STAT3 suppresses the AMPKα/ULK1-dependent induction of autophagy in glioblastoma cells.
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ABSTRACT:
SUBMITTER: Bhattacharya S
PROVIDER: S-EPMC9279602 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Muscle wasting is the key manifestation of cancer-associated cachexia, a lethal metabolic disorder seen in over 50% of cancer patients. Autophagy is activated in cachectic muscle of cancer hosts along with the ubiquitin-proteasome pathway (UPP), contributing to accelerated protein degradation and muscle wasting. However, established signaling mechanism that activates autophagy in response to fasting or denervation does not seem to mediate cancer-provoked autophagy in skeletal myocytes. Here, we show that p38β MAPK mediates autophagy activation in cachectic muscle of tumor-bearing mice via novel mechanisms. Complementary genetic and pharmacological manipulations reveal that activation of p38β MAPK, but not p38α MAPK, is necessary and sufficient for Lewis lung carcinoma (LLC)-induced autophagy activation in skeletal muscle cells. Particularly, muscle-specific knockout of p38β MAPK abrogates LLC tumor-induced activation of autophagy and UPP, sparing tumor-bearing mice from muscle wasting. Mechanistically, p38β MAPK-mediated activation of transcription factor C/EBPβ is required for LLC-induced autophagy activation, and upregulation of autophagy-related genes LC3b and Gabarapl1. Surprisingly, ULK1 activation (phosphorylation at S555) by cancer requires p38β MAPK, rather than AMPK. Activated ULK1 forms a complex with p38β MAPK in myocytes, which is markedly increased by a tumor burden. Overexpression of a constitutively active p38Tbeta; MAPK in HEK293 cells increases phosphorylation at S555 and other amino acid residues of ULK1, but not several of AMPK-mediated sites. Finally, ULK1 activation is abrogated in tumor-bearing mice with muscle-specific knockout of p38β MAPK. Thus, p38β MAPK appears a key mediator of cancer-provoked autophagy activation, and a therapeutic target of cancer-induced muscle wasting.
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Project description:Prototheca bovis, a highly contagious pathogen, causes bovine mastitis, resulting in premature culling of affected cows and severe economic losses. Infection with P. bovis caused oxidative stress and apoptosis in bovine mammary epithelial cells (bMECs); however, mechanisms underlying P. bovis-induced autophagy remain unclear. Therefore, the autophagy flux induced by P. bovis in bMECs was analyzed by Western blot and laser scanning confocal microscopy. Expression levels of proteins in the HIF-1α and AMPKα/ULK1 pathway, including HIF-1α, AMPKα, p-AMPKα, ULK1, p-ULK1, mTOR, and p-mTOR, plus expression of autophagy-related genes including SQSTM1/p62, Atg5, Beclin1, and LC3II/LC3I, were quantified with Western blot. Infection with P. bovis induced autophagosomes and LC3 puncta in bMECs that were detected using transmission electron microscopy and laser scanning confocal microscopy, respectively. In addition, lysosome-associated proteins Rab7 and LAMP2a, and lysosomal activity were measured with Western blot and laser scanning confocal microscopy. Infection with P. bovis induced an unobstructed autophagic flux, increased protein expression of LC3II/LC3I, and decreased SQSTM1/p62 protein expression at 6 hpi. Furthermore, P. bovis upregulated protein expression in the HIF-1α and AMPKα/ULK1 pathway and increased the ratio of LC3II/LC3I, implying autophagy was activated in bMECs. However, deletion of AMPKα or ULK1 decreased LC3II/LC3I expression levels and LC3 puncta numbers, suggesting that autophagy was inhibited in bMECs. Additionally, deficiency of HIF-1α decreased protein expression of AMPKα and ULK1 as well as LC3 puncta numbers, and autophagy induced by P. bovis was also inhibited in bMECs. At 6 hpi, lysosome-associated protein Rab7 was decreased and LAMP2a was increased, indicating normal autophagy. In contrast, at 12 hpi, expression of Rab7 and LAMP2a proteins indicated that autophagy was inhibited in bMECs at that time. Therefore, we confirmed that P. bovis infection induced autophagy in bMECs via the HIF-1α and AMPKα/ULK1 pathway, with involvement of lysosome-associated protein Rab7 and LAMP2a.
