GAPDH在乳腺癌细胞EMT及乳腺癌转移中的表达与功能研究

Metastases are the leading factor of cancer-related mortality. Epithelial-mesenchymal transition (EMT) is a process in which adherent epithelial cells shed their epithelial traits and acquire mesenchymal properties, which is thought to be important step for the progression of tumor cells to invasive and metastatic cells. Malignant cells characteristically exhibit altered metabolic patterns when compared with normal mammalian cells with increased reliance on anaerobic metabolism of glucose to lactic acid through glycolysis even in the presence of abundant oxygen. The inefficiency of the anaerobic pathway is compensated by increased glucose flux, a phenomenon first noted by Otto Warburg called aerobic glycolysis. Our previous study shows that the protein level of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), one of the enzymes involved in glycolysis, is upregulated during the Snail as well as TGF-beta-induced EMT process. The migration of breast cancer cells are partially blocked by knock down of GAPDH, suggesting that GAPDH may play an import role in breast cancer cell migration. We plan to investigate the molecular mechanism of the upregulation of GAPDH in the EMT of breast cancer cells. Meanwhile the mechanism through which GAPDH taking part in the migration and metastasis will also be deciphered. Moreover, through the relationship study of GAPDH expession and the metastasis status of clinical breast cancer patients, the character of Glucose metabolism in the EMT and breast cancer metastasis will be futher deciphered. The above research should shed new lights to the mechanism study of EMT and metastasis in breast cancer.

肿瘤转移是恶性肿瘤导致患者死亡的首要因素。上皮-间质转化(Epithelial-mesenchymal transition, EMT)是指上皮细胞通过特定程序转化为间质细胞的过程，是恶性肿瘤细胞获得转移能力的重要步骤。肿瘤细胞的代谢在整体上发生改变，其显著特征是肿瘤细胞在有氧状态下也优先进行糖酵解，而不是通过氧化磷酸化提供能量，即Warburg效应。我们在前期研究中发现Snail和TGF-B诱导细胞发生EMT时，参与糖酵解的3-磷酸甘油醛脱氢酶（GAPDH）蛋白水平显著升高， RNA干扰实验初步证明其参与乳腺癌细胞的转移，在此基础上，我们拟对GAPDH蛋白在EMT中上调的分子机制及其参与乳腺癌转移的途径和机制进行深入研究，结合临床乳腺癌病人GAPDH水平与肿瘤转移相关性的研究，旨在揭示EMT和肿瘤转移过程中葡萄糖代谢改变的原因和结果，为明确肿瘤EMT和转移发生的分子机理提供新的理论依据。

肿瘤转移是恶性肿瘤导致患者死亡的首要因素。上皮-间质转化(Epithelial-mesenchymal transition, EMT)是指上皮细胞通过特定程序转化为间质细胞的过程，是恶性肿瘤细胞获得转移能力的重要步骤。我们在前期研究中发现Snail和TGF-B诱导细胞发生EMT时，参与糖酵解的3-磷酸甘油醛脱氢酶（GAPDH）蛋白水平显著升高， RNA干扰实验初步证明其参与乳腺癌细胞的转移，在此基础上，我们对GAPDH蛋白在EMT中上调的分子机制及其参与乳腺癌转移的途径和机制进行深入研究，我们深入研究其机制时发现，在Snail和TGF-诱导细胞发生EMT过程中溶酶体的自噬功能有所改变，尤其是分子伴侣介导的自噬 (chaperone mediated autophagy, CMA)显著下降，从而导致了CMA降解底物蛋白的累积，而GAPDH蛋白是经典的CMA降解底物，其在上皮间质转化过程中蛋白水平的升高正是CMA下降的结果。除了GAPDH以往，我们还发现了新的CMA降解底物- NF-κB转录因子p65。我们首次证明了p65是一个新的分子伴侣介导自噬底物蛋白，在上皮间质转化过程中p65通过CMA降解过程受到明显抑制，从而导致p65蛋白质累积和NF-κB活性升高。除了p65蛋白质累积，我们还发现CMA活性在间质样细胞中降低，同时伴随多种CMA底物蛋白的累积。进一步，我们通过功能模拟以及功能回复实验证明CMA通过选择性调控p65蛋白质降解参与调节细胞EMT过程的发生以及间质样细胞表型和功能的维持。除了分子伴侣介导自噬活性降低，我们还在基因表达谱分析的基础上，并通过一系列实验证明在多种细胞发生EMT过程中溶酶体数目减少、溶酶体相关基因转录受抑制。我们发现调控溶酶体相关基因合成的关键转录因子TFEB在EMT过程中表达显著降低，并初步证明TFEB表达与Snail呈现负相关，这为EMT过程中溶酶体数目和生物合成减少提供了可能机制。