ebook img

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

162 Pages·2015·1.3 MB·English
by  
Save to my drive
Quick download
Download
Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.

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

i 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 ii Dedication This thesis is dedicated to my late grandmother, Mrs. Bharati Sen, who taught me to go out there and search for the truth. iii 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. iv 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. v 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 vi 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. vii 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. viii 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 ix 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 x Bibliography 127

See more

The list of books you might like

Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.