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Molecular Mechanisms of Esophageal Squamous Cell Carcinoma

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Molecular Mechanisms of Esophageal Squamous Cell Carcinoma University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2015 Molecular Mechanisms of Esophageal Squamous Cell Carcinoma Apple Long University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Molecular Biology Commons Recommended Citation Long, Apple, "Molecular Mechanisms of Esophageal Squamous Cell Carcinoma" (2015). Publicly Accessible Penn Dissertations. 1861. https://repository.upenn.edu/edissertations/1861 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1861 For more information, please contact [email protected]. Molecular Mechanisms of Esophageal Squamous Cell Carcinoma Abstract MOLECULAR MECHANISMS OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA Apple Long Anil K. Rustgi Esophageal squamous cell cancer (ESCC) is the 6th leading cause of cancer related deaths amongst American men. Although rare, this disease has a high mortality rate with a 5-year survival of 17%. Incidentally TP53 mutation is the most common genetic alteration in ESCC, along with over expression of EGFR and CYCLIN D1. We have previously modeled the invasive features of ESCC, through an in vivo-like 3D organotypic culture system, utilizing primary epithelial cells that have been transformed by overexpression of mutant TP53 and EGFR. From this model, a RNA microarray was performed to determine critical genes that are upregulated at the front of invasion. WNT10A was found to be over 4-fold upregulated, along with a WNT-signaling gene signature in the invasive cells. We additionally found that increased WNT10A expression was associated with ESCC, compared to normal esophageal tissue and that increased WNT10A staining was associated with poor prognosis. Functionally, WNT10A was determined to induce increased proliferation, migration, invasion and stemness properties in transformed esophageal cancer cells. In addition to examining the oncogenic properties of WNT10A, we also focused on generating an in vivo model of mutant TP53 driven ESCC. We utilized a carcinogen, 4-nitroquinoline 1-oxide (4-NQO), as a challenge to induce genetic changes in the background of Tp53 mutation, specific to the oral squamous, esophagus and forestomach tissues. We found preliminarily that TP53 mutation, specifically the R172H mutant, can accelerate tumorigenesis as compared to loss of TP53 alone. Additionally, when RNA-seq was performed on the tumor derived TP53R172H/- and TP53-/- cells, we found an increase of genes associated with an epithelial to mesenchymal transition (EMT), in the TP53R172H/- cells. Mesenchymal genes such as Cdh2, Zeb1, Zeb2, Twist, and Snail were increased, while epithelial genes such as Cdh1, Epcam, and Krt4 were decreased. Altogether, we surmise that the TP53 mutant, R172H, can induce gain-of-function (GOF), oncogenic properties in order to promote tumorigenesis, potentially by inducing an EMT phenotype. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Anil K. Rustgi Keywords Cancer biology, Esophageal Squamous Cell Carcinoma, Mutant p53, Wnt signaling Subject Categories Molecular Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1861 MOLECULAR MECHANISMS OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA Apple Long A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2015 Supervisor of Dissertation Anil K. Rustgi, M.D. T. Grier Miller Professor of Medicine; Chief of Division of Gastroenterology, Perelman School of Medicine Graduate Group Chairperson Daniel S. Kessler, Ph.D. Associate Professor of Cell and Developmental Biology Dissertation Committee Gerd Blobel, Ph.D. Frank E. Weise III Professor of Pediatrics, Perelman School of Medicine Makoto Senoo, Ph.D. Assistant Professor of Developmental Biology, University of Pennsylvania School of Veterinary Medicine Ellen Puré, Ph.D. Grace Lansing Lambert Professor of Biomedical Science, University of Pennsylvania School of Veterinary Medicine Chair: Andy Minn, M.D., Ph. D. Assistant Professor of Radiation Oncology, Perelman School of Medicine ACKNOWLEDGMENTS I would like to thank all of the members of the Rustgi and Nakagawa lab, past and present for ideas, inspiration, experimental design help, and actual help with experiments and great advice. I would also like to thank our collaborators, Drs. Andres Klein-Szanto, Ju-Seog Lee, Shelley Berger, Morgan Sammons, and Jiajun Zhu for their instrumental help with several of our experiments and experimental design. ii ABSTRACT MOLECULAR MECHANISMS OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA Apple Long Anil K. Rustgi Esophageal squamous cell cancer (ESCC) is the 6th leading cause of cancer related deaths amongst American men. Although rare, this disease has a high mortality rate with a 5-year survival of 17%. Incidentally TP53 mutation is the most common genetic alteration in ESCC, along with over expression of EGFR and CYCLIN D1. We have previously modeled the invasive features of ESCC, through an in vivo-like 3D organotypic culture system, utilizing primary epithelial cells that have been transformed by overexpression of mutant TP53 and EGFR. From this model, a RNA microarray was performed to determine critical genes that are upregulated at the front of invasion. WNT10A was found to be over 4-fold upregulated, along with a WNT-signaling gene signature in the invasive cells. We additionally found that increased WNT10A expression was associated with ESCC, compared to normal esophageal tissue and that increased WNT10A staining was associated with poor prognosis. Functionally, WNT10A was determined to induce increased proliferation, migration, invasion and stemness properties in transformed esophageal cancer cells. In addition to examining the oncogenic properties of WNT10A, we also focused on generating an in vivo model of mutant TP53 driven ESCC. We utilized a carcinogen, 4-nitroquinoline 1-oxide (4-NQO), as a challenge to induce genetic changes in the background of Tp53 mutation, specific to the oral iii squamous, esophagus and forestomach tissues. We found preliminarily that TP53 mutation, specifically the R172H mutant, can accelerate tumorigenesis as compared to loss of TP53 alone. Additionally, when RNA-seq was performed on the tumor derived TP53R172H/- and TP53-/- cells, we found an increase of genes associated with an epithelial to mesenchymal transition (EMT), in the TP53R172H/- cells. Mesenchymal genes such as Cdh2, Zeb1, Zeb2, Twist, and Snail were increased, while epithelial genes such as Cdh1, Epcam, and Krt4 were decreased. Altogether, we surmise that the TP53 mutant, R172H, can induce gain-of-function (GOF), oncogenic properties in order to promote tumorigenesis, potentially by inducing an EMT phenotype. iv TABLE OF CONTENTS ACKNOWLEDGMENTS ............................................................................................................ II ABSTRACT ................................................................................................................................ III LIST OF TABLES ..................................................................................................................... VII LIST OF FIGURES .................................................................................................................. VIII CHAPTER 1: INTRODUCTION ............................................................................................... 1 Esophageal cancer .................................................................................................................................. 2 Embryogenesis of the esophagus ............................................................................................................ 5 TP53: A potent tumor suppressor ........................................................................................................... 7 Gain of function roles of mutant TP53 .................................................................................................... 9 Role of mutant TP53 in 3D organotypic culture of transformed esophageal cells .................................. 12 WNTs and the canonical WNT pathway ................................................................................................ 15 WNTs in development .......................................................................................................................... 19 WNT pathway in cancer ........................................................................................................................ 22 Figures and figure legends .................................................................................................................... 27 CHAPTER 2: WNT10A PROMOTES AN INVASIVE AND SELF-RENEWING PHENOTYPE IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA ................................ 29 Abstract ................................................................................................................................................ 30 Introduction ......................................................................................................................................... 31 Results .................................................................................................................................................. 34 Discussion ............................................................................................................................................
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