Non-mutated Regulators of Cancer Growth in Basal-like Breast Cancer and Transformed Colon ...

162 Pages·2015·1.3 MB·English

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Non-mutated Regulators of Cancer Growth in Basal-like Breast Cancer and Transformed
Colon Cells
by
Anwesha Ghosh

Submitted in Partial Fulfillment of the
Requirements for the Degree
Doctor of Philosophy

Supervised by
Dr. Helene R. McMurray, PhD

Department of Biology
Arts, Sciences and Engineering
School of Arts and Sciences
University of Rochester
Rochester, New York
2015
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Dedication
This thesis is dedicated to my late grandmother, Mrs. Bharati Sen, who taught me to go out there
and search for the truth.
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Biographical Sketch
The author was born in Kolkata, India, on February 16, 1988. She attended Heritage
Institute of Technology, under the West Bengal University of Technology (India), and graduated
with the Bachelor of Technology degree in Biotechnology in 2010. She came to the University of
Rochester, Rochester NY (USA) in the Fall of 2010 and began graduate studies in the Department
of Biology. She received her Master of Science degree in Biology from University of Rochester
in 2012. She has worked as teaching assistant at the Department of Biology from 2010 to 2015.
She pursued her research on the genetics and signaling biology of cancer under the guidance of
Dr. Helene R. McMurray.
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Acknowledgements
I owe the deepest gratitude to my advisor, Dr. Helene McMurray, who has been a true mentor
for me in the process of learning how to pursue science. She has been a constant source of
guidance, encouragement and support during the developmental years of my PhD. Alongside
science, she has been a source of inspiration on how to effectively manage time and be a better
communicator.
I am deeply indebted to my committee members: Dr. David Goldfarb, Dr. Matthew Hilton, Dr.
Douglas Portman and Dr. Andrei Seluanov. They have provided constant support and insightful
suggestions that have been invaluable for the progress of my PhD.
I would like to thank Dr. Hartmut “Hucky” Land for his numerous suggestions and invaluable
discussions that I had. I would also like to thank Dr. Craig Jordan for providing crucial support for
the RNA sequencing component of my project. I owe my thanks to Soumyaroop Bhattacharya for
helping me with the analysis of the RNA sequencing results.
A special thanks to Cynthia Landry, Jill van Atta and Daina Bullwinkel for making the grad
school process smoother for me. I also want to thank the past and present members of the lab,
especially Dorothy Heyer, for her constant technical support, Emily Walters, Dr. Pierre Candelaria
and Luca Iorga for their friendship. My special thanks to folks from the neighbouring labs,
especially Aslihan, Brad, Jordan, Mary, Nirmalya, Shweta and Vijaya, for brainstorming sessions,
technical support and personal motivation. I also remember my friends in Rochester and around
the world, who are too numerous to mention and have supported me in their own special ways.
Lastly, I would like to express my eternal gratitude to my parents and my aunt for showering
me with constant love and support and believing in me.
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Abstract
Cooperation Response Genes (CRGs) are non-mutant regulators of the malignant phenotype
in cancer cells. Distinct sets of CRGs contribute to transformation in different types of cancer, with
accumulating evidence for a set of CRGs regulated by cooperation of mutant p53 and mutant Ras
that are important in multiple epithelial cancers, including transformed colon cells and basal-like
breast cancer (BLBC). Among the CRGs is Notch3, a transmembrane receptor of the Notch family
that regulates cell proliferation and fate specification in multi-cellular organisms. Here, we
demonstrate a cancer-selective role of Notch3 in restricting cell growth of BLBC.
Through genome scale transcriptomic analysis, we discover that there is a cancer-specific
response to activation of Notch3, part of which is required for the cell growth inhibitory role of
Notch3 in BLBC. We specifically identify a novel interaction between Notch and Sfrp2, a known
Wnt pathway antagonist, and show that the genetic interaction is essential for Notch3 to control
BLBC cell growth, concomitant with Sfrp2 dependent changes in expression of cell cycle
regulatory genes.
We also observe that a similar architecture underlies Notch3-mediated growth inhibition in
growth inhibition in the transformed colon cells. Our work elucidates how Notch3 restricts cancer
cell proliferation in transformed colon cells through a genetic interaction between Notch3 and
Sfrp2 that is essential for the cancer-selective growth inhibitory action of Notch3 in transformed
colon cells.
Lastly, we describe a statistical method based on linear regression modeling that refines the
method for identifying CRGs. While 84 of the genes originally identified as CRGs are also
identified by the linear modeling method, twelve new genes are identified as CRGs in the
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transformed colon cells. We test the importance of one of the new CRGs, Clca1 and find that
suppressing Clca1 expression causes a significant reduction in tumor formation. Hence the newly
proposed linear modeling method is useful for identifying CRGs critical for tumor formation.
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Contributors and Funding Sources
All experiments described in this thesis were performed by the author except for the following:
Chapter II
Figure 2.4: The high throughput RNA sequencing and CuffDiff Analysis was performed by the
UR Functional Genomics Center.
Chapter III
Figure 3.1: Compound perturbations of Sfrp2 knock-down and NIC3 expression in mp53/Ras
cells was performed by Dorothy Heyer and cell count assay was performed by Helene
McMurray.
Chapter IV
Figure 4.3: CRGs were identified by linear modeling and the statistical method was analyzed by
Jesse Llop and Peter Salzman.
Figure 4.4: Comparison of linear modeling with the original synergy score was performed by
Jesse Llop and Peter Salzman.

Funding for the research was provided by the Wilmot Cancer Center and the Breast Cancer
Research Initiative Fund through grants awarded to Dr. Helene R. McMurray.
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Table of Contents

Chapter I. General Introduction 1
1.1 Oncogene Cooperation in Cellular Transformation 2
1.2 Approaches to Achieving Cancer Selectivity 8
1.3 Basal-like Breast Cancer 16
1.4 Colorectal Cancer 18
1.5 The Role of Notch Signaling and Wnt Signaling in Cancer 20
1.6 Rationale of the Current Study 24
1.7 Figures 26
Chapter II. Notch3 cancer-selectively controls proliferation via a network of genes 28
specifically in basal-like breast cancer
2.1 Abstract 29
2.2 Introduction 30
2.3 Results 32
2.4 Discussion 39
2.5 Figures 42
Chapter III. Conserved molecular interactions implicated in cancer selective 56
control of cell growth by Notch3 in mp53/Ras-transformed murine colon cells and
BLBC
3.1 Abstract 57
3.2 Introduction 58
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3.3 Results 60
3.4 Discussion 63
3.5 Figures 66
Chapter IV. A novel statistical model for identification of synergistically regulated 72
genes
4.1 Abstract 73
4.2 Introduction 74
4.3 Results 77
4.4 Discussion 80
4.5 Figures 82
Chapter V. Discussions 89
5.1 Summary of Findings 90
5.2 Significance of Findings 92
5.3 Future Directions 97
5.4 Conclusions 100
Chapter VI. Materials and Methods 101
6.1 Materials 102
6.1.1 Parental Cell Lines 102
6.1.2 Plasmids 104
6.1.3 shRNA Target Sequences 106
6.1.4 Real-Time PCR Primers 107
6.2 Methods 109
Chapter VII. Appendix 115
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Bibliography 127