heparin-binding EGF, HB-EGF; and Epiregulin, EPR) 3)
ErbB3 and ErbB4-specfic (Neuregulin 1, 2, 3 and 4, NRG1-4) (Figure 1.2a). Following
ligand binding, the receptor undergoes a conformational change which accommodates
either homo or heterodimerizations with a neighboring erbB receptor (eg. EGFR/EGFR
or EGFR/erbB2) [30, 31]. The erbB2 receptor has no known soluble ligand and its
ability to bind ligand is controversial. However erbB2’s native conformation is
conducive for dimer formation, which will allow dimer formation in the absence of
ligand [32]. The dimerization of the receptors leads to trans-phosphorylation of the
cytoplasmic tails, with the exception of erbB3. ErbB3 lacks a kinase domain and its
activation is dependent on the dimerization with another erbB receptor [33]. Upon
dimerization and phosphorylation the receptors mediate several downstream signaling
events, which are important in development and tumorigenesis. 20
Figure 1.2. ErbB Dimers and Adaptor Protein Binding Sites. Taken from [7]. 21
The trans-phosphorylation event is responsible for the initiation of many signaling
pathways that include: the Mitogen Activated Protein Kinase (MAPK), Akt/
Phosphoinositide 3-Kinases (PI3K), mammalian Target of Rapamycin (mTOR) and Janus
Kinase/Signal transducers and activators of transcription (JAK/STAT) pathways
[Reviewed in [5]] (Figure 1.3). The MAPK signaling pathway is important in cellular
proliferation by propagating a signal from the cell surface to the nucleus, where it
promotes the transcription of c-Jun and c-fos [34]. EGFR induced activation of the
Akt/PI3K signaling pathway can inhibit apoptosis by indirectly blocking the release of
the pro-apoptotic cytochrome C [35]. Inhibiting the release of cytochrome C prevents the
assembly of the pro-apoptotic caspase-9 dependent apoptosome and ultimately prevents
the cell from undergoing programmed cell death. It is important to note that EGFR lacks
a binding domain for p85 (subunit of PI3K), but is able to activate the pathway indirectly
through erbB3 dimers or the adaptor molecule Gab1 [36, 37]. Akt/PI3K signaling is
repressed by phosphatase and tensin homolog (PTEN) protein, which is responsible for
removing phosphates from membrane associated phosphotidylinositols. Removal of
phosphates prevents the interaction of PI3K with Akt by altering their spatial localization.
mTOR acts downstream of the PI3K/Akt pathway to promote cell proliferation and
survival by upregulating translation via eIF4E of survival and anti-apoptotic proteins
[38]. JAK/STAT signaling plays role in development and cancer progression by
promoting activating the transcription of genes important in cellular differentiation,
proliferation, and apoptosis [Reviewed in [39]]. Activated EGFR promotes the kinase
activity of JAK, which results in the phosphorylation and dimerization of STATs. STAT 22
dimers translocate to the nucleus and directly activate transcription of genes such as,
cyclin D1 (cell cycle regulator/proliferation), MMPs (invasion/metastases), VEGF
(angiogenesis) and cytokines (immune modulation) [Reviewed in [40]]. Aberrant
activation of all these signaling cascades can further promote normal cell transformation
and cancer progression.
Figure 1.3. EGFR Signaling Pathways and Biological Outputs. Taken from [1] 23
Most of these signaling pathways are initiated as result of post-translational
modifications, such as phosphorylation (tyrosines, serines and theronines), ubiquitination
(lysines), and N-glycosylation. The variety of modifications and different dimer pairs
(EGFR/erbB2, EGFR/erbB3, etc) allows for the propagation of specific signaling
pathways. For example, the tyrosines that are phosphorylated on EGFR can mediate a
specific signal to proliferate (MAPK; Y992, Y1068, and Y1148) or inhibit apoptosis
(Akt/PI3K, Y1101) [Reviewed in [41]] (Figure 1.2b). Frequently, erbB receptors will
form a dimer with a neighboring erbB2 receptor, because of erbB2 native conformation.
This demonstrates that different dimer formations, phosphorylation events and specific adaptor/receptor interactions can promote or inhibit a diverse number downstream signaling event.
In breast cancer, the overexpression of erbB2 and its constitutively active conformation allows it to act as a potent oncoprotein in promoting tumorigenesis [42,
43]. Therefore erbB2 is a preferential dimer because it promotes receptor activation in a
ligand-independent fashion. This has made erbB2 a highly effective anti-cancer target in
breast cancers that overexpress it, but in the basal-like subtype erbB2 expression is commonly lost. In basal-like breast cancers, EGFR is overexpressed 60% of cases, but anti-EGFR tyrosine kinase inhibitors have shown limited efficacy in the treatment of basal-like breast cancers. Aberrant EGFR trafficking to the nucleus can promote cell proliferation, radioresistance, and metastases [44-47]. This indicates that although kinase activity is important the oncogenic function of EGFR is not limited to its effect on 24
signaling but aberrant trafficking of EGFR also is important in conveying a tumor-
promoting phenotype.
ErbB Receptor Trafficking
Regulation of total erbB receptor levels and downstream signaling is heavily dependent on the trafficking of the receptors following ligand stimulation. Normally prior to ligand stimulation EGFR is frequently found associated with portions of the
plasma membrane rich in lipids and protein (caveolae) [48]. Following EGF binding to
EGFR the resulting dimerization and receptor activation leads to the recruitment of the
regulatory protein, Casitas B-lineage lymphoma (Cbl) protein. Recruitment of Cbl to
EGFR is mediated by the phosphorylated tyrosine on EGFR (Y1045) and the subsequent
Src-homolgy domain (SH2) found on Cbl. Following recruitment, Cbl’s E3 ubiquitin
ligase activity mediates the addition of ubiquitins to lysines found on EGFR’s
cytoplasmic tail. EGFR ubiquitination promotes the recruitment of the Epidermal growth
factor receptor Substrate-15 (Eps15) through its ubiquitin interacting motif (UIM) and
will mediate it translocation to a clathrin-coated pit which promotes internalization [49].
Recently, the ring-finger domain of Cbl has been demonstrated to be vital in the
regulation of EGFR internalization and subsequent trafficking to the early endosome
[50]. Dependent on the poly or multi-ubiquitination status of EGFR, the receptor is then
trafficked to late endosome and is ultimately degraded in the lysosomal compartment.
Trafficking and degradation of EGFR in the lysosome are major regulatory mechanisms 25
for controlling total EGFR levels and signaling [51]. Mutations and alterations in the
EGFR trafficking pathway can lead to overexpression and overactivation of the receptor
which can promote aberrant signaling and oncogenic function (Figure 1.4A).
However, in the last decade the trafficking of EGFR to novel intracellular
compartments has been described. The presence of transmembrane proteins in the
nucleus has been established for all of the ErbB receptors, but the biological significance
of this altered trafficking is still being investigated [52-55]. Retrograde trafficking of
EGFR from the plasma membrane to the nucleus has recently been described [52, 56].
Following EGFR internalization, the receptor undergoes retrograde trafficking and is
transported back to golgi or directly to the endoplasmic reticulum (ER) where it interacts
with the Sec61 translocon. Normally, the Sec61 translocon is responsible for moving
mis-folded proteins from the ER membrane to cytosol for subsequent proteosomal
degradation [57]. Once in endoplasmic reticulum EGFR interacts with the Sec61
translocon, which results in EGFR’s release into the cytosol. In the cytoplasm, the
hydrophobic regions of EGFR interact with Hsp70 to prevent protein aggregation. EGFR
then interacts with Importin-β1 through its tri-partite nuclear localization signal (NLS,
RRRHIVRKRTLRR) [56]. Importantly, the NLS located on the juxtamembrane domain
on EGFR, ErbB2, and ErbB4 and the C-terminus of ErbB3 is well-conserved [58].
Importin-β1 mediates the trafficking of EGFR into the nucleus and the EGFR/Importin-
β1 interaction is necessary for EGFR nuclear translocation, however the fundamental
interaction with nuclear pore complex has not yet been described for EGFR [59].
However, nuclear translocation of erbB2 also requires Importin-β1 interaction and this 26
complex interacts with Nucleoporin-358 (Nup358) and the small GTPase, Ran [60].
Because of the similarities in the NLS of EGFR and erbB2 it is hypothesized that Nup358
is also important in EGFR’s entry into the nucleus (Figure 1.4B). Chromosome region
maintenance 1 (CRM1) has been proposed as the protein responsible for nuclear export
of both EGFR and erbB2 [59, 60]. An increase of nuclear EGFR trafficking is often
observed following ligand stimulation of the receptor [56]. Indicating that
overexpression of a ligand (EGF) or hyperactivation of EGFR could promote aberrant
trafficking.
A recent report demonstrated that EGFR being trafficked to the nucleus is bound
to ligand and that nuclear EGFR remains tyrosine phosphorylated. The authors utilized a
radioactive (I125) labeled EGF and subsequently cross-linked the labled EGF to
membrane EGFR and following high-salt nuclear extraction the EGFR-EGF-I125 complex
was detected in the nucleus [13]. The presence of EGFR in the nucleus is a poor
prognostic indicator in several types of cancers including, breast and ovarian [44, 47].
Once in the nucleus EGFR directly or indirectly interacts with promoter regions to
activate transcription of genes such as cyclin D1, b-myb, iNOS, Aurora kinase A, and
COX-2 [52, 61-64]. All of these genes and their resulting proteins are important in the
development and progression of breast cancer. 27
Figure 1.4. EGFR Trafficking. A) In non-transformed cells, EGFR (green) is trafficked and degraded in lysosome. B) In cancer cells, EGFR interacts with other protein, such as MUC1 (red), and this results in aberrant trafficking of EGFR to the nucleus and increased membrane recycling. 28
Recently, altered EGFR trafficking leading to its accumulation and activity in the
nucleus has been demonstrated to be important in conveying chemotherapeutic
resistance. Hsu et al. discovered that following treatment with a DNA damaging agent
(cisplatin) and ionizing radiation the presence of EGFR in the nucleus activated DNA protein kinase (DNA-PK). This activation lead to the increase of DNA repair and resulted in increased cell survival [45]. Another report found MDA-MB-468 breast
cancer cell line was resistant to an EGFR kinase inhibitor and that nuclear EGFR in these
cells was unchanged [65]. The altered trafficking of EGFR to the nucleus commonly found in cancer could be playing a significant role in chemoresistance and cancer
progression (Figure 1.5). However, the function of EGFR in the nucleus to act as a
transcription factor and other proteins that could be modulating this process has yet to be
fully elucidated. 29
Figure 1.5. EGFR oncogenic functions: tyrosine kinase-dependent signaling (left) and direct transcriptional activator (right). Modified from [6]. 30
ErbB Receptors in Breast Cancer
The transformation of a normal cell to cancerous cell requires the acquisition of
several hallmarks of cancer, which include sustained angiogenesis, self-sufficiency in
growth signals, metastatic potential, limitless replicative potential, insensitivity to anti-
growth signals, and evading apoptosis [66]. ErbB receptors have become favored
therapeutic targets because of their prominent role in all of these hallmarks of cancer. In
addition, transgenic mouse models of breast cancer have been established that are
dependent on erbB receptors for tumorigenesis demonstrating their oncogenic potency.
These mouse models of breast cancer function by overexpression of the receptor
(MMTV-Neu) or receptor hyperactivation (WAP-TGFα) in the mammary gland [67, 68].
Several anti-EGFR therapeutics have been developed and most show efficacy in mouse
models of breast cancer, but this efficacy has not significantly translated to human breast
cancers setting [Reviewed in [21]]. In some cases targeting both EGFR and erbB2 at the same time was shown to accelerate cancer progression [69]. The limited success of these targeted therapies indicates the need for a better understanding of how EGFR functions in cancerous conditions.
EGFR is overexpressed in approximately 30% of all breast cancers, but in the basal-like subtype it is closer to 60%. This overexpression can be a result of several
different events including gene amplification, activating mutations and altered
trafficking. EGFR (7p12) gene amplification has been found in approximately 6% of
breast cancers and results in increased protein expression [70]. Loss of EGFR regulation 31
can also be a result of mutation. For example in glioma, non-small lung, and breast
cancer a mutant EGFR (EGFRvIII) which has an in-frame deletion of the extracellular
domain results in a constitutively active receptor [71]. Proteins that are important in
EGFR trafficking have also been implicated in overexpression of the receptor. For
example, the tumor suppressor, Sprouty, is important in the normal trafficking of EGFR,
but loss of this protein results in increased EGFR expression [72]. In several cancers, the
aberrant trafficking can lead to EGFR overexpression or accumulation of EGFR in the
nucleus, where it directly promotes the transcription of other oncogenes [52]. Up until
recently the sole focus of anti-EGFR therapies has been the signaling pathways downstream of EGFR activation, but in breast cancers these therapies have had limited
success.
ErbB Receptor Therapeutics
Several anti-EGFR therapeutics have been developed for the treatment of a
variety of cancers and the target of most of these drugs is the receptor’s tyrosine kinase
activity (Table 1.1). For example, Gefitinib (Iressa) is a small molecule tyrosine kinase
inhibitor, which attenuates downstream signaling. Gefitinib’s efficacy as an anti-tumor
agent was first demonstrated in a mouse xenograft models of breast, lung, and prostate cancers [73]. However, resistance to gefitinib has been determined to be the result of egfr gene mutations, compensation from other receptor tyrosine kinases (IGFR), upregulation of downstream effector proteins (Akt) or downregulation of inhibitory 32
proteins (PTEN) [74]. Additionally, most patients that initially respond to gefitinib will
develop resistance within 6-12 months [Reviewed in [75]]. Another anti-EGFR therapy
recently developed was the monoclonal antibody, Cetuximab (Erbitux), directed against
EGFR’s ectodomain, which prevents dimerization and subsequent downstream signaling.
Preclinical in vitro and in vivo studies for cetuximab show significant efficacy in inhibiting metastases, proliferation, and angiogenesis [Reviewed in [76]]. However, the results observed in preclinical trials have not translated to the clinic. Interestingly, the expression level of EGFR in the tumor cells is a poor indicator of drug response for both the tyrosine kinase inhibitor and monoclonal antibody [77, 78]. Although it is important to target EGFR’s kinase activity, recently a kinase-dead mutant of EGFR was able to inhibit autophagic cell death in cancer cells [79]. Authors discovered that EGFR stabilized glucose transporters to promote cell survival under cytotoxic conditions. This indicates an EGFR oncogenic function that is independent of its kinase activity and a possible mechanism of resistance. Although some mechanisms of anti-EGFR therapy resistance have been established, evidence for altered EGFR trafficking to the nucleus as mode of resistance is becoming apparent [45, 80]. An initial step in the progression of breast cancer that results in this aberrant trafficking of EGFR to the nucleus is the loss of cellular polarity. Maintenance of the apical-basolateral border is essential in normal cell function and deterioration of this border will result in aberrant protein localization,
regulation, expression, and activity.
33
Compound Type Target Company Trastuzumab (Herceptin) Humanized mAb ErbB2 Genentech/Roche Pertuzumab (Omnitarg) Humanized mAb ErbB2 Genentech Cetuximab (Erbitux) Chimeris mAb EGFR ImClone/Merck
Matuzumab Humanized mAb EGFR Merck
Panitumumab Humanized mAb EGFR Abgenix Gefitinib (Iressa) Tyrosine Kinase Inhibitor EGFR AstraZeneca Erlotinib (Tarceva) Tyrosine Kinase Inhibitor EGFR Genentech
Lapatinib Tyrosine Kinase Inhibitor EGFR/ErbB2 GlaxoSmithKline
AEE788 Tyrosine Kinase Inhibitor EGFR/ErbB2/VEGFR Novartis Irreversible Tyrosine CI-1033 Kinase Inhibitor EGFR/ErbB2 Pfizer Irreversible Tyrosine EKB-569 Kinase Inhibitor EGFR/ErbB2 Wyeth-Ayerst EXEL 7647/EXEL 0999 Tyrosine Kinase Inhibitor EGFR/ErbB2/VEGFR EXELIXIS
Table 1.1. Anti-ErbB Therapies. Tyrosine Kinase Inhibitors, TKI and monoclonal antibody, mAb.
Modified from [7]. 34
Cellular Polarity
Maintenance of the apical and basolateral borders has recently been described as a
“non-canonical tumor suppressor” because loss of this border promotes cellular
transformation. Normally, the cell polarity is maintained by three different complexes of
protein: the Pars complex (Par3-Par6-atypical PKC (aPKC), apical), Crumbs complex
(Crb, PALS1, and PATJ), and the Scribble complex proteins (Discs large, Dlg and lethal giant larvae, Lgl) [Reviewed in [81]] (Figure 1.6). The apical membrane is maintained by the Crumbs and Pars complexes as a result of aPKC (Pars complex) phosphorylation
of the Scribble (Scr) complex [82]. Phosphorylation of the Scribble complex promotes
its localization to the basolateral membrane. As a result of this event the Crumbs/Pars
and Scribble complexes distinguishes the apical and basolateral membranes, which is
vital in normal cellular function. Additionally, cellular polarity is also maintained by the
presence of tight junctions and adherens junctions, but the localization of these junctions is highly dependent on the activity of the Pars/Crumbs/Scribble interactions [Reviewed in
[81]]. Overall, regulation of polarity is a vital component in cellular homeostasis, which
upon disruption could promote catastrophic side-effects. 35
Figure 1.6. Maintenance of Cell Polarity. Several complexes of proteins are important in maintaining polarity, including the Pars, Scribble, and Crumbs complexes. Adherens and Tight junctions (AJC) also a play significant role in this process. Taken from [4]. 36
The relationship of aberrant EGFR trafficking and cell polarity in cancer has been
hypothesized to be a major component in EGFR-dependent breast cancers. Under normal
circumstance, EGFR basolateral trafficking is highly regulated, but following cellular
transformation cell polarity is lost and EGFR is localized throughout the cell [8]. In a
kidney epithelial cell line, there is altered downstream signaling as a result of
mislocalized apical EGFR [83]. Interestingly, EGFR and other erbB receptors have been
determined to directly affect cellular polarity by several different aspects including
inhibiting the function of the Pars complex and promoting the dissociation of β-catenin
from adherens junctions [84-86]. These events will ultimately results in the degradation
of cellular polarity thereby promoting initiation and transformation. Importantly, the loss
of polarity promotes novel EGFR-protein interactions, which results in a more severe
oncogenic phenotype [8]. For example, EGFR interacts with another oncogene, MUC1,
in a cancer-dependent fashion and this interaction promotes altered EGFR trafficking and
function [8, 87]. Inhibition of EGFR’s interaction with MUC1 inhibited the growth of
tumors in vivo, which demonstrates the importance of cell polarity maintenance and the
consequence of its deterioration.
37
MUC1
MUC1 expression has been correlated with poor prognosis in several different
cancers including cholangiocarcinoma [88], breast [89], colon [90], gastric [91], lung
[92], and renal cell carcinoma [93]. MUC1 was first characterized when a laboratory
generated mouse antibodies against human milk-fat globules, which could be utilized for
diagnosing breast cancer [94]. MUC1 has been observed to be overexpressed in about
90% of all breast cancers and its loss of apical localization is a poor prognostic indicator
[95, 96]. Overexpression of MUC1 in the 3Y1 rat fibroblast cell line and in the
mammary gland of mice (MMTV-MUC1) both resulted in transformation and
tumorigenesis [97, 98]. Importantly, MUC1 expression is important in the development
of tumors in EGFR and Wnt-dependent tumorigenesis mouse models [8, 98]. Table 1.2
provides the description of MUC1’s role in several different transgenic mouse models.
Mouse Genotype Transformation Phenotype Reference MMTV-MUC1 Mammary Gland Tumor Formation {Schroeder, 2004 #42}
MMTV-Wnt-1/Muc1-/- Delayed Tumor Progression {Schroeder, 2003 #41}
WAP-TGFα/Muc1 -/- Delayed Tumor Onset/Reduced Lung Lesions {Pochampalli, 2007 #35}
MMTV-pyMT/Muc1-/- Delayed Tumor Progression/ Reduction of Lung Metastases {Spicer, 1995 #44} No Change (Note: Found that erbB2 overexpression lead to MMTV-ErbB2/Muc1-/- {Adriance, 2004 #245} transcriptional repression of Muc1) Table 1.2. Role of MUC1 in mouse model of breast cancer. Mouse mammary tumor virus –MMTV, Whey acid protein-WAP, and polyoma virus middle T antigen- pyMT
Additionally, knock-down of MUC1 in several breast cancer lines resulted in the differential transcription of genes that are important in both proliferation and invasion. 38
Authors discovered that loss of MUC1 expression resulted in a decrease in genes
involved in MAP- kinase (MAP2K1) signaling and extracellular matrix interactions
(ITGAV) resulting in a significant decrease in proliferation and invasion [99]. Recently,
aberrant trafficking of MUC1 into nucleus promoted the interaction of MUC1 and the
transcription factor, NF-kB, resulting in transcriptional activation [100]. This
demonstrates that MUC1 function as an oncogene is not only dependent on it signaling
effector role, but its role in the nucleus as a transcriptional regulator. These studies
demonstrate MUC1’s impact on tumorigenesis and its importance in further
understanding the mechanism driving its oncogenic function.
MUC1 Structure
Mucin1 (MUC1) is part of a larger family which is composed of three subtypes:
gel-forming, soluble, and transmembrane mucins. There are approximately 20 genes
(MUC1-MUC20) which make up this larger family, but the majority of mucins are in the
gel-forming and transmembrane subtypes. MUC1 is >300kDa heterodimeric
transmembrane mucin and consist of a single pass hydrophobic region, which anchors the
protein to the plasma membrane (Figure 1.7). MUC1’s extracellular domain (ED) consists of a SEA (Sea urchin, Enterokinase, and Agrin) domain, which allows for the interaction of carbohydrates [101]. Additionally, MUC1’s ED is composed of several serine/threonine tandem repeat regions and the number of tandem repeat regions is highly variable as a result of genetic polymorphisms (Variable Number Tandem Repetition, 39
VNTR). The abundance of serine and threonines allows for extensive O-glycosylation of
the ED, which can account of up to 50% of the mucin’s mass [Reviewed in [102]].
Glycosylation of MUC1’s VNTR is important in its intracellular trafficking to the apical
membrane and following proper trafficking glycosylation aids in MUC1’s function.
