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Role of Ceramide Kinase in Breast Cancer Progression

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Role of Ceramide Kinase in Breast Cancer Progression University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2014 Role of Ceramide Kinase in Breast Cancer Progression Ania Warczyk Payne University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cell Biology Commons, Molecular Biology Commons, and the Pharmacology Commons Recommended Citation Payne, Ania Warczyk, "Role of Ceramide Kinase in Breast Cancer Progression" (2014). Publicly Accessible Penn Dissertations. 1402. https://repository.upenn.edu/edissertations/1402 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1402 For more information, please contact [email protected]. Role of Ceramide Kinase in Breast Cancer Progression Abstract Recurrent breast cancer is typically an incurable disease and, as such, is disproportionately responsible for deaths from this disease. Recurrent breast cancers arise from the pool of disseminated tumor cells (DTCs) that survive adjuvant or neoadjuvant therapy, and patients with detectable DTCs following therapy are at substantially increased risk for recurrence. Consequently, the identification of pathways that contribute to the survival of breast cancer cells following therapy could aid in the development of more effective therapies that decrease the burden of residual disease and thereby reduce the risk of breast cancer recurrence. We now report that Ceramide Kinase (Cerk) is required for mammary tumor recurrence following HER2/neu pathway inhibition and is spontaneously up-regulated during tumor recurrence in multiple genetically engineered mouse models for breast cancer. We find that Cerk is apidlyr up-regulated in tumor cells following HER2/neu down-regulation or treatment with adriamycin and that Cerk is required for tumor cell survival following HER2/neu down-regulation. Consistent with our observations in mouse models, analysis of gene expression profiles from over 2,200 patients revealed that elevated CERK expression is associated with an increased risk of recurrence in women with breast cancer. Additionally, although CERK expression is associated with aggressive subtypes of breast cancer, including those that are ER-negative, HER2-positive, basal-like, or high grade, its association with poor clinical outcome is independent of these clinicopathological variables. Together, our findings identify a functional oler for Cerk in breast cancer recurrence and suggest the clinical utility of agents targeted against this pro- survival pathway. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacology First Advisor Lewis A. Chodosh Keywords Breast Cancer Recurrence, Ceramide Kinase, Ceramide Metabolism Subject Categories Cell Biology | Molecular Biology | Pharmacology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1402 ROLE OF CERAMIDE KINASE IN BREAST CANCER PROGRESSON Ania Warczyk Payne A DISSERTATION in Pharmacology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2014 Supervisor of Dissertation __________________________________ Lewis A. Chodosh, M.D., Ph.D., Professor of Cancer Biology and Medicine Graduate Group Chairperson __________________________________ Julie A. Blendy, Ph.D., Professor of Pharmacology Dissertation Committee Margaret M. Chou, Ph.D., Associate Professor of Pathology and Laboratory Medicine Anil K. Rustgi, M.D., T. Grier Miller Professor of Medicine Ben Z. Stanger, M.D., Ph.D., Assistant Professor of Medicine Ian A. Blair, Ph.D., A.N. Richards Professor of Pharmacology This work is dedicated to my husband, Dennis, whose loving support has been unwavering throughout the years. ii ACKNOWLEDGMENTS I am very grateful for the contributions of many mentors, colleagues, and friends to my work and to my development as a scientist. Above all, I would like to express my thanks to my mentor, Lewis Chodosh, for his guidance and support. I have learned innumerable lessons and have grown enormously as a scientist and a critical thinker thanks to his teachings. I would like to thank my committee, Margaret Chou, Anil Rustgi, Ben Stanger, and recently Ian Blair. I would also like to thank Emer Smyth, who served on my committee for the majority of my tenure as a graduate student and recently left the University of Pennsylvania. All members of my committee provided me with instrumental advice and were extremely supportive throughout my time as a graduate student. I am also indebted to those who have taught me and collaborated with me scientifically along the way, particularly Nate Snyder, Bobby Basu, and Tony Mancuso. Thanks also to Jianping Wang from the morphology core, who has assisted me enormously by her work sectioning