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Transforming Growth Factor Beta-Induced Tyrosine Phosphorylation of STAT3 and Cellular Invasion is Mediated by IL-6 Secr...

Permanent Link: http://ufdc.ufl.edu/UFE0041020/00001

Material Information

Title: Transforming Growth Factor Beta-Induced Tyrosine Phosphorylation of STAT3 and Cellular Invasion is Mediated by IL-6 Secretion in Breast Cancer Cell Lines
Physical Description: 1 online resource (126 p.)
Language: english
Creator: Parker, Nicole
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: breast, cancer, carcinogenesis, interleukin6, invasion, signal, signaling, stat3, tgfbeta, tumorigenesis
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Transforming Growth Factor Beta-Induced Tyrosine Phosphorylation of Signal Transducer and Activator of Transcription 3 and Cellular Invasion is Mediated by Interleukin-6 Secretion in Breast Cancer Cell Lines A hallmark of breast cancers is the overexpression or constitutive activation of various oncoproteins. Two examples are the proteins Signal Transducer and Activator of Transcription 3 (STAT3) and Transforming Growth Factor Beta (TGF-beta). Although each of these signaling molecules and their respective pathways have been implicated in cancer development, the mechanism of cross-signaling between these pathways has not been established in the cancer setting. We hypothesize that cross-talk between the TGF-beta and STAT3 pathways contributes to cancer invasiveness through an autocrine signaling loop. Preliminary data in nontransformed mouse mammary NMuMG cells indicate that exogenous TGF-beta treatment results in phosphorylation of STAT3 on its activating Tyrosine (705) site. Strikingly, this effect is observed only after several hours of TGF-beta treatment and appears to be mediated by the cytokine Interleukin-6 (IL-6). TGF-beta induced IL-6 mRNA upregulation occurs concomitantly with TGF-beta-stimulated STAT3 tyrosine phosphorylation. Blockade of IL-6 function with a mouse IL-6 receptor fusion protein abrogates this effect, thereby implicating IL-6 as the factor responsible for TGF-beta-stimulated STAT3 tyrosine phosphorylation in NMuMG cells. TGF-beta confers an invasive phenotype on NMuMG and Mv1Lu cells that is IL-6 dependent. Examination of this mechanism in human breast cancer cell lines suggests that TGF-beta-mediated secretion of IL-6 is responsible for the constitutive STAT3 tyrosine phosphorylation observed in many breast cancer cell lines. The invasive human MDA-MB-231 basal-like breast cancer cell line exhibits TGF-beta and IL-6 dependent invasion, while the noninvasive human MDA-MB-361 luminal breast cancer cell line is capable of attaining IL-6-dependent cellular invasiveness in response to TGF-beta treatment.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Nicole Parker.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Law, Brian K.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041020:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041020/00001

Material Information

Title: Transforming Growth Factor Beta-Induced Tyrosine Phosphorylation of STAT3 and Cellular Invasion is Mediated by IL-6 Secretion in Breast Cancer Cell Lines
Physical Description: 1 online resource (126 p.)
Language: english
Creator: Parker, Nicole
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: breast, cancer, carcinogenesis, interleukin6, invasion, signal, signaling, stat3, tgfbeta, tumorigenesis
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Transforming Growth Factor Beta-Induced Tyrosine Phosphorylation of Signal Transducer and Activator of Transcription 3 and Cellular Invasion is Mediated by Interleukin-6 Secretion in Breast Cancer Cell Lines A hallmark of breast cancers is the overexpression or constitutive activation of various oncoproteins. Two examples are the proteins Signal Transducer and Activator of Transcription 3 (STAT3) and Transforming Growth Factor Beta (TGF-beta). Although each of these signaling molecules and their respective pathways have been implicated in cancer development, the mechanism of cross-signaling between these pathways has not been established in the cancer setting. We hypothesize that cross-talk between the TGF-beta and STAT3 pathways contributes to cancer invasiveness through an autocrine signaling loop. Preliminary data in nontransformed mouse mammary NMuMG cells indicate that exogenous TGF-beta treatment results in phosphorylation of STAT3 on its activating Tyrosine (705) site. Strikingly, this effect is observed only after several hours of TGF-beta treatment and appears to be mediated by the cytokine Interleukin-6 (IL-6). TGF-beta induced IL-6 mRNA upregulation occurs concomitantly with TGF-beta-stimulated STAT3 tyrosine phosphorylation. Blockade of IL-6 function with a mouse IL-6 receptor fusion protein abrogates this effect, thereby implicating IL-6 as the factor responsible for TGF-beta-stimulated STAT3 tyrosine phosphorylation in NMuMG cells. TGF-beta confers an invasive phenotype on NMuMG and Mv1Lu cells that is IL-6 dependent. Examination of this mechanism in human breast cancer cell lines suggests that TGF-beta-mediated secretion of IL-6 is responsible for the constitutive STAT3 tyrosine phosphorylation observed in many breast cancer cell lines. The invasive human MDA-MB-231 basal-like breast cancer cell line exhibits TGF-beta and IL-6 dependent invasion, while the noninvasive human MDA-MB-361 luminal breast cancer cell line is capable of attaining IL-6-dependent cellular invasiveness in response to TGF-beta treatment.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Nicole Parker.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Law, Brian K.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041020:00001


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TRANSFORMING GROWTH FACTOR BETA-INDUCED TYROSINE PHOSPHORYLATION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3 AND CELLULAR INVASI ON IS MEDIATED BY INTERLEUKIN-6 SECRETION IN BREAST CANCER CELL LINES By NICOLE N. TEOH PARKER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1

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2009 Nicole N. Teoh Parker 2

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To my family and friends, who always support me; To my husband, who is my best friend and so much more 3

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ACKNOWLEDGMENTS Acknowledgments are a strange beast. You acknowledge all those w ho have contributed to your success, to your development as a person andin this caseas a sc ientist, and to whom you owe thanks. I do not know where to start: I think I need to thank every major person that I have come across in my life for helping shape who I am. In addition, so many circumstances and events have formed me, for better or worse, and I am thankful for all of them. First, I thank my parents, w ho overcame adversity in many forms and lead successful lives variously as nuclear physicists, a roboticist, a contract or specialist and insp ired mathematics and computer science teachers. From such a background I had the role models of education, hard work and perseverance, and was therefore taught to keep things in perspective and not take anything for granted. I would like to next thank my brother Eric, for being himself. Not only do we share incredibly similar DNA, but we are also alike in many ways that makes us able to support each other and be such good friends. Tha nks Eric, for having a good sense of humor and for being driven and successful! You are a great model for me to follow. Martin and Sheila, thank you for being amazing in-laws and supporters, and believing in me when I didnt believe in myself. I am so glad that we are family! I am thankful to the University of Florida Co llege of Medicine for accepting me into their program, and for seeing potential in me that was worth confe rring an Alumni Fellowship onto me. I am grateful for this chan ce, and for their confidence in me. Next I am grateful to my former supervisor s and mentors, including Dr. Maria Ragland, for getting me started in research and showing me a love for science; Dr. Laura Nilson for giving me a job in college and being an incredible role model of a female academic scientist and teacher; and in particular, Dr. Bertrand Jean-Claude, who got me started in cancer research and was an incredible source of inspiration for me. All thes e past mentors helped show me what it was like 4

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to be a researcher and scientist, and have contributed to my academic and personal knowledge. In addition, I would like to th ank past lab members and coworkers who supported me: Rima, Athanasia, Monica, Angie, Dr. Qiu, Nuria, Lucia, Tahilia, and everyone else that I have forgotten. Your friendship is sti ll greatly appreciated and valued! Similarly, my friends are always supportive of me, whether it is emotional, psychological, or entertaining support. Thanks to Shelby, Mel, Ada, Amanda, Michele, Shermi, and Star. Special thanks go out to our group of friends, including Ben and Shelby, Mel and Adam, Steve and Monica, Steve and Lindsay, Cort and Erin, and everyone else. Ada, thanks for visiting me in lab so many times: you have helped keep me grounded! A special thanks is due to my current mentor, Dr. Brian Law, and my lab mates. Dr. Mary Law deserves a special thank you because she trai ned me in many things, including how to be a patient scientist. Dr. Pat Corsino taught me al most all the techniques I know that I learned in graduate school; his attention to detail and a love of perfection was an inspiration for me, and helped me improve the quality of my work. Brad Davis kept everything running and was a stabilizing factor in my everyda y lab life: because of his excellence I was able to do so many experiments without regard to ma ny details, for which I thank his efficiency and patience. If I could be as good-natured and hardworking as he is I will be proud to say s o. Particular thanks are due to my committee members, especially Dr. Dietmar Siemann, who let me rotate in his lab and offered me a spot as a graduate student. I thank him for his guidance and good nature, sense of humor, and cynicism that implies a sense of re alism tinged with sarcasm. This is all in line with my own ways of thinking and viewing th e world. Thank you, Dr. Harrison and Dr. Luesch, for agreeing to be members of my graduate committee you guys have seen the same data over and over and always have a fresh perspective for me. You are both amazing role models of 5

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scientists and thinkers whom I look up to. Thank you for your support and guidance. Also I thank Sharon and Chris for giving me incredib le emotional support and teaching me during my rotation. Penultimately, I thank my graduate mentor, Dr. Brian Law. You gave me a chance to succeed in your lab, and I hope that I have lived up to your expectations and performed as well as you believed I could. I have learned an incredible amount from you, especially in conversations about science. I ha ve also learned other things, such as how to be a better writer and more analytical, as well as how to keep my sense of humor and optimism when things arent working. Youre a great boss and I am lucky to have been your graduate student. I hope to continue learning from you throughout my career. Finally, the most special acknowledgment is due to my husband Matt. Who else could understand my frustrations in everything better than him? Matt has been through it all. Not only has he provided me with an exemplary model of how to succeed in graduate school, he has done it with humor and a good nature that is amazing to emulate. I should be so lucky to display the same levels of goodness and productivity that he has shown, and I thank him for being incredible. He is a constant source of humor, comfort, love and support, and I hope I can return the same to him. He makes our home a place of joy and I am lu cky to be sharing my life with him. 6

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF ABBREVIATIONS ........................................................................................................14 ABSTRACT ...................................................................................................................................17 CHAPTER 1 INTRODUCTION................................................................................................................. .19 Breast Cancer: A Continuing Disease ....................................................................................19 Breast Cancer Incidence ..................................................................................................19 TGF and STAT3 Each Exhibit Proand Anti-Tumorigenic Roles in Cancer ...............20 Transforming Growth Factor ...............................................................................................21 Mechanisms of TGF Signaling ......................................................................................21 The Paradoxical Role of TGF in Carcinogenesis ..........................................................22 TGF and Cancer: Diagnostic and Therapeutic Implications .........................................23 Signal Transducer and Activ ator of Transcription 3 ..............................................................24 Cytokine Induced STAT3 Signaling a nd Mechanisms of STAT3 Activation ................24 Phosphorylation of STAT3 on Serine (727): Transcriptional significance .....................24 Regulation of STAT3 Signaling ......................................................................................25 IL-6 Signaling: Normal Cytokine Si gnaling Versus sIL-6R Trans-Signaling ................26 STAT3 and Cancer ..........................................................................................................27 Diagnostic and Therapeutic Implications ........................................................................28 STAT3 and TGF : Published Mechanisms of Cross-Talk .....................................................29 Epithelial-To-Mesenchymal Transition (EMT) and Breast Cancers ......................................29 TGF Induction of EMT Involves Diverse Pathways and Mechanisms .........................30 The Cadherin Switch: Mediating Changes in Cell-Cell Adhesion and Motility/Invasion ..........................................................................................................31 Regulation of Eand N-Cadherin Expr ession: A Potential Role for STAT3 ..................31 Rationale and Study Outline...................................................................................................32 Clinical Implications and Other Considerations .....................................................................33 2 GENERAL MATERIALS AND METHODS........................................................................37 Tissue Culture, Maintenance, and Plating of Cell Lines ........................................................37 Isolation of Mouse Mammary Tumor Associat ed Myofibroblast Cells and Maintenance of the TDF Cell Line ...........................................................................................................37 Tritiated (3H)-Thymidine Incorporation Assays .....................................................................37 Constructs, Drugs, and Reagents ............................................................................................37 Inhibition of Actin Polymerization and of Various Signal Transduction Pathways ..............38 Reverse Transcription Polymerase Chain Reaction (RT-PCR) ..............................................38 SDS-PAGE and Westen Blot Analysis ...................................................................................39 Conditioned Medium Experiments .........................................................................................40 7

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Transient Transfections and Luciferase Reporter Assays ......................................................40 Immunofluorescence Microscopy ..........................................................................................40 Functional Inhibition of IL-6 Function w ith the Receptor fusion protein mIL-6-RFP ..........41 Establishment of Clonal Cell Lines Stably Expressing the IL-6 Receptor fusion protein .....41 Antibody Array Analysis ........................................................................................................41 Boyden Chamber Cellular Invasion Assays ...........................................................................42 Image Acquisition and Processing, Data Quantification, and Statistical Analyses ................42 3 TGF-BETA INDUCTION OF STAT 3 TYROSINE PHOSPHORYLATION: MECHANISTIC DATA.........................................................................................................44 Introduction .............................................................................................................................44 Published Cross-talk Between Members of the TGF and STAT3 Signaling Pathways ......................................................................................................................44 Lysophosphatidic Acid Signaling ....................................................................................45 Results .....................................................................................................................................46 TGF Treatment Causes STAT3 Tyrosine P hosphorylation in Various Epithelial Cell Lines .....................................................................................................................46 TGF Stimulation of STAT3 Tyrosine P hosphorylation Does Not Occur Through Cell Cycle Arrest ..........................................................................................................46 TGF -Induced STAT3 Tyrosine Phosphorylation Requires Intact TGF Jak, and Smad3 Signaling, But Does Not Require NFB Signaling ........................................47 TGF Stimulation of STAT3 Tyrosine P hosphorylation Induces STAT3 Dependent Transcription ................................................................................................................48 TGF -Induced STAT3 Tyrosine Phosphorylatio n Requires the Presence of Serum ......48 Inhibition of Various Signaling Pathways Does Not Block TGF Stimulation of STAT3 Tyrosine Phosphorylation; Only Actin Depolymerization with Swinholide A Abrogates TGF -Induced STAT3 Tyrosine Phosphorylation ..............49 Lysophosphatidic Acid (LPA) Treatment Can Fulfill the Serum Requirement for TGF Stimulation of STAT3 Tyrosine Phosphorylation .............................................50 LPA Receptor Inhibitor VPC51299 Inhibits Serum and TGF Co-Stimulation of STAT3 Phosphorylation, But Not TGF -Induced IL-6 Upregulation .........................50 Conditioned Medium from TGF -Treated NMuMG Cells Induces STAT3 Tyrosine Phosphorylation in the T R1-Inactive R-1B Cell Line ...............................................51 Leukemia Inhibitory Factor (LIF) Does Not Induce STAT3 Tyrosine Phosphorylation ...........................................................................................................52 TGF Induction of STAT3 Tyrosine Phos phorylation Is Mediated by IL-6 Secretion ......................................................................................................................52 IL-6 Treatment is Not Sufficient to Indu ce Morphological Changes Associated with EMT.............................................................................................................................52 Functional Inhibition of IL-6 with the Receptor fusion protein mIL-6-RFP Abolishes TGF -Induced STAT3 Phosphorylation....................................................53 TGF Treatment Confers an IL-6 Dependent Invasive Phenotype on the Nontransformed NMuMG and Mv1Lu Cell Lines ......................................................54 TGF Treatment Induces IL-6 Dependent Upregulation of the Cell Adhesion Molecule N-Cadherin ...................................................................................................54 IL-6 Dependent N-Cadherin Transcription is Not Necessary or Sufficient for EMT .....55 8

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TGF Induces VEGF and GM-CSF Secretion ................................................................56 Matrix Metalloproteinase Inhibition Does Not Block TGF Stimulation of STAT3 Tyrosine Phosphorylation............................................................................................57 TGF Treatment Induces IL-6R Expression ...................................................................57 Exogenous sIL-6R Does Not Recapitulate TGF Stimulation of STAT3 Tyrosine Phosphorylation ...........................................................................................................58 Conclusions and Discussion ...................................................................................................58 4 EVIDENCE FOR TGF-BETA INDUCTION OF STAT3 TYROSINE PHOSPHORYLATION IN HUMAN BREAST CANCER CELL LINES...........................73 Introduction .............................................................................................................................73 Results .....................................................................................................................................75 Constitutive STAT3 Tyrosine Phosphorylation and IL-6 Upregulation Varies Across Breast Cancer Cell Lines .................................................................................75 Conditioned Medium (CM) from Breast Cancer Cell Lines Variously Induces STAT3 Tyrosine Phosphorylation a nd STAT3-Dependent Transcription ..................76 A TGF Signaling Component Contributes to Maintenance of STAT3 Tyrosine Phosphorylation in the MDA-MB -231 Breast Cancer Cell Line .................................77 Interruption of the TGF -IL-6-pSTAT3 Loop Blocks STAT3 Phosphorylation, IL6 Upregulation, and STAT3-Dependent Transcriptional Activ ity in MBA-MB231 Cells ......................................................................................................................78 Abrogation of p-STAT3 Abolishes the In trinsic Invasiveness of MDA-MB-231 Cells .............................................................................................................................78 Exogenous TGF Induces STAT3 Tyrosine Phosphorylation Through IL-6 Upregulation in MDA-MB-361 Cells ..........................................................................79 Mouse Mammary Tumor-Derived Fibr oblast (TDF) Conditioned Medium Recapitulates Exogenous TGF Treatment in MDA-MB-361 Cells ...........................79 TGF Treatment Confers the Invasive Phenotype on the Otherwise Noninvasive MDA-MB-361 Cell Line, Possibly Through TGF Induction of N-Cadherin Upregulation ................................................................................................................80 Conclusions and Discussion ...................................................................................................81 5 TOWARDS AN OVERALL CONCLUSION FOR TGF-BETA STIMULATION OF STAT3 TYROSINE PHOSPHORYLATION........................................................................87 Overall Findings and Significance ..........................................................................................87 Discussion and Future Work ..................................................................................................88 Conclusions .............................................................................................................................94 LIST OF REFERENCES ...............................................................................................................95 BIOGRAPHICAL SKETCH .......................................................................................................126 9

