HERC3-Mediated SMAD7 Ubiquitination Degradation Promotes Autophagy-Induced EMT and Chemoresistance in Glioblastoma

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 2019 American Association for Cancer Research. cells to obtain certain features of mesenchymal cells, such as

3602 Clin Cancer Res; 25(12) June 15, 2019

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 in various human cancer cells (15). (Guangzhou, China) at the time of surgery before any other These studies thus indicate that E3 ubiquitin ligases may play therapies. All the research was carried out in accordance with the indispensable roles in regulating the EMT status in cancer cells. provisions of the declaration of Helsinki of 1975. Fresh samples HECT E3 ubiquitin-protein ligases are characterized by a were immediately preserved in liquid nitrogen. All specimens had C-terminal homologous to the E6-AP C-terminus (HECT) confirmed pathologic diagnosis and were classified according to domain (approximately 350 amino acids in length), which med- the 2016 World Health Organization Classification of Tumors of iates interaction with cognate E2 ubiquitin conjugating enzymes the Central Nervous System. The use of human brain tumor and acts as the catalytic domain in the final attachment of specimens and the database was written consent and ethically ubiquitin to substrate proteins (16, 17). In turn, the substrate approved by the Institutional Review Board at Nanfang Hospital specificity of HECT E3s is assumed to be determined by their of Southern Medical University (Guangzhou, China). respective N-terminal extensions. On the basis of the presence of distinct amino acid sequence motifs within these N-terminal RNA isolation and real-time qRT-PCR extensions, human HECT E3s can be grouped into three subfa- Details are provided in Supplementary Materials and Methods; milies: HERC E3s (HECT and RCC1-like domain), Nedd4/ a detailed list of primers is provided in the Supplementary Nedd4-like E3s (WW domains), and SI (ngle)-HECT E3s (neither information available online. containing RCC1-like nor WW domains; ref. 18). HERC E3s comprise six members including "large" HERCs (HERC1 and Cell lines and culture conditions HERC2) and "small" HERCs (HERC3-6); limited studies of HERC The human GBM cell lines (U87MG, LN229, T98G, A172, and E3s have focused on the "large" HERCs. The deletion of HERC1 in U118) and the human embryonic kidney cell line 293T were acute lymphoblastic leukemia enhances MSH2 degradation to purchased from the ATCC. U251 cells were obtained from Shang- cause DNA mismatch repair deficiency and drug resistance (19). hai Institutes for Biological Sciences, Chinese Academy of Sciences Moreover, ubiquitination and proteolysis of XPA by HERC2 has (Beijing, China). The human glial cell line HEB was obtained from been confirmed in lung cancer cells, whereas stabilization of XPA Sun Yat-Sen University (Guangdong, China). We also established by downregulation of HERC2 moderately enhances excision five pairs of patient-derived primary GBM cell lines including

www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 3603

Li et al.

GBM131212 and GBM141119. The details of the primary cell Immunofluorescence (colocalization) culture and single-cell clone purification are provided in the Cells were grown on glass coverslips in 6-well plates with Supplementary Materials and Methods available online. The cells corresponding treatment. Then, cells were fixed with 4% parafor- were normally cultured in DMEM (containing 4.5 g/L glucose, maldehyde for 30 minutes and incubated with primary antibo- Gibco 11995065) with 10% FBS (Gibco, 16140071) and 1% dies, fluorescence dye–conjugated secondary antibodies, and penicillin/streptomycin (Gibco, 10378016). All cell lines were DAPI according to standard protocols. The details of these anti- cultured at 37 C in a humidified atmosphere of 5% CO2. For low- bodies are provided in the Supplementary information available glucose culture, the DMEM contained only 1.0 g/L glucose online. Confocal laser microscope scanning of fixed cells was (Gibco, 11885084). performed using a Laser Scanning Microscope (Carl Zeiss, LSM 880).

Western blotting and antibodies Cell viability assay Details are provided in the Supplementary Materials and Cell viability was assessed by using CCK-8 assays. First, cells Methods; a detailed list of antibodies is provided in the Supple- were seeded in 96-well plates at a density of 8,000 cells per well. mentary information available online. After an overnight incubation, the cells were treated under the indicated conditions. At the end of the treatment, the Cell Count- Transient knockdown of SMAD7 and HERC3 ing Kit-8 (CCK-8, Dojindo) was used to calculate the number of siRNAs targeting SMAD7 and HERC3 were provided by Ribo- viable cells by measuring the absorbance at 450 nm using the bio. Cells were transfected using Lipofectamine 2000 reagent BMG Microplate reader (BMG Labtech, CLARIOstar). Temozolo- (Invitrogen, 11668-019) 24 hours after plating. Transfection mide IC50 was calculated using SPSS version 20.0 (IBM SPSS). complexes were prepared according to the manufacturer's instructions and added directly to the cells to a ﬁnal oligonucleotide Cytoplasmic and nuclear protein extraction concentration of 100 nmol/L. Transfection medium was replaced Details of cytoplasmic and nuclear protein extraction are pro- 6 hours posttransfection. The siRNA sequences are available in the vided in the Supplementary Materials and Methods available Supplementary information available online. online.

Immunoprecipitation and ubiquitination assays Stable HERC3 overexpression and knockdown For ubiquitination assays, T98G cells were transfected with Cells were prepared and infected with control or HERC3-over- Myc-HERC3; to detect the ubiquitination of endogenous SMAD7, expressing LVs (Genechem, Shanghai; HERC3 NCBI Reference the cells were treated with 50 nmol/L chloroquine for 8 hours Sequence: NM_014606.2). The transfection and the evaluation of prior to harvest. Cells were lysed in IP lysis buffer (Thermo Fisher transfection efficiency were performed as described previous- Scientific, 87787), and the lysates were immunoprecipitated ly (28). The shHERC3 LV and control (Obio Technology) were using an anti-SMAD7 antibody (Santa Cruz Biotechnology, sc- generated via the same approach to infect the cells. The sequences 101152) and Protein A/G Magnetic Beads (Bimake, B23202) and contained in the viruses are available in the Supplementary subjected to Western blotting. Ubiquitination was detected using information available online. an antibody against HA-Tag. 293T cells were transfected with Myc- HERC3, Flag-SMAD7, and HA-Ubiquitin (WT) to detect the ubi- Cell migration and invasive assay (wound-healing assay) quitination of exogenous SMAD7, and the subsequent steps were Cell migration and invasion assays were performed as performed using the same approach as described above, although described previously (29). Details are provided in the Supple- the lysates were immunoprecipitated using an anti-Flag antibody. mentary Materials and Methods available online. The Branched Ubiquitin Antibody Sampler Kit (Cell Signaling technology, 33959T) was used to analyze the polyubiquitin chain Use of NIH ImageJ for cell morphology quantification. For measure- bound to SMAD7 in LN229 cells. 293T cells were transfected with ments of the length, width, and area of cells, we used ImageJ Myc-HERC3, Flag-SMAD7, and HA-Ubiquitin (WT), HA-Ubiquitin (Bethesda, MD; http://rsb.info.nih.gov) according to a previous K48R,orHA-Ubiquitin K63R to thoroughly assess the polyubi- report (29). quitin chain bound to SMAD7. For ubiquitination site analysis, 293T cells were transfected with Myc-HERC3, HA-Ubiquitin (WT), and Flag-SMAD7, Luciferase reporter assay Flag-SMAD7 K64A, Flag-SMAD7 K70A, Flag-SMAD7 K101A,or LN229 cells were cotransfected with plasmids including pRL- Flag-SMAD7 K220A, and the protein level of SMAD7 and HERC3 TK and SBE-luc plasmid, and siRNAs targeting HERC3. T98G cells was detected by pulldown and immunoblotting assays. were cotransfected with pRL-TK, SBE-luc, and Myc-HERC3 plasmids. At 48 hours after the transfection, cells were treated with Histologic evaluation and IHC staining 2 ng/mL recombinant human TGFb1 (PeproTech, 100-21) for 8 Tissue section staining was performed as described previous- hours. Luciferase activity was determined using a Dual Luciferase ly (29), and the details of the staining and the score system for the Reporter Assay System (Promega, E1910) and the BMG Micro- percentage of positive cells and staining intensity are available in plate reader (BMG Labtech, CLARIOstar) according to the man- the Supplementary Materials and Methods available online. ufacturer's instructions. To reduce luciferase level errors generated by the different efficiencies of plasmids transfected into cells, we Xenograft studies chose the luciferase levels of pRL-TK as a control. All experiments Intracranial xenograft model of nude mice was generated as were performed in triplicate and repeated at least three times. described previously (29). Mouse experiments were conducted in