Importantly, MUC1 expression is not isolated to glandular epithelial of the breast, but
expression has been associated with over 36 different tissue and cell types [Reviewed in
[103]]. Normal functions of MUC1’s ED consist of hydration and lubrication of the
lumen and microbial protection, but it is also able to interact with other extracellular
proteins [104]. The extracellular MUC1-protein interactions include galectin-3,
intercellular adhesion molecule-1 (ICAM-1), and selectins [105-107]. Galectin-3 is a
membrane associated galactoside-binding protein, which interacts with various O-glycans
on MUC1’s ED to promote adhesion. Recently, the over-expression of MUC1 has been
shown to upregulate galectin-3 expression, which promoted cancer-endothelial cell
interactions, a step that is vital in the metastatic process [108, 109]. Interestingly,
galectin-3 is also responsible for mediating the EGF-dependent extracellular interaction
of MUC1 and EGFR [108]. MUC1’s ED also interacts with ICAM-1 on endothelial cells
which provides another possible insight into MUC1’s role in intravasation and cancer
metastases [106]. Although MUC1’s extracellular interactions are important, its
intracellular protein interactions play a significant role in MUC1’s oncogenic function. 40
Figure 1.7. Structure of MUC1. MUC1 has a heavily O- glycosylated extrace llular domain (ecto-domain), a single pass transmembrane domain (TM) and a short cytoplasmic tail (CT) [2]. 41
MUC1 consist of a 72 amino acids cytoplasmic tail (CT), which has a plethora of
MUC1-protien interaction domains (Figure 1.8). Although the ED is important in the normal function the CT has been demonstrated to the main mediator of MUC1’s oncogenic function [110, 111]. The CT interacts with several proteins including, p53, estrogen receptor alpha, IKKβ, IKKγ, Src, Hsp90, Hsp70, β-catenin, EGFR, GSK3-β,
PKCδ, and Lck (Figure 1.8) [Reviewed in [4]]. Although it is important to note that some of these interactions are cancer-dependent as a result of disrupted cellular distribution and polarity. MUC1’s interaction with these others proteins is further discussed on page 43. The CT also has several domains important in post-translation trafficking such as the apical targeting, nuclear localization and oligomerization [112,
113].
MUC1 Transcriptional Regulation And Trafficking
MUC1’s transcription is regulated by several different transcription factors
including the estrogen receptor (ERα), signal transducer and activator of transcription
(STAT), nuclear factor –kappa B (NF-κB), and GATA3 [100, 114-116]. Following
transcription of the MUC1 gene, the mRNA is translocates to the endoplasmic reticulum,
where the protein is translated and inserted into the plasma membrane. Still in the
endoplasmic reticulum MUC1 is cleaved into an extracellular and membrane product,
following this proteolysis the two non-covalently linked subunits are trafficked through
the golgli to the apical membrane [117]. Apical membrane trafficking is achieved by the 42
presence of an apical targeting sequence (Cys-Gln-Cys) on MUC1’s juxtamembrane
domain [113]. During golgi trafficking, the extracellular domain is heavily O-
glycosylated, which will allow MUC1 to lubricate and hydrate the lumen [2, 118]. While
at the plasma membrane MUC1 will undergo constitutive internalization in order to
maintain O-glycosylation [119]. However in cancer MUC1’s level of O-glycosylation is
greatly reduced and its cellular distribution is altered. Recently, it has been determined
that following cleavage of the ED, the CT of MUC1 can translocate to the nucleus by
interacting with importin-β1 and nucleoporin p62 (Nup62) [112]. Additionally, this
nuclear translocation is dependent on MUC1’s ability to oligiomerize with Nup62 and
mutation of the amino acids important in this oligomerization results in a reduction of
transcriptional co-activation and tumorgenicity [112].
The most important attribute of MUC1’s cellular processes and function is it
cellular localization. Normally, MUC1’s apical localization is regulated by the apical
targeting sequence, but in cancer this regulation is lost and MUC1 can be detected in
several cellular compartments (basolateral plasma membrane, mitochondria, nucleus and
cytosol) [8, 120, 121]. Aberrant cellular localization promotes novel protein-protein
interactions that have been determined to be vital for MUC1’s oncogenic function.
Importantly, the loss of apical localization and accumulation of MUC1 in the cytosol
significantly correlates with a poor prognosis [122]. Although the importance of
maintaining cellular polarity is well-established the role of MUC1expression in this
maintenance has yet to be determined. 43
MUC1 Cytoplasmic Tail-Protein Interactions
The loss of MUC1 apical localization is poor prognostic indicator and is
commonly seen in breast cancer [122]. Aberrant cellular localization allows for novel
MUC1-protein interactions, which have been demonstrated to be important in MUC1’s
oncogenic function [123]. The cytoplasmic tail of MUC1 has been shown to contain
binding sites for many proteins that are implicated in transformation and progression of
cancer. These proteins include: GSK3β, c-Src, EGFR, β-catenin, and PKCδ [Reviewed
in [4] and [3]] (Figure 1.8).
Figure 1.8. MUC1’s Cytoplasmic Tail and its Protein Interactions. Taken from [3].
44
Loss of MUC1 in Wnt-1/β-catenin-dependent tumorigenesis significantly delays
tumor onset, which indicates that MUC1 is able to directly alter β-catenin function [124].
In vitro studies have concluded that MUC1’s cytoplasmic domain can promote β-catenin
dissociation from adherens junctions, which could also play a role in the breakdown of
cell polarity. Additionally, MUC1’s interaction with β-catenin blocks the degradation of
cytosolic β-catenin by inhibiting its GSK3β mediated serine/threonine phosphorylation
[124, 125]. Additionally, GSK3β interactions and serine phosphorylation of MUC1’s
cytoplasmic tail inhibits β-catenin/MUC1 interactions and initiates β-catenin degradation.
This indicates that the presence of MUC1 could act as switch to promote or inhibit b-
catenin stabilization, but this is highly dependent on other MUC1-protein interactions
(eg. MUC1-EGFR). Additionally, tyrosine phosphorylation on the MUC1 cytoplasmic
domain tyrosine, Y46, has been shown to increase the binding of β-catenin, Hsp70,
Hsp90 and c-Src [120, 126]. Additionally, the MUC1/β-catenin interactions have also
been detected in the nucleus, which promotes β-catenin dependent transcription [121].
The regulation of MUC1/β-catenin interaction that is mediated by MUC1’s tyrosine
phosphorylation is a possible mechanism to explain how activated EGFR promotes
MUC1/β-catenin interactions.
MUC1’s cytoplasmic tail also interacts with another oncogene, EGFR, in a
cancer-dependent fashion. Importantly, this interaction promotes the tyrosine
phosphorylation of MUC1’s CT (YEKV) and an increase in MAPK signaling [110].
Additionally, expression of MUC1 in an EGFR-dependent mouse model of tumorigenesis 45
promoted the formation tumors and lesions in the lung [8]. The authors discovered that
that expression of a vital cell cycle regulator, cyclin-D1, in these tumors was MUC1-
dependent. In vitro the presence of MUC1 following EGFR stimulation with EGF
resulted in a significant reduction in EGFR ubiquitination and increased recycling [87].
Additionally, inhibiting MUC1/EGFR interactions in both in vitro and in vivo models of breast cancer resulted in significant anti-proliferative and anti-oncogenic effects [123]
(Refer to chapter V).
Another oncogene, c-Src, interacts and tyrosine phosphorylates MUC1’s cytoplasmic tail and this interaction is important in tumorigenesis. In a c-Src dependent mouse model of breast cancer (MMTV-pyMT) the loss of Muc1 resulted in a significant delay in tumor onset and a reduction of pulmonary metastases [127]. The authors determined that expression of Muc1 promoted c-Src stability which promoted the interaction of c-Src with β-catenin and PI3K. Recently, a study using transgenic mice that over express MUC1 demonstrated that MUC1 was responsible for the development of mouse mammary tumors [98]. One mechanism driving MUC1-dependent transformation was discovered to be an increase in anti-apoptotic signaling such as, the
PI3K/Akt pathway [128]. Also, it is proposed that MUC1 inhibits the intrinsic apoptotic pathway through the interactions with Hsp70/90, which promote mitochondrial trafficking of MUC1’s CT [120, 129].
Overexpression of MUC1 alone is sufficient to drive tumorigenesis in a mouse model of breast cancer and the loss of MUC1 in other models (WAP-TGFα and MMTV- 46
Wnt-1) significantly delays tumor onset. In addition, our laboratory demonstrated that
the presence of MUC1 in an EGFR-dependent tumorigenesis mouse model of breast
cancer promoted tumor formation. Pochampalli et al found that in these tumors the expression of the cell cycle regulator/oncogene, cyclin D1, was dependent on MUC1 [8].
Additionally, these studies demonstrated that MUC1’s interaction with other known oncogenes (EGFR and β-catenin) is a cancer-specific event which promotes tumorigenesis and progression.
β-catenin Structure and Function
β-catenin signaling has been reported to be upregulated in greater than 50% of all breast tumors [Reviewed in [130]]. β-catenin is a 92kDa protein that has been shown to be conserved among several different organisms. The protein is made up of 12 armadillo domains, which are 42 amino acid repeats that form a superhelix [131, 132]. Normally, β-
catenin interacts with epithelial-cadherin (E-Cadherin) and α-catenin to form adherens
junctions which maintain cell-cell interactions [133]. E-Cadherin is calcium-dependent
transmembrane protein that functions by interacting with E-Cadherin from neighboring
cells to promote cell-cell adhesions. Additionally, E-Cadherin also functions to inhibit
receptor tyrosine kinases, by preventing dimerization and ligand binding [134]. β-catenin
has three highly conserved tyrosines that are critical in maintaining cell-cell adhesion
function. Tyrosines 142 and 654 are important in the interactions with α-catenin and E-
Cadherin. Tyrosine 489 is not in a known protein binding domain, although 47
phosphorylation of any three of these tyrosine results in β-catenin dissociation from
adherens junction [Reviewed in [135]]. The β-catenin/E-Cadherin complex of proteins
is responsible for binding the actin cytoskeleton, which promotes maintenance of cellular
and tissue organization. Adherens junctions can be stabilized through several serine
phosphorylations on E-Cadherin and p120 catenin [132]. p120 catenin is associated with
the juxtamembrane domain of E-Cadherin and loss of p120 results in the total loss of cell-cell adhesion [136].
In normal cells, once β-catenin dissociates from E-Cadherin it forms a complex with Axin and Adenomatous Polyposis Coli (APC) and then β-catenin is serine or
threonine phosphorylated by the Glycogen Synthase Kinase 3β (GSK3β).
Serine/threonine phosphorylation of β-catenin promotes the recruitment of the ubiquitin
ligase β-transducing repeat-containing protein (βTrCP). Ubiquitination of β-catenin
marks it for degradation by promoting its proteosomal trafficking, which degrades β-
catenin and prevents it from accumulating in the cytoplasm [137]. Both in normal and
cancer cells, Wnt signaling stabilizes cytoplasmic β-catenin by inhibiting the action of
the GSK3β/Axin/APC complex [138].
β-catenin/Canonical Wnt Signaling
The canonical Wnt/β-catenin signaling pathway is mediated by the binding of a
Wnt ligand (eg. Wnt-1) to the Frizzled (Frz) receptor/ Lipoprotein Receptor-Related 48
Protein (LRP) complex. Following ligand interaction with receptor complex, casein
kinase I/II is recruited, which activates the dishevelled (Dsh) protein. Activated Dsh will
inhibit GSK3β activity and prevent the serine/theronine phosphorylation of β-catenin
[139] (Figure 1.9). Normally, the Wnt signaling pathway is important in development
and is reported to induce differentiation and cell survival [Reviewed in [140]]. However,
in most transformed tissues β-catenin is not degraded, because of specific mutations
within β-catenin, upregulation of Wnt ligands, downregulation of Wnt inhibitors,
mutations in the GSK3β/Axin/ APC degradation pathway and/or deregulation of critical
signaling pathways [141, 142].
49
Figure 1.9. Canonical Wnt Signaling. Taken from [5].
50
β-Catenin in Breast Cancer
In breast cancer, EGFR is able to directly tyrosine phosphorylate β-catenin, which
results in a 6-fold reduction in E-Cadherin binding [143]. Recently, an indirect
mechanism of an EGFR-dependent β-catenin/E-Cadherin release was described, where
following transactivation of EGFR through treatment with Wnt-1 subsequent Erk2/CK2α
activation lead to decreased α-catenin/β-catenin association [144, 145]. Loss of α-
catenin/β-catenin association promoted the dissociation of β-catenin from E-Cadherin.
The reduced β-catenin/E-Cadherin association mediates the buildup of β-catenin in the
cytoplasm and this accumulation of stabilized β-catenin promotes its translocation into the nucleus [143]. The mechanism of β-catenin’s nuclear translocation is mediated by several mechanisms including direct interaction with nuclear pore proteins, Bcl-9-
dependent and/or TCF/LEF-1-dependent trafficking [146-148]. In the nucleus, β-catenin
can act as a transcriptional coactivator binding to the lymphoid enhancing factor/T-cell
factor (LEF/TCF) and eventually to TCF response elements in DNA. Later studies
reported that β-catenin/TCF response genes that were up-regulated were mostly pro-
proliferation (cyclin-D1), pro-survival (myc) and pro-migration (uPAR). It is also
important to note that β-catenin/TCF signaling plays an important role in mammary gland
development and maintenance [Reviewed in [140]].
The dissociation of β-catenin from adherens junctions results in down-regulation
of E-Cadherin and an up-regulation of neural-cadherin (N-cadherin). This cadherin
switch is in part a result of β-catenin-dependent transcriptional activation and SLUG1/2- 51
dependent transcriptional repression [149, 150]. In addition, the cadherin switch is a
common trait observed in metastatic cancers and is also utilized to determine the
occurrence of the epithelial to mesenchymal transition (EMT) [151]. Cancer cells that
have undergone EMT are often more migratory and invasive, which results in metastatic
tumors [Reviewed in [152]]. These secondary tumors will often result in organ failure
and are the main cause of cancer related deaths.
Peptide Therapy
The goal of understanding the basic science behind breast cancer development
and progression is to design less toxic and more effective targeted chemotherapies. For
example, the tyrosine kinase activity of erbB receptors has become a favored therapeutic
target (Refer to page 33). With respect to EGFR targeted small molecule tyrosine kinase
inhibitors (Iressa or Lapitinib) or antibody therapies (Cetuximab and Matuzimab), in vitro
and in vivo breast cancer demonstrated studies these drugs were highly effective.
However, in breast cancer clinical trials with these therapies have resulted in limited
efficacy and toxic side-effects (heart damage and skin damage). Recently, the use of cell-
penetrating peptide-based therapies has offered the benefit of targeted therapy with
minimal side-effects.
Cell penetrating peptides (CPPs) are 9-35mer cationic and/or amphipathic peptides that are rapidly endocytosed across cell membranes [153, 154]. Importantly, they can be linked to a variety of cargo, including anti-cancer therapeutics, making CPPs 52
an efficient, effective and non-toxic mechanism for drug delivery [123, 155-157]. CPP
conjugated therapies (CTTs) target a number of different processes specific to cancer
progression, including proliferation, survival and migration. In addition, many of these
CTTs also increase sensitivity to current anti-cancer therapy modalities, including
radiation and other DNA damaging chemotherapies, thereby decreasing the toxic dosage
required for effective treatment [158, 159]. Mechanistically, these CTTs function in a
dominant-negative manner by blocking tumor-specific protein-protein interactions with
the CPP-conjugated peptide or protein. Treatment of both cell lines and mouse models
demonstrate that this method of molecular targeting results in equal if not greater efficacy
then current standards of care, including DNA damaging agents and topoisomerase
inhibitors. For the treatment of invasive carcinoma, these CTTs have significant clinical
potential to deliver highly targeted therapies without sacrificing the patient’s quality of
life. The use of anti-cancer target CTTs is further discussed in chapter VI.
53
STATEMENT OF THE PROBLEM
Independently, β-catenin, EGFR, and MUC1 have all been shown to be
responsible for carcinogenesis. EGFR is overexpressed in ~ 60% and MUC1 is overexpressed in ~90% of basal-like breast cancers and β-catenin activity is aberrant in
~50% of breast cancer. In mouse models of breast cancer the co-expression of these
oncogenes promotes tumor onset and progression. In normal cells, these proteins are spatially separated, which prevents their interactions, but as cells transform and lose
polarity they are able to interact. The aberrant cellular localization of these proteins plays an important role in the development and progression of breast cancer. Specifically,
MUC1 has been shown to affect EGFR trafficking and β-catenin localization, which
further promotes their oncogenic function (Figure 1.10). For example, increased MUC1-
dependent EGFR recycling promotes aberrant tyrosine kinase activity, which can
contribute to the dissociation of β-catenin from adherens junctions and EMT.
Additionally the treatment of basal-like breast cancers with tyrosine kinase inhibitors
have had limited success indicating that kinase activity should not be the sole target in
future development of anti-EGFR therapeutics. The hypothesis is that MUC1/EGFR and
MUC1/β-catenin interactions promote the oncogenic activity of each protein and that targeting their interaction is a viable therapeutic option. To address this hypothesis I asked the following questions:
I) What is the role of MUC1 in EGFR or β-catenin trafficking to the nucleus?
a. Does MUC1 alter nuclear EGFR or β-catenin accumulation? 54
b. Does MUC1 alter nuclear EGFR function?
II) Evaluate the use of a CPP conjugated therapy (PMIP) to inhibit MUC1/EGFR
and MUC1/β-catenin interactions.
a. Utilize in vitro and in vivo models of breast cancers to determine
PMIP’s efficacy
III) Examine the role of EGFR activity in a Wnt-dependent mouse model of
tumorigenesis
Figure 1.10 Working Model. Epithelial cell polarization is lost during the progression of normal breast tissue (A) to a hyperplastic state (B) to breast cancer (C). This allows novel protein-protein interactions to occur, such as MUC1 (red)/EGFR (green) and MUC1/β-catenin (blue). MUC1 inhibits
degradation of EGFR and promotes recycling to the membrane. MUC1/EGFR promotes β-catenin dissociation from adherens junctions resulting in upregulation of oncogenic β-catenin transcriptional activation. Ultimately, these interactions promote breast cancer proliferation and metastases. It is important to note that epithelial cells are surrounded by an extracellular matrix (Background image: SEM of a collagen I matrix) that play an important mechanical and signaling role (Modified from [9]). 55
II. MATERIALS AND METHODS
Cell Lines
MCF10A, MDA-MB-468, BT20, and MDA-MB-231 cells were obtained from
American Tissue Culture Collection. MCF10A is an immortalized breast epithelial cell line, whereas MDA-MB-468, BT20 and MDA-MB-231 are transformed breast epithelial cell lines. MCF10A cells were maintained in RPMI with 10% FBS (Omega Scientific),
1% Penicillin-streptomycin (Cellgro), 10ng/ml cholera toxin (Sigma), 0.5 μg/ml
o hydrocortisone and 5ng/ml EGF at 37 C and 5% CO2. Cells were grown in RPMI
(MDA-MB-468 and MDA-MB-231, Cellgro) or EMEM (BT20, ATCC) with 10% FBS
o and 1% Penicillin-streptomycin at 37 C and 5% CO2.
Transfection with siRNA
Transfection with siRNA was performed as in [87]. The MUC1-1 siRNA targets
the extracellular domain (31045’-AAGACTGATGCCAGTA-GCACT-3’) [160] and the
siGENOME SMARTpool (M-004019-02-0020, Dharmacon/ThermoScientific) MUC1-2
siRNA targets the 3’UTR and open-reading frame (1-5’-
ACCAAGAGCUGCAGAGAGA-3’, 2-5’-GAUCGUAGCCCUAUGAGA-3’, 3-5’-
GCCGAAAGAACUACGGGCA-3’ AND 4-5’-CCACCAAUUUCUCGGA-CAC-3’).
Additionally, the control siRNA has no known mammalian gene targets (5’-
AATTCTCC-GAACGTGTCACGT -3’)(Qiagen). The transfections were performed with Lipofectamine 2000 (Invitrogen) as per the manufacturers’ specifications. Cells 56
were transfected, incubated for a specific time (MDA-MB-468, two days and MCF10A,
three days) and used for experiments.
Antibodies and Growth Factors
Table 2.1 PrimaryAntibodies Antibody Species Use Manufacturer MUC1 (CT-2) Hamster WB, IP, IF Neomarkers MUC1 (H295) Rabbit IF Santa Cruz EGFR (1005) Rabbit WB, IP, IF Santa Cruz EGFR (Ab-13) Mouse IP, ChIP Neomarkers EGFR (Ab-1) Mouse IF Neomarkers Phospho-EGFR (1173) Rabbit WB Cell Signaling ERK (9102) Mouse WB Sigma dpERK (M-8159) Mouse WB Sigma β-catenin (C-18) Goat WB Santa Cruz β-catenin (C-19220) Mouse IF BD Scientific β-actin (AC-15) Mouse WB Sigma Affinity BAP31 (MA3-002) Rat WB Bioreagents Histone H3 (C-16) Goat WB Santa Cruz Myosin IIa (D-16) Goat WB Santa Cruz IGF-R1β Rabbit WB Santa Cruz Phosphorylated Tryosine (PY99) Mouse WB Santa Cruz EEA1 (H-300) Rabbit WB Santa Cruz Phosphorylated Serine- 2 RNA polymerase II (H-5) Mouse ChIP Abcam
Secondary Antibodies Antibody Species Tag Manufacturer Anti-mouse Donkey 488, 584, HRP Invitrogen Anti-rabbit Donkey 488, 594, HRP Invitrogen Anti-goat Donkey 488, 594, HRP Invitrogen Anti-Armenian HRP, Texas Hamster Red, 488 Jackson Labs
57
Epidermal growth factor (EGF) was stored at -20oC at a concentration of 100ng/μl
(Invitrogen).
Immunoprecipitation and Immunoblotting
Protein was treated as in [87]. Nuclear protein was isolated via differential
detergent fractionation and the protein concentration was assessed by a Bicinchoninic
Acid assay. Protein (400 μg) was incubated in TNEN buffer (50mM Tris pH 7.5,
150mM NaCl, 1mM EDTA pH 8.0, 0.5% Igepal CA630, 2.0mM Sodium orthovanadate,
50 M ammonium molybdate, 10mM sodium fluoride, and Complete protease inhibitors)
and primary antibody (EGFR, Ab-13, Neomarkers, 2 μg) or mouse IgG for 1.5 hours at
4oC. The protein/TNEN/antibody mixture were tumbled at 4oC with agarose G beads
(Molecular Probes, Invitrogen) for 4 hours. The precipitations were washed three times
with TNEN buffer and then incubated with 2%SDS-PAGE buffer. Immunoprecipitations
were separated on an SDS-PAGE (Running buffer: 1X Tris-Glycine/0.1%SDS),
transferred (15% Methanol, 1X Tris-Glycine, 0.04% SDS) to PVDF (Millipore, Billerica,
MA). The membrane was blocked in either a 3% Milk/PBS/0.1% Tween solution or a
3% BSA/TBS/0.1% Tween and then used for immunoblot. Protiens on the membrane were treated with Super Signal Chemoilluminescent Substrate (Pierce), visualized on
Imagetech-B film (American X-ray, Louisville, TN) and developed with a Konica SRX-
101C. Densitometry of immuno-blots was performed with Image J software (NIH).