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LIST OF FIGURES Figure page 1-1 The TGF signaling pathway. ...........................................................................................34 1-2 Cytokine stimulated STAT3 pathway. ...............................................................................35 1-3 Mechanisms of IL-6 trans-signaling. .................................................................................36 1-4 The classical cell cycle model. ...........................................................................................36 2-1 Design of Conditioned Medium (CM) experiments. .........................................................43 2-2 Mouse Cytokine I antibo dy array chip (Ray Biotech). ......................................................43 3-1 TGF treatment stimulates STAT3 Tyr ( 705) in various ep ithelial cell lines. ..................59 3-2 TGF -stimulated STAT3 tyrosine phosphoryl ation is doseand time-dependent. ...........60 3-3 Cell cycle arrest is not suffi cient for STAT3 tyrosine phosphorylation. ...........................60 3-4 TGF treatment does not affect Src phos phorylation status in NMuMG cells. .................61 3-5 TGF stimulation of STAT3 tyrosine phosphorylation requires intact TGF /Smad3 and Jak signaling. ...............................................................................................................61 3-6 NF-kB activity is not required for TGF stimulation of STAT3 tyrosine phosphorylation. .................................................................................................................61 3-7 TGF activates STAT3 dependent gene tran scription as measured by luciferase constructs. ..........................................................................................................................62 3-8 TGF stimulation of STAT3 tyrosine phosphorylation requires the presence of serum. .................................................................................................................................62 3-9 Reintroduction of various growth factors does not fulfill the serum requirement necessary for TGF induced STAT3 phosphorylation. .....................................................63 3-10 Inhibition of various sign aling pathways does not block TGF -induced STAT3 tyrosine phosphorylation. ...................................................................................................63 3-11 Swinholide A treatment, but not treatment with other actin depol ymerizing agents, is sufficient to abrogate TGF induction of STAT3 tyrosine phosphorylation. ....................63 3-12 Lysophosphatidic Acid (LPA) can replace serum in TGF stimulation of STAT3 tyrosine phosphorylation. ...................................................................................................64 3-13 Treatment with Swinholide A is sufficient to inhibit LPA and TGF -induced STAT3 tyrosine phosphorylation. ...................................................................................................64 10

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3-14 The LPA receptor antagonist VPC51299 blocks serum and TGF costimulation of STAT3 tyrosine phosphorylation. ......................................................................................64 3-15 TGF treatment of NMuMG cells yields Conditioned Medium (CM) containing some factor capable of inducing STAT3 tyrosine phosphorylation in a T RI-inactive cell line. ..............................................................................................................................65 3-16 Leukemia Inhibitory Factor (LIF) does not induce STAT3 tyrosine phosphorylation in NMuMG cells................................................................................................................65 3-17 TNF and IL-6 Both Induce STAT3 Tyrosine Phosphorylation in NMuMG Cells. .........65 3-18 TGF induces Interleukin-6 (IL-6) upregulation. ..............................................................66 3-19 TGF induced Interleukin-6 (IL-6) upregulation is time-dependent. ................................66 3-20 IL-6 does not recapitulate mor phological changes associated with TGF -stimulated EMT. ..................................................................................................................................66 3-21 Tentative pathway describing TGF stimulation of STAT3 tyrosine phosphorylation. ...67 3-22 Characterization of the mouse IL-6 receptor fusion protein, mIL-6-RFP. ........................67 3-23 Establishment of 293A ce ll lines stably expressing the IL-6 receptor fusion protein. ......67 3-24 Isolation of clonal cell lines stably expressing the IL-6 receptor fusion protein. ..............67 3-25 IL-6 functional inhibition by the r eceptor fusion protein mIL-6-RFP blocks TGF induced STAT3 tyrosine phosphorylation. ........................................................................68 3-26 IL-6 treatment is sufficient to induce cellular invasiveness in NMuMG cells. .................68 3-27 TGF treatment confers invasive potentia l on the otherwise nonmigratory NMuMG cell line. ..............................................................................................................................68 3-28 TGF treatment confers invasive potentia l on the otherwise nonmigratory Mv1Lu cell line. ..............................................................................................................................69 3-29 TGF induces upregulation of N-Cadhe rin that is IL-6 dependent. ..................................69 3-30 Inhibition of IL-6 function does not bl ock the morphological changes associated with TGF -induced EMT..........................................................................................................70 3-31 TGF induces N-Cadherin upregulation and E-Cadherin mislocalization in NMuMG cells; TGF stimulation of N-Cadherin upregulation is IL-6 dependent...........................70 3-32 TGF induces secretion of Vascular Endot helial Growth Factor (VEGF) and Granulocyte Macrophage ColonyStimulating Factor (GM-CSF)....................................71 11

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3-33 Exogenous VEGF and GM-CSF do not induce STAT3 tyrosine phosphorylation in NMuMG cells. ...................................................................................................................71 3-34 Broad-range inhibition of Matrix Metalloproteinases (MMP s) with the small molecule inhibitor GM6001 does not inhibit TGF stimulation of STAT3 tyrosine phosphorylation. .................................................................................................................71 3-35 TGF treatment induces gene upregulation of the IL-6 receptor. .....................................72 3-36 Exogenous soluble IL-6 receptor (sIL-6R ) does not recapitulate TGF stimulation of STAT3 tyrosine phosphorylation. .................................................................................72 4-1 STAT3 expression and tyrosine phosphorylation vary across nontransformed epithelial and carcinoma cell lines. ....................................................................................82 4-2 IL-6 upregulation appears to corr elate with constitutive STAT3 tyrosine phosphorylation. .................................................................................................................82 4-3 Treatment with conditioned medium fro m various breast cancer cell lines results in differential STAT3 tyrosine phosphorylation in a T RI-inactive cell line. .......................82 4-4 Treatment with CMs of various breast cancer cell lines induces varying levels of STAT3-responsive m67-luciferase ac tivity that appears to be TGF dependent in MDA-MB-231 and Tumor-Deriv ed Fibroblasts (TDFs). ..................................................83 4-5 TGF drives maintenance of ST AT3 tyrosine phosphorylation. .......................................83 4-6 The invasive basal-li ke MDA-MB-231 breast cancer line exhibits constitutive STAT3 tyrosine phosphorylation that is TGF and IL-6 dependent.................................84 4-7 The MDA-MB-231 breast cancer line exhi bits constitutive IL-6 upregulation that is TGF and IL-6 dependent. .................................................................................................84 4-8 The invasive basal-li ke MDA-MB-231 breast cancer li ne displays STAT3 dependent luciferase transcription activity that is IL-6 dependent. .....................................................84 4-9 MDA-MB-231 invasion is TGF and IL-6 dependent. .....................................................85 4-10 Tumor-derived fibroblast conditioned medium induces TGF and Jak-dependent STAT3 tyrosine phosphorylation in the MDA-MB-361 cell line. .....................................85 4-11 IL-6 and TGF treatment induce IL-6 gene upregulation in the MDA-MB-361 human breast cancer cell line. ............................................................................................85 4-12 MDA-MB-361 cells acquire the invasive phenotype upon TGF treatment; this invasion is dependent on IL-6. ...........................................................................................86 4-13 IL-6 and TGF treatment induces N-Cadherin up regulation in MDA-MB-361 cells; Blockade of IL-6 function blocks N-Cadherin upregulation. ............................................86 12

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4-14 Proposed model for TGF and IL-6 dependent si gnaling in MDA-MB-231 and MDA-MB-361 human breast cancer cell lines. .................................................................86 13

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LIST OF ABBREVIATIONS ADAM A Disintegrin And Metalloprotease ALK Activin-Like Kinase ATX Autotaxin bFGF basic Fibrobla st Growth Factor BMP Bone Morphogenic Protein cEBP CCAAT Enhancer Binding Protein CK Cytokeratin CREB Ca 2+ /cAMP-Response Binding Element CM Conditioned Medium DMEM Dulbeccos Modified Eagle Medium DMSO Dimethyl Sulfoxide EGF Epidermal Growth Factor EGFR Epidermal Growth Factor Receptor EMT Epithelial to Mesenchymal Transition FAK Focal Adhesion Kinase FBS Fetal Bovine Serum GF Growth Factor gp130 Signal transducing cytokine receptor GM-CSF Granulocyte Macrophage Colony Stimulating Factor HGF Hepatocyte Growth Factor Id1, Id2 Inhibitor of Differentiation 1, 2 IL-6 Interleukin-6 IL-6R Interleukin-6 Receptor 14

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IL-10 Interleukin 10 Jak Janus kinase LAP Liver-enriched transcri ptional Activator Protein LIF Leukemia Inhibitory Factor LIFR LIF Receptor LPA Lysophosphatidic Acid m67-Luciferase Reporter construct c ontaining 4 STAT3 binding (m67) sites mIL-6-RFP mouse IL-6 Receptor Fusion Protein Inhibitor MMP Matrix Metalloproteinase NFNuclear Factor Kappa Beta PA Phosphatidic Acid Pai-1 Plasminogen Activator Inhibitor-type I PDGF/R Platelet-Derived Growth Factor (Receptor) PIAS Protein Inhibitor of Activated STATs PLD Phospholipase D RT-PCR Reverse Transcription Polymerase Chain Reaction SDS-PAGE Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis SH2 Src Homology 2 SHP-2 Src Homology 2-contai ning tyrosine phosphatase sIL-6R soluble Interleukin-6 Receptor siRNA small interfering ribonucleic acid SIS3 Specific Inhibitor of Smad3 SMA Smooth Muscle Actin SOCS Suppressor of Cytokine Signaling STAT3 Signal Transducer and Activator of Transcription 3 15

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STAT3-C Constitutively Activated STAT3 T RI (or II) Transforming Growth Factor Receptor I (or II) T RKI Transforming Growth Factor Receptor I Kinase Inhibitor TDF/TDMF Tumor-Derived Fi broblasts/Myofibroblasts TGF Transforming Growth Factor TGF Transforming Growth Factor TIMP Tissue Inhibitor of MetalloProteases TMA Tissue Microarray TNF Tumor Necrosis Factor VEGF Vascular Endothelial Growth Factor 16

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy TRANSFORMING GROWTH FACTOR BETA-INDUCED TYROSINE PHOSPHORYLATION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3 AND CELLULAR INVASI ON IS MEDIATED BY INTERLEUKIN-6 SECRETION IN BREAST CANCER CELL LINES By Nicole N. Teoh Parker December 2009 Chair: Brian K. Law Major: Medical SciencesPhysiology and Pharmacology A hallmark of breast cancers is the overexpre ssion or constitutive activation of various oncoproteins. Two examples are th e proteins Signal Transducer a nd Activator of Transcription 3 (STAT3) and Transforming Growth Factor (TGF ). Although each of these signaling molecules and their respective pathways have been implicated in cancer development, the mechanism of cross-signaling be tween these pathways has not b een established in the cancer setting. We hypothesize that cross-talk between the TGF and STAT3 pathways contributes to cancer invasiveness through an autocrine signaling loop. Preliminary data in nontransformed m ouse mammary NMuMG cells indicate that exogenous TGF treatment results in phosphorylation of STAT3 on its activating Tyrosine (705) site. Strikingly, this effect is observed only after se veral hours of TGF treatment and appears to be mediated by the cytokine Interleukin-6 (IL-6). TGF induced IL-6 mRNA upregulation occurs concomitantly with TGF -stimulated STAT3 tyrosine phosphorylation. Blockade of IL-6 function with a mouse IL-6 receptor fusion protein abrogates this effect, th ereby implicating IL-6 as the factor responsible for TGF -stimulated STAT3 tyrosine phosphorylation in NMuMG cells. TGF confers an invasive phenotype on NMuMG a nd Mv1Lu cells that is IL-6 dependent. 17

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Examination of this mechanism in human breast cancer cell lines suggests that TGF mediated secretion of IL-6 is responsible for the constitutive STAT3 tyrosine phosphorylation observed in many breast cancer cell lines. The invasive human MDA-MB-231 basal-like breast cancer cell line exhibits TGF and IL-6 dependent invasion, while the noninvasive human MDAMB-361 luminal breast cancer cell line is capab le of attaining IL-6-dependent cellular invasiveness in response to TGF treatment. 18

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CHAPTER 1 INTRODUCTION Breast Cancer: A Continuing Disease Breast Cancer Incidence Among women breast cancer is the most freque ntly diagnosed cance r and is the second leading cause of cancer-related deaths, with an estimated 27% of new cancer cases in women in the United States in 2009 (1). Risk factors include diet (2), BRCA1/2 mu tations (3), and drastic changes in body weight (4). Carcinogenesis, or the development of cancers from epithelial tissues in the body, results in th e development of bulky tumor masses. A hallmark of cancers is the overexpression or hyperactiva tion of various oncogenic protei ns. Although cancer etiologies differ widely, a successful cancer must possess the following abilities (5): 1) Heightened cell cycling and reproductive abilit y, with a loss of cell cycle checkpoints and regulation, and development of autonomous growth factor producti on; 2) Anti-apoptotic be havior; 3) Increase in angiogenesis and the associated 4) Invasive a nd metastatic ability. Common chemotherapies concentrate on eliminating tumor burden a nd therefore reducing th e cell population that possesses metastatic ability. Strikingly, a majo rity of cancer patients succumb not to primary cancers but to metastatic lesions, which are more difficult to detect and eliminate (1). Despite numerous advances in chemotherapeutics, the mechanisms involved in tumorigenesis are still being characterized. The basis for the development of new therapies relies on targeting receptors or other oncogenic proteins that are overactivated in cancers. However, the observation that there is oncogenic overactivation or cons titutive activation of proteins in certain cancer types is correlative at best, and these dysregulations are not necessarily the driving event behind the development of a can cer. Therefore, therap ies targeting proteins that help maintain a cancers survival, but not necessarily its progression per se, can augment 19

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anticancer regimens. However, such strategies ma y be regarded as insufficient due to a potential inability to inhibit the molecular fo rces that drive tumorigenesis. A critical part of breast cancer research relies not only on the development of speci fic agents to target cancers, but also on basic research that elucidates the mech anisms of carcinogenesis. In so doing, the particul ar etiology of a cancer may be uncovered and a potentially pr eventative or inhibitory pharmacological approach can be taken. For example, treatment w ith an anti-oncogenic ther apy in early phases of carcinogenesis may prevent the cancers developm ent into a big bulky mass, and therefore halt its invasion and metastatic ability, th ereby improving the patients prognosis. TGF and STAT3 Each Exhibit Proand Anti-Tumorigenic Roles in Cancer Two examples of oncogenic proteins are the growth factor Transforming Growth Factor (TGF ) and Signal Transducer and Activator of Tr anscription 3 (STAT3). The transcription factor STAT3 is required for Src-mediated tran sformation (6, 7). Overexpression of STAT3 has been observed in many cancers (8) and more recen tly has been demonstrated to play a positive role in tamoxifen response (9) a nd to correlate inversel y with overall patient survival (10, 11). Conversely, STAT3 contributes to other confounding effects such as chemoresistance in breast cancers (12). STAT3, therefore, is a factor whose precise role in breast cancer is still being elucidated. Similarly, TGF is a factor that has an elusive role in cancer. Expression of TGF protein correlates with shorter di sease-free survival in patients with early stage primary breast cancer (13). TGF can cause estrogen insensitivity, lead ing to refractory responses to such hormone-based breast chemotherapies as tamoxifen (14). Although TGF causes growth inhibition in the MDA-MB-231 breast cancer cell line (15), it is al so required for metastasis of this same cell line into bone (16). Many studi es have demonstrated the significance of serum 20

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TGF concentrations, as serum may be obtained with little invasive ness to the patient. Higher TGF serum concentrations were found to correlate with decreased survival in breast cancer patients (17, 18) and in breast cancer patients ex pressing a polymorphism in the Her2 gene (19). Transforming Growth Factor Mechanisms of TGF Signaling The cytokine TGF was first identified as a molecule with mitogenic properties, but this effect was later attributed to a different factor, TGF which is structurally and functionally similar to Epidermal Growth Factor (20,21). TGF was responsible for the pro-growth effects observed previously, while TGF was shown to be growth inhibitory in many systems (22). The TGF molecule belongs to a larger group of ligands termed the TGF Superfamily (23). This family includes many molecules, notably the Bone Morphoge nic Proteins (BMPs), that are involved in development, tissue diffe rentiation, and some cancers (24-27). The BMPs and other molecules signal in a manner analogous to TGF signaling, but interact with a separate group of BMP-specific receptors an d downstream effectors (28). TGF -mediated signaling occurs through the interaction of TGF with the Activin-Like Kinase (ALK5) protein (TGF Type I receptor, or T RI), which heterodimerizes with the Type II TGF receptor (T RII) (2932). These receptor-TGF oligomers associate in higher-order structures, forming a complex with a stoichiometric TGF -T RI-T RII ratio of 2 : 2 : 2 (31, 33). The TGF Type I (T RI) and Type II (T RII) Receptors are both required for intact TGF ligand-stimulated signaling (34). The TGF -T RI-T RII complex results in T RII transactivation by T RI (31), whereupon the Smad proteins are recruited to the receptors (35) (F igure 1-1). The TGF receptor-associated Smads are Smads 2 and 3 (36). These Smads are recruited to activated receptor dimers, where they are phosphorylated on key serine/threonine sites (37). 21