3604 Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer Research

HERC3 Promotes Autophagy-Induced EMT and Chemoresistance

accordance with the U.S. Public Health Service Policy on Humane respectively. Notably, the plasminogen activator inhibitor-1 Care and Use of Laboratory Animals (2015 reprint). The (PAI-1), an effector of the TGFb pathway, was also significantly study procedures were approved by the Institutional Animal Care upregulated. In contrast, SMAD7, known as an inhibitory Smad and Use Committee of Nanfang Hospital, Southern Medical (I-Smad), was significantly downregulated (Fig. 1C and E). University (Guangzhou, China). The details of tumor detection Together, these results indicated that the activation of the TGFb are provided in the Supplementary Materials and Methods avail- signaling pathway may play an important role in the autop- able online. hagy-induced EMT. In addition, HERC3 was also found to have an extremely high Reagents expression following rapamycin treatment, with its expression at The details of reagents used in this study are available in the 12 hours being approximately eight times (8.79 0.86) that at 0 Supplementary Materials and Methods available online. hours (Fig. 1D and E). Western blot results verified the upregulation of HERC3 by rapamycin or LG treatment, with expression Gene expression and Kaplan–Meier analysis of survival peaks at 16 and 12 hours after treatment in U87MG cells, probability using TCGA datasets (http://cancergenome. respectively (Supplementary Fig. S2A and S2B). Immunofluores- nih.gov) cence at 12 hours after treatment revealed the highest expression Level 3 RNA-SeqV2 data (containing data on gene, isoform, of HERC3, which was distributed in both the cytoplasm and exon, and junction levels), level 3 Agilent microarray gene nucleus (Supplementary Fig. S2C and S2D). Similarly, after expression data, and clinical data for multiple cancers were treatment with 200 mmol/L temozolomide, HERC3 was also downloaded from TCGA database using the TCGAbiolinks upregulated with an expression peak at 12–16 hours (Fig. 1F). (30) package on R platform in February 2018. RNA-Seq Immunochemical detection also showed similar cellular locali- expression-level read counts were normalized using two relat- zation in both the cytoplasm and nucleus at 12 hours after ed methods: FPKM and FPKM-UQ (the upper quartile FPKM), temozolomide treatment (Fig. 1G). according to the website of GDC (https://docs.gdc.cancer.gov/ Next, the autophagy inhibitors (3-MA and chloroquine) Data/Bioinformatics_Pipelines/Expression_mRNA_Pipeline/). and siRNA of ATG7 were used to interrupt autophagy in the The scan cut-off mode based on the median HERC3 expression early and late stages, respectively. These inhibitors signi- was selected without specifying a track subset. ficantly suppressed the overexpression of HERC3 induced by autophagy (Fig. 1H). In addition, the upregulation of SMAD7 Statistical analysis and downregulation of p-SMAD2/3 and two effectors of the All analyses were performed using SPSS version 20.0 (IBM TGFb pathway (PAI1 and NOTCH1) by autophagic inhibitors SPSS) and STATA software version 14.0 (StataCorp LP). See revealed the deactivation of this pathway. These results thus Supplementary Materials and Methods available online for indicated that autophagy could upregulate HERC3, downregu- details. late SMAD7, and activate the TGFb/SMAD2/3 pathway in GBM.

HERC3 overexpression is observed in the pseudo-palisade Results cells surrounding tumor necrosis and in tumor-adjacent Autophagy upregulates HERC3 expression, downregulates tissue, and correlates with poor prognosis especially in SMAD7, and activates the TGFb pathway in GBM the classical type GBM In our previous study, we reported that the autophagy induced To verify HERC3 expression in clinical glioma specimens, we per- by rapamycin, low glucose (LG), and temozolomide promoted formed IHC staining using a tissue microarray (HBraG090PG01), the EMT status in GBM cell lines, which could be attenuated by which included 3 normal tissue samples and 87 cases of glioma. autophagic inhibitors (26). In this study, we further confirmed the Higher expression of HERC3 correlated with the increase in EMT induced by autophagy in GBM. The longest cellular projec- malignant grade of the gliomas (Supplementary Fig. S3A). tions (pseudopodia) and the highest invasive ability could be Moreover, high HERC3 expression was identified in pseudo- observed at 12–16 hours after rapamycin (200 nmol/L) treatment palisade cells surrounding areas of tumor necrosis in U87MG cells. Furthermore, Western blotting indicated that the (Supplementary Fig. S3B), which have previously been con- expression peak of autophagy-related proteins (LC3B-II, ATG5, firmed to exhibit high EMT status (31). In addition, as described and ATG7) presented between 6 and 12 hours, accompanied by in our previous publication (28), we separately selected core the highest expression of mesenchymal markers (CDH2 and lesion tissues (T1 gadolinium-enhancing zone in the MRI; EC) SNAI1) and the lowest expression of an epithelial marker and peripheral tissues of GBM (related "normal" edema zone (CDH13) at approximately 12 hours after treatment surrounding the margin of ECs; PNCs; Supplementary Fig. S3C). (Supplementary Fig. S1). In addition, proteomics analysis EC regions suggested the enrichment of newborn vessels with using isobaric tags for relative and absolute quantitation high oxygen supplementation; however, the PNC regions were (iTRAQ) indicated that rapamycin upregulated the expression considered to reflect a hypoxia condition. Western blotting of of autophagy-related and mesenchymal-related proteins, but HERC3 showed significantly higher expression in PNC than in downregulated that of epithelial-related proteins (Fig. 1A and B). EC tissues (Supplementary Fig. S3D). Moreover, tumor samples iTRAQ results also revealed a significant upregulation of of a patient with GBM (ID: Glio-12), who experienced contra- TGFb signaling pathway activation-related proteins, albeit the lateral frontal lobe recurrence after total tumor resection and downregulation of TGFb pathway inhibitory proteins (Fig. 1C). radio-temozolomide chemotherapy (Supplementary Fig. S3E), Among these proteins, SMAD3 and SMAD5 (receptor-regulated were taken from the PNC and several EC regions. HERC3 was Smads, R-Smads) were upregulated and exhibited the highest overexpressed in PNC tissue compared with its expression in expression at 48 and 24 hours after rapamycin treatment, multiple tissues from ECs (Supplementary Fig. S3F).

www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 3605

Li et al.

Figure 1. Autophagy upregulates HERC3 expression, downregulates SMAD7, and activates the TGFb pathway in GBM. A, iTRAQ proteomics analysis indicates the upregulation of several autophagic key proteins by rapamycin treatment. B, iTRAQ indicates the upregulation of mesenchymal markers and downregulation of epithelial markers. C, iTRAQ indicates the upregulation of several effectors of the TGFb signaling pathway and R-Smads, albeit the downregulation of I-Smads. D, iTRAQ indicates the signiﬁcant upregulation of HERC3. E, Line graph showing protein levels (mean SD) of SMAD3, SMAD7, PAI1, and HERC3 at 6, 8, 12, 24, and 48 hours after rapamycin treatment compared with those at 0 hours. F, Western blotting of HERC3 with time gradients after temozolomide (200 mmol/L) treatment. G, IHC detection of HERC3 at 12 hours after temozolomide (200 mmol/L) treatment in U87 and T98G cells. Scale bar, 100 mm (100 )and25mm (400 ). H and I, Protein levels of two effectors of the TGFb pathway (PAI1 and NOTCH1); several Smads and HERC3 were detected by Western blotting following application of autophagy inhibitors, 3-MA, CQ, and siATG7 with or without temozolomide treatment.