Immunofluorescence 58
Cells were grown on cover slips, treated with siRNA, and incubated with EGF as described above. Cells were fixed with a 4% Paraformaldehyde /PBS solution and permeabilized with 0.5% Triton X-100/0.02% NaN3/PBS. Following fixation, cells were
o blocked with 20% Fetal Bovine Serum (FBS)/ 0.02% NaN3/PBS and incubated at 4 C for
18 hours with primary antibody (1:100) in 10% Fetal Bovine Serum (FBS)/ 0.02%
NaN3/PBS (stock buffer). Secondary antibody (1:200) was incubated at room
temperature for 45 min in stock buffer. The slides were visualized with a Zeiss Axiovert
200 confocal microscope (Molecular and Cellular Biology Microscopy Facility,
University of Arizona), images were captured using a Zeiss AxioCam and analyzed with
the Zeiss Meta software.
Growth Factor Treatment
Prior to growth factor treatment, cells were serum-starved overnight. Cells that
were to be used to study the effect of EGF treatment were incubated with 10 ng/ml or
20ng/ml receptor grade EGF (Invitrogen) for 10 minutes on ice. Free EGF was then
washed twice with PBS and serum-free media was added. Cells were then incubated at
37ºC in serum-free media for the required time period before being lysed and
fractionated.
Differential Detergent Fractionation 59
The differential detergent fractionation protocol followed was modified from
[161]. Prior to lysis, cells were washed, collected, and resuspended in cold PBS. The
cell suspension was then centrifuged for 2 minutes at 200 x g, and the PBS was removed.
Cytoplasmic Extraction. Cells obtained from the previous step were resuspended in 300 uL of digitonin lysis buffer [0.01% digitonin, 100mM NaCl, 300mM sucrose, 10mM
PIPES (pH 6.8), 3 mM MgCl2, 5mM EDTA, 2.0 mM Sodium orthovanadate, 50.0 μM
ammonium molybdate and 10.0 mM sodium fluoride and Complete protease inhibitors
(Roche)] and incubated on ice for 5 minutes. The insoluble material was then pelleted for
10 min at 400 x g and the supernatant was stored as the cytosolic fraction.
Membrane Extraction. The pellet obtained from the cytoplasmic centrifugation was resuspended in 300uL of TritonX-100 lysis buffer [0.5% TritonX-100, 100Mm NaCl,
300mM sucrose, 10mM PIPES (pH 7.4), 3mM MgCl2, 3mM EDTA, 2.0 mM Sodium
orthovanadate, 50.0 μM ammonium molybdate and 10.0 mM sodium fluoride and
Complete protease inhibitors (Roche)] and incubated on ice for 5 minutes. Cells were
then subjected to centrifugation for 10 min at 5000 x g and the supernatant was stored as
the membrane fraction.
Nuclear Extraction. The pellet obtained from the membrane centrifugation was resuspended in 500uL of Tween 40/ DOC lysis buffer [1% Tween-40, 10mM NaCl, 0.5%
Deoxycholate, 10mM PIPES (pH 7.4), 1mM MgCl2, 2.0 mM Sodium orthovanadate, 50.0
μM ammonium molybdate and 10.0 mM sodium fluoride and Complete protease
inhibitors (Roche)]. The pellets were then dounced 20 times with a B-dounce and 60
sonicated 3 times for 10 seconds each. This was followed by centrifugation for 10 min at
7000 x g and the supernatant was removed as the nuclear fraction.
Chromatin-Bound Protein Isolation
Chromatin-bound nuclear protein fractions were extracted per the manufacturers’
protocol using the Subcellular Protein Fractionation Kit from Thermo Scientific. After,
the soluble nuclear fraction was isolated the chromatin-bound protein fraction was
isolated by treating the DNA/protein complexes with Micrococcal nuclease, which
digested the DNA thereby releasing the protein into the supernatant. Protein
concentrations for all fractions were determined by Bicinchoninic Acid assay (Thermo
Scientific) and all of the samples were stored at -80ºC until use.
High Salt Nuclear Extraction
Protein was extracted as in [13]. Cells were collected in non-nuclear extraction
buffer [20mM HEPES, pH 7.0, 10mM KCl, 2mM MgCl2, 0.5%NP40, 2.0 mM Sodium
orthovanadate, 50.0 μM ammonium molybdate and 10.0 mM sodium fluoride and
Complete protease inhibitors (Roche)] and dounced 30 times. Lysates were centrifuged
for 5min at 1,500g and supernatant was removed. Pellet (nuclei) was washed 3 times
with non-nuclear extraction buffer, resuspended in high salt nuclear extraction buffer
[500mM NaCl, 20mM HEPES, pH 7.0, 10mM KCl, 2mM MgCl2, 0.5%NP40, 2.0 mM
Sodium orthovanadate, 50.0 μM ammonium molybdate and 10.0 mM sodium fluoride
and Complete protease inhibitors (Roche)] and incubated at 4oC for 10min. Suspension
was centrifuged for 10min at 15000g and supernatant was collected as nuclear fraction. 61
Chromatin Immunoprecipitation
Epithelial cells were grown in 100mm dishes and treated with MUC1 or controls siRNA. Cells were exposed to EGF treatment and allowed to incubate for 120 minutes.
Cells were then incubated with 1% paraformaldehyde /PBS for 10 minutes at room temperature and then treated 0.125M glycine for 5 minutes at room temperature. Cells were washed with PBS, resuspended in PBS with 2.0mM Sodium orthovanadate, 50μM
ammonium molybdate, 10mM sodium fluoride, and Complete protease inhibitors and
counted. Cells were then snap frozen and kept at -80oC. These cells were subjected to
chromatin immunoprecipitation based on the Millipore EZ-ChIP protocol (17-371).
Briefly, cells were lysed and the protein/DNA lysates were sonicated (five-10sec bursts)
and pre-cleared with DNA blocked agarose G beads (16-201, Millipore) for 30 minutes at
4oC. The pre-cleared lysates were incubated overnight at 4oC with 5 μg of antibody for
EGFR (Santa Cruz, 1005 and Neomarkers, Ab-13), Mouse IgG, Rabbit IgG, and RNA
Pol II (Upstate, clone CTD4H8). The antibody/protein/DNA complexes were isolated by incubating agarose G beads for 60 minutes at 4oC. The agarose G
bead/antibody/protein/DNA complexes were washed several times and the crosslink was
reversed by incubating with NaCl overnight at 65oC. DNA was treated with RNase A,
proteinase K, and isolated with a Qiagen spin column. DNA was used for PCR to
amplify the ATRS region of cyclin D1 (F: 5’-TGGTCTTGTCCCAGGCAGAG-3’ and R:
5’-GATGGTGGCCAGCATTTC-3’) and GAPDH (F: 5'-
TACTAGCGGTTTTACGGGCG-3’ and R: 5'-
TCGAACAGGAGGAGCAGAGAGCGA-3). The cyclin D1 PCR was performed for 35 62
cycles at 98oC for 10sec, 65.3oC for 10sec, and 72oC for 25sec. The GAPDH PCR was
performed for 32 cycles at 98oC for 10sec, 62oC for 10sec, and 72oC for 25sec. The PCR
products were analyzed on a 2% agarose gel and visualized using a Kodak Gel Logic
2000.
Invasion Assay
Collagen matrix (0.9 mg/ml Type I Rat tail collagen (BD Bioscicences, Billerica,
MA), 83.0% (v/v) M-199 medium (Life Technologies), and 0.18% NaHCO3 (Fisher,
Hampton, NH)) was poured into a 96 well plate. Prior to collagen polymerization a
0.8μm pore transwell insert (Corning Inc., Corning, NY) was placed on top of the matrix.
The polymerized collagen was rehydrated using a chemoattractant (20% FBS/EMEM).
BT-20 cells were treated with 10µM of hPMIP, CP, PTD4 peptides or water in serum free EMEM media overnight. Prior to loading the cells onto the transwell inserts they
were fluorescently labeled with Calcein-AM (Invitrogen) for 30 minutes and then washed
with PBS. The cells were then resuspended in a 10µM peptide/EMEM serum free media and loaded onto the inserts in each well (15,000 cells/well). The cells were allowed to
o invade into the matrix for 10hrs at 37 C with 5% CO2 in a humidified incubator. After
10hrs, the collagen matrix was treated with a 0.25% Collagenase in 40% FBS/PBS and
the inserts were removed (Calbiochem, San Diego, CA). The number of fluorescently
labeled cells that had invaded into the collagen were measured using a Molecular Devices
spectrophotometer (Ex:485, Em:538, and Cutoff:530). For each treatment group, a set of 63
cells were placed directly into the bottom chamber and used as a reference for 100% of
cell invasion. Invasion is then indicated as percent invasion (invaded cells/total cells).
MTT Assay
BT20 cells were plated into a 96 well plate (5 X 103 cells per well). The cells
were allowed to adhere and then they were treated daily with 10μM of hPMIP, CP, PTD4
peptides or water (treatments diluted in 10% FBS/EMEM) for seven days. After treatment the Vybrant MTT Cell Proliferation Assay (Molecular Probes) was performed following the manufacturers’ protocol.
Peptide Synthesis
hPMIP, Biotin-hPMIP, msPMIP, FITC-msPMIP, CP and PTD4 polypeptides were synthesized by GenScript (Scotch Plains, NJ) and delivered lyophilized. The peptides were resuspended at a concentration of 500μM in water and stored at -80oC in single-use aliquots. Peptide sequences are shown in Figure 5.1.
Mice
Mouse Genotyping. The genotypes of the mice were determined by extracting DNA from a small portion of the tail. The tail was incubated in a lysis buffer (10mM Tris, pH 8.0,
50mM NaCl, 25mM EDTA, pH 8.0, 0.5% SDS, and 0.5mg/ml Proteinase K) overnight and the DNA was extracted. Subsequently the DNA was utilized for the MMTV-Wnt-1
PCR (IMR513 - 5’-GGACTTGCTTCTCTTCTCATAGCC-3’ and IMR514 - 5’-
CCACACAGGCATAGAGTGTCTGC-3’) and waved-2 PCR (Wa2 F – 5’- 64
CCCAGAAAGGGATATGCG-3’ and Wa2 R – 5’-GCAACCGTAGGGCATGAG-3’, 45
cycles of 30 seconds at 94oC, 30 seconds at 55 oC, and 30 seconds at 72 oC). Note the
product of the waved-2 PCR (170bp, Egfrwildtype) was treated with a restriction enzyme
(FokI) to confirm the presence of a 95bp and 75bp piece ( Egfrwa2). The digested PCR products were separated on a 2% agarose gel to confirm size.
Mouse Protein Lysates
Protein lysates from mouse tissues were generated as in [8]. Mammary glands were removed from the mouse and immediately homogenizing in a tissue lysis buffer
(20mM HEPES, pH 7.0, 150mM NaCl, 1% Triton X-100, 2mM EDTA, pH 8.0, 2mM
EGTA, pH 8.0, 2.0 mM Sodium orthovanadate, 50.0 μM ammonium molybdate and 10.0 mM sodium fluoride and Complete protease inhibitors). Lysates were stored at -80oC.
Whole Mounts
As performed in [8]. Mammary glands were removed from the mice and fixed in
1:3 glacial acetic acid:ethanol for one hour. Fixed glands were washed with 95% and
100% ethanol for 15 minutes and then defatted with acetone for three days. Following
the acetone treatment, the glands were subsequently washed with 100%, 95%, and 70% for 15 minutes and briefly with distilled water to rehydrate the gland. Rehydrated glands were stained with 0.2% carmine/0.5% aluminum potassium sulfate overnight and then destained with subsequent 70%, 95%, 100%, 95% and 70% ethanol washes for 15 minutes each. Stained mammary glands were stored in glycerol solution, imaged with a
Leica dissecting microscope and analyzed using the MagnaFire software. 65
Xenograft Tumor Studies (PMIP)
Immunocompromised (scid) mice (Taconic, Rockville, MD) were tested for the
presence of serum IgG and found to be <20 μg/ml IgG. Female mice (four to six weeks
old) were injected with 1x107 cells embedded in Matri-gel (BD Biosciences) into the
mammary fat pad and allowed to grow to either 100mm3 or 500mm3, based on the
2 formula a x b/2 where a is the smaller diameter and b is the larger diameter. Mice were
injected intraperitoneally (i.p.) with 50 μg/g body weight of either PMIP or PTD4 control
peptide for 21 days and measured with calipers every two days. Either at the end of 21
days, or after the tumor had reached 800mm3, tumors were resected by injecting the mice
with buprenorphine (2.5 mg/kg body weight, Infusion Solutions, Totowa, NJ) at least one
hour prior to resection and anesthetizing the mice with isoflurane (Abbott, Abbott Park,
Il). Following surgery, mice were treated with buprenorphine (2.5mg/kg body weight) 8
and 16hrs post-surgery. Animals were then followed for 10 days to examine regrowth at
the primary tumor site or at secondary mammary glands, then sacrificed.
Genetically-Driven Tumor Studies (PMIP)
MMTV- pyV mT mice on an FVB background (W. Muller [67], obtained from
Jackson Laboratories) entered into study upon the development of tumors that measured
> 0.5cm in at least one diameter. Only mice greater than seven weeks of age were included in the study and mice that developed fluid cysts were excluded. Animals were injected i.p. with 50μg/g body weight of msPMIP or PTD4 once daily for 21 days. Each of ten mammary glands were measured using calipers every two days, and measurements 66
2 were used to determine tumor volume based on the formula a x b/2, with a being the
smaller diameter and b being the larger diameter. After treatment for 21 days, mice were
sacrificed by CO2 inhalation and tissues resected. Several msPMIP treated mice were
injected with FITC-msPMIP (50μg/g body weight) one or four hours prior to sacrifice
and intact tissues were visualized using a fluorescence MZFLIII Leica dissection
microscope. Both the xenograft and transgenic PMIP mouse studies were performed by
the Experimental Mouse Shared Service (University of Arizona, Tucson).
MMTV-Wnt-1 Transgenic Mouse Study
MMTV-Wnt-1 mice were obtained from Jackson Laboratories and waved-2 mice
were provided by Dr. Noreen Luetteke from the University of South Florida, Tampa.
The mice were maintained at the Arizona Health Science Center Animal facility (Tucson,
AZ) under conditions specified by the Institutional Animal Care and Use Committee.
MMTV-Wnt-1 mice were outbred (C57Bl6/FVB) onto the Egfrwa-2/wa2 background and at
least 14 virgin female mice from each genotype were generated (n=16, MMTV-Wnt-1
Egfr+/+ ; n=14, MMTV-Wnt-1 Egfrwa2/+ ; n=15, MMTV-Wnt-1 Egfrwa-2/wa2). After eight
weeks of age mice were palpated once a week until formation of a mammary gland tumor
and then the tumor volume was measured three times a week based on the formula a2 x b/2 where a is the smaller diameter and b is the larger diameter. Once the tumors grew to
larger than one centimeter the mouse was sacrificed and the tumors, contralateral
mammary, and lungs were collected. Protein lysates were generated for the tumors and
contralateral mammary glands. A small portion of the tumor and another contralateral 67
mammary gland were fixed in 10% buffered formalin and embedded in paraffin. Finally,
another contralateral mammary gland was used for whole mount.
All studies were performed under protocols approved by the Institutional Animal Care
and Use Committee of the University of Arizona.
Statistical Analysis
All statistics were performed in Excel (Microsoft) or STATA 10.0 (StataCorp). Test
implemented were two-tailed student t-test and one way analysis of varience (ANOVA).
68
III. MUC1 REGULATES NUCLEAR LOCALIZATION AND FUNCTION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR
Note: The work presented in this chapter is published in Journal of Cell Science (ADD FINAL CITATION). Except Figure 3.3
INTRODUCTION
Subcellular protein trafficking is a highly regulated process that is essential for
maintaining normal cell function and viability. In fact, the mislocalization of proteins
plays a fundamental role in promoting diseases such as cystic fibrosis, diabetes, and
several types of cancer [Reviewed in [162]]. In cancer specifically, aberrant nuclear
localization of receptor tyrosine kinases, such as, c-MET, ErbB1/EGFR, ErbB2, ErbB3,
ErbB4, FGF, and VEGF, results in increased transcription of genes that promote
chemoresistance, proliferation and disease progression [52-55, 163-165]. Notably, these
effects are not due to increased signal transduction, but are instead due to the ability of
these proteins to act as transcriptional cofactors [55]. A number of these proteins contain
nuclear localization signals (NLS), including the erbB family of receptors (ErbB1-4),
which share a highly conserved tri-partite NLS (Consensus Sequence: [RK](3)-x{2,3}-
[RK]{3,4}-x{2,3}-RR) [58]. While this NLS can drive nuclear translocation, the
receptors typically remain localized to the plasma membrane in polarized epithelium.
The Epidermal Growth Factor Receptor (ErbB1/EGFR) is an example of a protein
that has altered subcellular localization in cancer. In normal polarized epithelium, EGFR
is targeted to the basolateral membrane by well-conserved localization sequences 69
(KRTLRRLLQERELVEPLTPSGEAP) [29]. Under these circumstances, ligand stimulation induces receptor dimerization, ubquitination, endocytosis and degradation in the lysosome, thereby limiting EGFR expression and signaling [Reviewed in [166]].
However, in cancer cells it has recently been discovered that EGFR undergoes retrograde trafficking through the endoplasmic reticulum Sec61 translocon, which is followed by release into the cytosol [56]. Following this release from the membrane, EGFR’s tripartite nuclear NLS interacts with Importinβ1 allowing it to be shuttled through the nuclear pore complex into the nucleus [5]. Once inside the nucleus, it has been proposed that EGFR directly interacts with the endogenous promoter and initiates transcription of the cell cycle regulator, cyclin D1 [52]. Finally, the CRM1 exportin is capable of facilitating the nuclear export of EGFR [5]. Of note, the presence of EGFR in the nucleus of tumor cells correlates with a poor clinical outcome in breast, ovarian, oropharyngeal, esophageal squamous cell, and epithelial carcinomas [47].
The heterodimeric transmembrane mucin MUC1 is also highly expressed in epithelial carcinomas and inhibits the ubquitination of EGFR, altering normal EGFR trafficking [87]. In polarized ductal epithelium, MUC1 is constitutively internalized and recycled between the apical membrane and the golgi, which enables it to maintain a high level of O-glycosylation [119]. The apical localization of MUC1 is regulated by a well- conserved targeting sequence in the juxtamembrane domain (CQC) [113]. In non- polarized breast epithelium, MUC1 and EGFR interact at the plasma membrane resulting in increased EGFR internalization, the loss of lysosomal degradation and an increase in
EGFR recycling [87]. In addition to membrane localization, MUC1 is overexpressed and 70
mislocalized to the cytosol and nucleus in about 90% of breast adenocarcinomas, correlating with poor prognosis [89, 98, 110, 122]. Notably, the loss of Muc1 significantly delays tumor onset in an EGFR-dependent (WAP-TGFα) transgenic breast cancer model [8, 167]. In this study, loss of Muc1 within EGFR-dependent mammary gland tumors is also associated with a reduction of cyclin D1 expression [8].
MUC1 trafficking to the nucleus is due to the presence of a non-classical NLS
(RRK) that interacts with Nucleoporin 62 and Importinβ1, allowing for the nuclear co- translocation of numerous proteins, including γ-catenin, β-catenin and estrogen receptor
[97, 112, 121, 168]. In fact, the transcriptional activity of β-catenin is dependent on
MUC1’s nuclear localization, indicating that MUC1 is acting as co-factor [111, 112]. In addition, transcriptional targets of these proteins are activated in a MUC1-dependent manner. Given MUC1’s influence on EGFR trafficking following receptor activation and
EGFR’s newly described nuclear trafficking pathway, our current study was designed to determine if MUC1 functions as a regulator of EGFR nuclear translocation and transcriptional activation of the gene, cyclin-D1.
In this report, I demonstrate a novel role for MUC1 in EGFR regulation. Through biochemical and genetic analysis, I show that MUC1 alters the nuclear localization and function of EGFR in a manner that may have important consequences for EGFR- dependent tumors.
RESULTS
71
MUC1 promotes accumulation of nuclear EGFR
The loss of Muc1 expression significantly suppresses EGFR-dependent cancer
progression and cyclin D1 protein expression in the WAP-TGFα model [8]. In the
current study, I set out to determine if the mechanism by which MUC1 alters EGFR-
dependent cyclin D1 expression is via a novel regulation of nuclear trafficking and direct
transcriptional function of EGFR.
To examine the effect of MUC1 on EGFR cellular localization, I utilized two cell
lines expressing MUC1 (MCF10A and MDA-MB-468). Cells were transfected with either MUC1-specific siRNA to knock down MUC1 protein expression or a control
siRNA, and changes in EGFR localization were evaluated. Optimal knockdown of
MUC1 was observed after treatment with either MUC1-1 or MUC1-2 specific siRNAs after three days (MCF10A), as previously demonstrated with these MUC1 target sequences (Figure 3.1) [87]. For all fractionation studies, cells were serum-starved for
18 hours, treated with 10ng/mL EGF on ice for 10 minutes (to saturate membrane-bound receptors), and then excess ligand was removed. Cells were then incubated at 37oC for
120 minutes to induce receptor endocytosis and trafficking. Cells were then fractionated by differential detergent fractionation which takes advantage of the ionic properties of different detergents to isolate the membrane (Triton X100), cytosolic (Digitonin), and nuclear (Tween40) protein fractions [161]. Note that the membrane fractions include plasma membrane, mitochondria, golgi, endosomes, and the endoplasmic reticulum.
Proteins isolated from the membrane, cytosolic, and nuclear compartments were then separated by SDS-PAGE and examined by subsequent immunoblotting to determine the 72
effects of MUC1 expression on EGFR localization. To confirm fraction purity and equal
loading, expression of proteins known to localize in each cellular compartment were
analyzed (Plasma Membrane, IGF-1Rβ; Endoplasmic Reticulum Membrane, BAP31;
Endosomes, EEA1; Cytosolic, Myosin IIa; Nucleus, Histone H3) [161].
Analysis of protein lysates from MDA-MB-468 cells revealed that MUC1 and
EGFR are present in both the membrane and nuclear fractions under conditions of serum
starvation and EGF treatment (Figure 3.1A, top 2 panels). MUC1 expression did not
affect EGFR localization to the membrane fraction in the absence of EGF, although loss
of MUC1 resulted in decreased levels of EGFR in the membrane fraction in the presence
of EGF. This recapitulates the result of a previous study that described increased EGFR
membrane recycling in the presence of MUC1 (Figure 3.1A, top panel) [87]. The
maximal nuclear EGFR accumulation was observed after 120 minutes of EGF
stimulation, a finding similar to what was previously observed by another group [169].