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Phosphorylated Smads diffuse away from the re ceptor dimers and heterotrimerize with the coactivating (Co-Smad) Smad4 protein (38). After Smad heterotrimerization the complex translocates into the nucleus, where it can interact with cofactors such as p300 (39) and with Histone H3 (40) and alter the transcription of target genes (41), the promoter s of which typically contain CAGA repeats (42). Examples of TGF -Smad target genes include the Epidermal Grow th Factor Receptor (20, 41), Survivin (43), Myc (44), p21 (45), and Plasminogen Activator Inhibitor-type 1 (Pai -1) (46). Other examples of cancer-related genes that are regulated by TGF include Matrix Metallop roteinases (MMPs) (47, 48), Tissue Inhibitors of MMPs (TIMPs) (49, 50), and Vascular Endothelial Growth Factor (VEGF) (51, 52). Strikingly, this diverse array of genes falls into both an tiand pro-tumorigenic categories, which underscores the paradox of TGF in cancer [for reviews, see (53-55)]. The Paradoxical Role of TGF in Carcinogenesis As previously mentioned TGF is a cytokine that induces both tumor suppressive and oncogenic downstream functions. In nontransformed epithelial cell lines this cytokine induces growth arrest (56) and the pr ocess of Epithelial to Mesenc hymal Transition (EMT) (57, 58), although the latter event only a ppears to occur in some cell lines and not others (59). Key members of the TGF signaling pathway are mutated, downregulated, or absent in various cancers. Examples are the Smads (25, 60, 61), the TGF receptors (62, 63), and TGF itself (64). The Type III TGF receptor (T RIII) can act as a tumor suppressive molecule, binding and sequestering TGF Type I and Type II receptors a nd preventing their stimulation by TGF (65-70). The Type 1 isoform of the TGF ligand is present in th e serum of breast cancer patients with advanced disease (71) as compared to normal patients. TGF is overexpressed (72) and secreted by multiple cancer cell lines (72, 73) and tumor-derived cells, including fibroblasts 22

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(74), and is necessary for cross-talk between tumor-infiltrated fibroblas ts and neoplastic cells (75). However, the exact role of TGF in carcinogenesis remains ill-defined, due in large part to a TGF Switch that signifies the point at which TGF ceases tumor suppressive functions and becomes an oncogenic factor [(76, 77); reviewed in (78)]. Further complicating the issue is the fact that the observation of TGF present in cancers derived from patients is only correlative data: accrued mutations or changes in gene e xpression are observations made only after the tumor is formed, and no definite conclusions may be drawn about the tumors actual disease pathology. Therefore, understanding how TGF contributes to carcinogenesis will aid the discovery of more efficacious treatments in the clinic. TGF and Cancer: Diagnostic and Therapeutic Implications TGF is a factor displaying both antiand pro-tumorigenic downstream effects (79), and multiple members of the canonical TGF signaling pathway are mutated or dysregulated in various cancers (61, 80). However, despite seemingly contradictory roles in cancer TGF functions in late carcino genesis as a pro-tumorigenic factor, a nd its levels in serum are elevated in breast cancer patients whose disease is further progressed (71, 81) as well as in patients suffering other cancer types (82, 83). TGF is required for cancer cell invasiveness (57, 84) and inhibition of TGF signaling correlates with breast can cer patient outcome (85). The development of novel TGF inhibitors has formed a solid basis for identification of new cancer therapies (86, 87). Many approaches have been taken to abrogate TGF in various types of cancers (87-98), some of which have been indire ct. Some chemotherapies that are already clinically available but whose mechanisms were not well characterized have more recently been demonstrated to act thro ugh inhibition of the TGF pathway (90, 96, 97). This post-clinical 23

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elucidation of cytotoxic chemotherapeutic mechan isms holds promise for further characterization of other therapies that are efficacious in the clinic but whose mechanisms are not yet well understood. Signal Transducer and Activator of Transcription 3 Cytokine Induced STAT3 Signaling and Mechanisms of STAT3 Activation STAT3 is a latent cytoplasmic transcri ption factor and, upon phosphorylation of its activating Tyrosine (705) site, forms dimers with a tyrosine phosphorylated STAT3 or STAT1 molecule and translocates into the nucleus (99). STAT3 tyrosine phosphorylation can be induced by many cytokines and growth factors in cluding Interleukin-6 (IL6) (100), IL-10 (101), Leukemia Inhibitory Factor (LIF) (102, 103), Oncostatin M (104), Plat elet-Derived Growth Factor (PDGF) (105), basic Fibr oblast Growth Factor (bFGF) ( 102), Epidermal Growth Factor (20, 106, 107), and by intracellular kinase s such as Src (8) and Ras (108). STAT3 dimers then bind the promoters of targ et genes such as p21 and Cyclin D1 (109), MMP-9 (110), and the oncogene Pim1 (111). Many target genes of both activated and nonphosphorylated STAT3 have been identified that are necessary for such downstream functions as wound healing (112) and cancer (113), respective ly. Significantly, STAT3 can either activate or repress its ta rget genes (114) through its inter actions with other transcription factors such as JunB (115), or with cof actors such as p300/CREB (116). Further, STAT3 upregulates other genes necessa ry for additional cancer proce sses such as proliferation, angiogenesis and cell survival (117, 118). Phosphorylation of STAT3 on Serine (727): Transcript ional significance There is a large body of literat ure examining the Serine (727) site of STAT3 and its effect on STAT3 transcriptional activity (119-123). Some groups have demonstrated that phosphorylation of this site is re quired for maximal STAT3 transcri ptional activity (120), others 24

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have shown that this phosphosite represses STAT 3 transcription (124, 125) and still more have suggested that the Ser (727) site possesses no tr anscriptional significance (119, 122). While all of these studies and data ar e convincing, it is clear that regulation of the two STAT3 phosphositesand in particular the STAT3 Ser (727) siteis a complex and cell specific feature. The significance of this site lies be yond the scope of this study. Therefore, we will not provide detailed analys is of STAT3 Ser (727) signaling, although we have examined the regulation of this phosphorylation si te in the course of this study. Regulation of STAT3 Signaling There are several levels of STAT3 regulation that operate mainly through STAT3 dephosphorylation. STAT3 tyrosine phosphorylat ion is attenuated by the Src Homology 2Containing (SHP) tyrosine phosphatases in the nucleus (126), while the Suppressor of Cytokine Signaling (SOCS) proteins regulate STAT3 activ ity by binding the gp130 receptor and thereby preventing STAT3 occupancy of the same region (127). The Protein Inhibitor of Activated STATs (PIAS) proteins are responsible for dephosphorylating STAT3, rendering STAT3 monomers favorable for exportin binding, nuclear export and futu re reactivation (128-130). One group has shown that STAT3 binding domains in the SOCS3 pathway are essential for STAT3induced upregulation of SOCS3, implying autoin hibition of IL-6 induced STAT3 signaling under normal circumstances (131). Additional spatio temporal regulation of STAT3 can involve proteolytic processing of STAT3 protein (132), alternative splicing to obtain the STAT3-alpha (wild-type) and STAT3-beta (truncated) isofor ms (133), or p300/CREB-binding protein (CBP)mediated acetylation of a key ly sine residue for proper nuclear export (134), in addition to SUMO-mediated ubiquitination and degradation of the STAT inhibitory phosphatases SOCS and PIAS (130). The STAT3 isoform is naturally present, and acts as a dominant-negative form of STAT3 by binding and inhibiting phosphorylated STAT3 activity or by occupying the STAT3 25

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promoter binding sites and preventing dimer-medi ated transcription (135 ). STAT3 proteins, whether nonphosphorylated or tyrosine phosphorylated, reside in membrane lipid rafts before translocation into the nucleu s (136), and activated STAT3 exhibits a higher rate of nucleocytoplasmic shuttling than non tyrosine -phosphorylated STAT3 (1 37). Significantly, the coiled-coil domain of STAT3 is required for nucle ar translocation as well as nuclear retention (138, 139). STAT3 transport to the nucleus by endocytosis is anothe r physical level of regulation that may be disrupted by endocytic inhibitors (140). Once in the nucleus, STAT3 dimers must bind proper cofactors and coactiv ators such as p300/CBP (134) to properly transactivate target genes. Several studies sugge st that STAT3 transactivation of sites such as the LAP/cEBPbeta promoter ( 141) require STAT3 tethering to nearby DNA through cofactor activation and protei n complex formation. IL-6 Signaling: Normal Cytokine Signaling Versus sIL-6R Trans-Signaling Interleukin-6 (IL-6) cytokine si gnaling (Figure 1-2) occurs when IL-6 ligand binds to the IL-6 receptor (IL-6R), which then heterodimeri zes with the signal transducing receptor, gp130 (142). Upon dimerization the receptors become transa ctivated at key tyrosine sites, to which the soluble Jak kinases bind (143). These activated Jaks then phosphor ylate STATs at the receptor. After phosphorylation on key tyrosine sites the ST ATs diffuse away from the receptor and are free to dimerize and translocate into the nucleus (99). However, th is model is only one part of the IL-6 signaling pathway. Additional stimul ation can also occur th rough a trans-signaling mechanism, in which a soluble form of the IL-6 R (sIL-6R) is present in the extracellular milieu of the cell (144). The sIL-6R binds me mbrane-bound gp130 and potently activates gp130mediated signaling (145-151) (Fi gure 1-3). Interestingly, the ex tracellular domains D1-D3 of gp130 naturally occur as soluble receptors (152 -155), but potently inhibit IL-6R-gp130 mediated signaling (156, 157). 26

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The sIL-6R, which is the extracellular portion of the IL-6R, can be obtained by alternative splicing or by proteolytic cleavage. The ADAMs, or A Disintegrin a nd Metalloproteases (158, 159), cleave proteins such as the IL-6R with in trinsic MMP-like activity (159). In particular, ADAMs 10 and 17 (158) have been demonstrated to be important for cleavage and, therefore, activation of IL-6R and other receptors and ligands such as TNF(160) and Heparin-Binding Epidermal Growth Factor (HB-EGF) (161-163). STAT3 and Cancer STAT3 has varied downstream effects on th e various stages of carcinogenesis and metastasis. STAT3 has been demonstrated to be essential for Src-mediated transformation (6, 7) and IL-6 mediated transformation (164), and is overexpressed in many cancers (8). Exogenous expression of a constitutively activated form of ST AT3 revealed that this transcription factor is required for the development of sk in cancer (165). STAT3 largely contributes to upregulation of genes that are required for cancer pr ocesses such as prolif eration and evasion of apoptosis (117). In addition, STAT3 inhibits p53 function and ca n downregulate p53 at the gene transcription level, leading to abr ogation of p53-mediated DNA repair mech anisms and cell cycle arrest (166) (Figure 1-4). Further, as such a promiscuous agent in the cancer process, STAT3 appears to have a role in every step of carcinogenesis, and correlates with histologic grade in mammary cancers (167) and invasive potential in prostate cancers (168). Blockade of STAT3 in certain systems can not only restore apoptosis but can also induce cell cycle arre st and reduce cellular invasion (169) and cause growth suppression (170). Breast cancer metastasis requires only STAT3 phosphorylation on Tyr (705) and not on Ser (727) (122). STAT3 is important for maintenance of telomerase activity in prostate cance r cells (171) and, perhaps most clinic ally relevant, STAT3 is essential for hypoxia-induced angiogenesis (172-176) and fo r invasion and metastasis (177). IL-6 27

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contributes to multi-drug resistance (178), androg en-independent prostate cancer growth (179), is expressed at high levels in basallike breast cancers (180), and is abundant in the serum of breast cancer patients (181). Further, inhibition of IL -6 function inhibits breast cancer invasiveness (182-184). Other upstream molecules besides IL-6 can drive STAT3 oncogenic activity. In particular, cross-talk with other signaling pathwa ys such as the Epidermal Growth Factor (20) pathway can strongly induce STAT3 activity (185 186). EGF receptor (20) mediated STAT3 tyrosine phosphorylation contributes to cell survival, or evasion of apoptosis, in head and neck cancers (187), and also induces autocrine growth signaling in small cell lung carcinoma cells through STAT3-dependent Leukemia Inhibitory F actor (LIF) secretion (188). LIF expression also contributes to murine mammary carcinom as (189), while EGF signaling can stimulate activation of the soluble Src kinase (107). Diagnostic and Therapeutic Implications STAT3 has gained interest as a drug target due to its overexpression and constitutive activation in a large variety of cancers including breast (190), lung (191), colon (192), and ovarian cancers (193) as well as in Chr onic Myelogenous Leukemia and Acute Myelogenous Leukemia (194, 195), respectively [for a full review, see (196)]. Similarly, STAT3 inhibition can enhance the efficacy of established chemothe rapeutic agents and regimens (197) or in cancers that possess disrupted TGF signaling (198). Many types of STAT3 inhibitors have been developed thus far, including small mol ecule (199-201) and peptide (202, 203) inhibitors. However, no inhibitors targeting STAT3 have progr essed to clinical trials yet; rather, current chemotherapeutic agents have been explored and postclinically demonstrated to have antiSTAT3 mechanisms. Examples of this are Satr aplatin, a tetravalent platinum derivative (197), and Silibinin, an Erk1/Erk2 kinase inhibitor (204). It must be emphasized, however, that STAT3 28

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is a common downstream effector of many upstream receptor and soluble kinases; and therefore many targeted agents are specific for upstream ki nases of STAT3, rather than STAT3 itself. Many anticancer agents have been developed to target the IL-6 Receptor (IL-6R), Epidermal Growth Factor Receptor (20), Src, the Jak kinases, Tumor Necrosis Factor (TNF ), Plateletderived Growth Factor Receptor (PDGFR), and seve ral others. Furthermore, a large number of these agents have progressed to clinical trials (205-219) but no t to combination trials. Many future antitumor regimens are also po ssible that have not yet been tested. STAT3 and TGF : Published Mechanisms of Cross-Talk Although both TGF and STAT3 have been implicated in breast cancer growth and progression, the mechanism of cross-talk between these two pathways has not been extensively characterized. Several studies ha ve provided evidence suggesting TGF to be a factor that suppresses STAT3 tyrosine phosphorylation or Interleukin-6 (IL-6) signaling (220). However, the role of cross-talk between TGF and IL-6 induced STAT3 tyrosine phosphorylation has not been well established in the cancer m ilieu. One study reported TGF stimulation of STAT3 tyrosine phosphorylation (221), while a nother has demonstrated Smad4mediated inhibition of tyrosine phosphorylated ST AT3 in pancreatic cancers (222). However, the mechanisms responsible for these effects and that reconcile thes e various findings are currently not well characterized. Epithelial-To-Mesenchymal Transi tion (EMT) and Breast Cancers The process of EMT has been studied in breas t cancer cell lines (223) but remains elusive in vivo Strikingly, one group has demonstrated a role for the immune syst em in regulation of EMT in vivo promoting the development of breast can cer stem cells (224). Another study has demonstrated the effect of EGFR and STAT3 cro ss-talk and concluded th at this signaling can induce EMT due to STAT3 Ser (727) upregulation of the E-Cadherin repressor molecule Twist 29

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(225). Notably, EGFR/IL-6/STAT3 cross-talk drives EMT in ovarian carcinomas (226) and breast carcinoma lines (227) through IL -6 dependent N-Cadherin upregulation. TGF Induction of EMT Involves Di verse Pathways and Mechanisms Researchers described TGF induction of Epithelial Mesenchyma l Transition (EMT) first in the developmental process, in which EMT is critical for switching apical-basolateral polarity to differentiated tissue consisting of epithelial and mesenchymal cells (228). Later it became apparent that TGF stimulation of EMT was a process subverted by cancer cells to mediate invasion and metastasis (57). Du ring EMT epithelial cells lose th eir polarity and their ability to form cuboidal, tight colonies, and develop into fibroblastic nonpolar si ngle cells that poorly associate into colonies (229). In addition to ch anges in colony formation, the actin cytoskeleton is rearranged from cortical actin into parallel rigid F-actin fibers termed stress fibers (230). Subsequent studies have identifi ed various signaling molecules and pathways as being essential for both the molecular changes and for the change s in the cellular cytoskeleton induced by EMT. An important mediator of actin rearrangement RhoA, and its downstream kinase ROCK are critical for TGF stimulation of the EMT program (229). Intact Smad signaling is required for TGF initiation of EMT, as are Erk2 (231) and th e Erk pathway (232), NF kappa B (233), the EGF Receptor (234), and Snail (235) activity. A dditional molecules and pathways have been identified as critical for EMT-dependent orga nogenesis in other systems (236-240) but will not be discussed herein. TGF is a well-established inducer of stress fiber formation in cells that undergo EMT (57, 231, 241). This entails the reorganization of co rtical actin into filamentous, parallel-oriented actin fibers, termed stress fibers (232). Actin structure and cellular si gnaling are closely linked, as many signaling molecules reside at the sites of focal adhesi ons, where cells attach to a substratum (242). Examples include the solubl e Src kinase, which is well known to be an 30