Furthermore, two morphologically distinct cell strains, that is, publication (28), PNC cells exhibited significantly longer cell squamous-like and fibroblast-like, were isolated from EC and protrusions than EC cells (Supplementary Fig. S3G). Western PNC tissues, respectively, in 5 patients. Similar to our previous blotting further confirmed the relatively high expression of

3606 Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer Research

HERC3 Promotes Autophagy-Induced EMT and Chemoresistance

HERC3 and low expression of SMAD7 in PNC compared with expression. The results indicated that high expression of HERC3 those in EC cells (Supplementary Fig. S3H). correlated with poor prognosis of patients with GBM (overall To clarify the correlation of HERC3 with GBM prognosis, TCGA survival; OS), especially in the classical subtype (Fig. 2A). In database was used to perform survival analysis based on HERC3 proneural, neural, and mesenchymal GBM subtypes, HERC3

Figure 2. HERC3 correlates with poor prognosis of GBM, especially in the classical type of GBM. A and B, Overall survival analysis of patients with high (red) versus low (blue) HERC3 mRNA expression based on the TCGA database-GBM (left) and TCGA database-Classical type GBM (right; A) and also TCGA database-GBM with temozolomide therapy (B). C, Relative HERC3 expression in patient samples stratified according to the Verhaak classification based on TCGA database. Each bar represents mean SD. D, GBM paraffin sections (200 ) were used for IHC study of the protein expression of HERC3 and SMAD7. Scale bar, 100 mm. E, Percentage of patients showing low or high HERC3 protein expression in relation to the expression of SMAD7. F–H, Correlation of the expression levels of HERC3 and SMAD7 with the prognosis of patients with GBM. Kaplan–Meier analyses for overall survival of GBM based on HERC3 (F) or SMAD7 expression (G), and coincident HERC3 and SMAD7 expression (H). The log-rank test was used to calculate P values. There were significant differences at P < 0.05.

www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 3607

Li et al.

exhibited a similar tendency in predicting clinical outcome, but ing to previous reports (34, 35), T98G and U118MG proved to be no significant differences were found on the basis of the statistical MGMT-positive glioma cell lines; however, other four cell lines analysis (Supplementary Fig. S4A–S4C). Moreover, significant were MGMT-negative (Supplementary Fig. S4I). The above data correlation of high HERC3 level with poor curative effect of indicated that besides MGMT status, HERC3 level is also closely temozolomide was also found (Fig. 2B). Expression analysis correlated to temolzolomide sensitivity of GBM cells. For further indicated a relatively lower expression of HERC3 in the classical functional detection, LN229 (mesenchymal type) and T98G type of GBM than in the mesenchymal and proneural tumors (classical type) were selected to establish stable knockdown and (Fig. 2C). For comparison, we utilized 110 clinical samples from overexpression cell lines, respectively (Fig. 3B). HERC3 knock- patients with newly diagnosed GBM who had undergone surgery down in LN229 cells decreased the length of the pseudopodia; plus temozolomide chemoradiotherapy (Stupp regimen: radio- however, overexpression of HERC3 in T98G significantly therapy plus concomitant temozolomide 75 mg/m2; adjuvant increased the length (Fig. 3C and D). Both cell wound healing temozolomide 150 mg/m2 5/28 days for 6 cycles; ref. 2). IHC and Transwell assays also indicated that shHERC3 inhibited, detection was used to examine HERC3 and SMAD7 expression, whereas HERC3 overexpression (oeHERC3) enhanced cell migra- which was scored as 0–3þ according to the staining density and tion and invasion (Fig. 3E–H). Moreover, IC50 detection indicated percentage. Statistical analysis indicated that SMAD7 expression HERC3 overexpression significantly increased temolzolomide inversely correlated with HERC3 expression (Fig. 2D and E). resistance of GBM cells (Supplementary Fig. S4J and S4K). Moreover, the expression level of HERC3 and SMAD7 as tested Furthermore, shHERC3 increased the expression of an epithe- by qRT-PCR, together with other variables such as patient's age lial protein (CDH13/T-cadherin), but suppressed the mesenchy- and gender, tumor volume, and location (i.e., functional area or mal markers (fibronectin, vimentin, and CD44), with the EMT- not); and degree of tumor resection were recorded (Supplemen- related proteins ZEB1, SNAI1, and TWIST1 being inhibited as tary Table S1). First, a normal distribution test indicated that well. In contrast, oeHERC3 downregulated the expression of HERC3 and SMAD7 expression was not normally distributed. On CDH13 and upregulated the mesenchymal markers and EMT- the basis of Kaplan–Meier analysis of each variable, tumor vol- related proteins (Fig. 3I). These results indicated that oeHERC3 ume, functional area (P ¼ 0.0652), and expression level of HERC3 significantly promoted the occurrence of EMT, whereas being (P < 0.0001) and SMAD7 (P ¼ 0.0012) were selected for Multi- attenuated by shHERC3. This functional role was further dem- variate Cox regression analysis of OS. Nevertheless, scatter plot onstrated by Western blotting following temolzolomide and Spearman correlation coefficient for ranked data found a (200 mmol/L) treatment (Fig. 3J), confirming that the autop- significant negative correlation between HERC3 and SMAD7 hagy-induced EMT is dependent on HERC3 upregulation. More- expression (r ¼0.2929; P ¼ 0.0019). The final multivariate over, the elongated cellular morphologic changes induced by Cox regression analysis indicated that "functional area," along temolzolomide were also interrupted by shHERC3 (Fig. 3K). with HERC3 and SMAD7 expression level, affected the prognosis of OS (Supplementary Table S2A). However, as "functional area" HERC3 induces ubiquitination and degradation of SMAD7 to did not satisfy the proportional hazards assumption, which had to activate the TGFb/SMAD2/3 pathway in GBM be set as a stratification variable, repeated Cox multivariate To determine whether HERC3 regulated cellular invasion and regression analyses indicated that HERC3 (P < 0.001; HR ¼ EMT in GBM via activation of the TGFb pathway, the pSBE-luc 3.056; 95% CI, 1.893–4.934) and SMAD7 (P ¼ 0.002; HR ¼ plasmid with a TGFb-inducible SMAD binding element (SBE) was 0.502; 95% CI, 0.326–0.773) significantly influenced patient used to transfect LN229 and T98G cells. The results indicated that survival (Supplementary Table S2B and S2C). In addition, OS siRNA of HERC3 decreased the luciferase activity in parallel with curves consistently showed that patients with GBM demonstrat- the dosage of siRNA, whereas HERC3 overexpression enhanced ing higher HERC3 and lower SMAD7 expression exhibited poorer the activity (Fig. 4A). These results suggested that HERC3 pro- clinical prognosis (Fig. 2F–H). moted SMAD binding and activation of the TGFb pathway in a dose-dependent manner. Next, the higher mRNA levels of the two HERC3 level and MGMT status are correlated to the effectors PAI1 and NOTCH1 further indicated pathway activation temozolomide sensitivity of GBM cells, and autophagy-induced by HERC3 overexpression in T98G cells. In contrast, siHERC3 EMT is dependent on HERC3 upregulation decreased the level of these effectors, indicating blockage of the As HERC3 may more significantly influence the prognosis of pathway (Fig. 4B). the classical subtype of GBM, we tested molecular markers across In turn, Western blotting indicated that the inhibition of five established and two primary GBM cell lines. U87MG and HERC3 upregulated SMAD7 expression and downregulated LN229 cell lines along with G131212 primary GBM cells were p-SMAD2/3 level compared with those in the control regardless considered to be mesenchymal subtype, whereas T98G and U251 of recombinant human TGFb1 stimulation. In contrast, higher cell lines along with G141119 primary GBM cells expressed p-SMAD2/3 level and lower SMAD7 expression were observed classical type markers (Supplementary Fig. S4D–S4G); these when HERC3 was overexpressed (Fig. 4C). However, the ability of results were consistent with several previous publications (32, 33). shHERC3 to inhibit p-SMAD2/3 level was attenuated by siRNA of We next tested the baseline HERC3 expression in six GBM and SMAD7 (Fig. 4D). shHERC3 could also impede the extension one normal glial cell line (HEB) under normal culture conditions. of cellular pseudopodia induced by recombinant human TGFb1, High expression was identified in U87MG, LN229, A172, and with this effect also being attenuated by SMAD7 siRNA (Supple- U118 cells, with low expression in U251, T98G, and normal glial mentary Fig. S5A and S5B). Moreover, shHERC3 inhibited the HEB cells (Fig. 3A). By a website search (https://www.cancerrx number of migrating cells with or without induction by recom- gene.org), IC50 values of temozolomide for the six GBM cell lines binant human TGFb1 (Supplementary Fig. S5C). reflected the drug resistance induced by high HERC3 level, except Further qRT-PCR detection revealed no significant mRNA level U118MG and T98G (Supplementary Fig. S4H). Moreover, accord- change of SMAD7 upon rapamycin treatment, suggesting the

