This thesis submitted by Erik Freiter examines the role of integrin signaling and Src tyrosine kinase in cytoskeletal rearrangement during melanoma metastasis. Freiter hypothesized that Src activity reduces RhoA-mediated cytoskeletal rearrangement. Using cultured metastatic melanoma cells and primary melanocytes, Freiter altered Src activity exogenously. He observed an increase in GTP-bound RhoA when Src activity was decreased but no change in GTP-bound Rac1. The data suggest RhoA is involved in cytoskeletal remodeling downstream of Src in melanoma cells. Src may activate the regulator p190RhoGAP, reducing GTP-bound RhoA levels. Rac1 did not appear to be activated by Src in this model.

1. INTEGRIN SIGNALING AND CANCER
A thesis submitted in partial fulfillment of
The requirements for the degree of
Master of Science
By
Erik Freiter
To
Biology Department
Grand Valley State University
Allendale, MI
15 July 2008

2. GRAND VALLEY STATE UNIVERSITY
Biology Department
Allendale, MI 49401
College of Liberal Arts and Sciences
Graduate Student:__Erik Freiter_____
This thesis is accepted in partial fulfillment of the requirements for the degree of Master’s
of Science in Biology.
Graduate Committee Chair________________________________________Date______
Graduate Committee Member______________________________________Date______
Graduate Committee Member______________________________________Date______
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3. ACKNOWLEDGEMENTS
I would like to thank each of the members of my Graduate Committee for their support,
guidance, and consideration throughout the preparation of this work.
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4. ABSTRACT
INTEGRIN SIGNAL TRANSDUCTION
AND MELANOMA METASTASIS
By Erik Freiter
The αvβ3 integrin is thought to play an important role in melanoma metastasis.
We hypothesized that Src tyrosine kinase mediates integrin-initiated pathways
involved in cytoskeletal actin remodeling. Using a panel of cultured metastatic
melanoma cells and primary melanocytes as a model system, we exogenously
altered the cellular activity of Src. We witnessed an increase in GTP bound
RhoA-GTPase, but not GTP bound Rac1-GTPase. Our data suggest RhoA is an
effector molecule in cytoskeletal remodeling in our metastatic melanoma cell line.
Src appears to mediate a signal transduction cascade in which Src activates the
regulator protein p190RhoGAP which results in reduced GTP bound RhoA in
cultured metastatic melanoma. Our data do not point to Rac1 as an activated
substrate of Src in our melanoma model.
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5. TABLE OF CONTENTS
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii
CHAPTER
I. INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Cancer in the US: A disease in Flux. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
What is cancer? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Skin – Physiological Barrier to the Environment. . . . . . . . . . . . . . . . . . 4
Melanoma – a Deadly Skin Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Incorrect Signal Regulation – a Hallmark of Melanoma. . . . . . . . . . . . . 8
Integrins – Adhesion Receptor Proteins . . . . . . . . . . . . . . . . . . . . . . . . 11
Integrins in the Context of Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
The αvβ3 Integrin – Differential Migration and Invasion
of Cultured Melanoma Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Differential Regulation of the Tumor Cytoskeleton . . . . . . . . . . . . . . 18
RhoA GTPase in Cytoskeletal Arrangement . . . . . . . . . . . . . . . . . . . . 19
A Possible Role for Rac1 in Cytoskeletal Rearrangements . . . . . . . . . 20
Hypothesis: Src Activity Reduces RhoA Mediated
Cytoskeletal Rearrangement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
II. MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
SU6656 and PP2 Kinase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
Src Plasmid Constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Src Retrovirus Production and Infection . . . . . . . . . . . . . . . . . . . . . . . 25
Adhesion Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Production of GST beads for RhoA-GTP or Rac1-GTP
Affinity Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Preparation of GTP-bound Cell Lysates. . . . . . . . . . . . . . . . . . . . . . . . 30
Electrophoresis and Western Blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Antibodies and Associated Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
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6. III. RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Src Expression in NHEM, M14, and C8161.9 cell lines . . . . . . . . . . . 33
Src Effects on RhoA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Exogenous Src Expression in C8161.9 Cells . . . . . . . . . . . . . . . . . . . . 36
Src Alters RhoA-GTP Loading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Src Does not Effect Rac1 Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
IV. DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Literature Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
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7. LIST OF TABLES
TABLE PAGE
1. Percentage of US adults who were current, former, or never smokers . . . . . . . . 2
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8. LIST OF FIGURES
FIGURE PAGE
1. Skin is comprised of three layers the epidermis, dermis and hypodermis . . . . . . 5
2. Melanoma incidence rates from 1975 to 2004. . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3. B-RAF mutations in tissues collected from melanoma biopsy . . . . . . . . . . . . . 10
4. The Integrin Receptor Family. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5. Integrin engagement promotes various signaling events . . . . . . . . . . . . . . . . . . 15
6. The αvβ3 integrin exists as a dimer of two subunits . . . . . . . . . . . . . . . . . . . . . . 16
7. Proposed mechanism of Rho and Rac GTPase activation . . . . . . . . . . . . . . . . . 23
8. Src is more active in metastatic melanoma than in melanocytes . . . . . . . . . . . . 34
9. Treatment with PP2 or SU6656 decreases phosphorylation of p190RhoGAP. . 35
10. Kinase inhibitors PP2 or SU6656 increase GTP loading on Rho . . . . . . . . . . 36
11. Virus mediated overexpression of wild type or dominant inhibitory Src. . . . . 36
12. Dominant inhibitory Src decreases phosphorylation of p190RhoGAP . . . . . . 37
13. Dominant inhibitory Src increases loading of GTP on RhoA . . . . . . . . . . . . . 38
14. Dominant inhibitory Src does not increase loading of GTP on Rac. . . . . . . . . 39
viii