Interestingly, EGFR was observed in the nucleus in the presence or absence of EGF,
indicating that nuclear EGFR trafficking can occur in the absence of serum in these cell
lines (Figure 3.1A, top panel). Importantly, in both serum starved and EGF-treated cell lines, I observed that the absence of MUC1 inhibited the accumulation of EGFR in the
nucleus (Figure 3.1A, top panel). The effect of MUC1 on EGFR localization was found
to be similar in MCF10a cells with both MUC1-1 and MUC1-2 siRNAs (Figure 3.1B).
To determine if this result was an artifact of endoplasmic reticulum or post-lysis
contamination I performed a high-salt nuclear protein extraction on cells treated with
control or MUC1-1 siRNA. Cells were serum starved or stimulated with EGF for 0min 73
or 120min, subjected to high-salt protein extraction and proteins were analyzed by
immunoblotting. I discovered that after serum starvation and 0 min of EGF treatment there was a minimal difference in total nuclear EGFR with respect to MUC1 expression.
However, after 120 minutes of EGF treatment there was a significant MUC1-dependent accumulation of total EGFR in the nucleus (Figure 3.1C). This demonstrates that MUC1 promotes the accumulation of non-membrane bound nuclear EGFR.
74
Figure 3.1 MUC1 promotes nuclear EGFR Localization. A and C) MDA-MB-468 breast cancer cells or B) MCF10a immortalized breast epithelial cells were treated for three days with control (Ctrl) or MUC1 (MUC1-1 or MUC1-2) siRNA, were either serum starved (-Serum) for 18hrs or serum starved and then treated with EGF (10ng/ml, 120’ EGF). Cells were fractionated and protein (6μg) was separated by SDS-PAGE. After transfer to PVDF, proteins were immunoblotted (IB) for EGFR (Santa Cruz, 1005) and MUC1 (Neomarkers, CT2). Fraction purity and loading were determined by immunoblotting for BAP31(Affinity Bio-Reagents, MA3-002; Endoplasmic Reticulum Membrane, Mem), IGF-1Rβ (Santa Cruz, SC-713; Plasma Membrane), EEA1 (Santa Cruz, SC-33585; Endosomes), Myosin IIa (Santa Cruz, D16; Cytosolic, Cyto), Sp1 (Santa Cruz, 1C6), and Histone H3 (Santa Cruz, C-16; Nuclear, Nuc). White lines through blots indicate same samples and exposure but were non-contiguous. The molecular weights (kDa) are indicated on the right. Note that the cytoplasmic domain of MUC1 runs as multiple species 14-28 kDa, because of different glycosylation states [110].
75
To visualize the subcellular localization of EGFR and MUC1, control and MUC1 siRNA treated cells were analyzed by laser-scanning confocal microscopy. Cells were serum-starved and either left untreated (Figure 3.2, A-H’) or treated with 20ng/mL of
EGF for 120 minutes (Figure 3.2, I-P’), then immunolocalized for MUC1 (Figure 3.2, A,
E, I, M) and EGFR (Figure 3.2, B, F, J, N). I observed that in the absence of EGF, EGFR was mainly localized to the plasma membrane with limited cytosolic and nuclear localization, regardless of MUC1 expression (Figure 3.2, B and F). Under similar conditions, MUC1 localization was largely nuclear and cytosolic (Figure 3.2, A).
Following EGF treatment, punctate staining of EGFR was observed within the cytosol of cells from both treatment conditions (Figure 3.2, J and N, outlined arrows), but in MUC1 siRNA treated cells the cytosolic EGFR mainly localized to perinuclear regions (Figure
3.2, N and P’, outlined arrows) whereas in control siRNA treated cells that maintained
MUC1 expression, significant punctate EGFR staining was observed in the nucleus
(Figure 3.2, J and L’, arrowheads). These data corroborate our observation that MUC1 expression promotes EGFR nuclear accumulation.
76
Figure 3.2 MUC1 promotes nuclear EGFR accumulation. Immortalized breast epithelial cells (MCF10A) were treated with MUC1-1 (E-H’, M-P’) or Control (Ctrl) siRNA (A-D’, I-L’) and stimulated with EGF (20ng/mL) for 120 minutes. (I-P’) Cells were fixed, permeabilized, and used for immunostaining against EGFR (Neomarkers, Ab-1) (green, B, F, J, N) and MUC1 (Neomarkers, CT2) (red, A, E, I, M). (A-H’) Under serum starvation conditions (- serum) EGFR localized to the plasma membrane (white arrows), nucleus and cytoplasm. (I-L) In presence of EGF and MUC1, EGFR is localized within the nucleus (white arrowheads, L’). (M-P’) EGF treatment in the absence of MUC1 resulted in EGFR localization to perinuclear regions (white open arrows, P’). DAPI= Blue nuclei. A-P magnifications are 630X. Scale Bars=20μm
77
Inhibiting MUC1/EGFR interaction decreases nuclear EGFR
Knocking down MUC1 expression resulted in a significant decrease in nuclear
EGFR localization. To confirm that the interaction of MUC1 and EGFR promoted nuclear accumulation of EGFR I utilized the MUC1 mimetic peptide, PMIP, to inhibit the interaction. PMIP was observed to efficiently inhibit EGFR and MUC1 interactions in the BT20 breast cancer cell line resulting in a significant decrease in the proliferative and migratory potential (Figure 5.2 and 5.3) [123].
I next examined the effect of PMIP of the localization of EGFR. MDA-MB-468 cells were treated for 18 hrs with 20μM PMIP in serum-free media, stimulated with EGF for 120 minutes and the protein from cells was fractionated. Protein fractions were analyzed by immunoblotting as in previous experiment. I discovered that PMIP was able to inhibit nuclear EGFR accumulation compared to non-treated and control peptide treated cells (Figure 3.3). Lysate purity was confirmed by immunoblotting for endoplasmic reticulum membrane (BAP31) and nuclear (Histone H3). 78
Figure 3.3 Inhibiting MUC1/EGFR interaction inhibits nuclear EGFR accumulation. MDA-MB-468 cells were treated with PMIP (20uM) for 18hrs and then stimulated with EGF (10ng/mL) for 10 minutes at 4oC. Cells were then incubated for 120 minutes and subjected to differential detergent fractionation. Protein fractions were separated on an SDS-PAGE and used for immunoblotting against EGFR (Santa Cruz, 1005), BAP31 Affinity Bio-Reagents, MA3-002; Endoplasmic Reticulum Membrane, Mem) and Histone H3 (Santa Cruz, C-16; Nuclear, Nuc).
79
MUC1 and EGFR Interact in the Nucleus
The loss of MUC1 results in a significant decrease in nuclear EGFR. I next
wanted to determine if MUC1 and EGFR were interacting within the nuclear
compartment. MDA-MB-468 and MCF10A cells were treated as described in the
previous section and subjected to differential detergent fractionation. To determine if
MUC1 and EGFR were interacting within the nucleus of these cells, I isolated the nuclear
protein (400μg) and performed immunoprecipitation directed against EGFR and mouse
IgG, followed by immunoblotting against EGFR and MUC1. Following 120 minutes of
EGF treatment at 37oC, MUC1 and EGFR form a complex in the nucleus (Figure 3.4A,
top panel). Additionally, nuclear protein lysates were analyzed to confirm MUC1
expression and the resulting EGFR nuclear accumulation (Figure 3.1B).
To determine if interacting MUC1 and EGFR in the nucleus were also binding
nuclear DNA, I performed a fractionation that would allow us to distinguish between
soluble and chromatin-bound nuclear proteins. After EGF treatment, cells were
subjected to subcellular fractionation and the nuclear protein bound to chromatin was
isolated as a separate fraction from the soluble nuclear protein. This was accomplished
by first gently collecting protein from the nucleus and then collecting the
protein/chromatin complexes. These complexes were then treated with Micrococcal
nuclease, which released chromatin-associated protein into the supernatant. I found that
while EGFR was present at the same levels in the soluble nuclear fraction regardless of
MUC1 expression, EGFR was only found bound to chromatin in the presence of MUC1 80
(Figure 3.4B, Nuc CB). This data demonstrates that EGFR’s interaction with chromatin
is MUC1-dependent. Moreover, this data addresses the question of whether MUC1-
dependent changes in EGFR levels in the nucleus are a result of altered EGFR
degradation, as soluble nuclear EGFR was equivalent regardless of MUC1 expression
(Figure 3.4B, Nuc S). Therefore, the presence of MUC1 appears not to alter
accumulation of soluble EGFR in the nucleus, but appears to be vital in EGFR’s ability to
interact with the DNA.
81
Figure 3.4 MUC1/EGFR interaction in the nucleus promotes EGFR/chromatin interaction. A) MCF10A immortalized breast epithelial cells were treated with control or MUC1-1 siRNA. Cells were serum-starved and treated with EGF (10ng/ml, 120 minutes at 37oC). The nuclear protein fraction (400μg) was used for immunoprecipitation (IP) with antibodies against EGFR (Neomarkers, Ab-13) and mouse IgG, and the precipitated protein was separated on an SDS-PAGE and immunoblotted (IB) for EGFR (Santa Cruz, 1005) and MUC1 (Neomarker, CT2). SL= straight lysate (non-IP protein, 12μg). Fraction purity was confirmed by immunoblotting for Histone H3 (Santa Cruz, C-16) and Myosin IIa (Santa Cruz, D16). Note: Straight lysates for this experiment are shown in Figure 3.1B. B) MDA-MB-468 breast cancer cells were was fractionated into membrane (Mem), cytosolic (Cyto), nuclear soluble (Nuc S), and chromatin bound (Nuc CB) fractions and separated (15μg) on an SDS-PAGE and immunoblotted (IB) for EGFR (Santa Cruz, 1005) and MUC1 (Neomarker, CT2). Fraction purity and loading was determined by immunoblotting for BAP31 (American Bio-Reagents, MA3-002), Myosin (Santa Cruz, D16), Sp1 (Santa Cruz, 1C6) and Histone H3 (Santa Cruz, C-16). The molecular weights (kDa) are indicated on the side.
82
EGFR Colocalizes with Phosphorylated-RNA Polymerase II
Considering the ability of EGFR to interact with chromatin and ability to directly induce transcription, I next wanted to determine if MUC1 could affect EGFR-mediated transcriptional activation [52]. A classical marker for transcription initiation is phosphorylated serine-2 RNA polymerase II (pS2-CTD-RNA Pol II) [170]. Therefore, to visualize EGFR and pS2-CTD-RNA Pol II localization, MCF10A cells were transfected
with control or MUC1-1 siRNA and stimulated with 20ng/mL EGF for 120 minutes.
Following EGF treatment, cells were fixed, permeabilized, and pS2-CTD-RNA Pol II and
EGFR were immunolocalized. Under serum starvation conditions, EGFR was mainly
observed at the plasma membrane and there was no positive pS2-CTD-RNA Pol II
staining regardless of MUC1 status (Data not shown). Alternatively, in the presence of
EGF, pS2-CTD-RNA Pol II was readily observed in the nucleus in the presence of
MUC1 (Figure 3.5B, outlined arrows). Additionally, following 120 minutes of EGF
treatment in MUC1-expressing cells, there were a significant number of EGFR-positive
punctate spots within the nucleus (Figure, 3.5A arrows) as observed in figure 3.2J. This
nuclear EGFR was found colocalized with pS2-CTD-RNA Pol II in the presence, but not
in the absence of MUC1 (Figure 3.5, D’ compared to H’, white arrowheads). Note that
the colocalization indicates that EGFR is localizing to transcriptional start sites [170]. To
confirm that the activity of pS2-CTD-RNA Pol II localization was not MUC1-dependent,
I also treated cells with 50ng/mL of insulin. I found that insulin treatment for 120
minutes resulted in punctate pS2-CTD-RNA Pol II nuclear localization (Figure 3.5J,
outlined arrows) and did not affect membrane localization of EGFR (Figure 3.5I, white 83
arrowhead). These data suggest that MUC1 plays a regulatory role in both EGFR
trafficking to the nucleus and its interaction with active transcriptional elements.
84
Figure 3.5 EGFR colocalizes with phosphorylated RNA polymerase II. MCF10A cells were treated with MUC1-1 (E-H’) or control (Ctrl) siRNA (A-D’, I-L’) and stimulated with 20ng/mL of EGF for 120 minutes. Following treatment the cells were fixed, permeabilized and incubated with antibodies against EGFR (Santa Cruz, 1005) (green, A, E, I) and phosphorylated serine-2 RNA polymerase II (pS2-CTD-RNA Pol II)(Abcam, H5) (red, B, F, J). Cells were treated with DAPI to visualize nuclei (C, G, K) and composite colocalization of all three images are shown in D, H, and L. (A-D): Colocalization (yellow) is seen at the white arrowheads (D’). (E-H): Cytoplasmic EGFR staining is shown by a white arrow, E. (I-L): Cells were also treated with insulin (50ng/mL) to illustrate membrane EGFR localization (large white arrowhead, I) and pS2- CTD-RNA Pol II without EGF treatment (white outline arrow, L’). DAPI= Blue Nuclei. A-L’ magnifications are 630X. Scale Bars=10μm
85
MUC1 promotes EGFR-mediated cyclin D1 expression
EGFR’s ability to activate transcription of cyclin D1 and the importance of cyclin
D1 in breast cancer progression has previously been described [52]. EGFR has been
shown to interact with the Adenine/Thymine Rich Sequence (ATRS) promoter region of
the cyclin D1 gene on promoter constructs, although EGFR has not yet been reported as
actively engaging the endogenous cyclin D1 promoter. Additionally, in an EGFR-
dependent tumorigenesis mouse model, the protein expression of cyclin D1 was found to
be MUC1-dependent. Therefore, to determine MUC1’s role in EGFR induced
transcriptional activation of cyclin D1, I utilized chromatin immunoprecipitation (ChIP)
directed against EGFR.
MCF10A cells were treated with MUC1-specific siRNA and subsequently
stimulated with EGF to promote endocytosis and trafficking of EGFR. The EGFR/DNA
complexes were isolated and the ATRS region of the cyclin D1 promoter was amplified
by PCR to determine if EGFR association was altered by MUC1. Additionally, I
amplified the promoter of the housekeeping gene, GAPDH, to confirm RNA Pol II
interaction and to determine the purity of the EGFR ChIP. I discovered EGFR efficiently
interacts with the ATRS region of the cyclin D1 promoter and this interaction is enhanced
in the presence of MUC1 (Figure 3.6). This ChIP analysis was performed using
extracellular EGFR (Neomarkers, Ab-13) antibody, indicating that the EGFR’s
ectodomain is also translocating to the nucleus and engaging the promoter. Additionally,
in cells that express MUC1, a ChIP performed against RNA Pol II revealed that RNA Pol
II was interacting with the cyclin D1 promoter more frequently compared to cells that 86
lacked MUC1 (Figure 3.6). With respect to the GAPDH, I found that RNA Pol II was
associated with its promoter.
Previously, I demonstrated that the loss of MUC1 expression corresponded to a
significant decrease of the interaction of EGFR with the cyclin D1 promoter. To
determine if this increased interaction with the cyclin D1 promoter translated into
increased cyclin D1 protein, I extracted protein lysates from cells treated with control or
MUC1 siRNA. The loss of MUC1 resulted in significant loss of cyclin D1 protein
expression (Figure 3.7A). In addition, the increase was EGF dependent, indicating that
although EGFR can be found in the nucleus of serum starved cells, EGF treatment is
necessary to promote cyclin D1 expression. Densitometry analysis of three separate
experiments indicated there was a significant 1.4 fold increase in the expression of cyclin
D1 (Figure 3.7B). These observations demonstrate that EGFR in the nucleus interacts
with MUC1 to promote the transcription of cyclin D1.
87
Figure 3.6 EGFR interaction with the cyclin D1 promoter is MUC1-dependent. MCF10A cells were transfected with either MUC1-1 or control (Ctrl) siRNA, serum starved, treated with 10ng/mL EGF, and incubated for 120 min. Protein/DNA complexes were collected and chromatin immunoprecipitations were performed against EGFR (Neomarker, AB-13), Mouse IgG, and RNA Pol II (Millipore, clone CTD4H8). DNA was isolated from the immunoprecipitations and used for PCR against the ATRS region of the cyclin D1 promoter and the GAPDH promoter.
Figure 3.7 MUC1 promotes Cyclin D1 Expression. A) MCF10A cells were treated with control (Ctrl) or MUC1-1 siRNA and stimulated with 20ng/mL of EGF for 120 minutes. Cytosolic protein was isolated, analyzed on an SDS-PAGE and immunoblotted for cyclin D1 (Cell Signaling, 2922), MUC1 (Neomarker, CT2), and β-actin (Sigma, AC- 15). The molecular weights (kDa) are indicated on the right. B) Relative Cyclin D1 expression levels were determined by densitometry and two-tailed student t-test was performed based on three experiments.
88
DISCUSSION
This report identifies a novel regulatory function of MUC1 on EGFR’s cellular
localization and nuclear activity. In breast cancer cells, I observed that both MUC1 and
EGFR localize to the nucleus and that MUC1 expression promotes the nuclear
accumulation of EGFR regardless of exogenous ligand. Focusing on the nuclear proteins,
I were able to efficiently immunoprecipitate EGFR/MUC1 complexes and found that the
presence of MUC1 regulated the interaction of EGFR and chromatin. After EGF
stimulation in MUC1-expressing cells, I also detected significant colocalization of
nuclear EGFR and phosphorylated RNA Pol II. Subsequent examination of chromatin-
bound EGFR with an extracellular EGFR antibody revealed that MUC1 expression
promoted the interaction of EGFR with the ATRS region of the cyclin D1 promoter. The
loss of the cyclin D1 promoter/EGFR interaction directly translated to a MUC1-
dependent decrease in the expression of cyclin D1.
Previously, MUC1 and EGFR have been reported to interact in a cancer-
dependent fashion at the plasma membrane and this interaction inhibited the lysosomal
degradation of EGFR [8, 87]. In addition, overexpression of EGFR leads to altered
trafficking of internalized receptor [56]. Altered trafficking can include the loss of
lysosomal targeting, increase recycling to the plasma membrane, and retrograde
trafficking. Retrograde trafficking can occur when transmembrane receptors are
endocytosed and returned to the endoplasmic reticulum (ER). Once in the ER, EGFR
associates with the Sec61 translocon and is released into the cytosol. EGFR then
associates with Importinβ1 via its tri-partite NLS and is imported into the nucleus [5]. In 89
the current report I demonstrate that MUC1 plays a novel regulatory role in EGFR
trafficking to the nucleus, by promoting accumulation of nuclear localized chromatin-
bound EGFR. This implies that both EGFR and MUC1 undergo retrograde trafficking
and that MUC1 promotes EGFR interaction with chromatin. Additionally, a recent study has demonstrated that MUC1 directly modulates the interaction of a transcription factor with DNA, which results in MUC1-dependent transcriptional activation [100]. Therefore it is possible that the protein-protein interaction between MUC1 and EGFR is altering
EGFR’s conformation in a way that allows EGFR to interact with DNA. In addition,
MUC1 and EGFR could be serving as transcriptional cofactors, and this will be the subject of future studies.
Interestingly, I found that the nuclear translocation of EGFR was not dependent upon the addition of exogenous ligand. Alternatively, I found that the ability of MUC1 to influence EGFR/chromatin interaction did require exogenous EGF. In addition, I observed that EGFR’s ability to colocalize with pS2-CTD-RNA Pol II and interact with the cyclin D1 promoter was both MUC1 and EGF-dependent. Although some reports have described that EGFR’s nuclear localization is dependent on EGF stimulation, others have determined that overexpression of EGFR or autocrine ligand production can drive nuclear accumulation in the absence of exogenous ligand [52, 56]. Our data suggest that both EGF-dependent and independent mechanisms may be utilized during nuclear translocation.
Recently, the importance of EGFR’s kinase activity was examined by utilizing a kinase-dead mutant of EGFR. The authors demonstrated that this kinase-dead EGFR was 90
able to inhibit autophagic cell death in human cancer cells [79]. This study demonstrated
a mechanism of EGFR oncogenic action that is independent of EGFR’s kinase activity.
It also suggests that targeting the kinase domain should not be the only focus of EGFR
therapeutics. The widely-held concept that the tyrosine activity of EGFR confers its role
in tumor formation has resulted in several therapeutic approaches that specifically target
EGFR’s kinase domain. For example, gefitinib was designed to inhibit EGFR
dimerization and the resulting receptor activation. These types of therapies were shown
to be effective in vitro and in mouse models of tumorigenesis; however the response rates
were not recapitulated in human studies [Reviewed in [21]]. Interestingly, one of the
cell lines used in this study (MDA-MB-468) is gefitinib-resistant [65]. The authors
discovered that in MDA-MB-468 cells treated with gefitinib, EGFR’s nuclear
accumulation was unchanged. I have demonstrated that in MDA-MB-468 cells, MUC1
alters EGFR’s ability to interact with chromatin and it is possible that this interaction is
promoting gefitinib resistance.
Signal transduction cascades starting at the plasma membrane and utilizing
intracellular signaling proteins such as MAPK are considered to be the main regulatory
mechanism of cyclin D1 expression. Recently, it has been proposed that the expression
of cyclin D1 can be regulated through the direct interaction of EGFR and cyclin D1’s promoter. Understanding the regulation of cyclin D1 expression is essential to clarifying its role in cancer progression. Aberrant expression of cyclin D1 has been observed in
30% of breast cancer, which in both in vivo and in vitro models of breast cancer leads to increased proliferation and genetic instability [171, 172]. Loss of cyclin D1 expression in 91
transformed human cells results in decreased tumorigenicity in nude mice [173]. In
addition, the overexpression of cyclin D1 correlates with gefitinib resistance in head and
neck squamous carcinoma cells [174]. In breast cancer, the overexpression of EGFR and
cyclin D1 is not associated with gene amplification, but postulated to be a consequence of
an unknown stimulatory mechanism [171]. Our data suggest that expression of MUC1
allows EGFR to bind the cyclin D1 promoter, driving tumor progression (Figure 3.8).
In conclusion, this report offers a mechanism of transformation as a result of aberrant trafficking and mislocalization of a receptor tyrosine kinase. This novel regulatory function of MUC1 to mediate EGFR’s role as a transcriptional cofactor sheds important light on tumor-dependent alterations in trafficking.