3608 Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer Research

HERC3 Promotes Autophagy-Induced EMT and Chemoresistance

Figure 3. Autophagy-induced EMT was dependent on the upregulation of HERC3. A, Western blotting of HERC3 indicated high expression in U87MG, LN229, A172, and U118, but low expression in U251, T98G, and HEB (human normal glioma) cells. B, Several shHERC3 and oeHERC3 sequences were used to downregulate and upregulate HERC3 protein level. C, In LN229 (high HERC3 level) and T98G (low HERC3 level) cells, cellular morphologic changes were recorded by inhibiting or upregulating HERC3 expression, respectively. Scale bar: 100 mm (100 ), 50 mm (200 ). D, Pseudopodia length, cellular width, and area of C were measured and analyzed. E, Transwell assay was used to evaluate the migration ability of LN229 and T98G cells with shHERC3 and oeHERC3. Scale bar, 100 mm. F, Histogram indicates the analysis of Transwell assay results in E. G and H, Wound-healing assay of LN229 and T98G cells with shHERC3 and oeHERC3 (G). Scale bar, 200 mm, and the analysis result (H). I, Using shHERC3 and oeHERC3 for LN229 and T98G cells, respectively, the protein levels of EMT markers were detected. J, Under temozolomide (TMZ) stimulation condition in LN229 cells, the level change of EMT markers was detected. K, Under temozolomide and shHERC3 conditions, the cellular morphologic changes were recorded and analyzed (L). Scale bar, 50 mm. Each bar represents the mean SD of three independent experiments. Signiﬁcance was denoted as #, P > 0.05; , P < 0.05; , P < 0.01.

www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 3609

Li et al.

Figure 4. HERC3 induces ubiquitination and degradation of SMAD7 to activate the TGFb/SMAD2/3 pathway. A, pSBE-luc plasmid containing the TGFb-induced SMAD binding element (SBE) was used to transfect LN229 and T98G cells. The luciferase activity was detected after treatment with the dosage gradient of TGFb1and siHERC3. B, mRNA levels of NOTCH1 and PAI1, effectors of the TGFb pathway, were detected by qRT-PCR upon treatment with TGFb1 and siHERC3 in LN229 cells, or TGFb1 and oe HERC3 in T98G cells. C, Western blotting detected the protein level of P-SMAD2/3, several SMADs, and HERC3 upon treatment with TGFb1 and downregulation or upregulation of HERC3 in LN229 and T98G cells, respectively. D, After inhibiting SMAD7 expression, the protein level of P-SMAD2/3, several Smads, and HERC3 was detected to evaluate HERC3 functional dependence on SMAD7 level. E, In the pulldown assay of endogenous SMAD7 by overexpression of HERC3 in T98G cells, the protein levels of Ub, SMAD7, and HERC3 were evaluated by Western blotting. F, Flag- SMAD7, Myc-HERC3, and HA-Ub were transfected into 293T cells to establish a ubiquitination model in vitro. The levels of Ub, Myc, and Flag were detected to further confirm the expression change in SMAD7 and HERC3 and the ubiquitination level. G, The downregulation of SMAD7 by HERC3 was further confirmed by Western blotting, when CHX was used to block endogenous SMAD7 protein synthesis. H, SMAD7 and HERC3 protein level was detected upon treatment with a dosage gradient of HERC3 overexpression. I, Further detection of the ubiquitination level of SMAD7 and HERC3 protein level by pulldown assay and Western blotting, upon treatment with 200 and 300 mmol/L temozolomide. Each bar represents the mean SD of three independent experiments. Significance was denoted as #, P > 0.05; , P < 0.05; , P < 0.01; , P < 0.001.

3610 Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer Research

HERC3 Promotes Autophagy-Induced EMT and Chemoresistance

possibility of posttranscriptional regulation of SMAD7 by HERC3 tosis, with final degradation occurring through the K63-linked (Supplementary Fig. S5D). Bioinformatics analysis showed that ubiquitin chain–mediated lysosome pathway. HERC3 (aa321–653 in the N-terminal) shared a common To verify the binding site of SMAD7 to HERC3, we generated sequence with SMURF2 (aa138–636), which included the bind- SMAD7 mutants bearing single or multiple Lys-to-Ala sub- ing site of SMAD7 (aa208–217) in the WW domain of SMURF2 stitutions at every potential ubiquitination site as reported (Supplementary Fig. S5E). These results suggested that, as a kind previously (ref. 38; Supplementary Fig. S6A). As shown in of E3 ligase, HERC3 might utilize SMAD7 as its substrate protein Fig. 5E, Lys-to-Ala substitutions at K64A of SMAD7 (SMAD7- to promote the ubiquitin-mediated degradation of SMAD7. K64A) abolished the HERC3-catalyzed ubiquitination and deg- Pulldown of SMAD7 was next used to further detect ubiquiti- radation of SMAD7. Notably, Ub conjugation to the SMAD7- nation in T98G cells. Immunoprecipitation analysis indicated K64A substitution was also substantially reduced compared that HERC3 significantly increased the ubiquitination of endog- with that of any other mutant (Fig. 5E). We next generated enous SMAD7 (Fig. 4E). In addition, we transfected Flag-SMAD7, mutations of HERC3 in which each of the two RLD domains Myc-HERC3, and HA-Ub (wild-type; WT) into 293T cells, which was deleted (Supplementary Fig. S6B and S6C). Deletion of confirmed the ubiquitination of SMAD7 by HERC3 (Fig. 4F). the RCC1–3domains(DRLD1, aa156–1050) did not interfere Moreover, when cycloheximide (CHX) was used to block the with the RCC4-7 domain at the amino terminus; however, protein synthesis of endogenous SMAD7, Western blotting deletion of all RLD domains (DRLD2, aa366–1050) abolished showed that HERC3 downregulated the expression of SMAD7 in the complex formation (Fig. 5F). Thus, the RCC4–7domains a time- and dose-dependent manner (Fig. 4G and H). Pulldown (aa156–366) in HERC3 appeared to be required to mediate the experiments following treatment with two dosages of temozolo- binding to SMAD7. Moreover, to further confirm the E3 ubi- mide (200 and 300 mmol/L) further illustrated the concomitant quitin ligase function toward SMAD7, the HERC3 HECT increase in SMAD7 ubiquitination level induced by HERC3 domain was deleted (DHECT, aa1–951), which abolished the upregulation (Fig. 4I). ubiquitination of SMAD7.