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activator of STAT3, as well as Focal Adhesion Kinase (92, 243). Forces such as mechanical shear stresses can alter cellular signaling in the ab sence of other ligand stimulation (244). One of the key morphological ch aracteristics of TGF -stimulated EMT is a switch from cuboidal epithelial colonies to mesenchym al, elongated spindle-shaped cells that do not readily form colonies. Therefore, such a dramatic switchwhich is due to TGF treatment in some systemsmust necessarily engage a dramatic reorganization of the actin cytoskeleton (245, 246). The Cadherin Switch: Mediating Changes in Cell-Cell Adhesion and Motility/Invasion As part of the switch from an epithelia l to a mesenchymal cellular phenotype, cells downregulate the adhesion molecule E-Cadherin and upregulate N-Ca dherin (228). This process is mediated by the E-Cadherin repressor protei ns Snail and Slug (247) and the Inhibitor of Differentiation proteins Id1 and Id2 (248), but is still poorly understood. E-Cadherin expressors form tight homophilic bonds and therefore form tighter colonies, while cells expressing NCadherin poorly associate into colonies and are more motile (249). In development the so-called Cadherin Switch is sufficient to induce segregat ion of cells expressing these molecules, but in cancers this event is not well understood. Severa l studies in nontransformed mouse epithelial cells have demonstrated that N-Cadherin expr ession, regardless of E-Cadherin expression levels, is sufficient for cellular invasion (250, 251). Regulation of Eand N-Cadherin Exp ression: A Potential Role for STAT3 The process of Epithelial to Mesenchyma l Transition (EMT) is one that is well characterized in terms of the Cadherin Switch, or the downregulation of the epithelial adhesion molecule E-Cadherin, with concurrent upregula tion of the mesenchymal adhesive N-Cadherin protein (249). These homophilic adhesion molecu les, when expressed on their respective cell types, exhibit differential adhesiveness, with E-Cadherin junctions f acilitating tight colony 31

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formation (252). Conversely, mesenchymal cells expressing N-Cadherin do not readily form colonies (253), and many studies have been perf ormed to elucidate the exact role of these two cadherins in migration and invasiveness in vitro (251, 254-256). For example, the presence of N-Cadherin appears to drive ad hesive behavior, regardless of E-Cadherin expression status (250), at least in certain cell lines. It is important to note that a major system for studying EMT has until recently been via TGF treatment of nontransformed mouse mammar y NMuMG cells (58, 257). However, with the discovery that differe nt signals (225, 226, 233, 235, 238, 258261) can induce an EMT-like phenotype in multiple cell types (227, 236-238, 246, 262-270), scientists have gained more perspective on EMT. Several studies have observe d a correlation between STAT 3 tyrosine phosphoryation and downregulation of E-Cadherin (165, 226, 227, 263, 271273) and that activated STAT3 is also sufficient for upregulation of N-Cadherin (227, 272, 274). Further understanding of this mechanism is complicated by the fact that homotypic cell adhesions, and in particular ECadherin homodimeric complexes, can regulat e STAT3 tyrosine phos phorylation (275). However, the mechanism responsible for STAT3-dependent upregulation of N-Cadherin has not been characterized in detail. In particular, the specific cytokine s and growth factors responsible for these effects have not been implicated, nor has STAT3-mediated N-Cadherin upregulation been placed in the larger context of EMT or in the still more complex part EMT plays in carcinogenesis. Rationale and Study Outline Because of STAT3s varied roles in br east cancer promotion and progression, we hypothesized that tr eatment with TGF which is an important component of the cancer milieu, would result in activation of the STAT3 pathwa y, thereby modeling a possible signaling scenario 32

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in the carcinogenesis process. Thereafter, data suggesting this pathwa y occurs in invasive human breast cancers will emphasize the importance of TGF -STAT3 cross-talk. A major part of this study was to understand some of the crosstalk that occurs in breast cancers, particularly in the critical stages of its development, with the hope that future chemot herapeutic regimens and therapies may be developed based on our research. With these objectives in mind, we designed our study with the purpose of characterizing the mechanism by which TGF induces STAT3 tyrosine phosphorylation. By examining the downstream characters responsible for TGF /Smad and IL-6/STAT3 signaling, we hypothesized that critical members of each pathway were requir ed, and that rather than acting in a manner of cross-signaling, TGF was linearly inducing STAT3 tyro sine phosphorylation through the upregulation of Interleukin-6. Through examinati on of each of these inte ractions we sought to understand the relationship between TGF and STAT3. Clinical Implications and Other Considerations Due to the varied roles of TGF and STAT3 in carcinogenesis, particularly in the later stages of invasion and metastasis, many inhibito rs of these two pathways have already been developed, and some are in various stages of c linical trials (93, 94, 212, 219). However, because cancer development occurs through overexpressi on and cross-activation of many different signaling pathways, it is more than probable that different signaling cross-talk occurs aberrantly. TGF and STAT3 cross-talk can occur in nontrans formed mouse mammary epithelial cells as well as in a luminal human breast cancer cell line. Further, it is endogenous to a basal-like human breast cancer cell line, suggesting a role for TGF stimulation of STAT3 tyrosine phosphorylation in human breast cancer invasiveness. The observation that TGF stimulates STAT3 tyrosine phosphorylation in human breast cancer cell lines has several clinical imp lications. Current inhibitors of TGF and the STAT3 33

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pathway (including the IL-6 ligand and recepto r, Jak, and STAT3 itself) may be used in combination therapy for enhanced efficacy compared to either treatment alone. Further, development of future agents targeting components of both these pathwaysfor example, a dual inhibitor prodrug that is hydroly zed after entry into the cell, ma y thus target both the Jak and TGF kinase activity domains. Conversely, anothe r strategy could be to inhibit extracellular components of this pathway, namely secreted TGF and IL-6. In these ways and many others, we hope that our signaling studies will provide a basis for development of highly optimized, efficacious anti-cancer therapeutics in the future. Figure 1-1. The TGF signaling pathway. 34

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Figure 1-2. Cytokine stimulated STAT3 pathway. 35

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Figure 1-3. Mechanisms of IL-6 trans-signaling. Figure 1-4. The classical cell cycle model. 36

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CHAPTER 2 GENERAL MATERIALS AND METHODS Tissue Culture, Maintenance, and Plating of Cell Lines All cell lines were maintained in 10% FBS-DMEM at 37 C/5% CO 2 Cell cultures were maintained in the exponential growth phase. Cells were trypsinized and counted with a hemocytometer; for western blot experiments cel ls were typically plated at 350,000 cells per P 100 plate and incubated overnight at 37 C. MMTV-D1K2 tumor-de rived fibroblast lines (TDFs) were isolated as previously described (276). Isolation of Mouse Mammary Tumor Associated Myofibroblast Cells and Maintenance of the TDF Cell Line Mouse mammary tumors derived from a Cyclin D1-Cdk2 transgene (D1K2) driven by the Mouse Mammary Tumor Virus (MMTV) promoter we re harvested as previously published (276278). Tumor associated fibroblas ts were collected by differentia l trypsinization. The epithelial tumor cell component was maintained as tumor lines, while the tumor-derived myofibroblast (TDF) cells were characteriz ed as published (276). Tritiated ( 3 H)-Thymidine Incorporation Assays Cells were plated in 24-well pl ates. Treatments were for 24 h in triplicate. During the final two hours of treatment the cells were pulsed with 3 H-thymidine (Perkin Elmer, Boston, MA). Cells were fixed and washed twice with 10% trichloroacetic aci d. After neutralization with 0.2 N sodium hydroxide, suspension aliquots were quantified for radioactivity in a Beckman Coulter scintillat ion counter. Data were normalized to controls. Constructs, Drugs, and Reagents TGF (Millipore, Billerica, MA), Epidermal Growth Factor (EGF, Chemicon, Billerica, MA), Leukemia Inhibitory Factor (LIF, Millipore), Vascular Endot helial Growth Factor (VEGF, Peprotech, Rocky Hill, NJ), Granulocyte Macro phage Colony Stimulating Factor (GM-CSF, 37

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Peprotech), basic Fibroblast Gr owth Factor (bFGF, Biosource, Camarillo, CA), Hepatocyte Growth Factor (HGF, Chemicon), Tumor Necrosis Factor (TNF Chemicon), Leukemia Inhibitory Factor (LIF, Chemicon) Transforming Growth Factor (TGF, Chemicon), Lysophosphatidic Acid (LPA, Sigma, St. Loui s, MO), and sIL-6Ra (R&D Biosystems, Minneapolis, MN) were dissolved in PBS +1% BSA. Recombinant hIL-6 (Sigma) was dissolved in sterile water. The LPA receptor inhibitor VPC51299 was kindly provided by Dr. K. Lynch at the University of Virginia and was dissolved in PBS/DMSO. The inhibitors Stattic (Calbiochem, San Diego, CA), Jak Inhibitor I (Calbiochem), Jak2 inhibitor (AG490, Ca lbiochem), EGFR/Her2 Inhibitor (Calbiochem), T RKI (Calbiochem), SIS3 (Sigma-Aldrich), and GM6001 (Calbiochem) were dissolved in DMSO. Apratoxin A (279-281) was kindly provided by Dr. H. Luesch (University of Florida College of Ph armacy, Gainesville, FL). Wild-type STAT3 construct was obtained from Addgene (Cam bridge, MA) (courtesy of J. Darnell). Inhibition of Actin Polymerization and of Various Signal Transduction Pathways The actin depolymerizing agents Swinholide A, Cytochalasin D, Phalloidin, Latrunculin A, and Jasplakinolide (Calbiochem) were dissolved in DMSO according to the manufacturers instructions. Blockade of the Akt pathway (LY29002, Calbiochem), the Rho kinase ROCK (Y27632, Calbiochem), Hsp90 (Geldanamycin, Calbiochem), and Casein Kinase (CK)1 and CK1 (IC261, Calbiochem) were for 24 h at 37 C. All inhibitors were di ssolved according to the manufacturers instructions. Reverse Transcription Polymera se Chain Reaction (RT-PCR) Cells were plated as described in P 100 culture dishes. After treatments cells were harvested in Trizol reagent (Invitrogen, Carlsbad, CA) and RNA was isolated according to the manufacturers instruct ions. One microgram of RNA was re verse transcribed, and the resulting cDNAs were used for gene amplification by PCR with the following primers: Mouse -Actin, as 38

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in (254) (Forward 5-GTGGGCCG CCCTAGGCACCAG-3, Reverse 5CTCTTTGATGTCACGCACGATTTC-3); GAPDH (5-GAAATGAGCTTGACAAAG-3, Reverse 5CTGGCATGGCCTTCCGTG-3 ); mouse IL-6 (Forward 5ACCACTCCCAACAGACCTGT-3, Reverse 5 -TCCAGTTTGGTAGCATCCAT-3); human IL-6 (Forward 5-AGATTCCAAAGATGTAGCCG-3, Reverse 5TGCCTCTTTGCTGCTTTCAC-3); mouse TNF (Forward, 5ATCGGCTGGCACCACTAGTTG-3, Revers e 5-CCAGACCCTCACACTCAGATCAT-3); mouse/human IL-6R (Forward 5-CTGCCCACATTCCTGGTWG-3, Reverse 5GCTGWTGTCATAAGGGCTC -3, where W is A/ T); and human Cycloph ilin, as in (282). PCR reaction products were generated as follows: melt 96 C 1 min, anneal 60 C 1 min, extend 72 C 1 min; -Actin, as in (254); and mIL-6, melt 96 C 1 min, anneal 62 C 1 min, extend 72 C 15 sec. IL-6R PCR products were synthesi zed with the following to uchdown PCR protocol: 95 C (30 sec), 73.5 63.5 C (45 sec, decreasing 0.5 per cycle), 72 C (1 min), 15 cycles; 95 C (30 sec), 54 C (45 sec), 72 C (1 min), 25 cy cles. PCR products were visualized on a 2% agarose gel. SDS-PAGE and Westen Blot Analysis Cells were plated and treated, then extracts prepared as previously described (276). Proteins were resolved on sodium dodecylsulfate-polyacrylamide gels and transferred to nitrocellulose membranes. Immunoblotting was performed with the foll owing antibodies: Actin (Santa Cruz Biotechnologies, Santa Cruz, CA, 1:5000); STAT3 (Cell Signaling Technologies, Danvers, MA, 1:5000); pSTAT3-Y705 (Cell Signaling, 1:500); His 5 (Qiagen, Germantown, MD, 1:1000); Pim1 (Santa Cruz, 1:500); Src (s c-8995, 1:1000); pSrc-Y416 (Cell Signaling, #2101, 1:500); and pSrc-Y527 (Cell Signaling, #2105, 1:500). Goat anti-rabbit and anti-mouse IgG 39

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secondary antibodies conjugated with alkaline phosphatase (Santa Cruz) allowed band visualization. Conditioned Medium Experiments NMuMG cells were plated as described and treated for 24 h with various treatments. After treatment the conditioned medium (CM) was collected and cleared by centrifugation of cells and debris. NMuMG CMs were stored at 4 C for subsequent assays. The R-1B cell line (34) lacks a functional T RI and was treated with conditi oned medium for examination of TGF -independent signaling (Figure 2-1). Transient Transfections and Luciferase Reporter Assays Cells were transfected with Lipofectamine (Invitrogen), GeneJuice (EMD Biosciences, Madison, WI), or NanoJuice (EMD Biosciences) according to the manufacturers instructions. The STAT3-responsive m67-luciferase reporter construct (0.4 g; courtesy of J. Darnell) (283), pSTAT-luciferase construct (C lontech, Mountain View, CA), or MMP9-luciferase construct (284) (690 bp, courtesy of D. Boyd, MD Ande rson Cancer Center, TX) was transfected as indicated and treated for 24 h; 48 hours post-transfection, cells were lysed. Luciferase assays were performed in triplicate as previously desc ribed (285). Values were normalized to protein concentration. Immunofluorescence Microscopy Cells were plated on sterile gl ass coverslips at low density in 6-well plates. Treatments were for 24 h at 37 C. The next day the adhere nt cells were fixed with 1% paraformaldahyde followed by quenching with 50 mM ammonium chloride. All solutions were supplemented with 0.5% Triton X-100 detergent. Fixed cells were probed with prim ary antibodies (1:100) as for western blot analysis. Goat anti-mouse or rabb it secondary antibodies (1 :200) conjugated with Cy3 and/or Fluorophore 488 (Vector Laboratories, Burlingame, CA ), and DAPI staining (Vector 40

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Laboratories), permitted concurrent visualization of nuclei and proteins. Images were captured on a Leica TCS SP2 camera (Wetzlar, Germa ny) using the Openlab software suite. Functional Inhibition of IL-6 Function with the Receptor fusion protein mIL-6-RFP Transient transfection of the construct mIL6.Receptor Fusion Protein Inhibitor (mIL-6RFP) (286), consisting of the ligand-binding ex tracellular domains of the IL-6R and gp130, was carried out with Lipofectamine (Invitrogen) accord ing to the manufacturers instructions. The human form of this construct has been characte rized elsewhere (287, 288). Protein expression of the receptor fusion protein was monitored with immunoblot analysis with a His 5 antibody that binds the His 6 tag on the C-terminus of the fusion protei n. Functional validation of the construct was demonstrated in the presence of IL-6, w hose effect on STAT3 tyrosine phosphorylation was abrogated upon transfection and subsequent protein expression of the mouse IL-6 receptor fusion protein. Transient transfection of the recepto r fusion protein was sufficient for validation studies, but a more permanent method of recep tor fusion protein collection was required for subsequent assays. Establishment of Clonal Cell Lines Stably Ex pressing the IL-6 Receptor fusion protein 293A cells were transfected w ith Lipofectamine as described above to stably express either pcDNA3 vector control or the IL-6 recepto r fusion protein construct mIL-6-RFP. Positive clones were selected with 1.0 g /ml G418 medium. Clones were is olated by serial dilution and screened for construct expression by His 5 -antibody detection. Antibody Array Analysis Mouse Cytokine Array I (Ray Biotech, No rcross, GA) was purchased and performed according to the manufacturers inst ructions (Figure 2-2). Briefl y, NMuMG cells were plated at subconfluent levels in duplicate and incuba ted in the presence or absence of TGF for 24 or 48 h. After treatments, duplicate CMs and protein extrac ts were pooled. CM samples were diluted 1:1 41