92
Figure 3.8 Model A) Absence of EGF – 1. MUC1’s constitutive internalization to maintain glycosylation also results in EGFR being endocytosed. 2. EGFR and MUC1 both undergo retrograde trafficking through the endoplasmic reticulum and are released into the cytosol by the Sec61 translocon. 3. Both proteins are able to interact with Importin β1 which mediates entry in the nucleus. 4. MUC1 and EGFR’s interaction in the nucleus promotes nuclear EGFR accumulation. B) EGF Stimulation in the presence of MUC1 1. Following EGF treatment, EGFR is rapidly endocytosed in a clathrin-coated vesicle 2. EGFR and MUC1 undergo retrograde trafficking 3. Both proteins are imported into the nucleus and form a complex (4). 5. EGFR colocalizes with pRNA Pol II and associates with the cyclin D1 promoter to activate transcription. 6. Expression of MUC1 promotes the recycling of EGFR back to the plasma membrane. C) EGF stimulation in the absence of MUC1 1. EGF treatment results in EGFR endocytosis 2. EGFR is either retrograde trafficked or is degraded in the lysosome (5). 3. Cytosolic EGFR is imported in the nucleus and the absence of MUC1 inhibits EGFR nuclear accumulation and EGFR is exported out of the nucleus (4).
93
IV. EGFR ACTIVITY PROMOTES TUMORIGENESIS IN MMTV-WNT-1 MOUSE MODEL OF BREAST CANCER
INTRODUCTION
Breast cancer is the 2nd most common cancer among American women and the
role of oncogenes in the transformation of normal to cancerous tissue is still being
investigated. In mice the role of oncogenes to promote mammary gland transformation
has been well studied mainly through the use of the promoter isolated from the mouse
mammary tumor virus (MMTV). Utilization of the MMTV promoter allows for the
overexpression of the protein of interest specifically in the mammary gland with limited
expression in the salivary gland tissue, male reproductive organs, and lymphoid tissue
[175]. One of the first MMTV proto-oncogenes to be inserted into the mouse genome
was int-1 or Wnt-1 [176]. MMTV-Wnt-1 mammary gland tumors develop unifocally and
will infrequently metastasize in the presence of the primary tumor. Approximately 50% of female mice (SJL strain) develop adenocarcinoma within 6 months of being born and the remaining will develop tumors by one year [177]. Wnt-1 has been found to be associated with breast cancer, but is not normally expressed in normal breast tissue [178].
In addition, the expression of other Wnt ligands (e.g. Wnt -2, 3, 4 and 7b) has been demonstrated to be expressed in normal human breast tissue [179]. Overexpression of
Wnt-1 in the mammary gland of mice promotes transformation through the overactivation of the canonical Wnt/β-catenin pathway and its resulting targets genes. 94
The canonical Wnt/β-catenin signaling pathway is mediated by the binding of a
Wnt ligand (eg. Wnt-1) to the seven-pass transmembrane frizzled (Frz) receptor and this results in the inhibition of GSK3β by the Frz activated dishevelled (Dsh) protein
[Reviewed in [180]]. Normally, the Wnt signaling pathway is important in development and is reported to induce differentiation and cell survival [Reviewed in [140]]. However, in most transformed tissues β-catenin is not degraded, because of specific mutations
within β-catenin, upregulation of Wnt ligands, mutations in the GSK3β/Axin/ APC
degradation pathway and/or deregulation of critical signaling pathways. Although these
events are not common in breast cancer, the negative regulator of Wnt signaling secreted
Frizzled-related protein 1 (sFRP1) is often lost as a result of promoter methylation [181].
Importantly, the Wnt/β-catenin signaling has been reported to be upregulated in greater than 50% of all breast tumors [Reviewed in [130]]. Sabilization and transcription factor function of β-catenin has been proposed to be the primary mechanism of Wnt- dependent transformation.
β-catenin is a 92kDa protein that has been shown to be conserved among several different organisms and interacts with E-Cadherin and α-catenin to maintain cell-cell interactions [133]. β-catenin has three highly conserved tyrosines that are critical in maintaining cell-cell adhesion function, which upon phosphorylation will lead to reduced
β-catenin/E-Cadherin association. The reduced β-catenin/E-Cadherin association mediates the buildup of β-catenin in the cytoplasm [143]. In the presence of Wnt-1 signaling the accumulation of stabilized β-catenin in the cytoplasm promotes its translocation into the nucleus. In the nucleus, β-catenin can act as a transcription factor 95
binding to the lymphoid enhancing factor/T-cell factor (LEF/TCF) and eventually to TCF
response elements in DNA. Later studies reported that the genes that were up-regulated
in response to the β-catenin/TCF complex were mostly pro-proliferation (cyclin D1), pro-
survival and pro-migration (MMPs and uPAR) [182, 183].
A prior study has examined the effect of mammary gland tumorigeneis on increased β-catenin/Wnt signaling in the presence of epidermal growth factor receptor hyperactivation. The authors discovered that overexpression of Wnt ligands (Wnt-1 and
Wnt-3) in an epidermal growth factor receptor (EGFR)-dependent mouse model of
tumorigenesis (WAP-TGFα) enhanced mammary gland tumorigenesis compared to the
transgenic WAP-TGFα alone [184]. EGFR has been demonstrated to phosphorylate β-
catenin on three highly conserved tyrosines that are critical in maintaining cell-cell
adhesion function, which subsequent phosphorylation promotes adherens junction
dissociation [Reviewed in [135]]. In breast cancer EGFR is often over-activated and
over-expressed which leads to tyrosine phosphorylation of β-catenin, cytoplasmic
accumulation, and increased transcriptional activity. Schroeder et al. demonstrated in
MMTV-Wnt-1 mice that β-catenin tyrosine phosphorylation was tumor-dependent and
treatment with an EGFR specific ligand (TGFα and AR) resulted in the tumor-specific
phosphorylation of β-catenin [185]. The authors established that EGFR and β-catenin
interacted in both MMTV-Wnt-1 and MMTV-Neu derived tumors and they found
significant colocalization in both human primary and metastatic breast cancers [185].
Another report discovered that Wnt-1 treatment of breast cancer cells resulted in the
activation of ERK and ultimately led to the EGFR-dependent increase of cyclin D1 [144]. 96
ERK activation was a result of Wnt-1 induced transactivation of EGFR, which
demonstrates the potential oncogenic signaling crosstalk between Wnts and ErbBs.
The role of EGFR activity in these studies is somewhat unclear, because it is
unknown if overactivation of EGFR and Wnt ligands are acting independent of each
other. However, examining the role of EGFR activity in mouse models is difficult
because total EGFR knockdown is embryonic lethal [186]. Therefore to clarify the role
of EGFR activity versus Wnt signaling in the transgenic mouse models I utilized the
waved-2 EGFR mutant. These waved-2 mutant mice have a single nucleotide mutation in
the kinase domain of EGFR results in the same levels of total EGFR but an 80% loss of
activity [187]. In a mouse model of colon cancer (Apcmin) Roberts et al. examined the effect of the waved-2 EGFR mutation and they discovered that the loss of EGFR activity significantly reduced the number of pre-cancerous polyps [188].
These studies demonstrate the possible cooperation between EGFR and β-catenin signaling mechanisms in the promotion of tumorigenesis. Again, to more closely examine the effects of EGFR activity on this cooperation I utilized the β-catenin- dependent mouse model of tumorigenesis (MMTV-Wnt-1) and crossed it onto the EGFR tyrosine kinase activity deficient waved-2 background. I discovered that when MMTV-
Wnt-1 mice were either hetero or homozygous for the waved-2 genotype there was a significant delay in tumor onset and slower tumor growth alteration. Additionally, reduced EGFR activity in the MMTV-Wnt-1 mouse resulted in altered mammary gland development. Interestingly, total levels and cellular localization of β-catenin in the 97
tumors were unchanged compared across the different genotypes. This study
demonstrates that EGFR activity is important in the Wnt-1-dependent transformation of
mouse mammary glands.
RESULTS
EGFR kinase activity promotes Wnt-1 dependent tumorigenesis. MMTV-Wnt-1
transgenic mice were outbred (C57Bl6/FVB) onto an EGFR kinase deficient waved-2
background. Female mice were collected and after 8 weeks of age the mice were
examined weekly until mammary gland tumors were detected. After a tumor had become
established (>50mm3) the tumor volume was measured every other day and when one
edge exceeded one centimeter the mouse was sacrificed and the tissues were collected.
The loss of EGFR kinase activity resulted in the significant delay of mammary
gland tumor onset. Half of the MMTV-Wnt-1 mice with wildtype Egfr developed
mammary gland tumors in 22 weeks, similarly to the 24 weeks first described for the
Wnt-1 transgenic model (Figure 4.1). In contrast, Wnt-1 mice with either one or two
copies of the Egfr waved-2 gene displayed a significant delay in tumor onset. Tumors
that developed from the waved-2 mice grew at a similar rate until day 10 and then the
waved-2 grew a significantly slower rate compared to Wnt-1 Egfr wildtype derived
tumors (9.65mm3/day compared 41.1mm3/day)(Figure 4.2). 98
β-catenin and E-Cadherin protein expression is EGFR kinase independent. Wnt-1-
dependent downstream signaling results in the inhibition of β-catenin degradation
following dissociation from E-Cadherin and promotes β-catenin cytosolic accumulation.
Cytosolic β-catenin is then trafficked into the nucleus where it interacts with TCF/LEF to
activate transcription. To determine any alterations in total E-Cadherin and β-catenin
levels I examined proteins isolated from both MMTV-Wnt-1 Egfr wildtype and waved-2
tumors. Immunoblotting against both E-Cadherin and β-catenin I observed that there was
not a significant difference in expression levels (Figure 4.3).
β-catenin localization in MMTV-Wnt-1 tumors is EGFR kinase independent. In
breast cancer EGFR has been shown to tyrosine phosphorylate adherens junction
associated β-catenin, which will mediate the dissociation of E-Cadherin and cytosolic
release of β-catenin. I next examined if there were any differences cytosolic and nuclear
localization of β-catenin in tumors derived from EGFR wildtype and waved-2 mice. A
portion of each tumor was fixed, embedded in paraffin, and sectioned (5μM). Tumor
sections from each genotype were used to analyze changes in β-catenin localization and I
found that most of the β-catenin was associated with the plasma membrane independent
of EGFR’s kinase activity (Figure 4.4). 99
Figure 4.1 Loss of EGFR kinase activity significantly delays Wnt-1-dependent tumorigenesis. Outbred (C57Bl6/FVB) MMTV-Wnt-1 mice were crossed on to an EGFR tyrosine kinase mutant, waved-2 (wa2), and tumor onset was examined. (MMTV- Wnt-1 Egfr+/+, n=16; MMTV-Wnt-1 Egfrwa2/+, n=14; MMTV-Wnt-1 Egfrwa2/wa2, n=15).
The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again.
Figure 4.2 MMTV-Wnt-1 Egfr waved-2 tumors grow significantly slower. Outbred (C57Bl6/FVB) MMTV-Wnt-1 mice were crossed on to an EGFR tyrosine kinase mutant, waved-2 (wa2), and following tumor development (>50mm3) the growth rate was examined. n ≥ 3 for all genotypes and lines indicate moving average. (MMTV-Wnt-1 Egfrwa2/wa2, 9.65mm3/day compared to MMTV-Wnt-1 Egfr+/+, 41.1mm3/day) 100
Figure 4.3 Total β-catenin proteins is independent of EGFR tyrosine kinase activity. A) Tumors from MMTV-Wnt-1 Egfr wildtype and waved-2 were collected and protein from the tissue was isolated. Protein lysates were separated on an SDS-PAGE and transferred to a PVDF membrane. Proteins were analyzed by immunoblotting against β- catenin (SC-1496-R, Santa Cruz), E-Cadherin (610182, BD), and β-actin (Sigma) B) Densitometry performed of β-catenin relative to β-actin found no significant difference between genotypes.
Figure 4.4 β-catenin localization is independent of EGFR tyrosine kinase activity. Parrafin embedded tumors from each genotype were sectioned and used for immunofluorescence against β-catenin (green) and nuclei (blue). 101
EGFR kinase activity alters mammary gland development. The role of EGFR in the
developing mouse mammary gland has been described and its function in stromal associated cells (fibroblast) has been determined to be necessary for proper gland development [189]. At the time of sacrifice a normal contralateral mammary gland used
utilized to determine any developmental abnormalities. Upon staining the ductal
epithelium of the mammary glands, I examined gross differences of the epithelial
branching between genotypes (Figure 4.5). In the MMTV-Wnt-1 mouse that lacked
EGFR kinase activity secondary and tertiary branching was less distinguished compared
to wildtype mice. Again, this demonstrates that EGFR kinase activity is important in the
normal development of the mammary gland.
Figure 4.5 EGFR kinase activity is required for normal branching morphogenesis. Mammary glands were removed, fixed, defatted, and stained with carmine. Carmine stains the epithelium (black). Branching epithelia is clearly observed in the wildtype mice (left) compared to the less distinguishable epithelia observed in the waved-2 mice (right). 102
DISCUSSION
Wnt signaling cooperates with EGFR to promote β-catenin-dependent
transcriptional activation of TCF/LEF response genes, such as cyclin D1, VEGF, myc,
uPAR, and MMPs. All of these genes play a role in breast cancer progression to a
metastatic condition. In this current study, I utilized the MMTV-Wnt-1 transgenic mouse
model of breast cancer to determine the effect of EGFR tyrosine kinase activity in β- catenin-dependent tumorigenesis. Reduction of EGFR tyrosine kinase activity in the
Wnt-1 transgenic model resulted in a significant delay in tumor onset and tumor growth
rate.
EGFR is commonly overexpressed in the poorly differentiated triple-negative
breast cancer, which is the most chemoresistance and metastatic subtype. EGFR activity
and aberrant cellular localization in this subtype of breast cancer has been described as a
poor prognostic indicator [44, 46]. The loss of EGFR kinase activity in the Wnt-1 model
resulted in a slower growing tumor, which could possibly be explained through reduced
VEGF transcription and a lack of angiogenesis. Transgenic expression of Wnt-1 could
drive tumor formation and initial growth, but lack of nutrients would ultimately slow
tumor cell proliferation. This type of growth curve was observed in the waved-2 mice,
where after early growth the tumor became more cytostatic and took longer to reach at
least one centimeter in size.
Tumorigenesis in the MMTV-Wnt-1 model is heavily dependent on the
transcriptional activity of β-catenin and previous study has reported that Wnt ligand 103
expression can modulate EGFR-dependent tumorigenesis [14]. However, in MMTV-
Wnt-1 derived tumors I observed no difference in β-catenin localization and expression
with respect to reduced EGFR activity. Although EGFR kinase activity is reduced
compensatory activity and function of the other erbB receptors (erbB2 and erbB3) could
possibly explain the minimal b-catenin differences observed in the tumors. To address
this possible issue it will be important to examine the contralateral “normal” mammary
glands to see any alterations in β-catenin localization and expression. In addition,
determine if there are any preferential erbB dimers in the wildtype versus the waved-2
tumors. However, an obvious explanation could be that the delay in tumor onset
observed could be a result of other downstream EGFR signaling effectors, such as dual
phosphorylated ERK (ERK1/2).
A recent report discovered that EGF stimulation resulted in the activation of the
serine/theronine kinase CK2a by an ERK-2 dependent mechanism [145]. Following CK2
activation, it can serine phosphorylate α-catenin to promote adherens junctions disassembly and β-catenin stabilization. These events promoted the β-catenin dependent
transcriptional activation, increased cellular invasion and EMT. This reiterates the role of
EGFR activity and β-catenin stabilization but importantly provides a possible mechanism
of the delay of tumor onset observed in the EGFR activity deficient MMTV-Wnt-1 mice.
Although differences in β-catenin localization were not observed in the tumors between
genotypes examination of its localization in the contralateral normal mammary gland
could provide insight into possible early events driving tumorigenesis. 104
Although it is important to note that the mechanism driving the different tumor
onset and tumor growth rate is still unknown, the study does reveal that in an in vivo
model that EGFR tyrosine kinase activity and Wnt-1 signaling cooperate to drive tumorigenesis. These findings indicate that in triple negative breast cancer where EGFR is highly expressed the cotreatment of a Wnt and EGFR inhibitor could have additive or synergistic anti-cancer effects.
105
V. INTRACELLULAR MUC1 PEPTIDES INHIBIT CANCER
PROGRESSION
Note: The experiments described in this chapter have been published in [123].
Additionally, the experiment and resulting figure shown as Figure 5.2a was performed by
Dr. Ina Menzl. Xenograft and genetically-driven mouse studies were performed by
Experimental Mouse Shared Service (Tucson, AZ).
INTRODUCTION
MUC1 (DF3, CD227, episialin, PEM) is a heavily O-glycosylated heterodimeric
protein of >300 kDa, normally expressed abundantly on the apical surface of glandular
epithelia. In greater than 90% of human breast carcinomas and metastases, apical
localization is lost and MUC1 is overexpressed (by greater than 10 fold) and
underglycosylated [95, 190]. Deregulated expression of MUC1 is found in many other
types of adenocarcinomas as well, including cancers of the lung, pancreas, ovary and
prostate, in addition to being highly expressed in leukemias, myelomas and lymphomas
[191-193]. Studies in both genetic mouse models and cell line models have demonstrated
that MUC1 is an oncogene. A transgenic mouse model driving MUC1 (human)
overexpression to the mouse mammary gland (MMTV-MUC1) results in the
development of breast cancer and is accompanied by a failure of the mammary gland to
undergo complete postlactational regression via apoptosis [98]. Transfection of MUC1
into 3Y1 fibroblasts induces their transformation, and transfection of MUC1 into colon 106
cancer cells demonstrates that MUC1 overexpression inhibits drug-induced apoptosis
[120].
The cytoplasmic domain of MUC1 contains sites for multiple protein interactions, although these interactions go largely unformed in the polarized epithelium of the normal
breast, as the binding partners of MUC1 are typically found on the basolateral membrane
(Reviewed in [103] and [8, 87]. During cancer progression, when there is a loss of
cellular polarization, MUC1 is overexpressed and functionally interacts with src, GSK3β,
epidermal growth factor receptor (EGFR) and β-catenin, among others [87, 124, 126].
The sites for interaction between MUC1 and these proteins have been mapped to distinct
domains within the 72-amino acid cytoplasmic tail of MUC1 [126, 194, 195]. Both
EGFR and src can phosphorylate MUC1 on a YEKV motif, and this phosphorylation
results in increased binding of MUC1 to β-catenin through an SAGNGGSSLS domain
[126]. Recent evidence demonstrates that the interaction between MUC1 and EGFR can
significantly modulate EGFR biology and effect EGFR-dependent transformation [8, 87].
It has been established in both human breast cancer cell lines and transgenic mice
overexpressing MUC1 (MMTV-MUC1) that MUC1 and EGFR biochemically interact,
resulting in the potentiation of EGF-dependent p42/44 ERK activation during lactation
[110, 126]. Recently, our laboratory has demonstrated that MUC1 expression inhibits the
ligand-mediated ubiquitination and degradation of EGFR while enhancing its
internalization and recycling [87]. To evaluate the role of Muc1 in transformation,
Pochampalli et al generated WAP-TGFα mice on a Muc1 null background which 107
revealed that Muc1 expression has a dominant effect on TGFα-dependent transformation
of the breast, promoting both onset and progression [8].
While it is now established that EGFR is potently regulated by MUC1 expression,
transgenic mouse models have also implicated the cell adhesion protein, β-catenin, in
EGFR and MUC1 signaling. In a study of the WAP-TGFα transgenic model, Wnt1 and
Wnt3 were found to be selectively activated in the most aggressive breast tumors [184].
The Wnts are secreted glycoproteins that bind the transmembrane frizzled receptor,
resulting in a signaling cascade that inactivates the mechanism for β-catenin degradation
and results in transformation [183, 196-200]. Additionally, in MMTV-Wnt1 transgenic
mice, EGFR was found to interact with and phosphorylate β-catenin in a tumor-specific
manner [185]. These studies demonstrate that β-catenin and EGFR can affect their respective pathways to promote transformation. Finally, MUC1 is also implicated in β- catenin dependent transformation, indicating that these three proteins have the ability to cooperatively promote cancer progression. In MMTV-Wnt-1 transgenic mouse models crossed onto a Muc1-null background, loss of Muc1 corresponds to a significant reduction in tumor progression [124]. In a subsequent study, interactions between MUC1 and β-catenin were found to be highly increased in samples from human metastatic breast tumors, indicating that these interactions are clinically relevant [124].
Together, these studies demonstrate the strong potential for MUC1, EGFR and β-catenin to affect each other during transformation, including their striking co-upregulation during transformation and metastasis. MUC1 can inhibit the downregulation of EGFR and 108
promote the transforming ability of both EGFR and β-catenin. Additionally, genetically
derived mouse models implicate MUC1 in both EGFR- and β-catenin-dependent
transformation and metastasis. Interestingly, the interaction sites on MUC1 for both
EGFR and β-catenin lie in tandem on the MUC1 cytoplasmic domain. This study
demonstrates that targeting the interaction domain of MUC1 for both EGFR and β-
catenin through the utilization of MUC1 dominant negative peptides can significantly
affect breast cancer progression. Importantly, this dominant negative peptide can
significantly inhibit tumor progression in a genetically-driven mouse model of breast
cancer over a relatively short treatment time and may have important clinical
applications.
RESULTS
PMIP efficiently enters the cells, reduces EGFR protein levels and inhibits MUC1
colocalization with β-catenin.
The MUC1 cytoplasmic domain is composed of 72 amino acids, within which lies
a 15 amino acid domain containing sites of EGFR phosphorylation (human=YEKV and
mouse=YEEV) and β-catenin binding (human=SAGNGGSSLS and
mouse=SAGNGSSSLS,Figure 5.1a) [126, 201]. A 15 amino acid peptide was
synthesized to determine if it could act in a dominant negative fashion to block
interactions between endogenous MUC1 and EGFR/β-catenin. To allow this peptide to
gain entrance to the cell, a protein transduction domain was synthesized in tandem
[PTD4, Figure 5.1a [202], Reviewed in [203]]. To demonstrate that PMIP was entering 109
the cells, a biotin-labeled hPMIP (10μM) peptide (biotin-hPMIP) was pulsed onto cells in vitro. We found that the peptide was taken up and is retained in BT20 breast cancer cells
(Figure 5.1b).
To determine if the hPMIP peptide was able to block the interaction between
EGFR and MUC1, BT20 cells were treated overnight with either 10 μM hPMIP, 10 μM
control peptide (CP) or peptide vehicle (V), immunoprecipitated EGFR and
immunoblotted for MUC1 (Figure 5.2a). Coimmunoprecipitation between MUC1 and
EGFR in control treated cells was detected; however interaction was significantly
reduced in hPMIP treated cells (Figure 5.2a, top panel). In addition to protein interaction,
Pochampalli et al previously demonstrated that MUC1 expression inhibits the EGF- dependent degradation of EGFR [87]. To determine if PMIP was able to block the
MUC1-mediated effects on EGFR degradation, BT20 and MDA-MB-231 cells were treated with hPMIP or PTD4 control, and induced EGFR endocytosis by treatment with
EGF for 30 minutes. Examination of EGFR protein levels by immunoblotting demonstrated that hPMIP treatment resulted in a reduction of EGFR levels following
EGF treatment (Figure 5.2b and data not shown). Note that in the absence of EGF treatment, hPMIP treatment does not affect EGFR levels.
