Published OnlineFirst March 12, 2019; DOI: 10.1158/1078-0432.CCR-18-3791 Translational Cancer Mechanisms and Therapy Clinical Cancer Research HERC3-Mediated SMAD7 Ubiquitination Degradation Promotes Autophagy-Induced EMT and Chemoresistance in Glioblastoma Hong Li1, Junjie Li1, Lei Chen1, Songtao Qi1,2,3, Shishi Yu4, Zhijian Weng1, Ziyou Hu5, Qiang Zhou1, Zong Xin1, Linyong Shi1, Liyi Ma1, Annie Huang6, and Yuntao Lu1,2,3 Abstract Purpose: Glioblastoma, a common malignant intracranial Results: Autophagy inducers significantly upregulated tumor, has the most dismal prognosis. Autophagy was the expression of HERC3, which promotes ubiquitina- reported to act as a survival-promoting mechanism in gliomas tion-mediated degradation of SMAD7 in an autolyso- by inducing epithelial-to-mesenchymal transition (EMT). some-dependent manner. The corresponding increase in Here, we determined the critical molecules involved in autop- p-SMAD2/3 level and TGFb pathway activation finally hagy-induced EMT and elucidated the possible mechanism of induced EMT in cell lines and primary glioblastoma cells. chemoradiotherapy resistance and tumor recurrence. Moreover, HERC3 overexpression was observed in pseudo- Experimental Design: We used isobaric tags for relative and palisade cells surrounding tumor necrosis and in tumor- absolute quantitation to identify the critical proteins and adjacent tissue; high HERC3 and low SMAD7 levels pre- pathway mediating EMT via autophagy inducer treatment, dicted poor clinical outcome in glioblastoma; xenograft of and tested the expression of these proteins using tissue micro- nude mice and in vitro experiments confirmed these array of gliomas and clinical glioblastoma samples as well as findings. tissues and cells separated from the core lesion and tumor- Conclusions: Together, our findings reveal the indis- peripheral region. Analysis of the Cancer Genome Atlas data- pensable role of HERC3 in regulating canonical SMAD2/ base and 110 glioblastoma cases revealed the prognostic value 3-dependent TGFb pathway involvement in autophagy- of these molecules. The functional role of these critical mole- induced EMT, providing insights toward a better understand- cules was further confirmed by in vitro experiments and intra- ing of the mechanism of resistance to temozolomide and cranial xenograft in nude mice. peripheral recurrence of glioblastoma. Introduction widely used after surgical resection. Temozolomide has been evidenced to trigger autophagy-associated cell death to inhibit Autophagy is thought to play a double-edged sword role in tumor growth in GBM (3). However, the prognosis of patients cancer by either suppressing tumorigenesis via its quality con- with GBM remains poor despite such treatment, with a median trol function or promoting tumor survival under microenvi- survival of 14.6 months. Moreover, inhibition of autophagy by ronmental stress and increasing growth and aggressiveness (1). knockdown of autophagy related 12 (ATG12) in glioma cells As the standard Stupp therapy for glioblastoma (GBM; ref. 2), does not affect cell viability, proliferation, or migration, but the most common intracranial aggressive tumor, ionizing radi- rather significantly reduces cellular invasion in a three- ation plus concomitant and adjuvant temozolomide has been dimensional (3D) organotypic model. Thus, autophagy may play a critical role in the benign-to-malignant transition, which 1Department of Neurosurgery, Southern Medical University, Guangzhou, China. is central to the initiation of metastasis (4). In addition, 2Nanfang Neurology Research Institution, Nanfang Hospital, Southern Medical 3 autophagy has also recently been considered to be an impor- University, Guangzhou, China. Nanfang Glioma Center, Guangzhou, China. tant mediator of the epithelial-to-mesenchymal transition 4Editorial Department of the Journal of Southern Medical University, Guangz- hou, China. 5Research Center of Clinical Medicine, Nanfang Hospital, Southern (EMT) status in several epithelial carcinomas. In particular, in Medical University, Guangzhou, China. 