Goodman Cancer Research Center, McGill University, Montréal, Québec, Canada.
Department of Biochemistry, McGill University, Montréal, Québec, Canada.
Autophagy. 2021 Dec;17(12):3957-3975. doi: 10.1080/15548627.2021.1898748. Epub 2021 Mar 18.
Increased macroautophagy/autophagy and lysosomal activity promote tumor growth, survival and chemo-resistance. During acute starvation, autophagy is rapidly engaged by AMPK (AMP-activated protein kinase) activation and MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) inhibition to maintain energy homeostasis and cell survival. TFEB (transcription factor E3) and TFE3 (transcription factor binding to IGHM enhancer 3) are master transcriptional regulators of autophagy and lysosomal activity and their cytoplasm/nuclear shuttling is controlled by MTORC1-dependent multisite phosphorylation. However, it is not known whether and how the transcriptional activity of TFEB or TFE3 is regulated. We show that AMPK mediates phosphorylation of TFEB and TFE3 on three serine residues, leading to TFEB and TFE3 transcriptional activity upon nutrient starvation, FLCN (folliculin) depletion and pharmacological manipulation of MTORC1 or AMPK. Collectively, we show that MTORC1 specifically controls TFEB and TFE3 cytosolic retention, whereas AMPK is essential for TFEB and TFE3 transcriptional activity. This dual and opposing regulation of TFEB and TFE3 by MTORC1 and AMPK is reminiscent of the regulation of another critical regulator of autophagy, ULK1 (unc-51 like autophagy activating kinase 1). Surprisingly, we show that chemoresistance is mediated by AMPK-dependent activation of TFEB, which is abolished by pharmacological inhibition of AMPK or mutation of serine 466, 467 and 469 to alanine residues within TFEB. Altogether, we show that AMPK is a key regulator of TFEB and TFE3 transcriptional activity, and we validate AMPK as a promising target in cancer therapy to evade chemotherapeutic resistance. ACACA: acetyl-CoA carboxylase alpha; ACTB: actin beta; AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; AMPKi: AMPK inhibitor, SBI-0206965; CA: constitutively active; CARM1: coactivator-associated arginine methyltransferase 1; CFP: cyan fluorescent protein; CLEAR: coordinated lysosomal expression and regulation; DKO: double knock-out; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; DQ-BSA: self-quenched BODIPY® dye conjugates of bovine serum albumin; EBSS: Earle's balanced salt solution; FLCN: folliculin; GFP: green fluorescent protein; GST: glutathione S-transferases; HD: Huntington disease; HTT: huntingtin; KO: knock-out; LAMP1: lysosomal associated membrane protein 1; MEF: mouse embryonic fibroblasts; MITF: melanocyte inducing transcription factor; MTORC1: MTOR complex 1; PolyQ: polyglutamine; RPS6: ribosomal protein S6; RT-qPCR: reverse transcription quantitative polymerase chain reaction; TCL: total cell lysates; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TKO: triple knock-out; ULK1: unc-51 like autophagy activating kinase 1.
自噬和溶酶体活性的增加促进肿瘤生长、存活和化疗耐药。在急性饥饿时，AMPK（AMP 激活的蛋白激酶）的激活和 MTORC1（雷帕霉素靶蛋白激酶复合物 1）的抑制迅速启动自噬，以维持能量平衡和细胞存活。TFEB（转录因子 E3）和 TFE3（结合 IGHM 增强子 3 的转录因子）是自噬和溶酶体活性的主要转录调控因子，其胞质/核穿梭由 MTORC1 依赖性多位点磷酸化控制。然而，TFEB 或 TFE3 的转录活性是否以及如何受到调节尚不清楚。我们表明，AMPK 介导 TFEB 和 TFE3 上三个丝氨酸残基的磷酸化，导致营养饥饿、FLCN（多囊肾）耗竭和 MTORC1 或 AMPK 的药理学操作后 TFEB 和 TFE3 的转录活性。总的来说，我们表明 MTORC1 特异性控制 TFEB 和 TFE3 的胞质保留，而 AMPK 是 TFEB 和 TFE3 转录活性所必需的。MTORC1 和 AMPK 对 TFEB 和 TFE3 的这种双重和相反的调节类似于另一个自噬关键调节剂 ULK1（非典型蛋白激酶 1）的调节。令人惊讶的是，我们表明，化疗耐药是由 AMPK 依赖性 TFEB 激活介导的，这种激活可被 AMPK 的药理学抑制或 TFEB 中丝氨酸 466、467 和 469 突变为丙氨酸残基所消除。总的来说，我们表明 AMPK 是 TFEB 和 TFE3 转录活性的关键调节剂，并验证 AMPK 是逃避化疗耐药的癌症治疗中的一个有希望的靶点。ACACA：乙酰辅酶 A 羧化酶 α；ACTB：肌动蛋白 β；AICAR：5-氨基咪唑-4-甲酰胺核苷酸；AMPK：AMP 激活的蛋白激酶；AMPKi：AMPK 抑制剂，SBI-0206965；CA：组成型激活；CARM1：共激活物相关精氨酸甲基转移酶 1；CFP：青色荧光蛋白；CLEAR：协调的溶酶体表达和调节；DKO：双敲除；DMEM：杜尔贝科改良伊格尔培养基；DMSO：二甲基亚砜；DQ-BSA：牛血清白蛋白的自猝灭 BODIPY®染料缀合物；EBSS：Earle 平衡盐溶液；FLCN：多囊肾；GFP：绿色荧光蛋白；GST：谷胱甘肽 S-转移酶；HD：亨廷顿病；HTT：亨廷顿蛋白；KO：敲除；LAMP1：溶酶体相关膜蛋白 1；MEF：小鼠胚胎成纤维细胞；MITF：黑素细胞诱导转录因子；MTORC1：MTOR 复合物 1；PolyQ：多聚谷氨酰胺；RPS6：核糖体蛋白 S6；RT-qPCR：逆转录定量聚合酶链反应；TCL：总细胞裂解物；TFE3：结合 IGHM 增强子 3 的转录因子；TFEB：转录因子 EB；TKO：三重敲除；ULK1：非典型蛋白激酶 1。
