Published OnlineFirst August 20, 2012; DOI: 10.1158/0008-5472.CAN-12-0474 Cancer Tumor and Stem Cell Biology Research The Unfolded Protein Response Induces the Angiogenic Switch in Human Tumor Cells through the PERK/ATF4 Pathway Yugang Wang1, Goleeta N. Alam1, Yu Ning1, Fernanda Visioli1, Zhihong Dong2, Jacques E. Nor€ 2, and Peter J. Polverini3 Abstract Neovascularization is a limiting factor in tumor growth and progression. It is well known that changes in the tumor microenvironment, such as hypoxia and glucose deprivation (GD), can induce VEGF production. However, the mechanism linking GD to tumor growth and angiogenesis is unclear. We hypothesize that GD induces the angiogenic switch in tumors through activation of the unfolded protein response (UPR). We report that UPR activation in human tumors results in elevated expression of proangiogenic mediators and a concomitant decrease in angiogenesis inhibitors. cDNA microarray results showed that GD-induced UPR activation promoted upregulation of a number of proangiogenic mediators (VEGF, FGF-2, IL-6, etc.) and downregulation of several angiogenic inhibitors (THBS1, CXCL14, and CXCL10). In vitro studies revealed that partially blocking UPR signaling by silencing protein kinase RNA–like ER kinase (PERK) or activating transcription factor 4 (ATF4) significantly reduced the production of angiogenesis mediators induced by GD. However, suppressing the alpha subunit of hypoxia-inducible factors had no effect on this process. Chromatin immunoprecipitation (ChIP) confirmed binding of ATF4 to a regulatory site in the VEGF gene. In vivo results confirmed that knockdown of PERK in tumor cells slows down tumor growth and decreases tumor blood vessel density. Collectively, these results show that the PERK/ATF4 arm of UPR mediates the angiogenic switch and is a potential target for antiangiogenic cancer therapy. Cancer Res; 72(20); 5396–406. Ó2012 AACR. Introduction activate downstream signaling effectors, such as protein – Dividing tumors can rapidly outgrow their blood supply. kinase RNA like ER kinase (PERK), inositol requiring 1 This results in a toxic tumor microenvironment (TME) char- (IRE1), and activating transcription factor 6 (ATF6; ref. 5). acterized with hypoxia, acidic pH, glucose deprivation (GD), These effectors are maintained in an inactive state through and amino acid deficiency. Increasing evidence suggests that association with the molecular chaperone, glucose-regulated the TME contains stressors that promote accumulation of protein 78 kDa (Grp78; ref. 2). Upon ER stress Grp78 dis- misfolded proteins in the lumen of the endoplasmic reticu- sociates from these sensors, which activates UPR signaling lum (ER). This in turn activates intracellular signaling path- (6). After dimerization and transautophosphorylation, acti- ways termed as the unfolded protein response (UPR; refs. 1– vated PERK relays signal by phosphorylating the alpha a 3). Initially, the UPR is cytoprotective aimed at restoring subunit of eukaryotic initiation factor-2 (eIF-2 ), which in normal ER function (4). However, in the presence of severe turn inhibits general translation initiation and selectively or prolonged ER stress, cell death programs are activated translates several mRNAs including ATF4 (7). ATF4 trans- (3, 4). activates expression of several genes, such as C/EBP homol- CHOP Mammalian UPR is controlled by 3 ER-resident transmem- ogous protein ( ), a transcription factor implicated in Grp78 brane proteins that serve as proximal sensors of ER stress and apoptosis (8, 9), and (10). Upon UPR activation, IRE1 splices an unconventional intron from the X-box-binding protein 1 (XBP1) mRNA, producing an active transcription Authors' Affiliations: Departments of 1Biologic and Materials Sciences, factor XBP1-s (11, 12), which translocates into the nucleus 2Cariology, Restorative Sciences and Endodontics, and 3Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, and modulates the expression of several proteins involved in Michigan folding or clearance of aberrant proteins (2, 13). Finally, Note: Supplementary data for this article are available at Cancer Research during ER stress, ATF6 is processed into an active