Purpose: Glioblastoma, a common malignant intracranial tumor, has the most dismal prognosis. Autophagy was reported to act as a survival-promoting mechanism in gliomas by inducing epithelial-to-mesenchymal transition (EMT). Here, we determined the critical molecules involved in autophagy-induced EMT and elucidated the possible mechanism of chemoradiotherapy resistance and tumor recurrence.Experimental Design: We used isobaric tags for relative and absolute quantitation to identify the critical proteins and pathway mediating EMT via autophagy inducer treatment, and tested the expression of these proteins using tissue microarray of gliomas and clinical glioblastoma samples as well as tissues and cells separated from the core lesion and tumorperipheral region. Analysis of the Cancer Genome Atlas database and 110 glioblastoma cases revealed the prognostic value of these molecules. The functional role of these critical molecules was further confirmed by in vitro experiments and intracranial xenograft in nude mice.Results: Autophagy inducers significantly upregulated the expression of HERC3, which promotes ubiquitination-mediated degradation of SMAD7 in an autolysosome-dependent manner. The corresponding increase in p-SMAD2/3 level and TGFb pathway activation finally induced EMT in cell lines and primary glioblastoma cells. Moreover, HERC3 overexpression was observed in pseudopalisade cells surrounding tumor necrosis and in tumoradjacent tissue; high HERC3 and low SMAD7 levels predicted poor clinical outcome in glioblastoma; xenograft of nude mice and in vitro experiments confirmed these findings.Conclusions: Together, our findings reveal the indispensable role of HERC3 in regulating canonical SMAD2/ 3-dependent TGFb pathway involvement in autophagyinduced EMT, providing insights toward a better understanding of the mechanism of resistance to temozolomide and peripheral recurrence of glioblastoma.

Rationale: Glioblastoma (GBM) is the most common and aggressive brain tumor, characterized by its propensity to invade the surrounding brain parenchyma. The effect of extracellular high-mobility group box 1 (HMGB1) protein on glioblastoma (GBM) progression is still controversial. p62 is overexpressed in glioma cells, and has been associated with the malignant features and poor prognosis of GBM patients. Hence, this study aimed to clarify the role of p62 in HMGB1-induced epithelial-mesenchymal transition (EMT) of GBM both in vitro and in vivo . Methods: Immunoblotting, immunofluorescence and qRT-PCR were performed to evaluate EMT progression in both human GBM cell line and primary GBM cells. Transwell and wound healing assays were used to assess the invasion and migration of GBM cells. shRNA technique was used to investigate the role of p62 in HMGB1-induced EMT both in vitro and in vivo orthotopic tumor model. Co-immunoprecipitation assay was used to reveal the interaction between p62 and GSK-3β (glycogen synthase kinase 3 beta). Immunohistochemistry was performed to detect the expression levels of proteins in human GBM tissues. Results: In this study, GBM cells treated with recombinant human HMGB1 (rhHMGB1) underwent spontaneous EMT through GSK-3β/Snail signaling pathway. In addition, our study revealed that rhHMGB1-induced EMT of GBM cells was accompanied by p62 overexpression, which was mediated by the activation of TLR4-p38-Nrf2 signaling pathway. Moreover, the results demonstrated that p62 knockdown impaired rhHMGB1-induced EMT both in vitro and in vivo . Subsequent mechanistic investigations showed that p62 served as a shuttling factor for the interaction of GSK-3β with proteasome, and ultimately activated GSK-3β/Snail signaling pathway by augmenting the degradation of GSK-3β. Furthermore, immunohistochemistry analysis revealed a significant inverse correlation between p62 and GSK-3β, and a combination of the both might serve as a more powerful predictor of poor survival in GBM patients. Conclusions: This study suggests that p62 is an effector for HMGB1-induced EMT, and may represent a novel therapeutic target in GBM.

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