Nucleocytoplasmic shuttling of HERC3 suggests the functional HERC3 binding via its RCC4-7 domains is critical for SMAD7 role of HERC3 recycling ubiquitination, triggering SMAD7 degradation mainly through We first confirmed the intracellular localization of SMAD7 and the autolysosome degradation pathway HERC3 in T98G cells, which indicated that following temozolo- We next utilized the proteasome inhibitor MG132 to un- mide (200 mmol/L) treatment, HERC3 expression was upregu- cover the degradation pathway of HERC3-mediated ubiquiti- lated in both the cytoplasm and nucleus and reached the highest nation of SMAD7. We found that MG132 slightly increased level at 6–12 hours after treatment. In contrast, the level of SMAD7 the expression of SMAD7 under rapamycin treatment, whereas decreased constantly in both the cytoplasm and nucleus until 12 the autophagy inhibitors 3-MA and chloroquine significantly hours after treatment (Supplementary Fig. S6D). Western blotting attenuated the rapamycin-induced SMAD7 decrease (Fig. 5A). confirmed HERC3 upregulation in both the cytoplasm and nucle- The results suggested that the degradation of SMAD7 might us after 12-hour temozolomide (200 mmol/L) treatment, whereas occur mainly through the autolysosome rather than the protea- the level of p-SMAD2/3 significantly increased only in the nucle- some pathway. We therefore introduced the microtubule-asso- us, indicating the activation of TGFb signaling; similar results ciated protein 1 light chain 3, fused to the degradation-resistant were obtained following rapamycin treatment (Supplementary GFP protein (GFP-LC3) into LN229 cells to follow the flux. Fig. S6E and S6F). Furthermore, upon treatment with the nuclear The immunofluorescence detection of GFP-LC3 and SMAD7 export inhibitor, leptomycin B, HERC3 level in the nucleus was showed that protein colocalization could be observed in LN229 significantly increased. Correspondingly, total cellular SMAD7 cells with temozolomide 200 mmol/L treatment. After inhibit- and nuclear SMAD7 significantly increased as well, these results ing the expression of HERC3 by shHERC3, the SMAD7 level were also verified by immunofluorescence (Supplementary was increased and colocalization with LC3 was retained. In Fig. S6G and S6H). Together, these data suggest the nucleocyto- addition, p62/SQSTM1, an autophagy receptor that is known to plasmic shuttling role of HERC3 to transport SMAD7 outside of form complexes with cargo proteins (36), also exhibited clear the nucleus and then complete the ubiquitin-mediated degrada- colocalization with LC3 and SMAD7 (Fig. 5B). These results tion in autolysosomes, whereupon the E3 ligase HERC3 can be supported that HERC3 recruits SMAD7 for ubiquitination, thus recycled. promoting the recognition by the autophagy receptor. Moreover, whereas the Lys48-linked ubiquitin chain consis- Inhibition of HERC3 significantly decreases the tumor invasion tently guides proteolysis by the proteasome (37), the lysine and EMT in nude mice 63-linked ubiquitination of the substrate protein indicates pro- Two primary GBM cells, GBM141119 (low HERC3 expression) cessing by the endosomal-lysosomal–dependent degradation and GBM131212 (high HERC3 expression), were selected for an pathway (27). Therefore, we further used HA-Ub (WT) and intracranial tumorigenesis assay in nude mice. First, we verified Mutation HA-Ub (Ub-K48R and Ub-K63R) to analyze the degra- TGFb signaling activation and occurrence of EMT during over- dation pathway of SMAD7. Western blotting revealed that temo- expression and knockdown of HERC3 in vitro. In addition, the zolomide (200 mmol/L) significantly induced SMAD7 Ub-K63– cleavage of CD44 induced by the lentiviral (LV)-based stable cell linked polyubiquitin chain formation rather than Ub-K48–linked line LV-HERC3 was inhibited by LV-anti HERC3, suggesting that chains (Fig. 5C). In addition, Ub-K63R, but not Ub-K48R, was HERC3 enhanced the cellular invasive ability (Fig. 6A). Moreover, associated with the significantly reversed expression of SMAD7 CCK8 assay indicated that cellular proliferation was significantly (Fig. 5D). Our results indicated that the proteolysis of SMAD7 was increased by HERC3 overexpression, but decreased by down- closely related to autophagy level and LC3-associated phagocy- regulation of HERC3 (Fig. 6B). After intracranial tumor cell

www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 3611

Li et al.

Figure 5. HERC3 induces SMAD7 ubiquitination mainly by the autolysosome degradation pathway, and confirmation of the SMAD7-HERC3 binding site. A, Using a proteasome inhibitor (MG132) or early and later autophagic inhibitor [3-MA and chloroquine (CQ)], SMAD7 protein levels were detected and analyzed. B, Under temozolomide (TMZ) and/or shHERC3 treatment, the colocalization of P62, LC3, and SMAD7 was recorded by immunofluorescence assay under confocal microscopy. Scale bar, 10 mm. C, Immunoprecipitation (IP) assay of SMAD7 in LN229 cells treated with temozolomide (200 mmol/L); Western blotting (immunoblot; IB) was used to detect the expression of K48-linked and K63-linked polyubiquitin. D, HA-Ub mutation (Ub WT, Ub-K48R, and Ub-K63R) plasmids were transfected into 293T cells, and the protein level of SMAD7 and HERC3 was detected by pulldown assay and Western blot analysis. E, SMAD7-mutant plasmids bearing multiple Lys-to-Arg substitutions in every potential ubiquitination site were transfected into 293T cells. Western blotting detected the expression level of Ub, HERC3, and SMAD7. F, Plasmids containing the full-length RLD domain (aa1–1050), mutated RLD domains (aa156–1050, aa366–1050), and mutated HECT domain (aa1–951) of HERC3 were transfected into 293T cells. The protein levels of HERC3 and SMAD7 were detected by immunoprecipitation and Western blotting. Each bar represents the mean SD of three independent experiments. Significance was denoted as #, P > 0.05; , P < 0.01.

implantation, LV-HERC3 mice showed signiﬁcantly larger tumors expression was accompanied by a higher CD44 expression, along by MRI. In contrast, the downregulation of HERC3 signiﬁcantly with a more invasive cellular morphology (spindle cells with decreased the tumor volume (Fig. 6C). Survival analysis indicated longer pseudopodium). Ki-67 staining also indicated that HERC3 that high expression of HERC3 was closely correlated with poor overexpression increased the cellular proliferation (Fig. 6E). prognosis of nude mice (Fig. 6D). Moreover, we used a rabbit anti-human mAb of Nestin, a protein Moreover, immunohistologic detection indicated the inverse marker considered to be widely expressed in neuro-epithelial expression of HERC3 and SMAD7 in tumors. The higher HERC3 tumors such as GBM, but not in normal glial cells, to clearly

3612 Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer Research

HERC3 Promotes Autophagy-Induced EMT and Chemoresistance

Figure 6. HERC3 increases tumor invasion and EMT upon intracranial tumor transplantation in nude mice. A, In GBM141119 and GBM131212, two primary GBM cells, a stable HERC3-overexpressing cell line (LV-HERC3) was established in GBM141119 with lower HERC3 expression, whereas stable HERC3 knockdown cells (LV-anti HERC3) were generated in GBM131212 with HERC3 high expression. The protein levels of HERC3, several Smads, and two effectors of the TGFb pathway (PAI1 and NOTCH1), and some EMT markers (CD44 and CDH13) were detected by Western blotting. B, CCK8 assay was detected to evaluate the cellular proliferation of different cells. C, Coronal section of MRI scan was used to evaluate the tumor growth after intracranial tumor transplantation in nude mice. The tumor volume (mean SD) was statistically analyzed as a histogram. D, The follow-up data of the nude mice with en suite tumorigenesis were recorded to process the survival analysis. E, IHC detection was used to evaluate the expression of HERC3, SMAD7, CD44, and Ki-67 in intracranial tumors of different groups. Scale bar, 100 mm. F, Nestin expression was also detected by IHC assay. Scale bar, 200 mm. G, Schematic drawing indicating the mechanism by which autophagy upregulates HERC3 expression and induces ubiquitination and degradation of SMAD7 to activate the TGFb pathway and promote EMT in GBM. Significance was denoted as , P < 0.05; , P < 0.01. reveal the tumor boundary. Consequently, the tumors with lower of radio-chemoresistance and strong cellular invasiveness have HERC3 expression were found to exhibit a clear boundary been considered as the primary reasons for this dismal prog- between tumor and normal tissue, whereas the tumor cells with nosis. O-6-methylguanine-DNA methyltransferase (MGMT) high HERC3 expression demonstrated significant invasion into promoter methylation was believed to represent an indepen- the surrounding tissues with ill-defined margins (Fig. 6F). dent favorable prognostic factor for outcome and benefitfrom temozolomide chemotherapy, as temozolomide cytotoxicity is mediated mainly through methylation of the O6-position Discussion of guanine, whereas this DNA damage is rapidly repaired GBM constitutes the most refractory malignant intracranial by MGMT (39, 40); the median survival of patients with tumor with OS of approximately 14 months. Temozolomide, a tumors containing a methylated MGMT promoter was 21.7 new alkylating agent along with the Stupp regimen, which has months (41). However, as the OS was still less than two years, been widely used for chemo-radiotherapy of GBM, only slight- the results suggested the existence of another therapeutic ly increased the overall survival by 2 months (2). High levels resistance mechanism.

