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P53 mitigates the effects of oncogenic HRAS in urothelial cells via the repression of MCOLN1.

ABSTRACT: Inhibition of TRPML1, which is encoded by MCOLN1, is known to deter cell proliferation in various malignancies. Here, we report that the tumor suppressor, p53, represses MCOLN1 in the urothelium such that either the constitutive loss or ectopic knockdown of TP53-in both healthy and bladder cancer cells-increased MCOLN1 expression. Conversely, nutlin-mediated activation of p53 led to the repression of MCOLN1. Elevated MCOLN1 expression in p53-deficient cancer cells, though not sufficient for bolstering proliferation, augmented the effects of oncogenic HRAS on proliferation, cytokine production, and invasion. Our data suggest that owing to derepression of MCOLN1, urothelial cells lacking p53 are poised for tumorigenesis driven by oncogenic HRAS. Given our prior findings that HRAS mutations predict addiction to TRPML1, this study points to the utility of TRPML1 inhibitors for mitigating the growth of a subset of urothelial tumors that lack p53.

SUBMITTER: Jung J

PROVIDER: S-EPMC8243020 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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p53 mitigates the effects of oncogenic HRAS in urothelial cells via the repression of MCOLN1.

Jung Jewon J Liao Han H Coker Shannon A SA Liang Hong H Hancock John F JF Denicourt Catherine C Venkatachalam Kartik K

iScience 20210609 7

Inhibition of TRPML1, which is encoded by MCOLN1, is known to deter cell proliferation in various malignancies. Here, we report that the tumor suppressor, p53, represses MCOLN1 in the urothelium such that either the constitutive loss or ectopic knockdown of TP53-in both healthy and bladder cancer cells-increased MCOLN1 expression. Conversely, nutlin-mediated activation of p53 led to the repression of MCOLN1. Elevated MCOLN1 expression in p53-deficient ca ...[more]

PMID: 34222845

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Project description:Compelling evidence has demonstrated that macroautophagy/autophagy plays an important role in regulating multiple steps of metastatic cascades; however, the precise role of autophagy in metastasis remains unclear. This study demonstrates that autophagy inhibition induced by MCOLN1/TRPML1 suppresses cancer metastasis by evoking the ROS-mediated TP53/p53 pathway. First, we found that MCOLN1-mediated autophagy inhibition not only profoundly inhibits both migration and invasion in malignant melanoma and glioma cell lines in vitro, but also suppresses melanoma metastasis in vivo. Second, our study reveals that autophagy inhibition induced by MCOLN1 leads to damaged mitochondria accumulation followed by large quantities of ROS release. Third, we demonstrate that the elevated ROS resulting from autophagy inhibition subsequently triggers TP53 activity, which in turn modulates expression of its downstream targets that are involved in a broad spectrum of the metastatic cascade to suppress metastasis including MMP members and TWIST. In summary, our findings have established a mechanism by which autophagy inhibition suppresses metastasis via the ROS-TP53 signaling pathway. More importantly, our study demonstrates that autophagy inhibition through stimulation of MCOLN1 could evidently be one of the therapeutic potentials for combating cancer metastasis.Abbreviations: 3-MA: 3-methyladenine; AA: amino acid; ATG5: autophagy related 5; ATG12: autophagy-related 12; Baf-A1: bafilomycin A1; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CQ: chloroquine; DMEM: Dulbecco's Modified Eagle Medium; EMT: epithelial-mesenchymal transition; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; 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; MMP: matrix metallopeptidase; NC: negative control; NRK: normal rat kidney; PBS: phosphate-buffered saline; shRNA: short hairpin RNA; siRNA: short interfering RNA; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy-activating kinase 1.

Project description: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.

Dataset Information

P53 mitigates the effects of oncogenic HRAS in urothelial cells via the repression of MCOLN1.

Publications

p53 mitigates the effects of oncogenic HRAS in urothelial cells via the repression of <i>MCOLN1</i>.

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