The Underlying Effects Of CDK8and Srebp1in Endometiral Carcinoma

The number of women with newly diagnosed endometrial cancer (EC) increased by20%since1987; however the number of deaths posted a168%increase during the same time period. Estimated new cases and deaths from EC in the United States in2010are47,130and8,010, respectively. Although overall incidence of type Ⅱ cancers is approximately15%of all EC, it comprises a disproportional death in clinic (Sherman M et al.,2000). Epidemiological studies have identified obesity and diabetes as the most common risk factors for type Ⅰ EC, increasing the risk of EC up to4-fold. As a nearly universal feature of all cancers and obesity, aberrant lipogenesis is expected to have a prominent role in EC including those type Ⅱ cancers. As the population size affected by these diseases is expected to grow, particularly in developing countries, EC is expanding as a serious public health problem.Elevated lipogenesis is a metabolic hallmark of rapidly proliferating tumor cells. Although most normal cells acquire the bulk of their fatty acids from circulation, tumor cells synthesize more than90%of required lipids de novo. Consistent with a great demand for lipid synthesis, diverse human cancer cells express high levels of lipogenic enzymes, such as fatty acid synthase (FASN), the key rate-limiting enzyme in lipid biosynthesis. Aberrant lipogenesis is especially prominent in endometrial cancer (EC). However, molecular mechanisms underlying aberrant lipogenesis in both EC types are still unknown. In addition, it has not been explored whether targeting lipogenesis can provide any therapeutic value in treating EC.Cyclin-dependent kinases (CDKs) play fundamental roles in regulating cell proliferation and differentiation in eukaryotes. Dysregulation of CDKs and their regulatory partners, known as cyclins, disrupts proper control of cell proliferation, differentiation or apoptosis, thereby leading to abnormal development and diseases such as cancer. There are21CDK family members in mammals. Many studies in the past three decades have provided significant insights into understanding the roles of several CDKs, such as CDK1, CDK2, CDK4and CDK6, in regulating cell proliferation and how dysregulation of these CDKs and their regulatory cyclins contributes to tumorigenesis. However, much less is known about the potential roles of other CDKs and their deregulation in human cancers and other diseases.Upregulation of lipogenesis is a metabolic hallmark of proliferating tumor cells. Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate lipid homeostasis by controlling the expression of the key and rate-limiting enzymes required for cholesterol and fatty acid (FA) synthesis. As a master regulator of lipogenic genes, SREBPs contain three isoforms, SREBP-1a, SREBP-1c, and SREBP-2, have different roles in lipid synthesis. Several studies focus on the potential role of SREBPs in different types of tumor. In human liver hepatoma cell line HepG2, via activating SREBPs, the expression of functional LDL receptors can be induced, an event that may be triggered by activation of Nrf2. And loss of Nrf2-/-transgenic mice caused a marked steatosis in livers of ethanol-fed mice, and Srebpl was identified as a candidate transcription factor responsible for lipogenic enzyme induction. In breast cancer, ADD1/SREBP1pathway regulates the transactivation of the PPAR-y promoter during lipogenesis, and PPAR-y activation inhibits the proliferation of malignant cells. And ectopic expression of SREBP1in MCF10A cells significantly enhanced lipogenesis in stem-like cells and promoted cell growth as well as mammosphere formation. In type I EC, our previous study also confirmed that SREBP1protein overexpression and demonstrated increased nuclear distribution of SREBP1. And knockdown of SREBP1expression in EC cells induced significant cell death by apoptosis, suppressed cell growth, colonigenic capacity and tumor growth in vivo.Based on the reasons above, the project included two parts:Part Ⅰ Tumor-suppressive effects of CDK8in endometrial cancer cellsCDK8is either amplified or mutated in a variety of human cancers, and CDK8functions as an oncoprotein in melanoma and colorectal cancers. Previously, we reported that loss or reduction of CDK8results in aberrant fat accumulation in Drosophila and mammals, suggesting that CDK8plays an important role in inhibiting lipogenesis. Epidemiological studies have identified obesity and overweight as the major risk factors of endometrial cancer, thus we examined whether CDK8regulates endometrial cancer cell growth by using several endometrial cancer cell lines, including KLE, which express low levels of CDK8, as well as AN3CA and HEC-1A cells, which have high levels of endogenous CDK8. We observed that ectopic expression of CDK8in KLE cells inhibited cell proliferation and potently blocked tumor growth in an in vivo mouse model. In addition, gain of CDK8in KLE cells blocked cell migration and invasion in transwell, wound healing, and persistence of migratory directionality assays. Conversely, we observed the opposite effects in all of the aforementioned assays when CDK8was depleted in AN3CA cells. Similar to AN3CA cells, depletion of CDK8in HEC-1A cells strongly enhanced cell migration in transwell assay, while overexpression of CDK8in HEC-1A cells blocked cell migration. Furthermore, gene profiling of KLE cells overexpressing CDK8revealed genes whose protein products are involved in Lipid Metabolism, Cell Cycle, and Cell Movement pathways. Finally, depletion of CDK8increased the expression of lipogenic genes in endometrial cancer cells. Taken together, these results show a reverse correlation between CDK8levels and several key features of the endometrial cancer cells, including cell proliferation, migration and invasion, as well as tumor formation in vivo. Therefore, in contrast to the oncogenic effects of CDK8in melanoma and colorectal cancers, our results suggest that CDK8plays a tumor suppressive role in endometrial cancers. Part Ⅱ Prognostic relevance and potential tumorigenic role of SREBP1in type Ⅱ endometrial cancerType Ⅱ endometrial cancers, which composed of uterine serous papillary carcinoma and clear cell endometrial carcinoma, are the most aggressive type in endometrial cancer. SREBP1as a transcription factor regulates the synthesis of enzymes involved in sterol biosynthesis through binding to specific SRE DNA sequences, thus maintain proper levels of intracellular lipids environment. Herein, we evaluate a cohort of type Ⅱ endometrial cancer specimen and by using immunohistochemical staining to compare the expression pattern and prognostic relevance of SREBP1in type Ⅱ endometrial cancer. We found that the expression of nuclear SREBP1elevated in type Ⅱ endometrial carcinoma compared with the adjacent type Ⅰ endometrium cancer. And shRNA knockdown of SREBP1expression in spec-2cells significantly repress cell proliferation, cell migration, focus formation and in vivo tumor growth, indicate potential tumorigenic role of SREBP1in spec-2cells. Taken together, we found that SREBP1plays an essential role in type Ⅱ endometrial cancer cell growth both in vitro and in vivo, and SREBP1expression maybe a novel predictor for prognosis of type Ⅱ endometrial cancer.