www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 3613

Li et al.

Alternatively, our previous study demonstrated that temozo- In addition, SMAD7 was also shown to inhibit TGFb signaling lomide could induce autophagy and then mediate EMT to in the nucleus by specifically binding to the SMAD-responsive increase cell invasion and migration, which indicated another element within the promoter of PAI1 via its MH2 domain, thereby possible chemoresistance mechanism of GBM (29), with similar disrupting the formation of the TGFb-induced functional phenotype being observed following treatment with other classic SMAD2/3-DNA complex (52) and the consequent PAI1 expres- autophagy inducers such as rapamycin and LG. Here, we provided sion. Our data indicated that SMAD7 could be negatively regu- further evidence that temozolomide upregulates the expression of lated by HERC3 both in the cytoplasm and the nucleus. HERC3 the E3 ubiquitin ligase HERC3, which induces the ubiquitin- protein accumulation was observed in the nucleus when its export mediated degradation of SMAD7 and activation of the TGFb was inhibited by leptomycin B. Concordantly, the reduction of signaling pathway. In addition, in survival analyses for each SMAD7 protein level in the nucleus was abolished to a significant molecular subtype, we found that patients with the classical GBM extent. These results suggested that HERC3 recruits SMAD7 in the subtypes with low HERC3 levels survived longer. Although the nucleus and plays a degradative function in the cytoplasm similar predictive trend observed in the other three subtypes also through nucleocytoplasmic shuttling. hinted the correlation of HERC3 with poor prognosis, no signif- Although HERC3 was reported to indirectly bind to the NF-kB icant differences were found. This analysis showed that the RelA subunit after liberation from the IkBa inhibitor, leading to relationship of HERC3 with overall survival in GBM might its ubiquitination and protein destabilization, to our knowledge, be highly subtype-specific. Several studies have also reported the no study has previously indicated the nuclear localization of prognostic value of molecular subtypes in GBM. miR-222 is a HERC3 (23). However, another "large" HERC E3, HERC2, was prognostic factor in classical and neural GBM; miR-370 could found to shuttle between the nucleus and the cytoplasm to target predict the clinical outcome in neural GBM; and miR-34a, miR- BRCA1 for degradation in breast cancer (20). SMURF2 also 145, and miR-182 showed prognostic value in a proneural non-G- mediated SMAD7 binding and recruitment in the nucleus and CIMP group. These results may be related to the different genetic caused degradation of receptors via proteasomal and lysosomal backgrounds associated with each subtype (42). Furthermore, pathways (49). On the basis of our analysis, HERC3 may therefore tumor microenvironments could also be associated with these play double functional roles by mediating SMAD7 degradation, differences. The neural and mesenchymal subtypes have been which promotes the phosphorylation of SMAD2/3 and formation reported to harbor similar gene expression characteristics within of the R-SMAD/SMAD4 complex in the cytoplasm, while also normal neural and stromal tissues, respectively (43). Studies abolishing the binding of SMAD7 to the PAI1 promoter in the involving neural and stromal cells need to be undertaken to verify nucleus, thereby activating TGFb signaling. Consequently, this these results. In addition, "classical" subtypes of GBM are more promotes EMT to increase the invasion and migration of GBM sensitive to chemo-radiotherapy (44, 45). Our data also revealed cells (Fig. 6G). The above results were consistent with the histo- that HERC3 level was closely correlated to temozolomide sensi- logic findings, which indicated that HERC3 was highly expressed tivity of GBM based on TCGA database analysis and in vitro IC50 in the "normal" edema tissue (PNC), which was considered to detection, except the influence of MGMT status. Thus, in this exhibit hypoxia and low nutrition conditions. Our study thus study, HERC3/SMAD7 (key molecules) and the TGFb pathway suggested a possible mechanism underlying cell transition from were elucidated to be involved in autophagy-induced EMT, which tumor surrounding tissues and provided an explanation for GBM might allow the identification of novel therapeutic targets for recurrence at distant sites. Besides, HERC3 was also proved to overcoming the chemoresistance of GBM, especially in the classic increase cell proliferation and promote the tumor growth, which subtype. might be explained by the cellular dedifferentiation or tumor- The TGFb signaling pathway plays complex roles in regulating initiating ability induced by EMT. cell growth, differentiation, apoptosis, motility and invasion, Moreover, our data supported that the degradation of extracellular matrix production, angiogenesis, and immune SMAD7 by HERC3 occurred mainly in an autolysosome- response (46). Notably, the EMT mediated by the TGFb/SMAD dependent manner. Accumulating studies have proposed a role pathway has been identified as the key reason for the radio- for autophagy in the clearance of diffuse ubiquitinated proteins chemoresistance in breast cancer, lung cancer, hepatic carcino- delivered by p62/SQSTM1 (53–55). Similarly, the ubiquitin ma, and lymphoma (47, 48). However, I-Smads, such as ligase HACE1 was shown to ubiquitinate OPTN and promote SMAD7, can target the activated TGFBR1 for degradation, inhibits interaction with p62/SQSTM1 to form an autophagy recep- it the phosphorylation of SMAD2/3 (R-SMAD), and block the tor complex, thereby accelerating its degradation (56). More- complex formation with the common Smad, SMAD4, finally over, the different types of polyubiquitin (polyUb) chain interrupting the activation of the canonical TGFb/SMAD2/3 conjugation to substrates may induce a variety of degradation signaling pathway (49, 50). In turn, negative regulation of processes (27). In this study, K63R, but not K48R ubiquitin site SMAD7 by MIR-182 was shown to potentiate TGFb-induced mutation, significantly altered the expression of Smad7, which EMT and metastasis of cancer cells (51). In this study, we further confirmed the lysosomal degradation process. Our revealed that SMAD7 can be degraded in a ubiquitin-dependent results also provide the possible explanation for the recent manner by HERC3, which contains a HECT domain that med- report that pharmacologic inhibition of autophagy sensitized iates the interaction with E2 ubiquitin-conjugating enzymes GBM cells to temozolomide (57). and RLDs for substrate specificity. A series of truncated RLD In conclusion, our study clarified the molecular pathway of and HECT constructs were generated to confirm the substrate autophagy-induced EMT, indicating HERC3 and SMAD7 as the recognition of HERC3, which identified a binding site at aa key regulators of this biological process. In addition, we also 156–366. In addition, HERC3-SMAD7 recognition was abol- directly linked the autophagy induced by temozolomide to EMT ished by a SMAD7 mutant with Lys64-to-Ala (K64R), which along with increased cellular invasion and migration, which further confirmed the ubiquitination sites. suggested the possible mechanism of radio-chemoresistance in