tumors and performing histology. I am also deeply thankful to my lab mates, past and present, whose collaborative spirit and selflessness have made this experience so positive and have made it a joy to come to work every day. I am truly grateful for their scientific support as well as their friendship. Lastly, I am extremely fortunate to have a loving family who has supported me and loved me unconditionally. I am truly blessed to have such a wonderful husband, and wonderful parents and siblings. Thank you all. iii ABSTRACT ROLE OF CERAMIDE KINASE IN BREAST CANCER PROGRESSION Ania Warczyk Payne Lewis Chodosh, M.D., Ph.D. Recurrent breast cancer is typically an incurable disease and, as such, is disproportionately responsible for deaths from this disease. Recurrent breast cancers arise from the pool of disseminated tumor cells (DTCs) that survive adjuvant or neoadjuvant therapy, and patients with detectable DTCs following therapy are at substantially increased risk for recurrence. Consequently, the identification of pathways that contribute to the survival of breast cancer cells following therapy could aid in the development of more effective therapies that decrease the burden of residual disease and thereby reduce the risk of breast cancer recurrence. We now report that Ceramide Kinase (Cerk) is required for mammary tumor recurrence following HER2/neu pathway inhibition and is spontaneously up-regulated during tumor recurrence in multiple genetically engineered mouse models for breast cancer. We find that Cerk is rapidly up-regulated in tumor cells following HER2/neu down-regulation or treatment with adriamycin and that Cerk is required for tumor cell survival following HER2/neu down-regulation. Consistent with our observations in mouse models, analysis of gene expression profiles from over 2,200 patients revealed that elevated CERK expression is associated with an increased risk of recurrence in women with breast cancer. Additionally, although CERK expression is associated with aggressive subtypes of breast cancer, including those that are ER-negative, HER2-positive, basal-like, or high grade, its association with poor iv clinical outcome is independent of these clinicopathological variables. Together, our findings identify a functional role for Cerk in breast cancer recurrence and suggest the clinical utility of agents targeted against this pro-survival pathway. v TABLE OF CONTENTS ACKNOWLEDGMENTS ........................................................................................................... III ABSTRACT .................................................................................................................................. IV LIST OF ILLUSTRATIONS ..................................................................................................... VII CHAPTER 1: BREAST CANCER PROGRESSION, SPHINGOLIPIDS AND CERK ......... 1 CHAPTER 2: GAIN-OF-FUNCTION CERK EXPERIMENTS ............................................ 33 CHAPTER 3: LOSS-OF-FUNCTION CERK EXPERIMENTS ............................................ 60 CHAPTER 4: SUMMARY AND FUTURE DIRECTIONS ................................................... 97 REFERENCES.......................................................................................................................... 114 vi LIST OF ILLUSTRATIONS Chapter 1: Breast Cancer Progression, Sphingolipids and Cerk Figure 1.1. Ceramide synthetic and metabolic pathways Figure 1.2. C1P activates cPLA2 leading to eicosanoid synthesis and tumor-promoting signaling Figure 1.3. Ceramide and ceramide-1-phosphate signaling pathways Chapter 2: Gain-of-Function Cerk Experiments Figure 2.1. Cerk is spontaneously up-regulated in recurrent mammary tumors and following acute HER2/neu inhibition Figure 2.2. Cerk protects cells from apoptosis upon HER2/neu down-regulation in vitro Figure 2.3. Cerk promotes tumor cell survival in vivo following HER2/neu down-regulation Figure 2.4. Cerk promotes tumor recurrence in the MTB/TAN model. Figure 2.5. High CERK expression predicts decreased relapse-free survival in women with breast cancer. Figure 2.6. CERK expression in human breast cancer subsets Figure 2.7. Multivariate analysis of the association of CERK expression and relapse-free survival after adjusting for subsets Figure 2.8. Influence analysis Chapter 3: Loss-of-Function Cerk Experiments Figure 3.1. Cerk knockdown sensitizes tumor cells to apoptosis in vitro following HER2/neu down-regulation vii Figure 3.2. Cerk is required for tumor cell survival following HER2/neu down-regulation Figure 3.3. Cerk knockdown cannot be maintained in orthotopic recurrence assays Figure 3.4. Characterization of FLIPi conditional shRNA expression
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