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in Ray Biotech Sample Diluent and placed on the ar ray. Protein extracts were diluted as usual and immunoblotted with STAT3, pSTAT3, or Actin rabbit antibodies to confirm TGF -induced pSTAT3. Array slide images were obtained on a Typhoon 9410 laser scanner. Positive samples were bound to antibodies conjugated with a Cy3-equivalent signal, and this fluorescent signal was detected at 10 pixel resolution. Boyden Chamber Cellular Invasion Assays Invasion assay plates containing Matrigel-coated 8 m-por e membrane well inserts (BD Biosciences, San Jose, CA) were fed with cells ac cording to the manufacturers instructions. An FBS gradient was set up across the membrane, w ith low (0.2% FBS-DMEM) or complete (10% FBS-DMEM) medium in the insert and bottom wells respectively. All treatments were present in both chambers across the membranes. Cells a nd treatments were incubated for 24-72 h at 37 C; inserts were removed and the noninvaded ce lls removed with a cotton swab. The invaded cells were fixed with methanol and stained with crystal violet. Membranes were excised and mounted on microscope slides a nd invaded cells were counted. Image Acquisition and Processing, Data Qu antification, and Statistical Analyses Images of agarose gels were captured with ImageQuant software. Immunofluorescent microscopy samples were photographed with a Leica TCS SP2 camera. Antibody array slides were scanned with a Typhoon 9410 laser scanner. All images were edited with the Adobe Photoshop software suite or densitometrically analyzed with the Image J program (NIH, Bethesda, MD). All numerical data were analy zed with the Microsoft Office Excel program. Statistical analyses were performed with GraphPad Prism software or Microsoft Excel. Statistics were calculated with the tw o-tailed Students unpaired ttest. 42

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Treat for NMuMG cells Conditioned Medium (CM) collection Treat R-1B Cells with CMs R-1B T RI Figure 2-1. Design of Conditioned Me dium (CM) experiments. Figure 2-2. Mouse Cytokine I antibo dy array chip (Ray Biotech). 43

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CHAPTER 3 TGF-BETA INDUCTION OF STAT3 TYROSI NE PHOSPHORYLATION: MECHANISTIC DATA Introduction Published Cross-talk Between Members of the TGF and STAT3 Signaling Pathways Previously, only a handful of studies have demonstrated a link between STAT3 and TGF signaling, and few of those stud ies have investigated this phenomenon in cancers. Several groups have demonstrated an inhibitory role of TGF on IL-6 signaling (220, 289), with particular emphasis on immune system modulat ion (290, 291) and development of the Th17 phenotype (292). One study focused on the effect of TGF on IL-6 expression in a prostate system (293), and another explor ed cross-signaling between TGF and IL-6 pathways in human trabecular cells (294). TGF has varied effects, including imm unosuppression, fibrosis, and wound healing (295-298). However, the role of TGF in carcinogenesis is stil l under debate due to its seemingly contradictory antiand pro-tumorige nic functions (299-301). However, most data suggest a TGF Switch (302, 303) that signif ies the point at which TGF functions are no longer tumor suppressive but oncogenic. TGF signaling is mediated pr imarily through the Smad proteins, which are phosphorylated on key serine/threonine sites (23). Because STAT3 functions as an immunomodulator y factor, much of the research thus far has focused on this role. Quite separately, STAT3 has been heavily implicated in various stages and aspects of carcinogenesis (177, 187, 304) and is required for Src-mediated transformation (6, 7). STAT3 functions through phosphorylation of its activating Ty r (705) site (305). STAT3 phosphorylation induces formation of STAT3 homoor heterodi merization with other STAT family members (101, 306), after which these dime rs are translocated into the nucleus through importins and other proteins (138, 307). STAT 3 dimers bind to STAT3-inducible elements 44

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(SIEs), which are composed of the nuclear sequence AA(N 4-5 )TT, where N designates any base (308). Promoter-bound STAT3 dimers can cause either upregulation or downregulation of its target genes. Examples include upregulation of Bcl-2 (309), Myc and Survivin (118), p21 and Cyclin D1 (109), and downregulation of p53 ( 310) and Fas (311), which leads to enhanced growth rates, survival and evasion of apoptosis. Lysophosphatidic Acid Signaling Lysophosphatidic acid, or LPA, has been imp licated as a negative prognostic marker in several types of cancer, including breast (312, 31 3) and ovary (312, 314). Elevated LPA levels in serum correlates with poor patient prognosis (315-317), while autotaxin/Phospholipase D, the catalytic enzyme responsible for LPA production from Phosphatidic Acid (PA), is also abundant in serum of cancer patients (316, 317). LPA is a phospholipid that is produced from phosphatidic acid by the enzyme Autotaxin, also identified as Phospholipase D (PLD) (318). LPA binds its receptors, the LPA receptors LPA1-3, of the Edg family of receptors (319-321 ). Normal LPA signaling induces mitogenesis and differentiation through reorganization of th e actin cytoskeleton due to Rho, Rac and Cdc42 activation (242, 322, 323), which ar e also stimulated by TGF (324, 325). Considering the potential complexity of thes e pathways and potential downstream effects with regard to TGF and STAT3, we first examined the mechanism by which TGF induced STAT3 tyrosine phosphorylation. Initi al experiments suggested that TGF treatment caused stimulation of STAT3 phosphorylation in an in direct manner, supported in large part by time course data. In line w ith this observation we hypothesized that a growth factor or cytokine was upregulated upon TGF treatment, and was itself responsible for STAT3 tyrosine phosphorylation. The studies in the following secti ons demonstrate this mechanism, as well as other characteristics that we observed in the course of these experiments. 45

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Results TGF Treatment Causes STAT3 Tyrosine Phosphorylation in Various Epithelial Cell Lines Treatment with the growth factor TGF resulted in the phosphorylation of th e activating Tyr (705) site of STAT3 (Fi gure 3-1). Significantly, this effect was observed in both nontransformed cell lines (e.g. NMuMG and Mv1Lu ce lls) as well as in va rious carcinoma lines (breast cancer lines MDA-MB-361, MMTV-D1K2-T 1, and prostate cancer Du145 cells). We examined this mechanism primarily in nont ransformed mouse mammary epithelial NMuMG cells, in which the effects of TGF have already been characteriz ed in terms of Epithelial to Mesenchymal Transition (EMT) si gnaling (231). However, when R-1B cells (34), which lack a functional TGF Type I Receptor (T RI), were treated with TGF no STAT3 tyrosine phosphorylation was observed, indi cating that an intact TGF -T RI signaling interaction is required (Fig. 3-2). This cell lin e was later used to examine TGF -independent signaling, as will be discussed in detail later on. TGF stimulation of p-STAT3 occurred in a dose-dependent manner (Figure 3-2, left panel), with STAT3 tyrosine phosphoryl ation detectable at 0.1 ng/ml TGF treatment. In addition, treatment with 2.5 ng/ml TGF over time caused STAT3 phosphorylation after 4 h (Figure 3-2, right panel). Because direct growth factor signaling is us ually completed within shorter time periods, we hypothesized that an indirect mechanism was responsible for TGF induced STAT3 tyrosine phosphorylation. TGF Stimulation of STAT3 Tyrosine Phosphorylation Does Not Occur Through Cell Cycle Arrest Because TGF potently induces cell cycle arrest (326), we investigated whether cell cycle arrest was stimulating ST AT3 phosphorylation. Cell cycle arrest with low serum (0.2% FBS-DMEM), TGF or the microtubule inhibiting agent Nocodazole (500 nM) was quantified 46

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by 3 H-thymidine incorporation and STAT3 ty rosine phosphoryation was monitored by immunoblot analysis. Although each of these treatments markedly arrested cells, only TGF treatment was sufficient to stimulate tyrosi ne phosphorylation of STAT3 (Figure 3-3). Therefore, we ruled out the possibility that cel l cycle arrest was stimulating STAT3 tyrosine phosphorylation. One hypothesis was that TGF signaling caused Src activatio n, as has been previously demonstrated (327). Therefore, we treated NMuMG cells with TGF and examined Src phosphorylation on Tyr (416) and Tyr (527), the activating and inhibito ry Src phosphosites, respectively. We observed no significant increase in Tyr (4 16) phosphorylation, nor did we observe any decrease in Tyr ( 527) phosphorylation (Figure 3-4), indicating that Src does not significantly participate in TGF induction of STAT3 tyrosine phosphorylation. TGF -Induced STAT3 Tyrosine Phospho rylation Requires Intact TGF Jak, and Smad3 Signaling, But Does Not Require NFB Signaling We hypothesized that TGF treatment was signaling through Smad3 and stimulating STAT3 tyrosine phosphorylation, and that ST AT3 phosphorylation occurred through Jak kinase activity. We treated NMuMG cells with TGF in the presence or absence of a TGF Type I Receptor (T RI) kinase inhibitor (T RKI), a Jak inhibitor, or a specific Smad3 inhibitor (SIS3). Examination of STAT3 tyrosine phosphorylation by immunoblot revealed th at inhibition of any of these molecules was sufficient to abrogate TGF stimulation of STAT3 tyrosine phosphorylation (Figure 3-5). Similarly, because TGF has been demonstrated to induce Nuclear Factor kappa B (NFB) signaling (293), we trea ted NMuMG cells with TGF in the presence or absence of three NFB inhibitors (Bay 11-7082, SN -50, or Parthenolide). Treatment with one or all of these inhibitors was not sufficient to inhibit TGF induction of 47

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STAT3 tyrosine phosphorylation (F igure 3-6), suggesting that NFB signaling is not involved in this particular mechanism. TGF Stimulation of STAT3 Tyrosine Phos phorylation Induces STAT3 Dependent Transcription To examine downstream effects of TGF induction of STAT3 tyrosine phosphorylation, we transfected three STAT3-dependent luciferase constructs into NMuMG or HepG2 cells. The m67-luciferase construct (Courte sy of J. Darnell, (283)) was transfected into NMuMG cells, while the pSTAT (328) and MMP-9 (2.1 kb, courte sy of D. Boyd, MD Anderson Cancer Center (284)) luciferase constructs were transfected in to HepG2 cells, which di splay a high transfection efficiency. Treatment with TGF was sufficient to induce STAT3 dependent luciferase activation approximately two-fold in each case (Figure 3-7) (p=0.0109, p=0.011, and p=0.001 for m67, pSTAT, and MMP9 luciferase activities, respectively, as measured by a two-tailed unpaired Students t -test). These data suggested that TGF stimulation of STAT3 tyrosine phosphorylation induces STAT3-de pendent transcriptional activ ity. Examination of STAT3dependent genes was not performed because prom oter sites of STAT3 target genes are also activated by other transcription factors, incl uding the Smads, therefore complicating and potentially confounding upregulation data. Howe ver, these questions will be addressed in subsequent studies which lie beyond the scope of this particular project. TGF -Induced STAT3 Tyrosine Phosphorylati on Requires the Presence of Serum To investigate whether growth factors in the serum could be responsible for TGF stimulation of STAT3 tyrosine phosphoryl ation, we treated NM uMG cells with TGF in the presence of either complete medium (10% FBS-DM EM) as in previous experiments, or with low serum (0.2% FBS-DMEM). As shown in Figure 3-8, serum is required for TGF stimulation of STAT3 tyrosine phosphorylation. Closer ex amination of samples treated with TGF in the 48

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presence of low serum revealed marginal STAT3 phosphorylation, and complete medium alone was not sufficient to cause STAT3 tyrosine phos phorylation. We hypothesi zed that some factor present in serum was perhaps potenti ating (or was potentiated by) TGF signaling. However, the maximal effect is achieved upon TGF and serum co-treatment, indi cating that a threshold level of serum is required for significant levels of TGF induced STAT3 tyrosine phosphorylation. Inhibition of Various Signalin g Pathways Does Not Block TGF Stimulation of STAT3 Tyrosine Phosphorylation; Only Actin Depo lymerization with Swinholide A Abrogates TGF -Induced STAT3 Tyrosine Phosphorylation Because serum was required for TGF induction of STAT3 tyro sine phosphorylation, we hypothesized that there was a second particip ating pathway, perhaps induced by some serum factor, that together with TGF results in downstream STAT3 tyrosine phosphorylation. To examine this possibility, we screened a panel of growth factors for their ability to recapitulate STAT3 phosphorylation in the presence of TGF and low serum. As shown in Figure 3-9, it appeared that basic Fibroblast Gr owth Factor (bFGF), but not the other growth factors, was able to induce STAT3 phosphorylation in the presence of TGF and low serum. However, subsequent experiments did not su pport this finding, and we therefore ceased pursuit of this hypothesis. Following the latter observation, we screened a panel of inhibitors for their ab ility to inhibit TGF stimulation of STAT3 tyrosine phosphorylation in the presence of serum. Our rationale was that if an inhib itor of Pathway X abrogated TGF -stimulated STAT3 tyrosine phosphorylation, then Pathway X was involved. Therefore, as shown in Figure 3-10, we inhibited the following proteins and pathways: STAT3, Src, ROCK, Actin polymerization, Hsp90, and Casein Kinases delta and epsilon. Th e STAT3 inhibitor Apratoxin A (279-281) and various actin depolymerizing agents (Jasplakinolide, Cytochalasin D, Latrunculin A, Swinholide A, and Phalloidin) were sufficient to inhibit TGF -induced STAT3 tyrosine phosphorylation. 49

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We repeated the assay with the actin depolym erizing agents and found that only Swinholide A was sufficient to block STAT3 phosphorylation (Fi gure 3-11). The mechanism of action of this molecule involves depolymerization of actin filaments and sequestra tion of actin monomers in a 1:2 stoichiometric ratio (one molecule Swinho lide A per two actin monomers) (329, 330). This different mechanism of action may explain w hy Swinholide A, but not other depolymerizing agents that work through diffe rent mechanisms, inhibits TGF stimulation of STAT3 tyrosine phosphorylation. Further examination of other signaling pathways will clarify whether Swinholide A is affecting signaling on a global level, or if there is some level of specificity for this agent. However, such experime nts lie beyond the sc ope of this study. Lysophosphatidic Acid (LPA) Treatment Ca n Fulfill the Serum Requirement for TGF Stimulation of STAT3 Tyro sine Phosphorylation We hypothesized that Lysophospha tidic Acid (LPA) might be th e factor present in serum that is responsible for TGF induction of STAT3 tyrosine phosphorylation. LPA is abundant in fetal bovine serum used to supplement tissue cultu re growth media (315, 331). Therefore, we treated NMuMG cells with TGF and LPA in the presence of low serum (Figure 3-12). The results suggest that seru m depletion can be overcome by LPA treatment for TGF costimulation of STAT3 tyrosine phosphorylation. Notably, thes e effects were also blocked with Swinholide A (Figure 3-13). Although mode st STAT3 phosphorylation is present in low serum with TGF treatment, we hypothesize that a full or maximal effect is achieved only when serum or LPA is also present. LPA Receptor Inhibitor VPC-51299 Inhibits Serum and TGF Co-Stimulation of STAT3 Phosphorylation, But Not TGF -Induced IL-6 Upregulation Thanks to the generosity of Dr. Kevin Lync h (University of Virginia), we pre-treated NMuMG cells with the LPA antagonist VPC51299 fo r 1 h and subsequently treated the cells with TGF and complete medium (10% FBS-DMEM) for 24 h. We expected inhibition of LPA 50

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receptors and, therefore, abrogation of TGF induction of STAT3 phosphorylation. Indeed, LPA inhibition by VPC51299 pretr eatment appeared sufficient to prevent or inhibit TGF and seruminduced STAT3 tyrosine phosphorylation (Figure 3-14). Future experiments, however, will examine the mechanism by which LPA signaling is responsible for TGF co-stimulation of STAT3 tyrosine phosphorylation. Because LPA i nduced signaling is complex and has varied downstream effects, a complex series of genetic and pharmacologic experiments will be required to implicate which LPA receptor is responsible and which substrate downstream of that receptor(s) mediates STAT3 tyrosine phosphorylat ion. These experiments lie beyond the scope of this study. Conditioned Medium from TGF -Treated NMuMG Cells Induces STAT3 Tyrosine Phosphorylation in the T R1-Inactive R-1B Cell Line We hypothesized from time course data that TGF treatment was inducing upregulation of some gene that was itself responsible for STAT3 tyrosine phosphorylation. To confirm this hypothesis, we performed Conditioned Medium (CM) studies in the R-1B cell line, which lacks functional TGF Type I Receptors (T RI). We treated NMuMG cells with TGF or complete medium, collected the CM and treated R-1B mono layers with these CMs. Immunoblot analysis of the R-1B extracts suggested that NMuMG cells treated with TGF secrete some factor into the medium. Therefore, CM stimulation of R-1B cells allowed unknown factor-induced T RIindependent STAT3 tyrosine phosphor ylation (Figure 3-15). NMuMG-TGF -CM caused STAT3 tyrosine phosphorylation in R-1B cells whether R-1Bs were treated for 24 h (Figure 315, left panel) or 1 h (Figure 3-15, right panel) with the CMs. Therefore, TGF induction of STAT3 tyrosine phosphorylation involves some secreted factor that mediates downstream T RIindependent STAT3 phosphorylation. 51