3614 Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer Research

HERC3 Promotes Autophagy-Induced EMT and Chemoresistance

GBM. Moreover, HERC3 and SMAD7 levels closely correlated with Administrative, technical, or material support (i.e., reporting or organizing the prognosis of patients with GBM, especially the classic sub- data, constructing databases): H. Li, J. Li, L. Chen, S. Qi, S. Yu, Q. Zhou, Y. Lu types. Our research might therefore be helpful for identifying Study supervision: S. Qi, Y. Lu Other (statistical analysis of clinical data): S. Yu new therapeutic targets or regimens in combination with temozolomide to increase the sensitivity of GBM to chemotherapy. In addition, a better understanding of the mechanism of tumor Acknowledgments invasion and metastasis may facilitate the development of ther- We would like to acknowledge Chonglu Ren (Center for Genetic and apeutic strategies to overcome the issue of peripheral tumor Developmental Systems Biology, Department of Obstetrics & Gynecology, Nanfang Hospital) for his assistance in the analysis of TCGA databases. We recurrence to enhance patient prognosis. also thank Wenhua Liu (Clinic Research Center, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology) for the Disclosure of Potential Conflicts of Interest assistance in statistical analysis using SPSS and STAT software. This work was No potential conflicts of interest were disclosed. supported by the Chinese National Natural Science Fund (81772656), National Science Foundation of Guangdong Province (2016A030313563), Authors' Contributions National Key Clinical Specialty Project, and the Research Foundation of the Chinese Society of Neuro-oncology (CSNO-2016-MSD20). Conception and design: H. Li, A. Huang, Y. Lu Development of methodology: Z. Weng, Z. Hu, Q. Zhou, Y. Lu Acquisition of data (provided animals, acquired and managed patients, The costs of publication of this article were defrayed in part by the provided facilities, etc.): J. Li, L. Chen, Z. Weng, Z. Xin, L. Shi, L. Ma, Y. Lu payment of page charges. This article must therefore be hereby marked Analysis and interpretation of data (e.g., statistical analysis, biostatistics, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate computational analysis): H. Li, J. Li, L. Chen, S. Yu, Q. Zhou, Z. Xin, L. Shi, this fact. L. Ma, Y. Lu Writing, review, and/or revision of the manuscript: H. Li, J. Li, L. Chen, Received November 21, 2018; revised February 5, 2019; accepted February 27, A. Huang, Y. Lu 2019; published first March 12, 2019.

References 1. White E. The role for autophagy in cancer. J Clin Invest 2015;125:42–6. H3K56 acetylation promoting hypoxia-induced tumour progression. 2. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Nat Commun 2016;7:13644. et al. Radiotherapy plus concomitant and adjuvant temozolomide for 16. Verdecia MA, Joazeiro CA, Wells NJ, Ferrer JL, Bowman ME, Hunter T, et al. glioblastoma. N Engl J Med 2005;352:987–96. Conformational flexibility underlies ubiquitin ligation mediated by the 3. Hombach-Klonisch S, Mehrpour M, Shojaei S, Harlos C, Pitz M, Hamai A, WWP1 HECT domain E3 ligase. Mol Cell 2003;11:249–59. et al. Glioblastoma and chemoresistance to alkylating agents: involvement 17. Ogunjimi AA, Briant DJ, Pece-Barbara N, Le Roy C, Di Guglielmo GM, of apoptosis, autophagy, and unfolded protein response. Pharmacol Ther Kavsak P, et al. Regulation of Smurf2 ubiquitin ligase activity by anchoring 2018;184:13–41. the E2 to the HECT domain. Mol Cell 2005;19:297–308. 4. Macintosh RL, Timpson P, Thorburn J, Anderson KI, Thorburn A, Ryan KM. 18. Scheffner M, Staub O. HECT E3s and human disease. BMC Biochem 2007;8 Inhibition of autophagy impairs tumor cell invasion in an organotypic Suppl1:S6. model. Cell Cycle 2012;11:2022–9. 19. Diouf B, Cheng Q, Krynetskaia NF, Yang W, Cheok M, Pei D, et al. Somatic 5. Li J, Yang B, Zhou Q, Wu Y, Shang D, Guo Y, et al. Autophagy promotes deletions of genes regulating MSH2 protein stability cause DNA mismatch hepatocellular carcinoma cell invasion through activation of epithelial- repair deficiency and drug resistance in human leukemia cells. Nat Med mesenchymal transition. Carcinogenesis 2013;34:1343–51. 2011;17:1298–303. 6. Maycotte P, Thorburn A. Autophagy and cancer therapy. Cancer Biol Ther 20. Kang TH, Reardon JT, Sancar A. Regulation of nucleotide excision repair 2011;11:127–37. activity by transcriptional and post-transcriptional control of the XPA 7. Janku F, McConkey DJ, Hong DS, Kurzrock R. Autophagy as a target for protein. Nucleic Acids Res 2011;39:3176–87. anticancer therapy. Nat Rev Clin Oncol 2011;8:528–39. 21. Wu W, Sato K, Koike A, Nishikawa H, Koizumi H, Venkitaraman AR, et al. 8. Singla M, Bhattacharyya S. Autophagy as a potential therapeutic target HERC2 is an E3 ligase that targets BRCA1 for degradation. Cancer Res 2010; during epithelial to mesenchymal transition in renal cell carcinoma: an in 70:6384–92. vitro study. Biomed Pharmacother 2017;94:332–40. 22. Hochrainer K, Mayer H, Baranyi U, Binder B, Lipp J, Kroismayr R. The 9. Brabletz T, Jung A, Reu S, Porzner M, Hlubek F, Kunz-Schughart LA, et al. human HERC family of ubiquitin ligases: novel members, genomic orga- Variable beta-catenin expression in colorectal cancers indicates tumor nization, expression profiling, and evolutionary aspects. Genomics 2005; progression driven by the tumor environment. Proc Natl Acad Sci U S A 85:153–64. 2001;98:10356–61. 23. Hochrainer K, Kroismayr R, Baranyi U, Binder BR, Lipp J. Highly homol- 10. Brabletz T, Jung A, Spaderna S, Hlubek F, Kirchner T. Opinion: migrating ogous HERC proteins localize to endosomes and exhibit specific interac- cancer stem cells - an integrated concept of malignant tumour progression. tions with hPLIC and Nm23B. Cell Mol Life Sci 2008;65:2105–17. Nat Rev Cancer 2005;5:744–9. 24. Hochrainer K, Pejanovic N, Olaseun VA, Zhang S, Iadecola C, Anrather J. 11. Kahlert UD, Maciaczyk D, Doostkam S, Orr BA, Simons B, Bogiel T, et al. The ubiquitin ligase HERC3 attenuates NF-kappaB-dependent transcrip- Activation of canonical WNT/b-catenin signaling enhances in vitro motility tion independently of its enzymatic activity by delivering the RelA subunit of glioblastoma cells by activation of ZEB1 and other activators of epithe- for degradation. Nucleic Acids Res 2015;43:9889–904. lial-to-mesenchymal transition. Cancer Lett 2012;325:42–53. 25. Lopez-Alonso I, Aguirre A, Gonzalez-Lopez A, Fernandez AF, Amado- 12. Brabletz T. To differentiate or not–routes towards metastasis. Nat Rev Rodriguez L, Astudillo A, et al. Impairment of autophagy decreases ven- Cancer 2012;12:425–36. tilator-induced lung injury by blockade of the NF-kappaB pathway. Am J 13. Chaffer CL, Brueckmann I, Scheel C, Kaestli AJ, Wiggins PA, Rodrigues LO, Physiol Lung Cell Mol Physiol 2013;304:L844–52. et al. Normal and neoplastic nonstem cells can spontaneously convert to a 26. Yoo NJ, Park SW, Lee SH. Frameshift mutations of ubiquitination-related stem-like state. Proc Natl Acad Sci U S A 2011;108:7950–5. genes HERC2, HERC3, TRIP12, UBE2Q1 and UBE4B in gastric and 14. Wang H, Jiang Z, Na M, Ge H, Tang C, Shen H, et al. PARK2 negatively colorectal carcinomas with microsatellite instability. Pathology 2011;43: regulates the metastasis and epithelial-mesenchymal transition of glio- 753–5. blastoma cells via ZEB1. Oncol Lett 2017;14:2933–9. 27. Nathan JA, Kim HT, Ting L, Gygi SP, Goldberg AL. Why do cellular proteins 15. Wu HT, Kuo YC, Hung JJ, Huang CH, Chen WY, Chou TY, et al. K63- linked to K63-polyubiquitin chains not associate with proteasomes? polyubiquitinated HAUSP deubiquitinates HIF-1alpha and dictates EMBO J 2013;32:552–65.

www.aacrjournals.org Clin Cancer Res; 25(12) June 15, 2019 3615

Li et al.

