Effect of long-term metformin treatment in endometrial cancer development and progression

子宫内膜癌（EC）是全球最常见的妇科恶性肿瘤之一，分为雌激素依赖性I型和雌激素非依赖性II型癌症。I型占所有病例的75-85%。雌激素刺激子宫内膜细胞增殖，抑制细胞凋亡，支持EC的发展。胰岛素抵抗（IR;高胰岛素血症和正常胰岛素水平）与肥胖、II型糖尿病（DM II）和多囊卵巢综合征（PCOS）的组合也被认为是I型EC发展和进展的重要风险因素。在单纯IR的PCOS患者中，高胰岛素血症似乎是EC发生和进展的唯一促进因素。然而，高血糖症也被认为是一个独立的风险因素，是糖尿病II型患者中观察到的癌症发展的关键环节。因此，降低胰岛素和葡萄糖水平的胰岛素增敏剂被认为可以预防和治疗癌症。为了更好地了解二甲双胍在子宫内膜组织中的其他局部作用，我们阐明了在高胰岛素环境中暴露于高葡萄糖（反映糖尿病）和正常葡萄糖（反映IR）的雌激素暴露下，长期二甲双胍治疗下子宫内膜癌发生和进展期间的分子变化。通过使用与癌症发展和进展相关的已知基因的筛选阵列，我们计划在可能受二甲双胍影响的不同葡萄糖环境中定义高胰岛素血症下EC发展的靶基因。在此之后，我们将通过在不同培养条件下的基因和蛋白质表达变化分析来证实二甲双胍治疗的潜在机制。为了进一步了解高血糖和高胰岛素血症下EC发生和发展的分子变化，我们将使用siRNA敲除这些预选的靶基因以抑制其基因功能，然后分析不同条件下子宫内膜细胞增殖、迁移/侵袭和基因/蛋白表达的变化。这将有助于我们获得新的机制的见解，在长期的生理二甲双胍治疗子宫内膜癌的发展和转移的抗肿瘤作用。最后，但并非最不重要的是，使用敲除工具CRISPR/cas9，我们将评估子宫内膜癌细胞的行为是否由环境（胰岛素/高葡萄糖效应）重建，即使恶性状态的潜在驱动因素被敲除。本研究的长期目标不仅是进一步确定二甲双胍在高危患者中预防和/或治疗子宫内膜癌的作用，而且还阐明子宫内膜细胞在高胰岛素血症和高血糖症条件下如何变得恶性和侵袭性的未知机制，从而确定可能的新分子治疗靶点。

Endometrial cancer (EC) is one of the most common gynecological malignancies worldwide, and is classified into an estrogen-dependent type I, and an estrogen-independent type II cancer. Type I accounts for 75-85% of all cases. Estrogen stimulates endometrial cell proliferation and inhibits apoptosis supporting EC development. Insulin resistance (IR; hyperinsulinemia & normoglycemia), in combination with obesity, diabetes mellitus type II (DM II) and Polycystic Ovary Syndrome (PCOS) are also considered significant risk factors for the development and progression of type I EC. In PCOS patients with isolated IR, the hyperinsulinemia appears to be the sole promoting factor for EC initiation and progression. However, hyperglycemia is also considered an independent risk factor and is a critical link for cancer development observed in patients with DM II. Therefore insulin-sensitizing agents that reduce insulin and glucose levels are thought to prevent and treat cancer. In order to better understand additional local metformin effects within endometrial tissue, we elucidate the molecular changes during endometrial cancer development and progression under long-term metformin therapy in a hyperinsulinemic environment exposed to high glucose (reflecting diabetes) and normal glucose (reflecting IR) under estrogen exposure. By using a screening array of known genes relevant in cancer development and progression, we plan to define target genes of EC development under hyperinsulinemia in the different glucose environments that may be affected by metformin. After this we will confirm the suggested underling mechanisms of the metformin therapy by gene and protein expression change analysis under the different culture conditions. To understand further the molecular changes in EC development and progression under hyperglycemia and hyperinsulinemia we will knockdown these preselected target genes using siRNA to inhibit their gene function, followed by the analysis of the endometrial cell proliferation, migration/invasion and gene-/protein expression changes under the various conditions. This will help us gain novel mechanistic insights in the long-term physiological metformin treatment antitumor effects in endometrial cancer development and metastasis. Last, but not least, using the knockout tool CRISPR/cas9 we will evaluate if the endometrial cancer cell behavior is reestablished by the environment (insulin / high glucose effects), even when the potential drivers of the malignant state are knocked out. The long-term goal of this research is not only to further establish metformin in the prevention and/or treatment of endometrial carcinoma in patients at risk but also to elucidate unknown mechanisms how endometrial cells become malignant and aggressive in the conditions of hyperinsulimenia and hyperglycemia, therefore identifying possible new molecular therapeutic targets.