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The Pennsylvania State University the Graduate School Department The Pennsylvania State University The Graduate School Department of Pharmacology ALTERATIONS IN GENE EXPRESSION AS A RESPONSE OF TUMOR CELLS TO STRESSES A Dissertation in Pharmacology by Kathryn Joyce Huber-Keener 2012 Kathryn Joyce Huber-Keener Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2013 The dissertation of Kathryn Joyce Huber-Keener was reviewed and approved* by the following: Jin-Ming Yang Professor of Pharmacology Dissertation Advisor Chair of Committee Willard M. Freeman Associate Professor of Pharmacology Rongling Wu Professor of Statistics Robert G. Levenson Professor of Pharmacology Andrea Manni Professor of Medicine Jong K. Yun Director of the Pharmacology Graduate Program *Signatures are on file in the Graduate School ii ABSTRACT Solid cancers are the 2nd leading cause of death in adults in the United States. Understanding the mechanisms by which cancer cells survive under stress is pivotal to decreasing this statistic. Cancer cells are constantly under stress, a factor which needs to be taken into consideration during the treatment of solid cancers. Both intrinsic and extrinsic stresses impact the development and progression of neoplasms, down to the level of individual proteins and genes. Stresses like nutrient deficiency, hypoxia, acidity, and the immune response are present during normal tumor growth and throughout treatment. Tumor cells that survive these stresses are more adept at surviving hostile conditions and are more resistant to current therapies. Stress, therefore, shapes the tumor cell population. Gene expression alterations are the driving feature behind the adaptive ability of cancer cells to these stresses, and therefore, careful examination of these gene expression changes must be undertaken in order to develop effective therapies for solid cancers. In the present investigations, we first explore the global alterations of gene expression in tamoxifen resistance. Resistance to tamoxifen (Tam), a widely used antagonist of the estrogen receptor (ER), is a common obstacle to successful breast cancer treatment. While adjuvant therapy with Tam has been shown to significantly decrease the rate of disease recurrence and mortality, recurrent disease occurs in one third of patients treated with Tam within 5 years of therapy. A better understanding of gene iii expression alterations associated with Tam resistance will facilitate circumventing this problem. Using a next generation sequencing approach and a new bioinformatics model, we compared the transcriptomes of Tam-sensitive and Tam-resistant breast cancer cells for identification of genes involved in the development of Tam resistance. We identified differential expression of 1215 mRNA and 513 small RNA transcripts clustered into ERα functions, cell cycle regulation, transcription/translation, and mitochondrial dysfunction. The extent of alterations found at multiple levels of gene regulation highlights the ability of the Tam-resistant cells to modulate global gene expression. Alterations of small nucleolar RNA, oxidative phosphorylation, and proliferation processes in Tam-resistant cells present areas for diagnostic and therapeutic tool development for combating resistance to this anti-estrogen agent. After such a global exploration of cancer cell responses to stress, we next investigated a mechanism of cancer cell survival by exploring the alterations in protein synthesis inhibitor eEF-2K. Studies show that EF-2K plays a role in cell survival through this inhibition of protein synthesis and that its protein levels are increased in cancer. Post-translational modification of translation machinery is important for its regulation and could be critical for survival of cancer cells encountering stress. Thus, the purpose of our study is to examine the regulation of EF-2K during stress with a focus on the phosphorylation status and stability of EF-2K protein in cancer cells. Using two human glioma cell lines (T98G and LN229), we have found a 2-5 fold increase in EF-2K expression and activity under stress conditions of nutrient deprivation and hypoxia. mRNA levels are only transiently increased and shortly return to normal, while EF-2K iv protein levels continue to increase after further exposure to stress. This result could be explained by decreased turnover of EF-2K protein, which has a normal half-life of ~ 6-8 hours in glioma cells, so cycloheximide experiments were used to examine the effect of stress on EF-2K protein stability. A seemingly paradoxical decrease in EF-2K stability (t1/2 = 2-4h) was found when glioma cells were subjected to stress despite increased protein expression. Phosphorylation may play a role in this altered protein stability as EF-2K has multiple phosphorylation sites that are phosphorylated by the mTOR/S6 kinase (Ser78 and Ser366) and AMP kinase (Ser398), pathways which would be affected by stress. Therefore, phosphorylation-defective mutants of EF-2K were made to examine the effect of phosphorylation at these sites on EF-2K protein stability. We discovered that the AMP kinase site was pivotal to protein stability as the S398A mutant half-life increased to greater than 24 hours under both normal and stress conditions. Mutating the mTOR pathway sites made EF-2K protein more stable under normal conditions (t1/2 > 24h) but decreased to normal levels under stress conditions (t1/2 = 8h). Inhibiting the mTOR pathway with rapamycin treatment increased protein expression ~ 5 fold and increased EF-2K stability in these mutants under all culture conditions. These data indicate that EF-2K is regulated at multiple levels with phosphorylation playing an important role in protein turnover. The unexpected decrease in EF-2K protein stability during stress may be a compensatory mechanism for an additional level of regulation at the post-transcriptional level that increases EF-2K translation. Due to the importance of translation regulation during stress, it is reasonable to have increased translation of a regulator of protein synthesis while decreasing the same protein’s stability in order to v quickly adapt to changing nutrient levels. Further studies will examine the post- transcriptional regulation of EF-2K during stress as these data demonstrate its complex and tight regulation. Understanding the regulation of EF-2K could lead to therapeutics targeting EF-2K that could potentially render cancer cells intolerant to stress and susceptible to current treatments. Together, our results represent the global impact that stress can have on cancer cells. Our NGS study exemplifies the complexity of alterations caused by exogenous stresses on cancer cells. The wide variety of gene expression changes from genes involved in proliferation and survival to those that regulate energy metabolism and post- transcriptional indicate that stress selection can alter the overall functioning of the cancer cell. The focused eEF-2K study indicates how tightly and complexly the protein synthesis regulator is controlled, which has broad implications for global and specific translation of the numerous gene transcript alterations caused by stress. Understanding how gene expression and proteins are altered in cancer cells by different intrinsic and extrinsic stresses will help current and future researchers to develop novel and more effective methods of overcoming cancer cell survival and resistance to current treatments. vi TABLE OF CONTENTS ABSTRACT ............................................................................................................................. iii LIST OF FIGURES ................................................................................................................. xi LIST OF TABLES ................................................................................................................... xii LIST OF ABBREVIATIONS .................................................................................................. xiii ACKNOWLEDGEMENTS ..................................................................................................... xvi EPIGRAPH .............................................................................................................................. xx Chapter 1 .................................................................................................................................. 1 Cellular stress, gene expression, and cancer development and progression ............................ 1 1.1. Overview of Solid Cancers ...................................................................................... 1 1.1.1. Breast Cancer ................................................................................................. 3 1.1.1.1. Breast cancer statistics 3 1.1.1.2. Breast cancer progression 4 1.1.1.3. Breast cancer etiology 6 1.1.2. Glioma ............................................................................................................ 6 1.1.2.1. Glioma statistics 9 1.1.2.2. Glioma progression 9 1.1.2.3. Glioma etiology 10 1.2. Cellular stress ........................................................................................................... 11 1.2.1. Intrinsic stresses during tumor development and progression ....................... 13 1.2.2. Extrinsic stress caused by cancer treatments .................................................. 18 1.2.2.1. Surgery 19 1.2.2.2. Radiation 19 1.2.2.1. Chemotherapy 20 1.2.2.3. Targeted therapies 22 1.2.3. Molecular markers of stress ..........................................................................
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