28. Qiang Z, Jun-Jie L, Hai W, Hong L, Bing-Xi L, Lei C, et al. TPD52L2 impacts 42. Li R, Gao K, Luo H, Wang X, Shi Y, Dong Q, et al. Identification of intrinsic proliferation, invasiveness and apoptosis of glioblastoma cells via mod- subtype-specific prognostic microRNAs in primary glioblastoma. J Exp Clin ulation of wnt/beta-catenin/snail signaling. Carcinogenesis 2018;39: Cancer Res 2014;33:9. 214–24. 43. Lathia JD, Heddleston JM, Venere M, Rich JN. Deadly teamwork: neural 29. Lu Y, Xiao L, Liu Y, Wang H, Li H, Zhou Q, et al. MIR517C inhibits cancer stem cells and the tumor microenvironment. Cell Stem Cell 2011;8: autophagy and the epithelial-to-mesenchymal (-like) transition phenotype 482–5. in human glioblastoma through KPNA2-dependent disruption of TP53 44. Hall AW, Battenhouse AM, Shivram H, Morris AR, Cowperthwaite MC, nuclear translocation. Autophagy 2015;11:2213–32. Shpak M, et al. Bivalent chromatin domains in glioblastoma reveal a 30. Colaprico A, Silva TC, Olsen C, Garofano L, Cava C, Garolini D, et al. subtype-specific signature of glioma stem cells. Cancer Res 2018;78: TCGAbiolinks: an R/Bioconductor package for integrative analysis of 2463–74. TCGA data. Nucleic Acids Res 2016;44:e71. 45. Lee E, Pain M, Wang H, Herman JA, Toledo CM, DeLuca JG, et al. Sensitivity 31. Inukai M, Hara A, Yasui Y, Kumabe T, Matsumoto T, Saegusa M. Hypoxia- to BUB1B inhibition defines an alternative classification of glioblastoma. mediated cancer stem cells in pseudopalisades with activation of hypoxia- Cancer Res 2017;77:5518–29. inducible factor-1alpha/Akt axis in glioblastoma. Hum Pathol 2015;46: 46. Jakowlew SB. Transforming growth factor-beta in cancer and metastasis. 1496–505. Cancer Metastasis Rev 2006;25:435–57. 32. Mahabir R, Tanino M, Elmansuri A, Wang L, Kimura T, Itoh T, et al. 47. Neuzillet C, Tijeras-Raballand A, Cohen R, Cros J, Faivre S, Raymond E, Sustained elevation of Snail promotes glial-mesenchymal transition after et al. Targeting the TGFbeta pathway for cancer therapy. Pharmacol Ther irradiation in malignant glioma. Neuro Oncol 2014;16:671–85. 2015;147:22–31. 33. Yan X, Liao H, Cheng M, Shi X, Lin X, Feng XH, et al. Smad7 protein 48. Principe DR, Doll JA, Bauer J, Jung B, Munshi HG, Bartholin L, et al. TGF- interacts with receptor-regulated smads (R-Smads) to inhibit transforming beta: duality of function between tumor prevention and carcinogenesis. growth factor-beta (TGF-beta)/Smad Signaling. J Biol Chem 2016;291: J Natl Cancer I 2014;106:1–16. 382–92. 49. Kavsak P, Rasmussen RK, Causing CG, Bonni S, Zhu H, Thomsen GH, et al. 34. Murphy SF, Varghese RT, Lamouille S, Guo S, Pridham KJ, Kanabur P, et al. Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF Connexin 43 inhibition sensitizes chemoresistant glioblastoma cells to beta receptor for degradation. Mol Cell 2000;6:1365–75. temozolomide. Cancer Res 2016;76:139–49. 50. Massague J, Chen YG. Controlling TGF-beta signaling. Genes Dev 2000;14: 35. Chen Z, Wei X, Shen L, Zhu H, Zheng X. 20(S)-ginsenoside-Rg3 reverses 627–44. temozolomide resistance and restrains epithelial-mesenchymal transition 51. Yu J, Lei R, Zhuang X, Li X, Li G, Lev S, et al. MicroRNA-182 targets SMAD7 progression in glioblastoma. Cancer Sci 2019;110:389–400. to potentiate TGFbeta-induced epithelial-mesenchymal transition and 36. Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, et al. p62/ metastasis of cancer cells. Nat Commun 2016;7:13884. SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquiti- 52. Zhang S, Fei T, Zhang L, Zhang R, Chen F, Ning Y, et al. Smad7 antagonizes nated protein aggregates by autophagy. J Biol Chem 2007;282:24131–45. transforming growth factor beta signaling in the nucleus by interfering with 37. Satoh T, Sakata E, Yamamoto S, Yamaguchi Y, Sumiyoshi A, Wakatsuki S, functional Smad-DNA complex formation. Mol Cell Biol 2007;27: et al. Crystal structure of cyclic Lys48-linked tetraubiquitin. 4488–99. Biochem Biophys Res Commun 2010;400:329–33. 53. Myeku N, Figueiredo-Pereira ME. Dynamics of the degradation of ubiqui- 38. Chua JP, Reddy SL, Yu Z, Giorgetti E, Montie HL, Mukherjee S, et al. tinated proteins by proteasomes and autophagy: association with seques- Disrupting SUMOylation enhances transcriptional function and amelio- tosome 1/p62. J Biol Chem 2011;286:22426–40. rates polyglutamine androgen receptor-mediated disease. J Clin Invest 54. Dikic I. Proteasomal and autophagic degradation systems. Annu Rev 2015;125:831–45. Biochem 2017;86:193–224. 39. Gerson SL. Clinical relevance of MGMT in the treatment of cancer. J Clin 55. Varshavsky A. The ubiquitin system, autophagy, and regulated protein Oncol 2002;20:2388–99. degradation. Annu Rev Biochem 2017;86:123–8. 40. Gerson SL. MGMT: its role in cancer aetiology and cancer therapeutics. 56. Liu Z, Chen P, Gao H, Gu Y, Yang J, Peng H, et al. Ubiquitylation of Nat Rev Cancer 2004;4:296–307. autophagy receptor optineurin by HACE1 activates selective autophagy for 41. Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al. tumor suppression. Cancer Cell 2014;26:106–20. MGMT gene silencing and benefit from temozolomide in glioblastoma. 57. Yan Y, Xu Z, Dai S, Qian L, Sun L, Gong Z. Targeting autophagy to sensitive N Engl J Med 2005;352:997–1003. glioma to temozolomide treatment. J Exp Clin Cancer Res 2016;35:23.

3616 Clin Cancer Res; 25(12) June 15, 2019 Clinical Cancer Research

HERC3-Mediated SMAD7 Ubiquitination Degradation Promotes Autophagy-Induced EMT and Chemoresistance in Glioblastoma

Hong Li, Junjie Li, Lei Chen, et al.

Clin Cancer Res 2019;25:3602-3616. Published OnlineFirst March 12, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-3791

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2019/03/12/1078-0432.CCR-18-3791.DC1

Cited articles This article cites 57 articles, 13 of which you can access for free at: http://clincancerres.aacrjournals.org/content/25/12/3602.full#ref-list-1

Citing articles This article has been cited by 2 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/25/12/3602.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/25/12/3602. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.