Alterations In Gene Expression As A Response of Tumor Cells To Stresses
Open Access
- Author:
- Huber-keener, Kathryn Joyce
- Graduate Program:
- Pharmacology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 04, 2012
- Committee Members:
- Jin Ming Yang, Dissertation Advisor/Co-Advisor
Robert G Levenson, Committee Member
Rongling Wu, Committee Member
Willard Freeman, Committee Member
Andrea Manni, Committee Member - Keywords:
- eEF-2K
tamoxifen
glioma
breast cancer
protein synthesis
next generation-sequencing
protein stability - Abstract:
- Cancer cells are constantly under stress, a factor which needs to be taken into consideration during the treatment of 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, 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. 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), occurs in one third of patients treated with Tam within 5 years of therapy. A better understanding of gene 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, gen expression, and mitochondrial modification. 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 a protein called SIRT3 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 eEF-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 eEF-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), under stress conditions, we found a paradoxical increase in eEF-2K protein expression with a decrease in eEF-2K protein stability. 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 (S78 and S366) and AMP kinase (S398), pathways which would be affected by stress. Through phosphorylation-defective mutants, we discovered that S398 was pivotal to protein stability. Our data indicate that eEF-2K is regulated at multiple levels with phosphorylation playing an important role in protein turnover. 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 focused eEF-2K study indicates how tightly and complexly this 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.