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Leukemia Inhibitory Factor (LIF) Does Not Induce STAT3 Tyrosine Phosphorylation The IL-6 family cytokine Leukemia Inhibito ry Factor (LIF) has a significant role in mammary gland development, lactation, and postlactational involution (332, 333). LIF signals through the interaction of the LIF receptor (L IFR) with gp130, which transduces signals to downstream effectors (102, 334, 335). Because LI F is secreted by breast cancer cells (189, 336338), we hypothesized that TGF treatment could be inducing e xpression of this factor in NMuMG cells. We treated NMuMG cells with exogenous LIF and observed no STAT3 tyrosine phosphorylation, suggesting that the LI FR is not present at any apprec iable levels in this cell line (Figure 3-16). Therefore, we ru led out the possibility that TGF stimulation of STAT3 tyrosine phosphorylation acts through secret ion of the cytokine LIF. TGF Induction of STAT3 Tyrosine Phosphory lation Is Mediated by IL-6 Secretion We next treated NMuMG cells with two poten tial cytokine candidate s, Interleukin-6 (IL6) and Tumor Necrosis Factor (TNF ). Exogenous IL-6 or TNF treatment resulted in STAT3 tyrosine phosphorylation after 24 h (Figure 3-17). However, upon examination of IL-6 gene upregulation by RT-PCR, we observed that TGF treatment was sufficient to cause upregulation of IL-6, but not TNF (Figure 3-18). TGF treatment was also sufficient to induce IL-6 upregulation in a time-dependent manner (Figure 319), with initial IL-6 upregulation detectable after 4 h of TGF treatment and increasing thereafter. Si gnificantly, this pattern recapitulated the time-course of STAT3 tyrosine phosphorylation induced by TGF treatment. IL-6 Treatment is Not Sufficient to Induce Morphological Changes Associated with EMT Although IL-6 and TGF both induce phosphorylation of STAT3 on Tyr (705), only TGF has been demonstrated to indu ce Epithelial to Mesenchymal Tr ansition (EMT) in NMuMG cells in vitro (326). Therefore, we treated these cells with TGF or IL-6 and photographed the cells over subsequent days. We observed that TGF -treated NMuMG cells undergo overt 52

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morphological changes associated with EMT, specif ically a change from c uboidal epithelial cells forming well-delineated colonies to spindle-sh aped mesenchymal-like cells that do not form colonies. Significantly, IL-6 treat ment did not reproduce this effect, even after four days of treatment. Instead we observed that IL-6 treated NMuMG cells formed radially-arranged colonies rather than the classical cobblestone ap pearance displayed by epit helial cells (Figure 320). We concluded that IL-6 treatment, even at longer time points, does not induce morphological cellular changes associated with EM T. This conclusion was significant in our choice of cell line for subsequent invasion assays To distinguish betw een cells that have undergone EMT (which are generally more motile (57)), and those with TGF -stimulated STAT3 tyrosine phosphorylation, we used the M v1Lu cell line in addition to NMuMG cells for invasion assays. These results ar e discussed in detail later. Functional Inhibition of IL-6 with the Receptor fusion protein mIL-6-RFP Abolishes TGF -Induced STAT3 Phosphorylation Because data thus far indicated that TGF treatment induces STAT3 tyrosine phosphorylation and that TGF treatment causes IL-6 upregulation (Figure 3-21), we hypothesized that inhibition of IL-6 would abrogate TGF -induced STAT3 phosphorylation. We obtained a mouse IL-6 receptor fusion protein (courtesy of Dr. G. Mller-Newen, University of Aachen) consisting of the extracellular domai ns of gp130 and the IL-6 receptors (IL-6R), and which has been characterized elsewhere (286-288). After transient transfection of the mouse IL6 Receptor Fusion Protein (mIL-6-RFP) plasmid in to 293A cells, the receptor fusion protein was expressed at the protein level and was detectable by immunoblot due to the C-terminal His 6 tag (Figure 3-22). Further, expression of this pr otein was sufficient to in hibit IL-6 stimulated STAT3 tyrosine phosphorylation (Figure 3-19, right panel). We constructed a cell line stably expressing the mIL-6-RFP fusion protein, which is approximatel y 100 kilodaltons (Fig. 3-23). 53

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Further, we isolated clonal ce ll lines from the parental poly clonal population. Clone 7 of the 293A/mIL-6-RFP cell lines expressed the highest le vel of the His-tagged receptor fusion protein protein compared to the multiclonal (MC) popula tion (Figure 3-24), and this clonal line was maintained and used for all IL-6 in hibition experiments thereafter. Conditioned Medium (CM) from the 293A/mIL-6-RFP-CL7 cell line was collected and used to treat NMuMG cells in the presence or absence of TGF CM containing mIL-6-RFP was sufficient to abrogate TGF -stimulated STAT3 phosphorylation in NMuMG cells (Figure 3-25). Taken together, inhibition of IL-6 attenua ted STAT3 phosphorylation, suggesting that TGF stimulation of IL-6 causes downstream STAT3 phosphorylation. TGF Treatment Confers an IL-6 Dependent In vasive Phenotype on the Nontransformed NMuMG and Mv1Lu Cell Lines Mv1Lu and NMuMG cells were not inherently invasive as measured by cellular invasion through a Matrigel matrix coated 8 m-pore membrane; however, treatment with TGF significantly (Control versus TGF samples: NMuMG cells, p=0.0295; Mv1Lu cells, p=0.0023, as measured by unpaired Students t -test) increased cell invasiveness, which was abrogated with the IL-6 receptor fusion protei n in both NMuMG (Figure 3-26) and Mv1Lu (Figure 3-27) cell lines. Similarly, treatment with IL-6 resulted in increased invasiveness (Control versus IL-6 treatment, p=0.0043, Students t -test) that was abrogated by th e IL-6 receptor fusion protein (Figure 3-28), indicating that IL-6 is necessary and sufficient for NMuMG invasion in vitro. TGF Treatment Induces IL-6 Dependent Upregul ation of the Cell Adhesion Molecule NCadherin Treatment of NMuMG cells with TGF induces activation of Epithelial to Mesenchymal Transition (EMT). This program includes ch anges in morphology and in molecular marker expression, such as the adhesion molecules E-Cadherin and N-Ca dherin (228, 249). Upon treatment with TGF in the presence or absence of the IL -6 receptor fusion protein mIL-6-RFP, 54

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we observed an increase in N-Ca dherin expression that was dependent on IL-6 signaling (Figure 3-29). N-Cadherin expression increased with IL-6 tr eatment, indicating that IL-6 is sufficient for N-Cadherin expression. Further, IL-6 inhibition was sufficien t to inhibit basal and TGF induced N-Cadherin expression. No major cha nges in E-Cadherin expression were observed with TGF treatment and IL-6 blockade; however, because all treatments were for 24 h, it is possible that greater differences might be observe d with longer treatment times and therefore a EMT program that is further progressed. IL-6 Dependent N-Cadherin Transcription is Not Necessary or Sufficient for EMT Although IL-6 inhibition is sufficient to block TGF -induced upregulation of N-Cadherin, examination of the cellular morphology of NMuMG cells in the presence of different treatments indicated that IL-6 inhibition, and subsequently abrogation of N-Cadherin upregulation, was not sufficient to inhibit the morphological changes as sociated with EMT (Figure 3-30). Further, although N-Cadherin is a major factor in the EMT process (251, 339), overt blockade of NCadherin expression by IL-6 inhi bition does not block EMT, indi cating that other markers and processes are responsible for the majo r morphological changes induced by TGF in NMuMG cells. In addition, examination of the subcel lular localization of Nand E-Cadherin by immunofluorescence microscopy conf irmed that N-Cadherin upregulation is IL-6 dependent, as IL-6 treatment is sufficient to induce expres sion of this protein (Figure 3-31). TGF treatment induced upregulation of N-Cadherin, concomitant with internalization of E-Cadherin into the cytoplasm. Taken together, th ese studies indicated that TGF induction of N-Cadherin expression is IL-6 dependent, and that inhibition of N-Cadherin expression is dispensable for the progression of morphological cha nges associated with EMT. 55

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TGF Induces VEGF and GM-CSF Secretion We demonstrated earlier that TGF treatment induces STAT3 ty rosine phosphorylation in NMuMG cells, and that this phosphory lation is likely due to the s ecretion of IL-6. To quantify IL-6 secretion we performed antibody analysis of a mouse cytokine array chip from Ray Biotech. We observed that VEGF and GM-CSF were secr eted in a time-dependent manner after TGF treatment, both cytokines accumulating over 48 h (F igure 3-32). However, no IL-6 secretion was detected. This negative result may be due to assay error, although a mechanism driven by a homologous cytokine or factor effect was not ruled out by this finding. We therefore pursued other methods of implicating IL-6 protein upon TGF treatment, and did not rely on ELISA/antibody-array ty pe technologies. To confirm that VEGF and GM-CSF were not the cytokines responsible for TGF stimulation of STAT3 tyrosine phosphoryla tion, we treated NMuMG cells with exogenous VEGF or GM-CSF to determine whether NMuMG cells can respond to these cytokines. There was no indication that the NMuM G cells responded to these cy tokines in terms of STAT3 tyrosine phosphorylation (Figure 3-33), theref ore discounting the involvement of these two cytokines in our proposed mechanism. These data caused us to consider an alternativ e hypothesis: the secreted factor wasnt IL-6 but the soluble form of the IL-6R (sIL-6R), or the truncated extracellular IL-6R domain. This would account for TGF stimulation of STAT3 tyrosi ne phosphorylation through ligandindependent IL-6R trans-signaling, and this might account for the absence of IL-6 cytokine present in the antibody array result s. Moreover, if IL-6R could oligomerize with the mIL-6-RFP fusion protein, its activity or function might be blocked and therefore abrogate TGF induction of STAT3 tyrosine phosphorylation. 56

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Matrix Metalloproteinase Inhibition Does Not Block TGF Stimulation of STAT3 Tyrosine Phosphorylation Because we hypothesized that TGF stimulation of STAT3 ty rosine phosphorylation was acting through expression and cleav age to generate the soluble form of IL-6R, we opted to block the activity of ADAM-family proteases by using a Matrix Metalloproteinase (MMP) inhibitor. The ADAM proteins proteolytical ly generate such active prot eins as the IL-6R and TNF (158, 159). Therefore, we treated cells with the pa n-MMP small molecule inhibitor GM6001 (340). However, blockade with GM6001 did not abrogate TGF induction of STAT3 tyrosine phosphorylation (Figure 3-34), indi cating that the MMP family ADAM proteins, or other MMP family members, were not contributing to this signaling event. TGF Treatment Induces IL-6R Expression As previously mentioned, one possible m echanism of STAT3 phosphorylation was that TGF treatment causes upregulation of the IL-6 receptor (IL-6R). Numerous studies have already suggested that the soluble form of the IL-6R (sIL-6R) displays agonist activity when bound to IL-6 (148); this mechanism could also be occurring in TGF -treated NMuMG cells if a basal level of IL-6 is also present from serum. Indeed, TGF treatment did result in upregulation of IL-6R as examined by RT-PCR (Figure 3-35). Because alternativ e splicing generates a soluble IL-6R form in human but not mouse cells, we were able to conclude that any resulting IL-6R protein is membrane-localized, and that any soluble IL-6R woul d be obtained only by post-translational modification, i. e., proteolysis. Notably, the IL-6R that we observed by RTPCR was precursor mRNA, and is not yet sp ecified as membrane-bound or soluble IL-6R protein. Therefore, we were una ble to differentiate between these two localized forms at the RNA level. 57

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Exogenous sIL-6R Does Not Recapitulate TGF Stimulation of STAT3 Tyrosine Phosphorylation Treatment of NMuMG cells with recombinan t mouse soluble IL-6 receptor (sIL-6R ) did not fully recapitulate STAT3 tyrosine phosphorylation. Specifically, sIL-6R alone slightly increased STAT3 tyrosine phosphorylati on (Figure 3-36, left panel), but TGF and sIL-6R cotreatment did not induce a synergistic effect on STAT3 tyrosine phosphorylation. Further, cotreatment of NMuMG cells with the receptor fusion protein and sIL-6R did not inhibit STAT3 tyrosine phosphorylation (Figure 3-36, right panel), suggesting that sIL-6R does not oligomerize with the receptor fusi on protein; or, if it does, that it still retains signal transducing ability even in the presence of this fusion pr otein. Taken together these results do not corroborate previous data, which led us to reconsider our hypothesis. Based on these data we c oncluded that although TGF treatment causes upregulation of IL-6R at the gene transcription level, treatment with exogenous sIL-6R does not recapitulate the effects stimulated by TGF treatment. We therefore refocused our efforts on IL-6, and not IL6R, in this mechanism. Conclusions and Discussion We have demonstrated in this chapter that the nontransformed NMuMG and Mv1Lu cell lines respond to TGF treatment by phosphorylation of STAT 3 on its activating Tyrosine (705) site. The significance of this observation is twofold: not only is an indirect mechanism responsible for this phosphoryla tion event, but there is a si gnificant downstream effect on STAT3-dependent transcription and cellu lar invasion. Stimulation with TGF results in upregulation of IL-6, which is the main cytoki ne inducer of STAT3 tyrosine phosphorylation in many systems. We have demonstrated that bloc kade of IL-6 function with a specific receptor fusion protein abrogates TGF induction of STAT3 phosphorylation. IL-6 is also sufficient to 58

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induce cellular invasiveness in NMuMG cells Further, interruption of the TGF -IL-6-STAT3 signaling loop inhibits the cellu lar invasion conferred by TGF treatment in NMuMG and Mv1Lu cells. Finally, TGF treatment causes N-Cadherin upre gulation, which data suggest is IL-6 dependent. This upregulation may play a part in TGF (and therefore, in IL-6) dependent invasiveness. The significance of these observations holds many implications for human cancers. We have observed TGF to induce STAT3 tyrosine phosphorylation after 4 h of TGF treatment. Significantly, longer exposures to TGF as suggested by our datamay be a predisposing factor in the milieu of nontransformed cells in vivo to help promote neoplasia or a locally invasive and perhaps metastatic pheno type. That is, more chronic TGF exposures may be a contributing factor for neoplastic cell s to acquire invasive properties. One significant implication from these obser vations is the fact that the mechanisms contributing to a tumor cells transition from hyper plastic to neoplastic st ates is currently not well characterized. We hypothesize that TGF may play a significant part in this progression, as TGF is known to contribute to both migration (257) and cellular invasiveness (76, 341), as we have observed in this study. Figure 3-1. TGF treatment stimulates STAT3 Tyr (705) in various epithelia l cell lines. 59

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Figure 3-2. TGF -stimulated STAT3 tyrosine phosphoryl ation is doseand time-dependent. Figure 3-3. Cell cycle arrest is not su fficient for STAT3 tyrosine phosphorylation. 60

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Figure 3-4. TGF treatment does not affect Src phos phorylation status in NMuMG cells. Figure 3-5. TGF stimulation of STAT3 tyrosine phosphorylation requires intact TGF /Smad3 and Jak signaling. Figure 3-6. NF-kB activity is not required for TGF stimulation of STAT3 tyrosine phosphorylation. 61

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Figure 3-7. TGF activates STAT3 dependent gene tran scription as measured by luciferase constructs. Figure 3-8. TGF stimulation of STAT3 tyrosine phosphorylation requires the presence of serum. 62

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Figure 3-9. Reintroduction of various growth factors does not fulfill the serum requirement necessary for TGF induced STAT3 phosphorylation. Figure 3-10. Inhibition of various signaling pathways does not block TGF -induced STAT3 tyrosine phosphorylation. Figure 3-11. Swinholide A treatment, but not treatment with other actin depolymerizing agents, is sufficient to abrogate TGF induction of STAT3 tyrosine phosphorylation. 63

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Figure 3-12. Lysophosphatidic Acid (LPA) can replace serum in TGF stimulation of STAT3 tyrosine phosphorylation. Figure 3-13. Treatment with Swinholide A is sufficient to inhibit LPA and TGF -induced STAT3 tyrosine phosphorylation. Figure 3-14. The LPA receptor antagonist VPC51299 blocks serum and TGF costimulation of STAT3 tyrosine phosphorylation. 64

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Figure 3-15. TGF treatment of NMuMG cells yields Conditioned Medium (CM) containing some factor capable of inducing STAT3 tyrosine phosphorylation in a T RI-inactive cell line. Figure 3-16. Leukemia Inhibitory Factor (LIF ) does not induce STAT3 tyrosine phosphorylation in NMuMG cells. Figure 3-17. TNF and IL-6 Both Induce STAT3 Tyrosine Phosphorylation in NMuMG Cells. 65

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Figure 3-18. TGF induces Interleukin-6 (I L-6) upregulation. Figure 3-19. TGF induced Interleukin-6 (IL-6) upregulation is time-dependent. Figure 3-20. IL-6 does not recapitulate morphological changes associated with TGF -stimulated EMT. 66

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Figure 3-21. Tentative pathway describing TGF stimulation of STAT3 tyrosine phosphorylation. Figure 3-22. Characterization of the mouse IL-6 receptor fusion protein, mIL-6-RFP. Actin His5293A/pcDNA3 293A/mIL-6-RFP100 kDa Figure 3-23. Establishment of 293A cell lines stably expressing the IL-6 receptor fu sion protein. Figure 3-24. Isolation of clonal cell lines stably expressing the IL-6 receptor fusion protein. 67

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Figure 3-25. IL-6 functional inhibition by the receptor fusion protein mIL-6-RFP blocks TGF induced STAT3 tyrosine phosphorylation. Figure 3-26. IL-6 treatment is sufficient to induce cellular invasiveness in NMuMG cells. Figure 3-27. TGF treatment confers invasive potentia l on the otherwise nonmigratory NMuMG cell line. 68

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Figure 3-28. TGF treatment confers invasive potentia l on the otherwise nonmigratory Mv1Lu cell line. Figure 3-29. TGF induces upregulation of N-Cadhe rin that is IL-6 dependent. 69

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Figure 3-30. Inhibition of IL-6 function does not block the mo rphological changes associated with TGF -induced EMT. Figure 3-31. TGF induces N-Cadherin upregulation a nd E-Cadherin mislocalization in NMuMG cells; TGF stimulation of N-Cadherin upre gulation is IL-6 dependent. 70

