Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou, China.
Department of Pharmacology, Faculty of Medicine, Dalhousie University, Halifax, Canada.
Autophagy. 2021 Dec;17(12):4401-4422. doi: 10.1080/15548627.2021.1917132. Epub 2021 Apr 23.
Macroautophagy/autophagy is elevated to ensure the high demand for nutrients for the growth of cancer cells. Here we demonstrated that MCOLN1/TRPML1 is a pharmaceutical target of oncogenic autophagy in cancers such as pancreatic cancer, breast cancer, gastric cancer, malignant melanoma, and glioma. First, we showed that activating MCOLN1, by increasing expression of the channel or using the MCOLN1 agonists, ML-SA5 or MK6-83, arrests autophagic flux by perturbing fusion between autophagosomes and lysosomes. Second, we demonstrated that MCOLN1 regulates autophagy by mediating the release of zinc from the lysosome to the cytosol. Third, we uncovered that zinc influx through MCOLN1 blocks the interaction between STX17 (syntaxin 17) in the autophagosome and VAMP8 in the lysosome and thereby disrupting the fusion process that is determined by the two SNARE proteins. Furthermore, we demonstrated that zinc influx originating from the extracellular fluid arrests autophagy by the same mechanism as lysosomal zinc, confirming the fundamental function of zinc as a participant in membrane trafficking. Last, we revealed that activating MCOLN1 with the agonists, ML-SA5 or MK6-83, triggers cell death of a number of cancer cells by evoking autophagic arrest and subsequent apoptotic response and cell cycle arrest, with little or no effect observed on normal cells. Consistent with the results, administration of ML-SA5 in Patu 8988 t xenograft mice profoundly suppresses tumor growth and improves survival. These results establish that a lysosomal cation channel, MCOLN1, finely controls oncogenic autophagy in cancer by mediating zinc influx into the cytosol. Abbreviations: 3-MA: 3-methyladenine; AA: amino acid; ATG12: autophagy related 12; Baf-A1: bafilomycin A1; BAPTA-am: 1,2-bis(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; co-IP: coimmunoprecipitaion; CQ: chloroquine; DMEM: Dulbecco's Modified Eagle Medium; FBS: fetal bovine serum; GAPDH: glyceraldehyde- 3-phosphate dehydrogenase; HCQ: hydroxychloroquine; HEK: human embryonic kidney; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCOLN1/TRPML1: mucolipin TRP cation channel 1; MTORC1: mechanistic target of rapamycin kinase complex 1; NC: negative control; NRK: normal rat kidney epithelial cells; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; RPS6KB/S6K: ribosomal protein S6 kinase B; shRNA: short hairpin RNA; siRNA: short interfering RNA; SNARE: soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TPEN: N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; TTM: tetrathiomolybdate; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; Zn: zinc.
自噬作用会被上调以确保癌细胞生长所需的大量营养物质。在这里，我们证明了 MCOLN1/TRPML1 是癌症（如胰腺癌、乳腺癌、胃癌、恶性黑色素瘤和神经胶质瘤）中致癌自噬的药物靶点。首先，我们表明通过增加通道的表达或使用 MCOLN1 激动剂 ML-SA5 或 MK6-83 来激活 MCOLN1，可以通过扰乱自噬体与溶酶体之间的融合来阻断自噬流。其次，我们证明 MCOLN1 通过介导锌从溶酶体向细胞质释放来调节自噬。第三，我们揭示了锌通过 MCOLN1 流入阻断了自噬体中的 STX17（突触融合蛋白 17）和溶酶体中的 VAMP8 之间的相互作用，从而破坏了由两个 SNARE 蛋白决定的融合过程。此外，我们证明来自细胞外液的锌流入通过与溶酶体中的锌相同的机制阻断自噬，证实了锌作为膜转运参与者的基本功能。最后，我们揭示了用激动剂 ML-SA5 或 MK6-83 激活 MCOLN1 通过引发自噬停滞和随后的凋亡反应和细胞周期停滞，触发多种癌细胞死亡，对正常细胞几乎没有或没有影响。与结果一致，在 Patu 8988t 异种移植小鼠中给予 ML-SA5 可显著抑制肿瘤生长并提高存活率。这些结果表明，溶酶体阳离子通道 MCOLN1 通过介导锌流入细胞质来精细控制癌症中的致癌自噬。缩写：3-MA：3-甲基腺嘌呤；AA：氨基酸；ATG12：自噬相关 12；Baf-A1：巴弗霉素 A1；BAPTA-am：1,2-双（2-氨基苯氧基）乙烷-N，N，N'，N'-四乙酸四（乙酰氧甲基）酯；co-IP：共免疫沉淀；CQ：氯喹；DMEM：杜尔贝科改良伊格尔培养基；FBS：胎牛血清；GAPDH：甘油醛-3-磷酸脱氢酶；HCQ：羟氯喹；HEK：人胚肾；LAMP1：溶酶体相关膜蛋白 1；MAP1LC3/LC3：微管相关蛋白 1 轻链 3；MCOLN1/TRPML1：粘蛋白 TRP 阳离子通道 1；MTORC1：雷帕霉素靶蛋白激酶复合物 1；NC：阴性对照；NRK：正常大鼠肾上皮细胞；PBS：磷酸盐缓冲盐水；PtdIns3K：磷脂酰肌醇 3-激酶；RPS6KB/S6K：核糖体蛋白 S6 激酶 B；shRNA：短发夹 RNA；siRNA：小干扰 RNA；SNARE：可溶性 NSF 附着蛋白受体；SQSTM1/p62：自噬体相关蛋白 1；STX17：突触融合蛋白 17；TPEN：N，N，N'，N'-四（2-吡啶基甲基）乙二胺；TTM：四硫钼酸盐；ULK1：UNC-51 样自噬激活激酶 1；VAMP8：囊泡相关膜蛋白 8；Zn：锌。