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Project description:Glioblastoma (GBM) is the most common primary brain malignancy in adults with a dismal prognosis. Despite advances in genomic analysis and surgical technique and the development of targeted therapeutics, most treatment options are ineffective and mainly palliative. Autophagy is a form of cellular self-digestion with the goal of recycling intracellular components to maintain cell metabolism. Here, we describe some recent findings that suggest GBM tumors are more sensitive to the excessive overactivation of autophagy leading to autophagy-dependent cell death. GBM cancer stem cells (GSCs) are a subset of the GBM tumor population that play critical roles in tumor formation and progression, metastasis, and relapse, and they are inherently resistant to most therapeutic strategies. Evidence suggests that GSCs are able to adapt to a tumor microenvironment of hypoxia, acidosis, and lack of nutrients. These findings have suggested that autophagy may promote and maintain the stem-like state of GSCs as well as their resistance to cancer treatment. However, autophagy is a double-edged sword and may have anti-tumor properties under certain conditions. The role of the STAT3 transcription factor in autophagy is also described. These findings provide the basis for future research aimed at targeting the autophagy-dependent pathway to overcome the inherent therapeutic resistance of GBM in general and to specifically target the highly therapy-resistant GSC population through autophagy regulation.
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Project description:Glioblastoma is one of the most aggressive human cancers with poor prognosis, and therefore a critical need exists for novel therapeutic strategies for management of glioblastoma patients. Itraconazole, a traditional antifungal drug, has been identified as a novel potential anticancer agent due to its inhibitory effects on cell proliferation and tumor angiogenesis; however, the molecular mechanisms involved are still unclear. Here, we show that itraconazole inhibits the proliferation of glioblastoma cells both in vitro and in vivo. Notably, we demonstrate that treatment with itraconazole induces autophagic progression in glioblastoma cells, while blockage of autophagy markedly reverses the antiproliferative activities of itraconazole, suggesting an antitumor effect of autophagy in response to itraconazole treatment. Functional studies revealed that itraconazole retarded the trafficking of cholesterol from late endosomes and lysosomes to the plasma membrane by reducing the levels of SCP2, resulting in repression of AKT1-MTOR signaling, induction of autophagy, and finally inhibition of cell proliferation. Together, our studies provide new insights into the molecular mechanisms regarding the antitumor activities of itraconazole, and may further assist both the pharmacological investigation and rational use of itraconazole in potential clinical applications.
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Project description:ULK1 (unc-51 like autophagy activating kinase 1) is the key mediator of MTORC1 signaling to macroautophagy/autophagy. ULK1 functions as a protein complex by interacting with ATG13, RB1CC1/FIP200, and ATG101. How the ULK1 complex is regulated to trigger autophagy induction remains unclear. In this study, we have determined roles of Atg8-family proteins (ATG8s) in regulating ULK1 activity and autophagy. Using human cells depleted of each subfamily of ATG8, we found that the GABARAP subfamily positively regulates ULK1 activity and phagophore and autophagosome formation in response to starvation. In contrast, the LC3 subfamily negatively regulates ULK1 activity and phagophore formation. By reconstituting ATG8-depleted cells with individual ATG8 members, we identified GABARAP and GABARAPL1 as positive and LC3B and LC3C as negative regulators of ULK1 activity. To address the role of ATG8 binding to ULK1, we mutated the LIR of endogenous ULK1 to disrupt the ATG8-ULK1 interaction by genome editing. The mutation drastically reduced the activity of ULK1, autophagic degradation of SQSTM1, and phagophore formation in response to starvation. The mutation also suppressed the formation and turnover of autophagosomes in response to starvation. Similar to the mutation of the ULK1 LIR, disruption of the ATG13-ATG8 interaction suppressed ULK1 activity and autophagosome formation. In contrast, RB1CC1 did not show any specific binding to ATG8s, and mutation of its LIR did not affect ULK1 activity. Together, this study demonstrates differential binding and opposite regulation of the ULK1 complex by GABARAPs and LC3s, and an important role of the ULK1- and ATG13-ATG8 interactions in autophagy induction.Abbreviations: ATG5: autophagy related 5; ATG7: autophagy related 7; ATG8: autophagy related 8; ATG13: autophagy related 13; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; ATG101: autophagy related 101; BAFA1: bafilomycin A1; BECN1: beclin 1; Cas9: CRISPR associated protein 9; CRISPR: clustered regularly interspaced short palindromic repeats; EBSS: earle's balanced salt solution; DAPI: 4'-6-diamidino-2-phenylindole; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor-associated protein like 1; GABARAPL2: GABA type A receptor-associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescence protein; gRNA: guide RNA; KI: kinase inactive mutant; KO: knockout; LC3A: microtubule associated protein 1 light chain 3 alpha; LC3B: microtubule associated protein 1 light chain 3 beta; LC3C: microtubule associated protein 1 light chain 3 gamma; LIR: LC3-interacting region; MTORC1: mechanistic target of rapamycin kinase complex 1; PBS: phosphate buffered saline; PCR: polymerase chain reaction; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; qPCR: quantitative PCR; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RPS6KB1: ribosomal protein S6 kinase B1; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TALEN: transcription activator-like effector nuclease; TUBA: tubulin alpha; ULK1: unc-51 like autophagy activating kinase 1; WB: western blotting; WIPI2: WD repeat domain phosphoinositide interacting 2; WT: wild type.
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