Critical opposing roles for TFEB andTFE3 in melanoma progression

Unlike MITF-M, the melanocyte-specific isoform of MITF, TFEB, TFE3 and non-melanocyte MITF isoforms are regulated primarily by mTORC1-mediated phosphorylation at the lysosome that promotes their cytoplasmic retention. As low levels of glucose or amino acids trigger down-regulation of MITF, it is likely that on nutrient limitation some MITF functions are assumed by TFE3 and TFEB, enabling MITFLow melanoma cells to survive and proliferate. The role TFEB and TFE3 in melanoma biology and their impact on MITF-driven proliferation is poorly understood. TFEB and TFE3 KO of 501mel melanoma cell lines were generated using guides targetting TFE3 (GGTACTGTTTCACCTGCTGC) and TFEB (AGTACCTGTCCGAGACCTAT). KO cells were clonally isolated and validated prior to experiment.

与MITF-M（即小眼畸形相关转录因子（MITF）的黑色素细胞特异性同工型）不同，TFEB、TFE3以及非黑色素细胞来源的MITF同工型主要受溶酶体处雷帕霉素复合物1（mTORC1）介导的磷酸化修饰调控，该修饰可促进这些蛋白在细胞质内的滞留。当葡萄糖或氨基酸水平低下时，会触发MITF的表达下调，因此在营养匮乏条件下，部分MITF的功能可由TFE3与TFEB代偿，进而使MITF低表达（MITFLow）的黑色素瘤细胞得以存活并增殖。目前学界对TFEB与TFE3在黑色素瘤生物学中的作用，以及其对MITF驱动的细胞增殖的影响尚了解甚少。本研究通过靶向TFE3（向导序列：GGTACTGTTTCACCTGCTGC）与TFEB（向导序列：AGTACCTGTCCGAGACCTAT）的向导RNA（guide RNA），构建了501mel黑色素瘤细胞系的TFEB与TFE3双敲除（KO）细胞株。所有敲除细胞株均经过单克隆分离，并在正式实验前完成了验证。