To determine if PMIP could block the interactions between MUC1 and β-catenin that occur during transformation, the effects of hPMIP treatment on protein colocalization in the BT-20 breast cancer cell line was evaluated [124, 185]. Cells were serum starved and treated with either 10 μM hPMIP, 10 μM control peptide (CP), 10 μM PTD4, or 110
vehicle control (V) overnight, fixed and immunofluorescence was performed. While β-
catenin and MUC1 colocalization at the cell cortex could be identified in control
conditions (Figure 5.2 c-f, arrows), their interactions were significantly inhibited by
hPMIP treatment (Figure 2c).
These results demonstrate that PMIP treatment inhibits both MUC1/β-catenin and
MUC1/EGFR interactions. In addition, PMIP inhibits MUC1-mediated inhibition of
EGF-dependent degradation of EGFR.
111
Figure 5.1 a) MUC1 peptide (PMIP) can efficiently enter cells. a) MUC1’s cytoplasmic amino acid sequence (MUC1CT), which includes an EGFR phosphorylation site (underline) and β-catenin binding site (double underline). Human and mouse sequence for the PMIP peptide (human PMIP, hPMIP and mouse PMIP, msPMIP), the PTD4 protein transduction domain and the control peptide (CP) containing the last 12 amino acids of the human MUC1 cytoplasmic domain are shown. b) BT-20 cells were treated with 10μM Biotin-hPMIP for 4hrs (Green=Biotin-PMIP and Blue=DAPI (nucleus), 400x and 630x)
112
Figure 5.2 PMIP blocks MUC1/β-catenin and MUC1/EGFR interactions. a) BT-20 cells were treated overnight (-serum) with either 10μM hPMIP, 10μM control peptide (CP) or vehicle control (water; V), and protein lysates generated. Lysates (500μg) were immunoprecipitated (IP) with either anti-EGFR (Ab-13) (lanes 1-6, top and middle panel) or IgG (lane 7, top and middle panel) and immunoblotted (IB) with anti-MUC1 (top panel; CT2) or anti-EGFR (middle panel; 1005). Total levels of MUC1 (35 μg) are shown in the bottom panel and in right column of top panel (straight lysate, SL). White lines through blots indicate same gel and exposure but were non-contiguous. b) BT-20 cells were treated for 18hrs (-S) with hPMIP (10μM) or PTD4 (10μM). The cells were treated with EGF (30’ EGF, 10ng/ml) to induce endocytosis or left serum free (-S) and protein lysates were generated. The lysates were immunoblotted (IB) for EGFR (1005) and β-actin (AC-15) c-f) BT-20 cells were treated (-serum) overnight with (c) hPMIP (10μM), (d) control peptide (10μM), (e) PTD4 (10μM) or (f) water (V) and pulsed with the same peptides for an additional 30’ prior to fixation. The cells were probed for MUC1 (primary; H295: red) and β-catenin (primary; C-14: green). Co-localization (arrows) is designated with white pixels. Magnification=400x. 113
PMIP inhibits cellular invasion and proliferation in vitro
As hPMIP treatment demonstrated a strong biochemical inhibition of MUC1 interactions with both β-catenin and EGFR, potential effects of PMIP treatment on cell
growth and invasion were evaluated. To determine the effects of PMIP treatment on cell
proliferation, BT20 cells were treated with either 10 μM hPMIP, 10 μM control peptide
(CP), 10 μM PTD4 or peptide vehicle (V) for 7 days continually, then the number of cells was evaluated by quantifying the differences in formazan conversion through a colormetric MTT assay [204]. Following seven days of hPMIP treatment the growth of
BT20 cells was significantly inhibited compared to the control peptide (CP), PTD4 and water (V) (*, p<0.001, **, p<0.0002, ***, p<0.002,Figure 5.3a).
The effect of PMIP on cellular invasion was next evaluated. BT20 or MDA-MB-
231 breast cancer cells were treated overnight with either hPMIP or PTD4 control peptide
(10μM), then induced to invade through an 8.0μM filter into Type I collagen using 20% fetal bovine serum (FBS) as a chemoattractant. Cells that were able to migrate through the filter and invade into the gels were fluorescently labeled and quantified. PMIP treatment significantly inhibited the ability of cells to invade compared to cells treated with controls in both BT-20 cells and MDA-MB-231 (#p<0.0001 and data not
shown,Figure 5.3b).
114
Figure 5.3 PMIP inhibits cell proliferation and invasion of breast cancer cells in vitro. a) BT20 cells were cultured in a 96-well dish (5 x 103 cells/well) and treated daily for 7 days with either hPMIP (10μM), CP (10μM), PTD4 (10μM) or water (V) in EMEM 10% FBS. An MTT assay was performed to quantify cell number at the start of treatment (Day 0, DO) and after treatment was complete (Day 7, D7) (*, p<0.001, **, p<0.0002, ***, p<0.002, ANOVA). Error bars represent standard error. b) BT-20 cell lines were treated with either hPMIP (10μM), CP (10μM), PTD4 (10μM) or water (V) overnight, labeled with Calcein AM, allowed to invade through a transwell (8.0μM) insert into a Type I collagen gel, and the invaded cells were fluorescently measured. (#p<0.0001, ANOVA). The data from the invasion and proliferation assays represents four independent experiments with at least seven replicates per experiment. Error bars represent standard deviation.
115
PMIP inhibits tumor growth and recurrence in a xenograft breast cancer model
As hPMIP strongly inhibited cellular growth and invasion in vitro, the ability of
hPMIP to suppress tumor growth and metastasis in vivo was evaluated. In these
experiments, highly metastatic MDA-MB-231 breast cancer cells were implanted into the
mammary fat pad of severe combined immune deficiency (scid) mice and tumor growth
was evaluated.
To determine potential effects of hPMIP on primary tumor growth, scid mice
bearing MDA-MB-231 tumors (100mm3) were treated for 21 days with either hPMIP or
PTD4 control peptide (i.p. injections) (Figure 5.4a-c). Upon removal of drug, tumors were allowed to continue to grow until they reached a volume of 1000mm3, then surgical
resection of primary tumors was performed (Figure 5.4b). Treatment with hPMIP
resulted in a significant decrease in tumor size compared to control treated animals at 21
days when drug administration was stopped (*p=0.028, Figure 5.4a). Additionally, this
corresponded to a significant increase in the length of time required for hPMIP treated
mice to reach resection size of 1000mm3 (**p=0.03,Figure 5.4b). Although treatment
ended approximately 20 days prior to resection, hPMIP treatment substantially decreased the amount of tumor regrowth and spread 10 days after resection (Figure 5.4c). Also, a
decrease in the size of metastatic tumors in the hPMIP-treated animals was observed
compared to control, and next designed an experiment that would allow us to determine if
hPMIP was affecting tumor spread following tumor resection. 116
To evaluate the effects of PMIP on tumor spread (Figure 5.4a and b), MDA-MB-
231 breast cancer cells were allowed to establish a large tumor mass (500mm3) and mice
were injected (i.p.) for 21 days with hPMIP or control peptide (PTD4) (Figure 4.4d).
Immediately after the end of treatment, primary breast tumors were resected, and animals
were followed to examine rates of tumor regrowth and/or metastasis to secondary
mammary glands. While the presence of secondary mammary gland tumors were found
in equal number for both treatment groups, the tumor volume for the control treated
animals averaged 760mm3 while the PMIP treated averaged only 73mm3 (Figure 5.4e).
Note that mice were not treated with drug during the 10 days in which regrowth was
followed. These xenograft models revealed that PMIP treatment could have long lasting
anti-growth effect, which was observed in tumor regrowth following resection.
117
Figure 5.4 PMIP significantly inhibits tumor growth and recurrence in vivo. a) MDA-MB-231 cells in Matrigel were injected into the mammary fat pad of scid mice, and daily peptide treatment (50μg/g body weight of hPMIP or PTD4) began when tumors reached 100mm3 (hPMIP and PTD4 n=8 mice) and primary tumor growth was assessed 2 (*, p=0.028, ANOVA). Tumor burden represents the average volume (v= a x b/2) of all tumors at the stage indicated. b) After the end of treatment, the amount of time the tumors took to progress to 1000mm3 was measured (**, p=0.03, ANOVA). c) After resection, mice were observed for tumor regrowth at the primary site or secondary mammary glands (hPMIP n=7 mice and PTD4 n=8 mice). d) MDA-MB-231 cells in Matrigel were injected into the mammary fat pad of scid mice, and daily peptide treatment (50μg/g of hPMIP or PTD4) began when tumors reached 500mm3 (hPMIP n=6 mice and PTD4 n=4 mice) and primary tumor growth was assessed. e) Following 21 days of treatment the tumors were resected immediately and tumor regrowth at the primary site and spread to secondary mammary glands was monitored (hPMIP n=4 mice and PTD4 n=4 mice). Error bars represent standard error.
118
PMIP inhibits tumor growth and induces regression in genetically-driven breast
cancer
While the xenograft model demonstrated the effect of hPMIP treatment on growth
and progression of established cell lines, we wanted to determine how msPMIP would
affect tumor initiation and progression in a mouse model which better recapitulates
human breast cancer. The MMTV-pyV mT transgenic mouse model of breast cancer
strongly resembles human breast cancer by activating multiple signaling pathways,
including AKT, src and shc [67, 205]. Studies have demonstrated that the resulting breast
cancer pathologically and molecularly mimics the full progression of hyperplasia, ductal carcinoma in situ and adenocarcinomas observed in human disease [206, 207]. To first determine if peptide could be delivered to the mammary glands and tumors of these animals, FITC-labeled msPMIP was injected and analyzed peptide retention (Figure
5.5a). At two hours post-injection, FITC was detected throughout the animal’s body cavity, including all organs (data not shown). After four hours, FITC-msPMIP was found to be retained selectively in the mammary gland, mammary gland tumors and in the colon and skin (Figure 5.5a and data not shown).
To determine the effects of msPMIP on genetically-driven breast cancer progression, MMTV-pyV mT mice bearing at least one mammary gland tumor of >0.5cm
in diameter (in MMTV-pyV mT mice there are ten potential tumor sites) were treated for
21 days with either msPMIP or PTD4 control peptide. Treatment had a dramatic effect
on tumor growth, as msPMIP significantly slowed the total tumor growth from ~590% to 119
~194% over the 21 days of treatment (*p=0.039;Figure 5.5b). Additionally, msPMIP
treatment significantly decreased the tumor growth rate compared to control (PTD4)
treated tumors (**p=0.007;Figure 5.5c). Note that treatment of MMTV-pyV mT mice with hPMIP (as opposed to msPMIP) had no effect on tumor growth, emphasizing the amino acid specificity of PMIP.
Overall size of tumors that arose throughout the study were next analyzed. This analysis demonstrates that while 13% of the tumors in the control (PTD4) group grew larger than 500mm3 by the end of the study, only 1% of the tumors in the msPMIP treated
group reached that size (Figure 5.5d). As this transgenic model had continual expression of the polyoma middle T transgene driving tumorigenesis throughout the study, the
effects of drug treatment on the formation of new tumors were examined. Although both
msPMIP and control (PTD4) groups had a similar number of tumors sized 100-300mm3 at the beginning of treatment, this number nearly doubled by the end of treatment in the control group, but remained the similar in the msPMIP group (Figure 5.5d). These data indicated that msPMIP treatment inhibits tumor initiation in this model (Initiation equals percent of tumor transitions from 0mm3 to 100mm3). To analyze tumor initiation further,
the percent of tumors that were initiated during drug treatment were determined. This
analysis demonstrates that in the msPMIP group there was a significant decrease (12 vs.
20 initiated tumors, data not shown, p=0.0045) of tumor initiation during the study.
Although highly significant decreases in tumor formation and growth were
observed from treatment of tumor-bearing MMTV-pyV mT mice with msPMIP, not all 120
tumors in the study responded to treatment (Figure 5.6a). Analysis of each mammary
gland demonstrates that while most tumor growth rates slowed substantially in response
to msPMIP (white bars), a number of tumors continued to grow, indicative of the stochastic pathway activation in this model. Importantly, a subset of established tumors treated with msPMIP (four tumors) regressed completely under treatment, although none of the control treated tumors did so (Figure 5.6a, *). In addition, there was no detectable toxicity (no weight loss, organ failure or disorientation) following treatment with msPMIP or PTD4. Further investigation of tumor tissue sections by cleaved caspase-3 immuno-histochemistry and hemotoxylin-eosin staining of tumors that regressed or responded to msPMIP treatment did not reveal a difference in apoptotic cells compared to
PTD4 control peptide treated tumors (Figure 5.6b). 121
Figure 5.5 PMIP significantly slows progression of MMTV-pyV mT induced mammary gland tumors. a) MMTV-pyV mT transgenic mice were injected with FITC-msPMIP (50μg/g body weight), sacrificed four hours later and various tissues visualized using fluorescence microscopy. FITC-msPMIP localization in the mouse mammary gland tumors is shown (2.5x and 8x). b) Mammary gland tumors (>0.5 cm in diameter) were allowed to develop and mice were injected daily (50μg/g body weight, 21 day treatment, i.p. injection, 1X per day) with either msPMIP (7 mice) or PTD4 (6 mice). At the end of treatment, animals were sacrificed and proteins lysates made of the tumors for later analysis. In the course of treatment, total tumor growth for all tumor sites (msPMIP n=70, PTD4 n=60) was significantly lower in the msPMIP treated mice than in the PTD4 mice (193.8% ± 77.7% vs. 589.5% ± 283.6%, *, p=0.039, ANOVA). c) Mammary gland tumors of msPMIP treated mice grew at a significantly slower rate than PTD4 treated tumors (p=0.0076, ANOVA). d) Tumor size distribution for the msPMIP or PTD4 treated transgenic mice revealed that 47% (28 out of 60 possible tumor sites) of the PTD4 treated tumors were larger than 100mm3 compared to 27% (19 out of 70 possible tumor sites) of the msPMIP treated tumors. Numbers above data are numbers of tumors that meet the size criteria over the total potential tumor sites. 122
Figure 5.6 MMTV-pyV mT tumors have differential response to msPMIP. a) Individual growth of each tumor site (each bar is an individual tumor site) from animals described in figure 3 were treated daily with msPMIP (open bars) or PTD4 (grey bars) at 50μg/g body weight for 21 days. Tumor progression was observed every three days during the 21 day treatment (PTD4, n=60 tumor sites and PMIP, n=70 tumor sites). In four instances (*), msPMIP treated tumors completely regressed, however none of the control (PTD4) treated tumors regressed. Individual tumor sites are grouped by mammary fat pads (1-10) to allow for the comparison of similar anatomical sites. MFP=mammary fat pad b) Tumors from msPMIP (1, left 2 panels) and PTD4 (2, right 2 panels) treated mice were sectioned (3μm) and subsequently used for hemotoxylin-eosin staining and cleaved caspase-3 immunohistochemistry (200x). 123
msPMIP treatment results in a reduction of EGFR and Muc1 levels
To determine if msPMIP was affecting EGF-dependent degradation of EGFR in
vivo, protein lysates from tumors of MMTV- pyV mT animals injected with EGF and
peptide 30 minutes prior to animal sacrifice (after a standard 21 day drug treatment). A
striking reduction in the expression of EGFR and corresponding phosphotyrosine in the
msPMIP treated mouse compared to the control treated animal (Figure 5.7a). Note that
in this mouse, there were no frank tumors remaining after the 21 day msPMIP treatment
(remaining mammary fat pads were analyzed), while we obtained six tumors of greater
than 400mm3 from control treated animals. Analysis of EGFR expression and activation of the MDA-MB-231 xenograft tumors was restricted to tumors that were not treated with EGF. Nonetheless, a slight reduction in the levels of phospho-EGFR (Y1173) in hPMIP treated compared to control treated tumors was observed (Figure 5.8, top panel).
Tyrosine 1173 phosphorylation can result in the recruitment of shc to EGFR, resulting in activation of the MAP Kinase ERK. By examination of activated ERK, found that hPMIP treatment resulted in a strong downregulation of ERK activity compared to controls (Figure 5.8).
To determine if msPMIP blocked interaction between Muc1 and β-catenin, we began by establishing levels of Muc1 protein expression in the tumor lysates.
Interestingly, msPMIP treatment induced a loss of Muc1 protein in both the MMTV- pyV mT model (Figure 5.7a and b) and in the MDA-MB-231 xenograft model (Figure 5.7c and d). Total levels of β-catenin and its target genes cyclin D1 and Twist are unchanged
between PMIP and control (Data not shown). 124
Figure 5.7 PMIP is associated with reduced Muc1 expression. a) A representative MMTV- pyV mT msPMIP (P) treated mouse and a PTD4 mouse (C) were each injected 30 minutes prior to sacrifice with epidermal growth factor (1μg/g body weight) and with peptide. Following sacrifice the tumors were collected and protein lysates were generated. Protein (50μg) was separated by SDS-PAGE, transferred and immunoblotted for expression of phosphotyrosine (PY99), EGFR (1005), Muc1 (CT2), and β-actin (AC- 15). b) Lysates from MDA-MD-231 xenograft tumors (not treated with EGF; described in Figure 2) were similarly analyzed to determine levels of EGFR and MUC1 protein expression. Relative protein levels of Muc1 were measured by densitometry and graphed (Muc1/β-actin, *, p=0.014, ANOVA). Molecular weights are shown on the right. IB = immunoblot.
125
Figure 5.8 hPMIP treatment results in reduced levels of phosphorylated EGFR and dpERK. Protein lysates were prepared from the tumors described in Figure 5.4d. Protein (50μg) was separated by SDS-PAGE, transferred and immunoblotted for expression of 1173pEGFR (1173), EGFR (1005), dpERK (M-8159), ERK (9102), and β- actin (AC-15). White lines through blots indicate same gel and exposure but were non- contiguous.
PMIP Downregulates Met Expression
To better understand any possible alterations in downstream signaling and
transcriptional activation a whole genome cDNA microarray was utilized to determine
changes in transcripts following PMIP treatment. After extracting total RNA from BT20
cells treated with PMIP (10μM), PTD4 (10μM) or vehicle a cDNA reaction was
performed with fluorescently labeled nucleotides. The cDNA was then hybridized to a
Codelink Whole Genome microarray and the intensities were normalized with internal
controls. Several genes were differentially regulated after PMIP treatment compared to
controls (Table 5.1). Of interest, RT-PCR confirmed that the oncogenic hepatocyte
growth factor receptor (c-Met) c-Met was significantly downregulated greater than 3 fold
following PMIP treatment (Figure 5.9). 126
Table 5.1 Genes that are differentially regulated following PMIP treatment. BT20 cells were treated with PMIP (10μM) for 18hours and total RNA was extracted. RNA was processed into cDNA and was hybridized onto a Codelink Whole Genome microarray. These arrays were analyzed using Genespring. Genes downregulated (a) and upregulated (b) are listed in the table, with function and fold change compared to PTD4 and vehicle controls. 127
Figure 5.9 MET is significantly downregulated in response to PMIP. a) Microarray intensities from BT20 breast cancer cells treated with PMIP (10μM), PTD4 (10μM), or vehicle. b) RNA from PMIP, PTD4, and vehicle treated cells was extracted and used for RT-PCR against MET. RT-PCR was performed by Derrick Broka.
128
Discussion A MUC1 mimetic peptide (MIP) linked to a protein transduction domain (PTD4)
can freely enter transformed cells and inhibit their invasion in vitro. This same peptide
can inhibit primary tumor growth, tumor spread, and recurrence of tumors after resection
in an orthotopically implanted breast cancer model. PMIP can survive in circulation with
tissue specific retention and no detectable toxicity. Importantly, PMIP can
significantly inhibit tumor growth in a genetically-driven mouse model that mimics
human breast cancer. Mechanistically, this tumor-inhibitory effect is closely associated
with a decrease in EGFR and MUC1 expression. Together these data demonstrate that
this peptide-based intracellular drug shows strong efficacy as a non-toxic treatment for
breast cancer.
The over-expression of human MUC1 in mouse mammary glands promotes
transformation and the loss of MUC1 in several transgenic models can significantly delay
tumor onset [8, 98, 124]. This may be due to the number of oncogenic partners MUC1
has been demonstrated to interact with, namely β-catenin, src, and EGFR [Reviewed in
[103]]. This mimetic peptide is designed to block interactions between MUC1, β-catenin
and EGFR, and the study demonstrated that PMIP treatment does block MUC1
interactions with these proteins. In addition, a loss of MUC1 expression in response to
PMIP treatment under certain conditions, which may be the result of downregulation of
EGFR. While the mechanism of MUC1 loss is unknown, it would certainly result in a
loss of Muc1-dependent oncogenic signaling. It is tempting to speculate that in the
presence of PMIP, MUC1 and EGFR are alternatively trafficked and enter the lysosomal 129
degradation pathway, leading to their enhanced degradation. PMIP treatment of MDA-
MB-231 cells overexpressing MUC1 under a CMV promoter for only 3 hours in culture
induces a loss of MUC1 overexpression, indicating that the effect is not transcriptionally
regulated (data not shown). Loss of MUC1 in another study demonstrated a significant
decrease in activated ERK, which is a similar observation in PMIP treated tumors [99].
Importantly, PMIP treatment resulted in significant downregulation of the growth factor
receptor, MET. In non-small lung cancer EGFR and Met can act synergistically to
promote tumorigenesis, proliferation and invasion [208]. Therefore a loss of Met and
EGFR could be another mechanism of PMIP’s anti-tumor effect. However, future studies
will focus on determining the precise mechanism by which PMIP suppresses protein
expression and tumor progression.
One important observation is the lack of toxicity associated with PMIP treatment
(no weight loss, signs of distress or organ failure). While this is not an unexpected result with the use of an endogenous peptide, it points to a potentially low level of toxicity in patients. We have also examined the possibility that PMIP has activated an immune response in the immune-intact MMTV- pyV mT transgenic animals. Examining leukocytic infiltrates using protein levels of CD45 as a marker, there was increase in those tumors treated with PMIP versus control ([209]; data not shown). Additionally, studies by groups investigating the potential adjuvant activity of the protein transduction domain have found that the TAT protein transduction domain is not immunogenic [210].
