TFEB在黑色素瘤靶向药物耐药性中的作用机制

In 2015, there are 4.3 million new cancer cases and 2.8 million deaths in China, brought a heavy burden to our country and society, it is very urgent to find effective drugs for the treatment of cancer. Tumor targeting drugs which inhibit specific cancer gene mutation are able to suppress the growth of tumor cells. However, the tumor cells consistently evolve to activate other signaling pathways to maintain their growth, eventually develop drug resistance. Autophagy is a cell process that damaged proteins or organelles are packaged by autophagosome and delivered into the lysosome for degradation and reuse. Tumor cells make use of autophagy to fight drugs, inhibit tumor cell death, thereby generating drug resistance. BRAF is a serine/threonine kinase, its genetic mutation happens in a variety of cancers, including melanoma, lung cancer, colon cancer, gastric cancer, ovarian cancer and thyroid cancer. TCGA cancer genome database shows that BRAF(V600E) point mutation is the major mutation form, especially in melanoma, accounts for about 50% of all patients. BRAF (V600E) can activate the BRAF kinase activity, activating downstream MEK-ERK signaling pathway, so as to promote cancer cell growth and proliferation. Although the targeting drugs of BRAF (V600E), such as Vemurafenib, can effectively inhibit the growth of cancer cells and prolong the patient's survival, but they also have a severe problem of drug resistance. Using melanoma as a model, we found that BRAF (V600E) targeting drugs can activate autophagy by inducing TFEB translocate into the nucleus; Knockdown TFEB by shRNA can significantly inhibits cell autophagy, and effectively increases the effect of targeting drugs. Next, we will identify the key phosphorylation site of TFEB which is regulated by targeted drugs, and find the corresponding kinase and phosphatase. Then, we will choose specific activators or inhibitors of these kinases and phosphatases as small molecule drugs, combine with targeting drugs to test the treatment effects in both melanoma cells and mouse tumor model.

细胞自噬是细胞将损坏的蛋白或细胞器用自噬小泡包裹后，送入溶酶体进行降解并重新利用的过程。肿瘤细胞利用自噬抑制细胞凋亡并产生耐药性。BRAF是一个丝氨酸/苏氨酸激酶，在多种肿瘤中存在突变，V600E点突变是主要突变形式，可以激活下游MEK-ERK信号通路，促进肿瘤细胞生长。BRAF(V600E)的靶向药物维罗非尼虽然可以有效抑制肿瘤细胞生长，延长病人的生存期，但存在严重的耐药性问题。我们以黑色素瘤为模型发现V600E靶向药物可以促进自噬转录因子TFEB入核，从而激活细胞自噬。敲低TFEB表达可以显著降低细胞自噬水平，降低靶向药物的耐药性。下一步我们将鉴定靶向药物调控TFEB入核的关键磷酸化位点，并寻找对应的激酶和磷酸酶。再针对激酶和磷酸酶选择特异的激活剂或抑制剂作为小分子药物，与维罗非尼共同作用，在细胞和小鼠肿瘤模型中检验两种药物共同作用在肿瘤治疗中的效果。

肿瘤耐药性是肿瘤治疗领域的研究难题，肿瘤通过多种不同的方式激活生长信号，对化疗药物、靶向药物和免疫药物产生耐药性。本研究通过研究细胞自噬和细胞铁死亡中的关键基因，探寻肿瘤耐药的分子机制，发现新的解决肿瘤耐药的可成药靶点，并通过细胞和小鼠模型进行药物分子筛选。在一项发表于Nature Communication的工作中我们发现在携带BRAFV600E的黑素瘤中，自噬是由BRAF抑制剂(BRAFi)诱导的，作为TFEB转录因子介导的协调溶酶体生物发生/功能的转录程序的一部分。在异种移植黑色素瘤中，阻断BRAFi诱导的自噬-溶酶体功能的转录激活会导致肿瘤进展增强、emt转分化、转移性传播和化疗耐药性，抑制TGF-β信号通路可恢复黑色素瘤细胞的肿瘤分化和药物反应。在另一项发表于Nature Communication的工作中我们发现erastin通过诱导ROS应答转录因子 FOXM1 激活神经元前体细胞表达发育下调 4（Nedd4）的表达。Nedd4是真核生物 HECT 结构域 E3 连接酶家族的重要成员，在进化过程中保持保守性。Nedd4 通过其WW结构域与VDAC2/3的PPxY序列结合并促进VDAC2/3降解，从而降低了癌细胞对 erastin 的敏感性。通过抑制Nedd4和FOXM1的表达，以减少VDAC2/3蛋白质降解的方法，可以增加癌细胞对erastin的敏感性。该项研究阐明了一种基于FOXM1-Nedd4-VDAC2/3负反馈回路，揭示了肿瘤细胞如何适应新的小分子药物并维持细胞内稳态的分子机制，并提示了以Nedd4作为靶标治疗，通过细胞铁死亡治疗肿瘤的新思路。 此外，还有数篇相关工作发表在Cell Death and Differenciation, Cellular Signalling等杂志，为解析肿瘤耐药性提供了思路。