MCOLN1/TRPML1 finely controls oncogenic autophagy in cancer by mediating zinc influx.

ABSTRACT

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 in vitro 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.Abbreviation 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; Zn2+ zinc.