One potential reason for PMIP’s lack of toxicity may be due to the relatively fast clearance of small peptides from the body [211]. Importantly, while PMIP-FITC 130
appeared to be cleared overall from the body 4 hours after injection, it was retained in
specific organ sites, including the mammary gland and colon. This site specific retention
may be due to an increase in peptide binding partners in these particular tissues.
Previously Pochampalli et al demonstrated that MUC1 inhibits the ligand-
dependent degradation of EGFR, resulting in enhanced receptor stability [87].
Furthermore, Pochampalli et al have demonstrated that this interaction promotes the
oncogenic properties of EGFR [8]. Neither of the mouse models used in these
experiments were previously shown to be dependent upon EGFR for progression, and
yet, PMIP is significantly effective in each model. This indicates that PMIP may have
broad applications against tumors overexpressing MUC1, which encompasses most
epithelial neoplasias [212]. In addition, PMIP may serve as an important adjuvant
therapy with anti-EGFR treatments [Reviewed in [213]]. Our data indicate that MUC1
induces the internalization, altered trafficking and enhanced signaling of EGFR [87].
Therefore, if PMIP blocks these interactions, anti-EGFR therapy that relies on surface-
localization of EGFR could by enhanced by PMIP co-delivery.
This study demonstrates the efficacy of PTD4-linked peptide-based drugs and the
value of MUC1-directed targets in breast cancer. Importantly, these data indicate that
PMIP is a potent drug that is active at all stages of tumor progression; inhibiting growth,
inducing regression and inhibiting metastatic spread.
131
VI. ANTI-CANCER THERAPIES THAT UTILIZE CELL PENETRATING PEPTIDES
Note: The review presented in this chapter has been published in [214].
INTRODUCTION
Significant progress has been made in the identification of tumor specific cellular pathways which can be successfully blocked in vitro. Unfortunately, the safe, systemic delivery of these blocking agents to patients has been difficult and sometimes impossible due to difficulty with tissue penetration, toxicity and functional activity in vivo. The recent development of cell penetrating peptides (CPPs) has overcome these barriers, leading to the development of novel tumor-specific molecular therapeutics. Also known as protein transduction domains (PTD), CPPs consist of short amino acid sequences that are able to mediate the translocation of a conjugated cargo across the plasma membrane.
Once inside the cell, CPP-associated cargos are then able to interact with their intracellular targets, and block their tumorigenic properties. Importantly, the non-toxic mechanism of cell penetration allows for the safe and effective systemic delivery of cancer therapeutics.
Over the last decade, several laboratories have utilized naturally occurring and synthetic CPPs to deliver several types of therapeutic cargo including proteins, antibodies, peptides, and nucleic acids directly into the cell’s cytoplasm [123, 155-157].
CPPs are easily manipulated and optimized, and can be used to target cargo to a specific 132
cell-type or tissue [215]. For example, one sub-group of CPPs can cross the blood-brain
barrier, allowing for the development of therapies that target brain cancers which are
normally difficult to treat with conventional chemotherapies [154, 216]. Importantly,
CPP bound therapies offer a novel approach to treating cancer in a specific and non-toxic
manner, as damage to the cell membrane, receptors or transporters are not required for
entry into the cell. This chapter will discuss naturally occurring and optimized protein
transduction domains and their utilization in the delivery of anti-cancer therapies into
intracellular compartments of diseased cells. Additionally, this chapter will focus on
studies with pending or recently awarded patents whose functionality has been verified
and published, and discuss the very promising future of these therapeutics for the
treatment of cancer.
Cell Penetrating Peptides
CPPs were first discovered through the study of model organisms such as
Drosophila melanogaster (Antennapedia, Antp) and the Human Immunodeficiency Virus
(HIV Tat) retrovirus, and there are now two fundamental types of CPPs that have been
utilized for intracellular delivery of therapeutic molecules [153, 154]. The first and most
common are the cationic CPPs, which are short amino acid sequences that are mainly
composed of arginine, lysine, and histidine. These amino acids are responsible for the
cationic charge of the peptide that will mediate the interaction of the peptide with anionic
motifs on the plasma membrane. Both naturally occurring and synthetic cationic CPPs 133
have been described and are shown in Table 1. A second class of CPPs is the
amphipathic peptides, which have lipophilic and hydrophilic tails that are responsible for
mediating the peptide translocation across the plasma membrane Table 1. Although these
classes of CPPs are composed of different amino acids, the mechanism of intracellular
delivery is similar.
134
Type of Name Amino Acid Sequence CPP Ref
1 HIV Tat (47-58) YGRKKRRQRRR Cationic [154] 1a PTD-3 YARKARRQARR Cationic [202] 1b PTD-4 YARAAARQARA Cationic [202] 1c PTD-5 YARAARRAARR Cationic [202] 1d PTD-6 YARAARRAARA Cationic [202] 1e P3 YGRKKRRQR Cationic [217]
1f Polyarginine Rn Cationic [218]
Antennapedia 2 (Antp) RQIKIWFQNRRMKWKK Cationic [153]
DAATATRGRSAASRPTERPRAPAR- VP-22 3 SASRPRRPVD Cationic [219]
4 PTD-SN1 RKMLKSTRRQRR Cationic [220]
5 Bac RRIRPRPPRLPRPRPRLPFPRPG Cationic [221]
6 Transportan GWTLNSAGYLLGKINLKALAALAKKIL Amphipathic [222]
7 Pep-1 KETWWETWWTEWSQPKKKRKV Amphipathic [223]
Table 6.1. Commonly Utilized Cationic and Amphipathic Cell Penetrating Peptides. More specifically, this chapter will discuss recent patents that utilize CPPs 1, 2, 4 and 6. However, the remaining CPPs are also relevant to the field of peptide-based therapies. 135
Mechanism of Cell Penetrating Peptides The detailed mechanism of action by which CPPs transverse the plasma
membrane and interact with cytosolic proteins was recently reviewed, and readers are
encouraged to consult those publications for the details summarized here [224, 225].
Briefly, CPPs are composed of several cationic amino acids such as lysine or arginine,
and a group of these amino acids result in a highly positively charged peptide sequence.
These positively charged sequences are able to interact with acidic motifs on the plasma
membrane in a receptor-independent fashion. Acidic motifs can be several different
substrates found on the plasma membrane including proteoglycans and glycolipids [226,
227]. A recent patent has introduced the use of synthetic CPP, PTD-SN1, to improve the
efficiency of this interaction and ultimately the peptide’s cytosolic translocation [220].
Following interaction with these substrates, the peptides undergo cell-type independent
internalization [228]. The exact mechanism of this internalization process is yet to be
elucidated but it is dependent on several peptide attributes including type of CPP and the
size and charge of the cargo. A recent study has indicated that Antp and Tat CPP
internalization can be mediated by clathrin-dependent endocytosis, caveolin-dependent
endocytosis, macropinocytosis, or direct intracellular translocation [226] (Figure 6.1).
Another report used therapeutic cargo to alter the rate of endocytosis, which allowed the
therapy to act in an extracellular fashion [229]. Following endosomal internalization, the
CPP-tagged therapy (CTT) escapes lysosomal degradation by a process that is poorly
understood. It is known however, that endosomal escape is the rate-limiting step in
delivering therapeutic doses of CTTs to the cytosol [224]. Once the CTT is released into 136
the cytosol, it is able to directly interact with its target. In vitro, a CPP (Tat) linked
therapy has been demonstrated to enter the cell and start affecting their target within five
minutes of treatment [230]. In a mouse tumor model, a GFP-CTT (Tat) was observed to
be localized at the tumor site within 30 minutes of treatment, thus illustrating CPPs’ uptake speed and efficiency [123].
137
Figure 6.1 Mechanism of Peptide Membrane Transduction. 1) CPP-Cargo peptide is delivered to site of disease. 2) The CPP’s highly cationic amino acids interact with proteoglycans (left) or glycolipids (right) on the cell surface. Note: There is evidence that the CPP-cargo directly enters the cell (center arrow) 3) The peptide is endocytosed via clathrin or caveoloae or it is macropinocytosed. 4) Endosomal escape of peptide. 5) Interaction with target protein.
138
Non-Immunogenic
One of the benefits of using CPPs for therapeutic delivery is the lack of toxicity
compared to other cytoplasmic delivery devices, such as liposomes, etc. One potential
drawback to using exogenous peptides, though, is the ability to induce a humoral immune
response against the therapy, which can be highly detrimental for subsequent treatments.
A commonly used CPP derived from HIV’s Transcriptional Transactivator (Tat) was
investigated for immunogenicity, and the portions of the Tat protein involved in eliciting
a humoral immune response were determined. The authors discovered that the portion of
Tat that conveys protein transduction, PTD, failed to induce an immune response [210].
In addition, our group has evaluated the induction of an immune response following
repeated PTD-4 injection in immune intact mice, and observed no significant change in
CD45-positive lymphocyte infiltration into tumors [123]. These studies indicate that the
use of Tat’s PTD to deliver anti-cancer therapy does not induce a measurable immune
response, although long-term toxicity studies in patients have not yet been performed.
With respect to another PTD, Antp, several studies have determined that Antp efficiently
elicits an immune response to the peptide cargo following processing by an antigen presenting cell [231, 232]. These examples demonstrate the possibility of modulating the immune response to the CTT based on the PTD used to deliver the therapeutic peptide.
Anti-Cancer Cell Penetrating Peptides
Since 1988, when the first protein transduction domains were described, the use of CPPs to deliver anti-cancer therapies (CTTs) to the cytosol and nuclear compartments has become more common. Over the last decade numerous reports and patents have 139
been published demonstrating the potential of CTTs for the treatment of leukemia and
breast, lung, pancreatic, ovarian, colon cancers [123, 155, 159, 217, 229, 233-235]. With respect to anti-cancer therapies, CTTs have been used to inhibit oncoprotein nuclear translocation, modulate oncoprotein signaling, and block metastases-promoting cell-ECM interactions (Figure 6.2) [123, 217, 229, 233]. The mechanism of inhibition is similar in most cases: a target interaction is determined and a portion or the whole interacting protein is attached to a CPP. The CTT is then transduced across the plasma membrane and delivered into the cytosol or nucleus to block the endogenous pro-cancerous interactions from occurring.
140
Figure 6.2 Mechanism of CTT Anti-Cancer Therapy. 1) The CPP portion of the CTT attaches to the plasma membrane and directly inhibits ECM Receptor/ECM interaction. This inhibits the ability of metastatic cancer cells to establish secondary tumor sites. 2) CTTs block cancer dependent protein-protein interactions, resulting in the inhibition of proliferation, angiogenesis, and chemoresistance. 3) Additionally, inhibition of protein interactions can modulate nuclear translocation of oncogenic transcription factors. 141
Nuclear Translocation
Protein trafficking is a common cellular mechanism that regulates signal
transduction and transcriptional activation. Following oncoprotein signaling, the
translocation of activated proteins into the nucleus often results in the transcription of
genes that are responsible for conveying many of the hallmark of cancers [66]. Increases in oncoprotein nuclear translocation are often observed in cancer cells and can further promote transformation and metastases.
Following DNA damage, there are a number of cell-cycle inhibitor proteins that become activated and are translocated to the nucleus. For example, in normal cells, DNA damage induces p21 nuclear translocation and cell cycle arrest, however, the extent of
DNA damage determines whether the cell undergoes caspase-dependent apoptosis or is able to repair its DNA [236, 237]. Current anti-cancer modalities such as radiation and
most chemotherapies cause irreparable DNA aberrations, which induces the cell to
undergo apoptosis. However, these treatments can also result in the generation of a sub-
population of cancer cells that aberrantly upregulate p21, which results in the inhibition
of caspase 3-dependent apoptosis, thereby allowing the cells to survive the treatment
[158, 159]. Blocking p21 nuclear translocation could inhibit the ability of p21 to convey
chemoresistance, resulting in sensitizing cancer cells and allowing for a reduction in
chemotherapy dosages. In an effort to sensitize cancer cells to the DNA damaging
effects of radiation and chemotherapy, a recent study utilized a CPP conjugated to an
anti-p21 antibody. The CPP-anti-p21 effectively blocked p21 translocation, allowing 142
cells to progress through the cell cycle following chemotherapeutic exposure and
replicate severe DNA adducts, inducing apoptosis [233]. These authors demonstrated
that an IgG region could be conjugated to Tat’s PTD, (immunoconjugates; ICs) and the
tat-anti-p21 IC was shown to inhibit the translocation of p21 into the nucleus [233].
Inhibition of nuclear trafficking of p21 resulted in the loss of the p21 dependent anti- apoptotic effect, and sensitized breast cancer cells to both γ-radiation and Camptothecin, a Topoisomerase inhibitor [233]. This study offers an example of how the tat-anti-p21
IC, in combination with chemotherapies, could result in lower and more effective dosages as well as less discomfort for the patient. The use of CPPs to deliver antibodies directly into the cytosol and nucleus of cancer cells has tremendous potential, allowing for the intracellular targeting of cancer-specific protein mutations.
Aberrant nuclear translocation of proteins is also observed when cells are exposed to pro-inflammatory cytokines, an event frequently observed in epithelial cancers
[Reviewed in [238]]. A protein commonly activated and translocated to the nucleus in the presence of these cytokines is the transcription factor, NF-κB /p65. NF-κB is normally found in the cytosol as a complex of several different proteins (p50, p65, and
IκBα) and following an inflammatory signal, the inhibitor IκBα dissociates from the complex. Following dissociation, p65 is serine phosphorylated and translocated to the nucleus where it activates transcription of several pro-cancerous genes such as, cyclin-
D1, MMP-9 and COX-2 [239]. To inhibit the nuclear translocation of p65, a peptide was designed to block its serine phosphorylation sites. A recently approved patent utilized a p65 derived peptide conjugated with Antp (CPP) to inhibit p65 from activating 143
transcription [240]. The peptide, PTD-p65-1, mimics the amino acid sequence of the p65 protein that spans two serine residues essential to nuclear translocation. In KBM-5 leukemia cells, treatment with PTD-p65-1 was shown to inhibit p65 phosphorylation, p65 nuclear translocation and p50-p65 DNA binding. These PTD-p65-1 treated leukemia cells also demonstrated an increase in the cytotoxic sensitivity following exposure to a chemotherapeutic agent, doxorubicin. Additionally, PTD-p65-1 was able to block the p65-dependent transcription of cyclin-D1, MMP-9, and COX-2 [159]. This indicates that peptides like the PTD-p65-1 can be utilized to sensitize chemotherapy resistant cancer cells to apoptosis or be used to treat chronic inflammation.
Cancer cells often have deregulation of protein expression due to a variety of reasons, including gene amplification, epigenetic alterations, aberrant signaling or atypical protein translation. The altered protein expression of the eukaryotic initiation factor 4E (eIF4E) has been described to increase the translation of mRNAs with a long 5’ untranslated regions [241]. Increased eIF4E has been shown to be directly responsible for the altered protein expression of several oncogenes such as cyclin D1, VEGF, and
FGF2. Additionally, the overexpression of eIF4E has recently been described as an indicator of breast cancer recurrence [242].
eIF4E functions by entering the nucleus, interacting with mRNA, and assist in presenting the mRNA to the ribosome for translation. The nuclear translocation of eIF4E is vital for its function and blocking nuclear entry can promote cytoplasmic accumulation and apoptosis. A recent patent discusses the use of a CPP attached to a portion of the 144
potato virus Y VPg protein to inhibit eIF4E nuclear translocation [243]. In Drosophila
the authors have previously demonstrated that the VPg protein directly binds to eIF4e and
inhibits cell proliferation. The conserved nature of imitation factors allowed a similar
approach in mammalian cells. The authors demonstrated that treatment with CPP-VPg
inhibited eIF4E nuclear translocation, loss of cell proliferation and cell death [243].
Nuclear translocation also plays an important role in signal transduction,
transmitting signals from membrane-bound receptors to the nucleus. The erbB growth
factor receptor family (erbB1/EGFR, erbB2/Her2, erbB3, and erbB4) has been implicated
in the progression of several cancer types including breast, lung, gastrointestinal stromal,
and prostate [Reviewed in [244]]. For example, erbB2 is a common therapeutic target
that is amplified and overexpressed in approximately 30% of all breast cancers [245].
One of the downstream signaling events following erbB2 activation is the tyrosine
phosphorylation, dimerization, and nuclear trafficking of the signal transducers and
activators of transcription 3 (STAT3) transcription factor. STAT3 is been found
deregulated in most solid tumors (breast, ovarian, pancreatic, prostate, and melanoma)
and hematological diseases (lymphoma and leukemia) [246-251]. Following STAT3
nuclear translocation, it activates the transcription of several oncogenes that play a role in
conveying a chemoresistant phenotype, such as VEGF, Bcl-2, CyclinD1, and MMP9
[252]. Therefore, nuclear translocation of STAT3 mediated by ErbB2 is an attractive
therapeutic target. 145
In 2006, a report was published that demonstrated that the CPP Tat could be
manipulated to specifically target breast cancer cells that overexpress erbB2/Her2, by
targeting cells with an erbB2 binding peptide conjugated to Tat (P3-AHNP). By
concentrating the P3-AHNP peptide to the cells that were erbB2-positive, the authors
reported that cells overexpressing erbB2 preferentially absorbed the peptide [217]. P3-
AHNP was then additionally conjugated to a STAT3 binding peptide, STAT3BP,
resulting in a CTT that could specifically localize to erbB2 overexpressing breast cancer
cells and block STAT3 nuclear translocation, by blocking STAT3 dimerization [217].
Importantly, P3-AHNP-STAT3BP inhibited STAT3-DNA interactions and its resulting
oncogenic transcriptional activity. A significant decrease in proliferation and an increase
in apoptosis were observed in P3-AHNP-STAT3BP treated breast cancer cell lines and
xenograft tumors [217]. This is a novel example of how the protein expression profile of certain cancers can lead to the development of CTTs that specifically target certain cell- types.
Oncogenic Signaling Modulation
Oncogenic signaling as a result of cancer-dependent protein-protein interactions can promote many aspects of cancer progression, including cell cycle progression, inhibition of apoptosis and insensitivity to anti-growth signals. In addition, changes in
cellular polarity can result in novel protein-protein interactions that are present in cancer
cells, but do not occur in polarized epithelium due to junctional separations. In some
instances these cancer-dependent protein-protein interactions result in aberrant signaling 146
and trafficking. These interactions and signaling events thus provide potential targets for a cancer-specific therapy.
Our laboratory has focused on targeting those interactions that occur in tumor cells, but not in normal polarized epithelium. Basolateral proteins, such as the Epidermal
Growth Factor Receptor (erbB1/EGFR), and apical proteins, such as MUC1 are separated when epithelial cells maintain junctional formations. Loss of polarization in tumor cells, and in metastatic cancer specifically, results in novel protein-protein interactions that promote neoplasia. In the case of MUC1 and EGFR, interactions result in altered protein trafficking and oncogenic signal transduction events that promote breast cancer progression [87]. EGFR is a potent driver of tumor growth and when it interacts with
MUC1, EGFR signaling is amplified [87]. Targeting the interaction domains between these proteins, demonstrated that a CTT, PMIP, blocks MUC1 and EGFR interactions resulting in significantly slower tumor growth [253]. PMIP was designed from a small portion of the MUC1 cytoplasmic tail and conjugated with the CPP, PTD4 [202]. In in vitro studies with breast cancer cell lines, PMIP’s inhibitory effect corresponded to a significant decrease in invasion and proliferation. In the xenograft mouse model, PMIP treatment of the aggressive MDA-MB-231 breast cancer cell line resulted in slower growth and a reduction in distant metastases. Additionally, PMIP dramatically slowed tumor growth and in several instances caused regression of solids tumors in the genetically-driven mouse mammary tumor model (MMTV- py MT). Again, as others have observed, PMIP treated mice showed no weight loss or gross tissue damage, indicating a lack of toxic side-effects [123]. 147
Immune response modulation against cancer has been the main focus of many
cancer researchers because the concept that the body’s natural defense could target and
eliminate tumor cells is an ideal therapeutic approach. Though there are many drawbacks
to the immune modulation approach mainly autoimmunity. However, a recent patent has
utilized a peptide to promote potential tumors antigens to the cell surface of dying tumor
cells which elicits an anti-tumor response. The underlying concept of this patent is based
on the finding that following anthracyclin (common type of chemotherapy, a DNA
intercalating agent) the proteins calreticulin (CRT), KDEL receptor, and ERp57 are
found at the plasma membrane. The membrane localization of these protein results in an
immune response against these tumor antigens. However, this membrane localization is
inefficient in dying cancer cells so a peptide was designed to promote membrane
localization of these proteins. The authors found that inhibition of a protein phosphatase,
GADD34, with a CTT (DPT-PP1 inhibitor) in colon and cervical cancer cells lines
resulted in increased membrane localization of CRT, KDEL receptor, and ERp57.
Additionally, this elicited a greater immune response compared to peptide controls or no
treatment [254]. This a novel use of a CTT to alter intracellular pathways that play an
important role in extracellular processes, such as an immune response.
Most anti-cancer therapies used in the clinic take advantage of the replicative
nature of cancer cells by disrupting DNA replication and synthesis. Radiotherapy (IR) is
often used to cause double-strand breaks in proliferating tumor cells, thereby promoting
apoptosis. However, following radiation exposure the cell has an innate signaling
mechanism to arrest the cell cycle to allow for DNA repair mechanism. For example, 148
upon IR exposure the ataxia telangiectasia mutant (ATM) is activated through the
interaction with another protein, Nijmegen breakage syndrome 1 (NSB-1), and this
activation leads to downstream signaling events which result in cell cycle arrest [255].
The cell cycle arrest allows for DNA repair, which can promote the viability of the cell
and inhibition of apoptosis. Although in normal cells this mechanism is important in
preventing genomic instability however in cancer cells this pathway assists tumor cells in
becoming radioinsensitive. A recent patent describes the use of a peptide based approach
to inhibit the NSB-1/ATM interaction to sensitize tumor cells to IR [256]. The peptide
designed utilized the polyarginine (R9) PTD attached to a NSB-1 mimetic peptide
(wtNIP). A study found that treatment of cervical and prostate cancer cells with wtNIP
resulted in efficient cellular uptake and limited cytotoxicity. However, the authors found
that wtNIP treatment following radiation was able to sensitize these cells and increase
cell death compared to peptide controls [257].
The drawback to the previous approach would be the non-specific nature of the
CTT. In contrast, another patent has utilized a peptide-based therapy to specifically
target the increased availability of single-stranded DNA (ssDNA) in cancer stem cells. In
2006, a report was published demonstrating the presence of “bell-shaped” nuclei in
colorectal tumors and upon closer investigation the authors found this population of cells
contained stem cell like properties [258]. The authors reported that “bell-shaped” nuclei
undergo symmetrical and asymmetrical nuclear fission and this phenomenon results in
increased availability of ssDNA. This led to the design of a peptide that could enter cells,
bind ssDNA, and promote apoptosis of the cancer stem cells. This patent however, 149
mentions the use of CPP to deliver the peptide but does not provide a specific CPP [259].