6Brain Tumor Research Center, SickKids hepatocellular carcinoma cells, starvation-induced autophagy Hospital, Toronto, Canada. is critical for cellular invasion via the induction of EMT (5). Note: Supplementary data for this article are available at Clinical Cancer Furthermore, autophagy is thought to comprise a potential Research Online (http://clincancerres.aacrjournals.org/). molecular mechanism of chemoresistance in cancers (6, 7), H. Li, J. Li, L. Chen, and S. Qi contributed equally to this article. and has accordingly been assessed as a potential therapeutic target during EMT in renal cell carcinoma (8). Corresponding Author: Yuntao Lu, Nanfang Hospital, #1838 North Guangzhou EMT, a critical biological behavior involved in proper embry- Avenue, Guangzhou 510515, China. Phone: 8620-61641806; Fax: 8620-6164- 1806; E-mail: [email protected] onic development, has also been shown to mediate tumor invasion and metastasis in several tumors (9, 10), including Clin Cancer Res 2019;25:3602–16 gliomas. In GBM, the EMT stimulated by activators, including doi: 10.1158/1078-0432.CCR-18-3791 members of the SNAI family (e.g., ZEB1 and ZEB2), can cause Ó2019 American Association for Cancer Research. cells to obtain certain features of mesenchymal cells, such as 3602 Clin Cancer Res; 25(12) June 15, 2019 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst March 12, 2019; DOI: 10.1158/1078-0432.CCR-18-3791 HERC3 Promotes Autophagy-Induced EMT and Chemoresistance repair activity under cisplatin treatment (20). HERC2 also plays Translational Relevance an important role in breast carcinogenesis through ubiquitin- Autophagy-induced epithelial-to-mesenchymal transition mediated degradation of the breast cancer suppressor BRCA1 (21). (EMT) reportedly contributes indispensably to tumor invasion In comparison, rare studies regarding "small" HERCs have indi- and chemoresistance in gliomas. We previously proved cated that HERC3 mRNA exhibits high expression in brain tis- that temozolomide, a classic glioblastoma chemotherapeutic sue (22) and that "small" HERC proteins can interact with each agent that also functions as an autophagic inducer, can induce other and localize to the same cellular structures, which are EMT, which mediates drug resistance. Here, we uncovered that identified as late endosomes and lysosomes (23). HERC3 is also the E3 ubiquitin ligase, HERC3, promotes the ubiquitination- considered as a novel candidate for regulating the inflammatory mediated degradation of SMAD7 (I-Smad) in an autolyso- response initiated by NF-kB (24), which negatively regulates some-dependent manner, and consequently activates the autophagy (25). Although the functional role of "small" HERCs TGFb pathway, which plays indispensable roles in autop- in cancer remains unknown, frame shift mutations in HERC3 hagy-induced EMT; the differential expression of HERC3 and have been correlated with microsatellite instability in gastric and SMAD7 in the core lesions and peripheral tissues indicated the colorectal carcinomas (26). mechanism of the peripheral recurrence of chemoradiother- In this study, we found that HERC3 is the critical molecule apy-treated glioblastoma. Moreover, analysis of the clinical involved in autophagy-induced EMT and that it activates the database revealed a prognostic value of HERC3 in glioblasto- SMAD2/3-dependent TGFb signaling pathway by ubiquitin- ma. Targeting HERC3 via tumor xenografts significantly inhib- mediated degradation of SMAD7, which is indicative of the ited cellular invasion, reversed EMT, and increased overall endosomal-lysosomal–dependent degradation pathway (27). survival. Our data would be of remarkable value for designing Besides, HERC3 and SMAD7 level were differentially expressed new combination therapies that can overcome chemoresis- in tissues and cells separated from the core lesion and tumor- tance and that can be further tested in clinical trials. peripheral region. The levels of these two molecules were closely correlated with poor prognosis of patients with GBM, especially those with the classical subtypes, who were treated with the Stupp regimen after surgery (2) by The Cancer Genome Atlas longer cellular projections (pseudopodia) and higher invasive (TCGA) bioinformatics analysis and follow up data for 110 ability (11). In addition, EMT may also induce the cellular clinical cases. Our findings elucidate the possible mechanism of dedifferentiation of noncancer stem cells, allowing the cells to temozolomide chemoradiotherapy resistance and tumor recur- acquire self-renewal and tumor-initiating ability, as well as a rence of GBM. tolerance to chemoradiotherapy (12, 13). Recently, Parkinson protein 2 E3 ubiquitin protein (PARK2) was shown to partic- ipate in regulating the invasion–metastasis cascade in GBM Materials and Methods cells by downregulating ZEB1 expression to mitigate EMT and Clinical tumor sample collection acting as a metastasis suppressor in GBM progression (14). In In total, 110 patients with newly diagnosed GBM who had contrast, E3 ubiquitin ligase HectH9 was found to mediate undergone surgery plus standard chemoradiotherapy (Stupp K63-linked polyubiquitination of HAUSP and cause CBP-medi- regimen) were included in this study. All samples were collected ated H3K56 acetylation on HIF-1a target gene promoters to from the Nanfang Hospital of Southern Medical University promote EMT/metastasis