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Figure 3-32. TGF induces secretion of Vascular Endot helial Growth Factor (VEGF) and Granulocyte Macrophage ColonyStimulating Factor (GM-CSF). Figure 3-33. Exogenous VEGF and GM-CSF do not induce STAT3 tyrosine phosphorylation in NMuMG cells. Figure 3-34. Broad-range inhi bition of Matrix Metalloproteinases (MMPs) with the small molecule inhibitor GM6001 does not inhibit TGF stimulation of STAT3 tyrosine phosphorylation. 71

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Figure 3-35. TGF treatment induces gene upregulation of the IL-6 receptor. Figure 3-36. Exogenous soluble IL-6 receptor (sIL-6R ) does not recapitulate TGF stimulation of STAT3 tyrosine phosphorylation. 72

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CHAPTER 4 EVIDENCE FOR TGF-BETA INDUCTION OF STAT3 TYROSINE PHOSPHORYLATION IN HUMAN BREAST CANCER CELL LINES Introduction STAT3 is a transcription factor that has been implicated in various stages of the carcinogenesis process, including growth, inhibition of apoptosis angiogenesis, invasion, and metastasis. Isolation of human breast cancer lines from harvested tumors is one way to study the mechanisms of cancer progression, as well as to develop new therapies without the use of expensive and time-consuming animal models. Recent studies have implemented molecular classification for breast cancer tumor types to cla ssify tumors according to the molecular profile of cancer markers (342-344). For example, identif ication of triple-nega tive breast cancersthe basal-like subclassificationis significant b ecause of the lack of expression of Her2, Progesterone Receptor, and Estroge n Receptor (342). These three re ceptors are the basis of the development of many chemotherapeutic agents and, in the absence of these receptors, few clinical agents are available for patients of triple-negative breast cancers. Therefore, identification of tumor markers can give clues as to the particular pathoge nesis of breast tumors, as well as suggest specific therapies based on the tumors molecular composition. STAT3 is just such a marker. When phosphorylated on its Tyr (705) site, STAT3 correlates with poor patient prognosis (345-347) tumor grade (167, 348, 349), and metastasis (350) in various cancers. Significantly, many i nvasive breast cancer cell lines isolated from tumors display endogenous constitutive STAT3 phosphorylation, but so far the literature has outlined little reason for this phenomenon. Although two published studies (183, 184) indicate that sustained IL-6 secretion drives constitutive STAT3 phosphorylation, the mechanism was not fully characterized. Further, our data suggest that TGF contributes to secretion of IL-6 in the 73

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NMuMG and Mv1Lu cell lines, which is a novel signaling mechanism not previously examined in detail. Strikingly, the luminal-type human breas t cancer cell line MDA-MB-361 (351) does not display constitutive STAT3 tyrosine phosphorylati on, but the basal-like (3 51) breast cancer cell lines MDA-MB-231, BT549, and others do display this phosphorylation event. Using the MDAMB-361 and MDA-MB-231 cell lines as a model for TGF maintenance of IL-6 secretion and, therefore, sustained STAT3 phosphorylation, we have uncovered evidence supporting the presence of this mechanism in human breast cancer lines. To ultimately study the molecular basis behi nd human breast cancer treatment efficacies, one groups approach to understanding breast can cers was to categorize tumors according to molecular marker expression analys is. Srlie et al. ( 342) sorted 65 normal a nd neoplastic breast tissue samples into the following categories: normal breast, luminal A, luminal B, ErbB2overexpressing, and basal-like breast cancers. The human MDA-MB-231 and MDA-MB-361 cells lines have been described as basal-like and luminal, respectively (223). These cell lines were both derived from human breast tumors yet di splay varying levels of various epithelial and mesenchymal markers. Examples include Vi mentin and Smooth Muscle Actin (SMA), both expressed by the basal-like MDA-MB-231 cells; th e luminal marker E-Cadherin is expressed in MDA-MB-361 cells (223). A significant observati on was made regarding the basal-like breast cancer subtype: basal-like breast cancer cell lines display a set of markers that had previously been defined as EMT markers (352). Molecular profiling of breast cance r cells and cell lines enabled such comparisons. Striking similarities may be drawn between EMT and basal markers, offering a functional explanation for breast cancer pathology. The pr operties of basal-like breast cancer cells and having undergone an EMT process both correlate with poor patient prognosis. 74

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Taken together these observations suggest th at there may be some overlapping etiological similarities between the process of EMT and the formation of basal-like breast cancers. Indeed, we have observed some data to this effect in our laboratory, the details of which are still being characterized. Overall, due to the mechanism of TGF induction of STAT3 tyro sine phosphorylation that we characterized in the nontransformed m ouse mammary NMuMG cell line, we had good reason to believe that this mechanism may be either inducible or already o ccurring in human breast cancer cell lines. In line with this reasoning we sought to stimulate TGF /IL-6/STAT3 signaling in the luminal MDA-MB-361 cell line, whic h displays no endogenous STAT3 tyrosine phosphorylation but responds to both IL-6 and TGF in terms of STAT3 phosphorylation. Similarly, we hypothesized that the MDA-MB-231 cell line, which exhibits constitutive STAT3 tyrosine phosphorylation, c onstantly expresses the TGF /IL-6/STAT3 signaling loop, and that this loop may be responsible in large part for the high degree of inva sion observed in the MDAMB-231 cell line. Results Constitutive STAT3 Tyrosine Phosphorylation and IL-6 Upregulation Varies Across Breast Cancer Cell Lines We and others [Unpublished data, and (8)] have observed that differe nt breast cancer cell lines display varying levels of constitutive STAT3 tyrosine phosphorylation. Thus far few (182) papers have investigated the si gnificance of this observation. As indicated in Figure 4-1 and Figure 4-2, there appears to be a correlat ion between constitutive STAT3 tyrosine phosphorylation (Figure 4-1) and endogenous IL-6 upregulation (Figure 4-2) in these cell lines. The noninvasive mouse mammary NMuMG cell line and the human luminal MDA-MB-361 breast cancer cell line do not display STAT3 tyrosine phosphor ylation in the absence of 75

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stimulation, while the invasive and basa l-like BT549, MDA-MB-231, and MDA-MB-468 breast cancer cell lines exhibit constitutive STAT3 tyro sine phosphorylation. Similarly, the MDA-MB361 cell line displays low levels of endogenous IL-6 upregulation, while the MDA-MB-231 and BT549 cell lines produce high levels of IL-6 mRNA. We chose to use the MDA-MB-231 and M DA-MB-361 cell lines as a model for TGF maintenance of STAT3 tyrosine phosphoryl ation. MDA-MB-231 cells display strong constitutive STAT3 tyrosine phosphorylation and IL-6 upregulation, while the MDA-MB-361 cell line does not. Therefore, we subsequently sought to demonstrate stimulation of the TGF IL-6-STAT3 pathway in the MDA-MB-231 cell line and to induce these si gnaling events in the MDA-MB-361 cell line. Conditioned Medium (CM) from Breast Ca ncer Cell Lines Variously Induces STAT3 Tyrosine Phosphorylation and STAT3-Dependent Transcription Conditioned Medium (CM) was collected from th e panel of breast canc er cell lines that was previously examined. The nontransformed R-1B cell line was used to study TGF Type I Receptor (T RI) independent signaling mechanisms. We treated R-1B cells with the breast cancer CMs and examined STAT3 tyrosine phosphorylation by immunoblot analysis. The data indicate that there is a varying level of some cytokine or grow th factor present in the CMs of the breast cancer cells (Figure 4-3), a nd that this cytokine is capab le of inducing STAT3 tyrosine phosphorylation independently of TGF signaling. In addition, we cultured breast cancer cell lines in the presence or absence of a TGF Type I Receptor kinase inhibitor (T RKI) and collected the conditioned medium We transfected the m67-Lu c STAT3 responsive luciferase construct into HepG2 cells due to this cell lines high transfection efficiencies and low endogenous STAT3 tyrosine phosphorylation. Treat ment of the transfected HepG2 cells with the breast cancer CMs (from control or T RKI samples) resulted in varying levels of STAT3 76

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dependent luciferase activation (F igure 4-4, grey bars), corroborati ng the presence of some factor that is capable of inducing STAT3 de pendent transcription. Further, T RKI-CM of MDA-MB231 and Tumor-Derived Fibroblast (TDF) cells induced lower levels of STAT3-dependent luciferase activity (Figure 4-4, open bars), indicating that whatever cytokine is present in the CM of breast cancer cells is dependent on TGF signaling. These data suggest that TGF is inducing expression of a growth factor or cytokine, wh ich we hypothesized to be IL-6, that can induce STAT3 tyrosine phosphorylation. These data imply that this mechanism occurs in certain breast cancer cell lines, as we previously observed in the nontransformed mouse mammary NMuMG cell line. A TGF Signaling Component Contributes to Maintenance of STAT3 Tyrosine Phosphorylation in the MDA-MB-231 Breast Cancer Cell Line The basal-like human MDA-MB-231 breast can cer cell line displays constitutive STAT3 tyrosine phosphorylation, whereas the MDAMB-361 human breast cancer line does not. Therefore, treatment with a TGF Type I Receptor Kinase Inhibitor (T RKI) would indicate whether TGF signaling was involved with this phosphor ylation event. As demonstrated in Figure 4-5, blockade of TGF Type I Receptor kinase activity markedly reduced endogenous STAT3 tyrosine phosphorylation, indicat ing involvement of the Type I TGF Receptor in the potential TGF -IL-6-STAT3 signaling loop. It is signi ficant to note that MDA-MB-231 cells already display high levels of constitutive ST AT3 tyrosine phosphorylat ion, and only marginal increases were observed with TGF or IL-6 stimulation (data not shown); increased phosphorylation may not be possible because STAT3 molecules are already maximally phosphorylated in MDA-MB-231 cells. 77

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Interruption of the TGF -IL-6-pSTAT3 Loop Blocks ST AT3 Phosphorylation, IL-6 Upregulation, and STAT3-Dependent Transc riptional Activity in MBA-MB-231 Cells The IL-6 receptor fusion protein was used to inhibit IL-6 function in the MDA-MB-231 cell line, which produces constitutive IL-6 mRNA. Blockade of this protein would suggest proper IL-6 protein translation a nd regulation, which could contribute to this cell lines invasive and pro-growth properties. Indeed, blockade of IL-6 function with the IL-6 receptor fusion protein blocked both constitutive STAT3 tyrosi ne phosphorylation (Figure 4-6) and IL-6 upregulation (Figure 4-7), sugge sting that not only is TGF involved, but that STAT3 phosphorylation is maintained by IL-6 secretion in th is cell line. Therefore, these data point to a system in which TGF and STAT3 maintain IL-6 upregulat ion, which itself results in STAT3 tyrosine phosphorylation. In addition, transf ection of a STAT3 respons ive luciferase reporter gene (m67-luciferase) into the HepG2 cell lin e and treatment with MDA-MB-231 conditioned medium (CM) revealed that the MDA-MB-231 cells secrete a factor that induces STAT3dependent transcription (Figure 4-8). Further, IL-6 inhibiti on with the mIL-6-RFP receptor fusion protein markedly reduced MDA-MB-231 induced STAT3 dependent transcriptional activation of the m67-luciferase reporter, indica ting that IL-6 is secreted by MDA-MB-231 cells and is responsible for the STAT3-dependent tran scription induced by CM from this cell line. Abrogation of p-STAT3 Abolishes the Intr insic Invasiveness of MDA-MB-231 Cells Because the MDA-MB-231 cell line is intr insically migratory and invasive (353), abrogation at any point in the TGF -IL-6-STAT3 signaling loop should inhibit this cell lines motile and invasive behavior. As sh own in Figure 4-9, inhibition of TGF Type I Receptor kinase activity, Jak activity, IL-6 function, or STAT3 dimerization markedly reduced MDA-MB231 cell invasion (Control versus T RKI, p<0.0001, Students t -test) through a Matrigel-coated 78

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porous membrane. Taken together, STAT3 tyrosine phosphorylation by TGF -induced IL-6 upregulation is required for MDA-MB231 cell motility and invasiveness. Exogenous TGF Induces STAT3 Tyrosine Phosphoryla tion Through IL-6 Upregulation in MDA-MB-361 Cells MDA-MB-361 cells, if all receptors and signa ling are preserved, should result in STAT3 tyrosine phosphorylation upon treatment with exogenous TGF Indeed, treatment with TGF strongly induced STAT3 tyrosine phosphorylation in the MDA-MB -361 cell line (Figure 4-10). Notably, IL-6 treatment caused the same effect: IL-6 treatment stimulated STAT3 tyrosine phosphorylation in MDA-MB-361 cells (Figur e 4-10). Finally, treatment with TGF or IL-6 induced marked IL-6 upregulation in the MDAMB-361 cell line (Figure 4-11), suggesting that this may be responsible for TGF induction of STAT3 tyrosine phosphorylation observed in this cell line (Figure 4-10). Mouse Mammary Tumor-Derived Fibroblast (TDF) Conditioned Medium Recapitulates Exogenous TGF Treatment in MDA-MB-361 Cells It has been demonstrated in many cancers, both histologically a nd pathologically, that tumor-associated fibroblasts are heavily associat ed with the nearby cancer cells (354). Mouse mammary derived tumor associated myofibroblasts (TDFs) were isolated from a basal-like breast cancer mouse model (276, 277) and have been shown to secrete TGF (276). We maintained the stable TDF cell line and collected cond itioned medium (CM) periodically. TDF-CM treatment induced robust STAT3 tyrosine phosphorylation in the human MDAMB-361 breast cancer cell line (Figure 4-10). Strikingly, this effect was inhibited by Jak inhibition with the small molecule Jak inhibito r AG490, by Smad3 inhibition with the SIS3 small molecule inhibitor, a nd by inhibition of TGF receptor kinase activity with the small molecule kinase inhibitor T RKI. This effect is significant because MDA-MB-361 cells can respond to TDF-CM by exhibiting STAT3 tyrosine phosphor ylation, and because th ese tumor-associated 79

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fibroblasts are likely a significant source of TGF as we have observed to be the case in TDFs and which has been published for othe r fibroblastic cell lines (355). TGF Treatment Confers the Invasive Phenot ype on the Otherwise Noninvasive MDAMB-361 Cell Line, Possibly Through TGF Induction of N-Cadherin Upregulation The MDA-MB-361 cell line was plated with va rious treatments onto Matrigel-coated 8 m-pore membranes and incubated with an FBS gr adient for 72 h at 37 C. We observed that MDA-MB-361 cells are minimally invasive, but th at invasiveness is great ly enhanced (Control versus TGF samples, p=0.0007, as measured by unpaired Students t -test) with TGF treatment, and that this increase in invasion is markedly i nhibited by IL-6 inhibition (Figure 4-12). It is important to note that MDA-MB-361 cells possess a high degree of homotypic cell-cell adhesion, with formation of colonies due to ti ght junction formation (356), partially explaining the low intrinsic invasiveness: whole colonies cannot squeeze through an 8 m-pore as easily as a single, nonadherent cell. Along the same line of reasoning, we treated MDA-MB-361 cells with TGF or IL-6 and performed immunoblot analys is to examine N-Cadherin expression. We observed that TGF induced marked expression of N-Ca dherin, as did IL-6 (Figure 4-13). Inhibition of IL-6 function was sufficient to abrogate N-Cadherin upreg ulation induced by both TGF and by IL-6 treatment. Therefore, because N-Cadherin has been demonstrated to be sufficient for cellular motility and invasion (250), we hypothesize that a major mechanism of TGF -induced invasion in MDA-MB-361 cells is due to IL-6 dependent N-Cadherin upregulation. Finally, based on these data we constructed a model of TGF maintenance of IL-6 dependent STAT3 tyrosine phosphorylation in MDA-MB-231 cells (Figure 4-14), and a model for TGF induction of IL-6 dependent tyrosine p hosphorylation in MDA-MB-361 cells (Figure 4-14). These two cell lines demonstrate that the TGF -IL-6-STAT3 signaling loop we observed 80

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in nontransformed NMuMG cells is present or inducible in two human breast cancer cell lines, suggesting that these signaling events may be occurring in human breast cancer in vivo Conclusions and Discussion In previous chapters we have outlined the signif icance of the TGF -IL-6-STAT3 mechanism in the greater context of carcinogenes is literature, and have presented data supporting this mechanism in nontransformed mouse mammary epithelial cells. However, the question of whether this mechanism operates in human breast cancer cell lines remained unanswered. We have demonstrated in this chapter that not only do human breast cancer cell lines display varying levels of STAT3 tyrosine phosph orylation and IL-6 upregulation, but that the basal-like MDA-MB-231 breast can cer cell line displays TGF -dependent STAT3 phosphorylation and IL-6. The signi ficance of this observation ca rries several implications. First, because TGF maintenance of IL-6 secretion a nd STAT3 tyrosine phosphorylation is occurring in this cell line, some genetic or perhap s epigenetic mechanisms are present that enable this mechanism to occur unabated. One possibl e explanation could be the downregulation of such STAT3 regulators as the SOCS and SH P2 proteins, which are downregulated or dysregulated in breast cancers and other cancers (310, 357-362). In an immune setting TGF can lead to SOCS3 downregulati on (363), a potential mechanism that might be occurring in this system as well. The second implication for the presence of the TGF -IL-6-STAT3 signaling loop in MDA-MB-231 cells concerns invasiveness. Each of these three molecules contributes to cellular migration and invasion in some system, but no st udies have demonstrated that these are all involved in one pathway and are to gether responsible for inducing cellular invasiveness. To our knowledge this is the first study demonstrating th at this mechanism confers cellular invasion on otherwise noninvasive cells, as we have shown with the MDA-MB-361 cell line. 81