This a novel therapeutic approach which takes advantage of increased ssDNA to
specifically target cancer stem cells for apoptosis.
Other laboratories have targeted protein-protein interactions that are important in
anti-apoptotic signaling. An important step that leads to the inhibition of apoptosis in
cancer cells is the increased signaling and stabilization of inhibitors of apoptosis proteins
(IAP). One protein that has been found to stabilize IAPs is heat-shock protein 90
(Hsp90). Hsp90 is commonly deregulated in cancer and has been shown to assist the cell
in adapting to environmental stresses [260]. Importantly, Hsp90 is able to directly
interact with an IAP, survivin, and inhibit its proteosomal mediated degradation.
Recently approved patents propose the use of CTTs that specifically targets the
interaction between Hsp90 and survivin [261, 262]. The peptide, shepherdin, was designed to mimic a small domain of the survivin protein that interacts with the ATPase domain of Hsp90 and was then conjugated with the CPPs Antp or Tat. The authors demonstrated that in vitro treatment of several cancer cell lines (breast, prostate, colon, and cervical) with shepherdin caused caspase-dependent apoptosis and loss of membrane integrity [263]. In addition, shepherdin treated breast and prostate tumors grown superficially in immunocompromised mice showed slower tumor growth versus controls and no toxic side-effects were observed. Notably, this report revealed that shepherdin effects were cancer cell specific and did not have any effect on normal fibroblast [263].
Therefore, shepherdin demonstrates the inhibition of protein-protein interactions that 150
results in the non-toxic targeting of tumors via the sensitization of cells to apoptotic
signals.
The use of Hsp90 as a therapeutic peptide target has not been limited to IAPs.
Another recent patent application describes the use of an Hsp90 peptide to promote the
degradation of eNOS [264]. Activation of endothelial nitric-oxide synthase (eNOS) by
Hsp90 results in the increased generation of nitric oxide (NO) which allows the cancer cell to become insensitive to hypoxic and stress conditions. In this manner, activation of eNOS by Hsp90 is able to convey an anti-apoptotic response in several cancers, including oral squamous cell, colon, breast and prostate [265-268]. Therefore the interaction of
Hsp90 and eNOS provides a potential anti-cancer target. An Hsp90 directed CTT was generated, and treatment of endothelial cells resulted in a significant decrease in nitric-
oxide production [269]. The loss of nitric-oxide production has been observed to slow
the proliferation of a malignant melanoma cell line [270]. Thus, the targeting of Hsp90
may be useful in the treatment of multiple cancer types and further demonstrates that the
blocking of cancer-specific protein-protein interactions may be a viable therapeutic
approach.
Modulation of ECM Interactions
Aberrant interactions between tumor cells and the extracellular matrix is an
important component of tumor progression. The ability of tumor cells to form productive
interactions with components of the ECM is critical for movement from the primary 151
organ to distant sites. For example, cells from the primary tumor of the breast
disassociate from the tumor mass and utilize the surrounding ECM to migrate towards
blood and lymph vessels where they intravasate and are transported throughout the body,
eventually forming secondary tumor sites in many tissues, including liver, bone, and
brain [Reviewed in [271]]. This metastatic process is similar in most solid tumors and
demonstrates the critical role of the ECM interactions with the tumor in metastatic
progression [Reviewed in [272]]. A recent study has utilized the Tat derived CPP linked
with an elastin-like polypeptide (Tat-ELP) to inhibit ovarian cancer invasion, migration,
and metastases [229]. Elastin is an extracellular matrix protein commonly found in
connective tissue throughout the body that conveys elasticity to tissues such as blood
vessels, lungs, skin, and the bladder. This CTT was designed to block the interaction
between elastin and circulating tumor cells, resulting in the loss of secondary tumor site
establishment. The concept presented in this report is unique in the sense that the authors
attached a CPP to a large elastin-like polypeptide (800aa), which resulted in Tat-ELP
binding to the cell surface followed by a more gradual endocytosis [229]. In this way,
Tat-ELP is attached to the cell membrane and is functioning to inhibit ECM interactions
prior to internalization. Treatment of ovarian cancer cells with Tat-ELP resulted in an
inhibition of the circulating tumor cells from attaching and establishing secondary tumor
sites [229]. The use of Tat to bind a therapy to the cell surface is a novel approach to
inhibiting ECM Receptor/ECM interactions that could have potential in treating pre-
metastatic and metastatic disease.
152
Novel Therapeutic Targets
The complexity of cancer biology and our understanding of mechanisms
underlying cancer development are ever growing. As new areas of cancer research are
being explored the discovery of new therapeutic targets has been uncovered. Recently,
several patents applications have been submitted which plan on utilizing CPPs to deliver
anti-cancer therapies directed against these novel targets. For example, the role of
microRNA (miRNA) in cancer formation and progression is a relatively new field in
cancer biology so a recent patent describes a miRNA processing protein as a potential
therapeutic target [273]. Another patent plans to utilize CPPs to target a new area of cancer biology, epigenetics. Specifically, this patent describes the use of a CPP to deliver a antisense nucleic acid to block the translation of a splice variant of DNA methyltransferase 3B, which has been shown to promote hypermethylation and gene silencing in cancer [274-276].
In this chapter, I have examined the use of CTTs to target cancer in a manner that is non-toxic to normal cells. In vitro and in vivo CTTs have been shown to translocate across the plasma membrane, directly interact with their target in the cytosol or nucleus, and promote cancer cell death. Additionally, CTTs have also been used to induce sensitivities to chemotherapies and radiation. CTTs have shown to be effective in many cancer types, and while the shift from the laboratory to the clinic has been slow, recent progress in should promote this translation. 153
Endosomal Escape
As more CTTs are being developed and tested in the clinic setting, there is a great
need for a better understanding of how these peptides escape the endosomal
compartments, which limit their therapeutic potential. The ability to optimize endosomal escape would allow for higher therapeutic concentrations in the cytoplasm, resulting in an increase in the CTT/target interaction. However, several of the studies examined in this reviewed show a significant amount of CTT in the tumor several hours after injection.
One possible explanation for this phenomenon is the increased bio-availability of the target found within tumor cells. The CTT fails to be degraded because it is interacting with its target [277].
CPP Stability and Modifications
One concern with using peptides as therapeutics is the potential effects of serum proteases on peptide degradation prior to cellular internalization. A potential modification to CTTs could involve incubation with SDS or polysialic acid, which has been shown to increase peptide stability and diffusion throughout the body [231].
Inhibiting serum protease activity in the blood would also assist in maintaining a higher concentration of therapeutic peptide. Although few studies have attempted this, Yi et al. discovered that treatment of human serum samples with heparin or citrate could stabilize the serum peptides [278]. Another approach to stabilize peptides in the circulatory system is the manipulation of the amino-acid isomerization, which could have a dramatic effect on peptide bioavailability and potency. For example, the isomerization of an L- 154
isoleucine to a D-isoleucine in an anti-angiogenic thrombospondin-1 peptide (Mal II D-
Ile) resulted in better pharmocodynamic properties [279]. The proposed mechanism of
this result was the limited protease activity towards the D-amino acid, thereby increasing
the circulatory concentration of the peptide. Another biological characteristic of D-amino
acids is that processing and presentation to the immune system of D-amino acid peptide
is inefficient thereby limiting an immune response [280]. A recent patent describes the
use of a CPP containing D-amino acid to deliver a targeted anti-cancer therapy to
leukocytes and endothelial cells [281]. In addition to amino acid isomerization, cyclic
peptides that contain a disulfide or amide bonds show an increased therapeutic advantage
versus linear peptides [282]. These studies indicate that the amino acids of CTTs can be
manipulated to optimize peptide bioavailability and efficacy.
Another potential drawback of CTTs is that most of the time the CTT is a low
molecular weight peptide, which results in efficient renal clearance, and poor tumor
penetration. To address this issue one study utilized a hydrophilic polyethylene glycol
coat to act as an efficient “macromolecular drug carrier” [283]. More recently the use of
nanoparticles to delivery macromolecules drugs such as, peptides and genes, offers a potential solution to the drawbacks of just treating with a CTT alone [284]. Theoretically
these carriers would function by forming a “capsule” around the therapy, which would
able to deliver relatively larger doses of CTT through mucosal barriers to target tissues.
In conclusion, the use of CTTs to treat cancer is a relatively new field and there are still many areas that need to be explored. However CTTs show great promise in the 155
field of anti-cancer therapy because of their many benefits including low toxicity,
minimal modulation of immune system, tissue specificity, ability to target most pro- cancerous interactions and regulation of oncogenic signal transduction. The therapeutic
approaches/patents discussed in this chapter are only a snapshot of all new CTTs that have been reported, but it provides a glimpse of the potential these peptides have in the fight against cancer.
156
VII. CONCLUDING STATEMENTS
There are approximately 200,000 new patients diagnosed with breast cancer each
year and over 40,000 will succumb to the disease. Stage and subtype are major determinants of patient prognosis and survival. Breast cancers that are classified into the
basal-like subtype tend to more aggressive and metastatic, which results in decreased
patient survival. Basal-like breast cancers are often described based on their genetic and
proteomic profile. Commonly, these types of breast cancer are estrogen receptor,
progesterone receptor, and erbB2 negative, which means that therapies designed to target
these proteins are ineffective. However, there are several proteins that are highly
upregulated in basal-like breast cancer and these include EGFR and basal epithelium
cytokeratins.
Normally, EGFR expression and signaling is regulated by endocytosis and trafficking mechanisms, but in cancer, mutations in these pathways can result in aberrant
EGFR activity and localization. Loss of polarity in epithelial cells will promote the
mislocalization of EGFR and will result in novel EGFR-protein interactions. For
example, EGFR interacts with another oncogene, MUC1, in a cancer dependent fashion and this interaction is facilitated by the loss of spatial organization. Additionally,
EGFR’s interaction with an adheren junction protein, β-catenin, will promote dissociation
of cell-cell junctions and ultimately loss of cell polarity. 157
The role of MUC1/EGFR and MUC1/β-catenin cancer-dependent protein-protein
interactions in tumorigenesis was established by examining the loss of Muc1 in an EGFR
and an β-catenin-dependent mouse models of breast cancer. In both of these models, the
loss of MUC1 resulted in a significant delay in tumor onset. Mechanistically, in the
EGFR-dependent tumor model cyclin D1 protein expression with directly correlated with
MUC1’s expression status. Previously, our laboratory has observed that the presence of
MUC1 promotes EGFR internalization and recycling. The body of work presented here
demonstrates that the presence of MUC1 promotes EGFR nuclear localization and
mediates EGFR-dependent transcriptional activity. Specifically, we discovered that
MUC1’s interaction with EGFR within the nucleus mediated the EGFR’s interaction with
chromatin and the cyclin-D1 promoter. Upon the loss of MUC1, we found a significant
decrease in cyclin-D1 protein expression. Therefore to inhibit MUC1/EGFR and
MUC1/β-catenin interactions we developed a CPP-mimetic peptide, which mimics a
portion of the MUC1 CT (PMIP). We discovered that the CPP (PTD4) promoted the
efficient uptake of PMIP into breast cancer cells and blocked the target interactions. In
addition, blocking these interactions resulted in a significant decrease of EGFR levels
following EGF treatment. We also observed that by blocking EGFR/MUC1 interactions
in in vitro models of breast cancer we could inhibit EGFR nuclear translocation,
proliferation, and invasion. Importantly, the treatment of PMIP in a xenograft and a
transgenic model of breast cancer significantly inhibited primary tumor growth and the
establishment of secondary tumors. In some cases PMIP was observed to promote the
full regression of mammary gland tumors. This result indicates that although EGFR’s 158
signaling pathways are important in tumorigenesis, that MUC1 and EGFR’s oncogenic
activity could be dependent on each other (Figure 7.3).
MUC1 and EGFR’S Role In Autophagy
The role of autophagy of cancer is a relatively new field and the process of
autophagy can promote cancer cell survival or death. The autophagic outcome is highly
dependent on the extent of cell stress and nutrient bioavailability. Autophagy occurs
when starving cells redistribute nutrients to vital cellular machinery thereby promoting
cell survival in a nutrient depleted environment. In 2009, a new MUC1-dependent cell
survival role was described in which the overexpression of MUC1 promoted ATP
production and cell survival in a low glucose microenvironment. The mechanism
driving this phenotype was determined to be MUC1’s promotion of autophagy in starving
cancer cells [285]. In contrast, the overexpression of a kinase dead EGFR mutant
inhibited autophagy and promoted cell survival by stabilizing a sodium/glucose
transporter (SGLT1)[79]. Stabilization of SGLT1 promoted high levels of intracellular
glucose which prevented the cell from undergoing autophagic death. These studies
demonstrate that both MUC1 and EGFR could be utilized as an autophagic target to
promote cytotoxicity in starving cancer cells.
159
Targeting MUC1/EGFR Interaction and EGFR Activity In Combination
Recently, aberrant EGFR localization within the cell has been associated with
increased chemotherapeutic resistance. Additionally, overexpression of a kinase-dead
mutant of EGFR inhibited autophagic cancer cell death following treatment with a
cytotoxic agent [79]. However, EGFR tyrosine inhibitors do effectively block
downstream signaling and by blocking EGFR/MUC1 interactions we observed a
significant decrease in total EGFR levels. PMIP treatment of breast cancer cells also resulted in the loss of nuclear EGFR localization (Figure 3.3). Therefore we
hypothesized that co-treatment of breast cancer cells with a tyrosine kinase inhibitor and
PMIP could have synergistic affects. The basis of this hypothesis is that by inhibiting
MUC1/EGFR interactions with PMIP we are promoting EGFR degradation, inhibiting
nuclear EGFR function and that by inhibiting the kinase activity of remaining EGFR we
could further exacerbate the anti-tumor effects. Initial experiments using an EGFR
specific kinase-inhibitor (AG1478) and PMIP have resulted in a significant inhibition of
proliferation compared to the single agents (Figure 7.1). This demonstrates that targeting
EGFR kinase activity should not be the sole focus of future anti-EGFR therapeutic. In
the future, targeting EGFR’s aberrant localization in conjunction with EGFR-dependent
downstream signaling pathways (eg. PI3K/Akt) could result in greater anti-tumor
response in basal-like breast cancers.
160
Figure 7.1. EGFR Tyrosine kinase inhibitor (TKI) and PMIP combination significantly inhibit proliferation. An MTT assay using BT20 breast cancer cells treated with a combination of 100nM EGFR TKI (AG1478) and 20μM PMIP for 7 days demonstrated a significant inhibition of proliferation (61% compared to single agent alone (PMIP, 31% and AG1478, 40%)). Student t-test was performed by comparing treatments versus control peptide (CP). MTT assay was performed by Aarthi Goverdhan. 161
EGFR As A Biomarker
The use of EGFR as anti-cancer target is widely accepted, but the use of EGFR
expression in breast cancer diagnosis is still unclear. Overexpression of EGFR in the
basal-like subtype is a poor predictor of clinical outcome, but detection of EGFR expression is not standardize so its use as biomarkers has been limited. However, the
work presented here indicates that although EGFR expression is important the cellular localization of EGFR is also important. The presence of nuclear EGFR has ready been determined to be a poor prognostic indicator in ovarian and breast cancer [44, 46, 47].
Additionally, the mislocalization of MUC1 also is indicative of clinical outcome, the
more cytosolic MUC1 directly correlates with a poorer prognosis [122]. In the future of
breast cancer diagnosis examining the expression and localization of MUC1 with respect to EGFR could be a powerful biomarker.
Preventing Loss of Cell Polarity
This work again re-establishes the importance of MUC1/EGFR and MUC1/β-
catenin interactions, but the underlying fact that is often overlooked is that these
interactions are highly dependent on the loss of cellular polarity. Maintenance of cellular
polarity is an important “tumor suppressor” and breakdown of this cellular organization
promotes novel oncogenic protein-protein interactions. Although this step is critical in
tissue transformation, the mechanism of the loss of cell polarity is poorly understood. In
addition, there has been evidence that EGFR and β-catenin are playing roles in the loss of 162
cell polarity [85, 86]. Given MUC1’s ability to promote EGFR and β-catenin
tumorigenesis, MUC1 overexpression could also be playing a significant role in driving
the loss of polarity. To determine MUC1’s role in the loss of polarity ongoing studies in
our laboratory are examining the overexpression of an apical versus a basolateral
targeting MUC1. These studies will yield important insight into the role of MUC1 in the
early transformation events in polarized epithelial cells.
Role of Heat Shock Proteins In EGFR And MUC1-Dependent Tumorigenesis
Although MUC1’s role in early cancer progression has yet to be established,
MUC1’s interactions with other proteins in cancer cells is now known to promote
proliferation, invasion and chemoresistance. Hsp70/90 have both been determined to
independently interact with EGFR and MUC1. Heat shock proteins 70 and 90
(Hsp70/90) assist in the refolding of proteins, prevent protein aggregation, and are vital in
antigen presentation. Hsp70 interaction with EGFR prevents its aggregation in the
cytosol following Sec61-dependent retrotranslocation from the endoplasmic reticulum
[56]. Hsp70/90 interact with a phosphorylated tyrosine on MUC1’s CT, which promotes
MUC1 mitochondrial localization resulting in the inhibition of the intrinsic apoptosis
pathway [129]. Recently in our laboratory, the interaction of Hsp70 and EGFR has been
determined to be MUC1-dependent [286]. Additionally, the deacetylation of the N-
terminus of proteins has recently been described to inhibit lysosomal degradation [287].
Aberrant endosomal trafficking of EGFR has been observed to be perturbed in the 163
presence of the interacting heat shock protein, Histone Deacetylase-6 (HDAC-6). EGFR
interaction with heat shock proteins and HDAC6 resulted in a significant reduction of
acetylated EGFR and its lysosomal trafficking [288]. HDAC6 regulates this process by
binding to heat shock proteins associated with the aggresomes and will aid in the
chaperoning process [288]. Therefore in the presence of MUC1, Hsp70/90 could be
recruited to EGFR cytoplasmic tail, which increases HDAC6 interaction thereby reducing
EGFR degradation. This could be a possible mechanism of how MUC1 is promoting
EGFR recycling and signaling, but also a mechanism of increased nuclear EGFR in the
presence of MUC1. Importantly, the interactions of MUC1 and EGFR with HSPs are
novel therapeutic targets that could be targeted with a peptide-based therapy.
MUC1’s Role In EGFR Cytosolic Release
The mechanisms of MUC1 and EGFR nuclear translocation are still being
investigated and the work presented invokes the questions do MUC1 and EGFR traffic
together or are their paths to the nucleus independent? I hypothesize that MUC1 and
EGFR are being trafficked together and that the presence of MUC1 in this trafficking
plays an important role in the cytosolic release of EGFR. In the endoplasmic reticulum,
misfolded proteins are presented to Sec61 and this interaction will mediate the
ubquitination of misfolded proteins and subsequent proteosomal degradation [289].
However, if EGFR and MUC1 are being trafficked together, then MUC1 could inhibit
EGFR’s ubquitination as observed in an earlier study, which examined MUC1’s role in 164
aberrant EGFR trafficking [87]. Although this study examined the effect of MUC1 on
ubquitination of membrane associated-EGFR following EGF stimulation the principal
that the presence of MUC1 inhibits EGFR ubquitination could still be a valid mechanism
for increased EGFR cytosolic release and decreased proteosomal degradation.
Nuclear EGFR Trafficking: Anti-Cancer Target
Overexpression of EGFR is commonly seen in the basal-like subtype of breast
cancer, but therapeutic responses to anti-EGFR tyrosine kinase inhibitors has been
limited. The work presented here demonstrates the EGFR oncogenic function can be
multi-faceted, because although EGFR signaling is important, cellular localization in
cancer cells is starting to be appreciated as a mechanism of increased proliferation and
radioresistance. Trafficking of EGFR to the nucleus involves the interaction with several
other proteins including heat shock proteins and importins, therefore to inhibit nuclear
EGFR accumulation and function we have developed an EGFR/Importinβ1 inhibitory
peptide. We utilized the protein transduction domain (PTD4) and mimicked EGFR’s
NLS to create a peptide (eNLS) which would compete for importin interaction thereby
inhibit EGFR nuclear translocation. Preliminary results indicated that targeting EGFR
nuclear trafficking inhibits proliferation in cancer cells (Figure 7.2). Novel anti-cancer
therapies that target aberrant localization of EGFR and other proteins offers a new field
of cancer therapeutics.
165
0.2 Day 0 Day 3 0.15
0.1
0.05 Relative Absorbance Relative 0 50uM eNLS 50uM CP 50uM Water NT
Figure 7.2. Blocking nuclear EGFR translocation inhibits cell proliferation. BT20 breast cancer cells were treated with eNLS, control peptide (CP), vehicle (water), or non- treated for 3 days. Cells were then counted using an MTT assay. Student t-test was performed to determine that eNLS significantly inhibited proliferation (p=0.0001). Assay was performed by Aarthi Goverdhan. 166
The work presented in this dissertation offers a new insight into a novel
mechanism of EGFR activity in breast cancer development, progression, and chemo-
resistance. Additionally, a 'first-in-class' peptide-based therapy was developed that
demonstrates the oncogenic importance of MUC1/EGFR and MUC1/beta-catenin
interactions. In the future, peptide-based drugs may be viable options in the pursuit of
non-toxic cancer-specific therapies. For example, a peptide that could inhibit nuclear
translocation of EGFR could result increased chemotherapy sensitivity and cancer-
specific cytotoxicity. Overall, this body of works demonstrates the importance of
EGFR's activity and localization within cells and the use of mimetic peptides as potential
anti-breast cancer therapeutics.
167
Figure 7.3. Working Model with PMIP Treatment A) In a normal polarized epithelial cell
MUC1 (red, 1) is constitutively recycled, after EGF stimulation EGFR (green, 2) is degraded in the lysosome with minimal recycling (3), and b-catenin (blue) is associated with adherens REFERENCESjunctions. B) As cell undergoes transformation MUC1 expression and EGFR activity are increased, which promotes the loss of polarization by dissociation of β-catenin from adherens junctions (1). Loss of polarity promotes the mislocalization of proteins resulting in novel protein-protein interactions. C) These interactions promote EGFR recycling (1) and β- catenin-dependent transcriptional activation, and trafficking of EGFR into the nucleus (2). These cancer-dependent events result in increase metastases, proliferation and chemoresistance. D) Treatment with PMIP inhibits MUC1/EGFR (1) and MUC1/ β-catenin interactions (2), which results in decreased invasion, proliferation, cell growth, and chemoresistance. Additionally, PMIP inhibits EGFR nuclear accumulation, which downregulates the cell-cycle regulator, cyclin D1.
168
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