A Lysosome independent role for TFEB in activating DNA repair and inhibiting apoptosis in breast cancer cells

Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy with critical roles in several cancers. Lysosomal autophagy promotes cancer survival through degradation of toxic molecules and maintenance of adequate nutrient supply. Doxorubicin (DOX) is the standard of care treatment for triple-negative breast cancer (TNBC); however, chemoresistance at lower doses and toxicity at higher doses limit its usefulness. By targeting pathways of survival, DOX can become an effective antitumor agent. In this study, we examined the role of TFEB in TNBC and its relationship with autophagy and DNA damage induced by DOX. In TNBC cells, TFEB was hypo-phosphorylated and localized to the nucleus upon DOX treatment. TFEB knockdown decreased the viability of TNBC cells while increasing caspase-3 dependent apoptosis. Additionally, inhibition of TFEB-phosphatase calcineurin sensitized cells to DOX-induced apoptosis in a TFEB dependent fashion. Regulation of apoptosis by TFEB was not a consequence of altered lysosomal function, as TFEB continued to protect against apoptosis in the presence of lysosomal inhibitors. RNA-Seq analysis of TFEB knockdown MDA-MB-231 cells identified a significant downregulation in cell cycle and homologous recombination while genes involved in interferon-γ and TNFα signaling were upregulated. In consequence, knockdown of TFEB was found to disrupt DNA repair following DOX, as evidenced by persistent γH2A.X detection. Together, these findings describe in TNBC a novel lysosomal independent function for TFEB in responding to DNA damage.

转录因子EB（Transcription factor EB，TFEB）是溶酶体生物发生与自噬的主调控因子，在多种癌症中发挥关键作用。溶酶体自噬通过降解有毒分子并维持充足的营养供给，促进癌症细胞存活。阿霉素（Doxorubicin，DOX）是三阴性乳腺癌（triple-negative breast cancer，TNBC）的标准治疗药物，但低剂量下产生的化疗耐药性与高剂量下的毒副作用限制了其临床应用价值。若能靶向肿瘤细胞存活通路，阿霉素可成为高效的抗肿瘤制剂。本研究探讨了TFEB在三阴性乳腺癌中的作用，及其与阿霉素诱导的自噬和DNA损伤的关联。在三阴性乳腺癌细胞中，经阿霉素处理后，TFEB呈低磷酸化状态并定位于细胞核。敲低TFEB可降低三阴性乳腺癌细胞的存活率，同时增强半胱天冬酶-3（caspase-3）依赖型细胞凋亡。此外，抑制TFEB的磷酸酶钙调神经磷酸酶，可通过TFEB依赖的方式使细胞对阿霉素诱导的凋亡更为敏感。TFEB对细胞凋亡的调控并非溶酶体功能改变的继发效应，因为在溶酶体抑制剂存在的条件下，TFEB仍可发挥抗凋亡作用。对敲低TFEB的MDA-MB-231细胞进行RNA测序（RNA-Seq）分析发现，细胞周期与同源重组相关基因显著下调，而干扰素-γ（interferon-γ）及肿瘤坏死因子α（TNF-α）信号通路相关基因则显著上调。由此可见，敲低TFEB会破坏阿霉素处理后的DNA修复过程，这一点可通过持续检测到γH2A.X得到证实。综上，本研究揭示了三阴性乳腺癌中TFEB在响应DNA损伤过程中一种全新的、不依赖于溶酶体的功能。