TRPML1-induced autophagy inhibition triggers mitochondrial mediated apoptosis.

Previous studies have demonstrated that autophagy tightly regulates apoptosis. However, the underlying mechanism whereby autophagy regulates apoptosis remains unclear. Here, we discover a "autophagy inhibition-mitochondrial turnover disruption-ROS elevation-DNA damage-p53 transactivation-apoptosis" axis that explicates the process of autophagy modulating apoptosis. We found that autophagy inhibition induced by TRPML1, a cationic channel localized in the lysosome, results in accumulation of damaged mitochondria via blocking the mitophagic flux to lysosomes in human melanoma and glioblastoma cells. The disrupted mitochondria turnover leads to ROS elevation, which in turn causes severe damage to DNA in these cancer cells. Damage to DNA resulted from TRPML1-mediated autophagy inhibition subsequently activates p53, which ultimately triggers mitochondrial mediated apoptosis by modulating pro- and anti-apoptosis proteins in these cancer cells. As a result, by triggering apoptosis, TRPML1-induced autophagy inhibition greatly suppresses growth of human melanoma and glioma both in vitro and in vivo. In summary, our findings define the mechanism underling the regulation of autophagy inhibition in apoptosis and represent TRPML1 as a novel target for potentially treating melanoma and glioblastoma in the clinical setting.

Blunting TRPML1 channels protects myocardial ischemia/reperfusion injury by restoring impaired cardiomyocyte autophagy.

Accumulating evidence suggests that autophagy dysfunction plays a critical role in myocardial ischemia/reperfusion (I/R) injury. However, the underling mechanism of malfunctional autophagy in the cardiomyocytes subjected to I/R has not been well defined. As a result, there is no effective therapeutic option by targeting autophagy to prevent myocardial I/R injury. Here, we used both an in vitro and an in vivo I/R model to monitor autophagic flux in the cardiomyocytes, by exposing neonatal rat ventricular myocytes to hypoxia/reoxygenation and by subjecting mice to I/R, respectively. We observed that the autophagic flux in the cardiomyocytes subjected to I/R was blocked in both in vitro and in vivo models. Down-regulating a lysosomal cationic channel, TRPML1, markedly restored the blocked myocardial autophagic flux induced by I/R, demonstrating that TRPML1 directly contributes to the blocked autophagic flux in the cardiomyocytes subjected to I/R. Mechanistically, TRPML1 is activated secondary to ROS elevation following ischemia/reperfusion, which in turn induces the release of lysosomal zinc into the cytosol and ultimately blocks the autophagic flux in cardiomyocytes, presumably by disrupting the fusion between autophagosomes and lysosomes. As a result, the inhibited myocardial autophagic flux induced by TRPML1 disrupted mitochondria turnover and resulted in mass accumulation of damaged mitochondria and further ROS release, which directly led to cardiomyocyte death. More importantly, pharmacological and genetic inhibition of TRPML1 channels greatly reduced infarct size and rescued heart function in mice subjected to I/R in vivo by restoring impaired myocardial autophagy. In summary, our study demonstrates that secondary to ROS elevation, activation of TRPML1 results in autophagy inhibition in the cardiomyocytes subjected to I/R, which directly leads to cardiomyocyte death by disrupting mitochondria turnover. Therefore, targeting TRPML1 represents a novel therapeutic strategy to protect against myocardial I/R injury.

Stimulating TRPM7 suppresses cancer cell proliferation and metastasis by inhibiting autophagy.

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitous cation channel possessing kinase activity. TRPM7 mediates a variety of physiological responses by conducting flow of cations such as Ca2+, Mg2+, and Zn2+. Here, we show that the activation of TRPM7 channel stimulated by chemical agonists of TRPM7, Clozapine or Naltriben, inhibited autophagy via mediating Zn2+ release to the cytosol, presumably from the intracellular Zn2+-accumulating vesicles where TRPM7 localizes. Zn2+ release following the activation of TRPM7 disrupted the fusion between autophagosomes and lysosomes by disturbing the interaction between Sxt17 and VAMP8 which determines fusion status of autophagosomes and lysosomes. Ultimately, the disrupted fusion resulting from stimulation of TRPM7 channels arrested autophagy. Functionally, we demonstrate that the autophagy inhibition mediated by TRPM7 triggered cell death and suppressed metastasis of cancer cells in vitro, more importantly, restricted tumor growth and metastasis in vivo, by evoking apoptosis, cell cycle arrest, and reactive oxygen species (ROS) elevation. These findings represent a strategy for stimulating TRPM7 to combat cancer.

Autophagy inhibition mediated by MCOLN1/TRPML1 suppresses cancer metastasis via regulating a ROS-driven TP53/p53 pathway.

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.

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

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.

A Time-Delayed Mathematical Model for Tumor Growth with the Effect of a Periodic Therapy.

A time-delayed mathematical model for tumor growth with the effect of periodic therapy is studied. The establishment of the model is based on the reaction-diffusion dynamics and mass conservation law and is considered with a time delay in cell proliferation process. Sufficient conditions for the global stability of tumor free equilibrium are given. We also prove that if external concentration of nutrients is large the tumor will not disappear and the conditions under which there exist periodic solutions to the model are also determined. Results are illustrated by computer simulations.