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Finally, the clinical implications ar e of some significance. Current TGF and IL-6 family inhibitors are in various stages of clinical trials; however, comb ination therapy of these agents with current chemotherapeutic agents that have been post-clinically dem onstrated to act through the TGF signaling pathway holds encouraging promis e for greater efficacy against cancers. Such combination regimens, once optimized, may potentially target tumor cells with greater selectivity, and therefore with fewer harmfu l and toxic side effects to the patient. Figure 4-1. STAT3 expression and tyrosine phosphorylation vary across nontransformed epithelial and carcinoma cell lines. Figure 4-2. IL-6 upregulation appears to co rrelate with constitutive STAT3 tyrosine phosphorylation. Figure 4-3. Treatment with conditioned medium from various breast cancer cell lines results in differential STAT3 tyrosine phosphorylation in a T RI-inactive cell line. 82

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Figure 4-4. Treatment with CMs of various breas t cancer cell lines induces varying levels of STAT3-responsive m67-luciferase ac tivity that appears to be TGF dependent in MDA-MB-231 and Tumor-Deriv ed Fibroblasts (TDFs). Figure 4-5. TGF drives maintenance of STAT 3 tyrosine phosphorylation. 83

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Figure 4-6. The invasive basa l-like MDA-MB-231 breast cancer line exhibits constitutive STAT3 tyrosine phosphorylation that is TGF and IL-6 dependent. Figure 4-7. The MDA-MB-231 breast cancer line exhibits constitutiv e IL-6 upregulation that is TGF and IL-6 dependent. Figure 4-8. The invasive basallike MDA-MB-231 breast cancer li ne displays STAT3 dependent luciferase transcription activ ity that is IL-6 dependent. 84

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Figure 4-9. MDA-MB231 invasion is TGF and IL-6 dependent. Figure 4-10. Tumor-derived fibroblast conditioned medium induces TGF and Jak-dependent STAT3 tyrosine phosphorylation in the MDA-MB-361 cell line. Figure 4-11. IL-6 and TGF treatment induce IL-6 gene upregulation in the MDA-MB-361 human breast cancer cell line. 85

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Figure 4-12. MDA-MB-361 cells acqui re the invasive phenotype upon TGF treatment; this invasion is dependent on IL-6. Figure 4-13. IL-6 and TGF treatment induces N-Cadherin up regulation in MDA-MB-361 cells; Blockade of IL-6 function blocks N-Cadherin upregulation. Figure 4-14. Proposed model for TGF and IL-6 dependent si gnaling in MDA-MB-231 and MDA-MB-361 human breast cancer cell lines. 86

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CHAPTER 5 TOWARDS AN OVERALL CONCLUSION FOR TGF-BETA STIMULATION OF STAT3 TYROSINE PHOSPHORYLATION Overall Findings and Significance We have described herein a mechanism of TGF induction of STAT3 tyrosine phosphorylation. This phosphorylation is due to the upregulation of the cytokine IL-6; downstream effects of this upregulation include STAT3 tyrosine phosphorylation and acquisition of cellular invasiveness, poten tially due to IL-6 dependent N-Cadherin upregulation. TGF induction and maintenance of STAT3 tyrosine phosphorylation through IL-6 upregulation is present in a basal-like model of breast cancer, the human MDA-MB-231 cell line. Abrogation of the TGF -IL-6-STAT3 signaling loop i nhibits MDA-MB-231 invasion, and stimulation with TGF or IL-6 induces an IL-6 dependent increas e in STAT3 tyrosine phosphorylation in the human MDA-MB-361 breast cancer cell line, which does not display endogenous STAT3 tyrosine phosphorylation. There may be other aspects of TGF induction of STAT3 phosphorylation that are of importance: namely, the serum requirement of TGF stimulation of STAT3 phosphorylation, the exact role of the STAT3 Ser (727) site a nd its relationship to the tyrosine site and transcriptional significan ce, and whether LPA is involved in these mechanisms. It may be that these topics are artifacts of TGF -induced Epithelial to Mesenchymal Transition (EMT). In line with this hypothesis is our finding that IL-6 expression is responsible for N-Cadherin upregulation, possibly driving the invasive behavior that we have also demonstrated to be IL-6 dependent. In addition, IL-6 treatment is not sufficient to drive the morphological changes associated with EMT, while blockade of IL -6and ostensibly N-Cadherin expressionis not sufficient to block the morphological changes to a fibroblastic phenotype. Together, these data suggest that N-Cadherin expressi on is dispensable for morphological changes associated with 87

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EMT, and that it is insufficient to induce these changes; but that IL-6 induction of N-Cadherin expression appears to correlate with TGF induced invasiveness. Therefore, use of the IL-6R antibody Tocilizumab, or some other IL-6 targeted agent, might reduce the invasiveness of breast cancer cells in vivo In reality, however, EMT is a compli cated process and is only beginning to be well characterized using in vitro models. Many signaling path ways and molecules contribute to this program, which is inducible only in a few cell lines (59), and many researchers are attempting to investigate the process of EMT in the carcinogenic process in vivo Discussion and Future Work We have described a mechanism of TGF induction of STAT3 tyrosine phosphorylation through IL-6 upregulation and secretion. Protei n inhibition of IL-6 w ith the receptor fusion protein mIL-6-RFP blocks TGF stimulated STAT3 tyrosine phosphorylation. This new mechanism of STAT3 activation is significant because it appears to be necessary for cellular invasiveness of nontransformed mammary epithelia l cells. Although others have demonstrated that TGF induces cellular invasion (84), the mechanism responsible for this effect was not well characterized. Further, TGF treatment induces EMT in NMuMG cells; however, IL-6 treatment does not recapitulate the mesenc hymal phenotype, and is required for N-Cadherin expression and TGF stimulation of cellular invasion. TGF treatment in the presence of the IL-6 receptor fusion protein is sufficient to bl ock N-Cadherin expressi on and invasion, but is not sufficient to inhibit the morphological cha nges associated with EMT. There are several implications for these results Others have demons trated that N-Cadherin expression is sufficient for motility and invasion (250). However, our data suggest that NCadherin expression is not necessary for the morphological switch to a mesenchymal phenotype, but may mediate TGF -induced invasiveness. 88

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We have demonstrated the presence of this pathway in a human breast cancer cell line, the basal-like (351) MDA-MB-231 cells. Significantly, this cell line displa ys constitutive STAT3 tyrosine phosphorylation and IL-6 upre gulation, which both appear to be TGF dependent. Similarly, interruption of the TGF -IL-6-STAT3 pathway at any point resulted in abrogation of IL-6 upregulation and STAT3 tyrosine phosphoryl ation, and in marked inhibition of MDA-MB231 cellular invasiveness. The breast cancer subtypes, including basallike breast cancers, are defined based on the gene expression profiles of br east tumors (342, 364). Significantl y, basal-like breast cancers do not express Estrogen Receptor (ER), Progesterone Receptor (PR), or the Her2 receptor, and are referred to as Triple Negative breast cancers. Because many current breast cancer therapies target one of these three receptors, coupled with the fact that basal-like breast cancers predict a poor patient prognosis, basic understanding of the m echanisms of breast cancer development is more crucial than ever for the development of ne w therapies against this highly invasive breast cancer subtype. The overlap of basal-like breast cancer marker s with EMT markers in breast samples has only strengthened the hypothesis that development of basal-like breast cancers and the EMT phenotype might share si milar molecular underpinnings. Future studies of STAT3 signaling will c oncentrate on the characterization of its activation upon TGF treatment: co-immunoprecipitation an d complex formation experiments will elucidate the binding partners that are responsible for STAT3 tyrosine phosphorylation, which current data suggest are Jak kinases. Furt her, a relevant question that was examined in this study but not discussed is the role of the ST AT3 Ser (727) site: much debate persists over what role this phospho-site plays in STAT3 gene tr anscription. Future work may implicate this phospho-site in, for example, transc ription of certain sets of gene s that are distinct from other 89

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gene sets that are induced by tyrosine-phosphorylated STAT3. A large amount of work would be necessary to address this question, and is best served by future studies which lie beyond the scope of this project. Other relevant and immediate questions linger concerning TGF stimulation of STAT3 tyrosine phosphorylation. There may be othe r cytokines or growth factors induced by TGF that are also, or instead, responsible for STAT3 tyrosi ne phosphorylation, rather than IL-6; one set of experiments that address this question is the use of IL-6 promoter luciferase reporter constructs that have various transcription factor binding sites mutated to abolish binding ability (available from the Belgian Coordinated Collection of Microorganisms, LMBP/BCCM). In this way examination of the promoter region, and ther efore of the relevant structure-function relationships, can be attributable to Smad activity and not other fact ors. Removal of the relevant transcription factor binding s ite should produce no reporter ac tivity, and should also abolish TGF induced STAT3 tyrosine phosphorylation in tran sfected cells. Ideally, data would reveal that Smad activity is responsible for IL-6 gene transcription, and Chromatin Immunoprecipitation (ChIP) anal ysis would corroborate identif ication of the Smad binding regions of the promoter. If Smads are causing ac tivation of other transcri ption factors such as AP-1, then siRNA knockdown of the transcriptio n factors might constitute an experiment addressing this possibility. A second set of future experiments would address the question of EMT and N-Cadherin, and to what extent STAT3 and IL -6 are involved. For example, is IL-6 secretionand therefore STAT3 tyrosine phosphorylationnecessary for only N-Cadherin expression in TGF -induced EMT, or is it just an artifact of the morphologi cal changes and molecular upheaval that occurs? Further, are all these events present in tissues and neoplastic cells that undergo EMT in vivo ? An 90

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intriguing aspect of TGF -induced EMT is the role IL-6 a nd STAT3 tyrosine phosphorylation occupy in this complex process. Some studies have already identified a role for STAT5 in EMT (365). Because STAT5 and STAT3 share many oncogenic functions and both correlate with poor outcome in breast cancer patients (366, 367), it is plausible that STAT3 may participate in EMT as well. In line with this reasoning, our preliminary data have demonstrated that IL-6 is responsible for upregulation of the homophilic adhesion molecule N-Cadherin. However, blockade of IL-6 function, a nd therefore of N-Cadherin upreg ulation, does not inhibit the morphological changes associated with EMT, but does inhibit cellular inva siveness. Therefore, an interesting question is whet her N-Cadherin expression itself is sufficient for cellular invasion in this cell line. Indeed, Johnson and Wheelock (230, 249-251, 255, 339, 368-380) have examined this process in great detail in vari ous cell types, and have demonstrated that NCadherin, and not E-Cadherin, ex pression is essential for EMT induced motility and progression (250). However, it must be noted that am ong NMuMG cell lines, many clones exist that can greatly differ in char acteristics and TGF responsiveness between different laboratories. Therefore, caution must be exercised in examin ation of similar processes in the same cell line among different clones. We have also observed that NMuMG cells lose TGF responsiveness over longer tissue culture passages. The late-p assage NMuMG cells become spindle-shaped, begin to display endogenous STAT3 tyrosine phosphorylation, and appe ar to secrete TGF as measured by Pai-1 luciferase assays (data not shown), altogether rese mbling cells that have undergone EMT. Significantly, this brings up the question of whether more invasive and mesenchymal-type NMuMG cells can be induc ed by culturing cell populations over longer periods of time with many passages, or possibly with persistent TGF treatment until matricrine release. This model may be a more accurate representation of the cancer milieu, in which TGF 91

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resides over long periods. Establishment of such a cell line would imply a correlation between earlier-passage cells, which are epithelial in be havior and marker expression, and low cellular invasiveness; and conversely, cells th at have been cultured with TGF or that have undergone an EMT-like process over long culture periods, beco me more mesenchymal-like and therefore more invasive. In this way it might be possible to draw more parallels in vitro between the EMT process and the basal-like phenot ype, supporting gene array analysis data already published for several breast cancer ce ll lines (352). Similarly, but perhaps of more clinical relevance, future studies will be required that concentrate on the role of TGF induced STAT3 activation in breast cancer development and invasion. An important link with human breast cancers, moreover, should be forged for proper examination of this mechanism. For example, biochemical analysis of human breast cancer tissues would suggest whether constitutive STAT3 phosphorylation is present; correlative analysis by immunohistochemistry woul d demonstrate a link between TGF and IL-6-JakSTAT3 signaling. Tissue MicroArray (TMA) studie s would facilitate this observation. Mouse studies that examine this problem would require genetic alterations of breast cancer cell lines. For example, the human breast cancer MDA-MB231 cells might be altered to overexpress a dominant negative version of the Type I TGF Receptor (T RI), the mIL-6-RFP receptor fusion protein, or to express siRNA direct ed to IL-6. These cell lines, together with the parental MDAMB-231 cell line, would constitute a matched set of cell lines that could be used to examine tumor growth in nude mice. If, for example, mice injected with parental MDA-MB-231 cells reach a particular tumor volume sooner than mi ce injected with MDA-MB-231 cells engineered to overexpress IL-6 siRNA, then certainly tumor growth could be attr ibuted at least in part to IL6 secretion; and reintroduction of exogenous siRNA-resistant IL-6 would restore a pro-growth 92

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phenotype. Conversely, blockade of TGF and IL-6 function in mice implanted with parental MDA-MB-231 cells would abrogate not only tumor growth, but also tumor invasiveness and metastatic ability. In addition, genetic anal ysis would be performed on the MDA-MB-361 cell line. For example, constitutive expression of TGF IL-6, or a constitutively activated STAT3 construct (STAT3-C) should confer growth and invasive advantages, possibly through the upregulation of such factors as N-Cadherin. Furt her, mouse studies would confirm the presence of the TGF -IL-6-STAT3 signaling mechanism in vivo Examples of these mouse studies that would elucidate the relationship between Tumor-Derived Fibroblasts (TDFs) and cancer cells would be to inject the mammary glands of nude mice with either MDA-MB-361 or TumorDerived Fibroblasts alone, or to co-inject these cell lines as a mixed population. If there is crosstalk between the two cell types in vivo there should be a marked increase in tumor growth and local invasion for co-injected sites compared to either site alone. After harvesting tumors we would examine the levels of STAT3 tyrosine phosphorylation in tumor cell extracts; MDA-MB361 cells should respond to factors such as TGF and IL-6 that are secreted from the co-injected TDFs and are therefore present in the tumor microe nvironment. In this way we would be able to quantify the growth advantag e the MDA-MB-361 cells would have. Immunohistochemical analysis of tumor sections would allow identification of TDFs a nd cancer cells, and would reveal whether there is fibroblast infiltration into MDA-MB-361 tumors. If TDFs infiltrated amongst the cancer cells, this would suggest that the st rong homotypic adhesions normally present in MDA-MB-361 cells are disrupted, perhaps due to IL-6 depende nt N-Cadherin upregulation. Such tumor studies would yiel d valuable data about the in vivo implications of the TGF -IL-6STAT3 signaling loop, and whether it confers height ened invasiveness and growth advantages in a mouse model. 93

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Conclusions Significant portions of the lite rature have focused on TGF and STAT3 as oncogenic factors that contribute to carcinogenesis and cancer invasivene ss. However, few studies have focused on the interplay between th ese two pathways. Here we have presented data that suggest TGF induces STAT3 tyrosine phosphorylation th rough upregulation of the cytokine IL-6. Significantly, this occurs only with longer exposures to TGF and the effect increases thereafter, presumably due to accumulation of IL-6 in the ex tracellular milieu. This may be a model of the chronic growth factor exposures that are presen t in precancerous lesions. In addition, these chronic exposures confer a migratory and i nvasive phenotype in the otherwise non-motile Mv1Lu cells. Significantly, these effect s are highly dependent on intact TGF and IL-6-STAT3 signaling. Examination of these same signali ng pathways in a human model of breast cancer, the basal-like MDA-MB-231 cell line reveals the presence of this mechanism in this cell line. The prevalence of this mechanism is not know n among breast cancer cell lines; however, we hypothesize that it is active in cell lines that e xpress constitutively phos phorylated STAT3. Our hope is that this study and its associated publicat ions will one day help dr ive the development of anticancer agents and therapeutic regimens that possess higher selectivity and efficacy in patients with fewer associated side-effects and less collateral damage. 94

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BIOGRAPHICAL SKETCH Nicole Nadine Parker (ne Teoh) was born in 1982 in Fort Wayne, Indiana, to William and Nihal Teoh. She grew up in vari ous cities in Alabama, gradua ting from the Alabama School of Mathematics and Science in 2000. She attended McGill University and did her undergraduate research with Dr. Bertrand Jean-Claude in the Cancer Drug Research Laboratory. After graduating in December 2004 with a Bachelor of Ar ts in Economics with a minor in Biology, she moved to Gainesville, Florida and began workin g on osteoporosis in Dr. Tom Wronskis lab at the University of Florida. Upon her acceptance to the Interdisciplinary Program (IDP) in the College of Medicine, she received an Alumni Fellowship and has completed her graduate research under the mentorship of Dr. Brian Law. She plans to enter the field of science and medical writing and editing following her dissertation defense and subsequent degree conferral. 126