tran- Online (http://cancerres.aacrjournals.org/). scription factor, moves into the nucleus, and upregulates ER Corresponding Author: Peter J. Polverini, University of Michigan, 1011 chaperones and folding enzymes (2, 14). North University, Rm. 1234, Ann Arbor, MI 48109-1078. Phone: 734-763- Studies have shown that GD induces the expression of VEGF 3311; Fax: 734-763-5142; E-mail: [email protected] in different tumor cell lines (15–17), suggesting that besides doi: 10.1158/0008-5472.CAN-12-0474 hypoxia (18, 19), low concentration of nutrients play a role in Ó2012 American Association for Cancer Research. triggering angiogenesis (20). It is also known that the 5396 Cancer Res; 72(20) October 15, 2012 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst August 20, 2012; DOI: 10.1158/0008-5472.CAN-12-0474 UPR-Mediated Angiogenic Switch pathologic stimulus GD causes ER stress and alters gene Microarray analysis expression through UPR signaling (20). However, mechanistic Total RNA was isolated from UM-SCC-81B cells treated with studies of UPR-mediated angiogenesis have been conducted or without GD (glucose, 0.1 mmol/L) using the RNeasy Plus for the most part using nonphysiologic agents, such as thapsi- Mini Kit (Qiagen). A Human gene chip U133 Plus 2.0 (Affyme- gargin (TG), focusing mainly on VEGF expression (21, 22). The trix) was used to analyze gene expression. Details and results mechanism underlying GD-induced UPR in tumor angiogen- can be accessed in Gene Expression Omnibus (GEO) repository esis has not been fully elucidated. (GSE38583). In this study, we report that activation of the UPR by GD plays a pivotal role in tumor angiogenesis by activating the Immunoblotting angiogenic switch and that the PERK/ATF4 pathway of the Whole-cell lysates were resolved by SDS–PAGE and trans- UPR is involved in this process. These results suggest that ferred onto polyvinylidene difluoride membrane (Pierce) and targeting the proangiogenic arm of the UPR may reveal new probed with the antibodies: Grp78, CHOP, ATF4, b-actin strategies for cancer treatment. (Santa Cruz), HIF1a (BD Pharmingen), PERK (Cell Signaling), and spliced XBP1 (Biolegend). Horse radish peroxidase- Materials and Methods conjugated secondary antibodies were from Santa Cruz. Super- Cells Signal West Pico Chemiluminescent Substrate (Pierce) was The head and neck squamous cell carcinoma (HNSCC) cell used to visualize immunoreactive bands. line UM-SCC-81B (from Dr. Thomas E. Carey, Departments of Otorhinolaryngology and Pharmacology, University of Michi- ELISA gan Medical School, Ann Arbor, MI), the breast cancer cell line ELISA was conducted according to the manufacturer's MCF7 (American Type Culture Collection), the glioma cell line instructions. Briefly, cell culture supernatants were diluted U87 (from Dr. Yi Sun, Department of Radiation Oncology, 1:10, applied to each well (100 mL), incubated at room tem- University of Michigan, Ann Arbor, MI), and mouse embryonic perature for 2 hours, and washed 3 times. The secondary fibroblast (MEF) cell lines (MEF–PERKþ/þ and MEF– antibody reaction was conducted at room temperature PERKÀ/À, from Dr. Andrew Fribley, Department of Pediatrics, (1 hour). Stabilized chromogen was used for colorimetric Division of Hematology/Oncology, Wayne State University, reactions. Optical density was measured at 450 nm using a Detroit, MI) were maintained in Dulbecco's Modified Eagle's plate reader (Spectra Max M2). Medium (DMEM) with high glucose (Invitrogen) with 10% FBS. All tumor cell lines were authenticated recently by DNA Lentivirus infection fingerprinting with short tandem repeat profiling. Primary GFP-expressing lentiviral constructs expressing short hair- human dermal microvascular endothelial cells (HDMEC; Cam- pin RNA (shRNA) against PERK, ATF4, and HIF1a were from 5 brex) were cultured in endothelial cell growth medium-2 Open Biosystems. For infection, 1 Â 10 cells were plated in 6- (EGM2; Cambrex). TG and tunicamycin (TM) were purchased cm plates, infected with the lentivirus and sorted with flow from Sigma. cytometry to ensure 100% positivity. Established stable cell lines were cultured with 2 mg/mL puromycin. Immunofluorescence and immunohistochemical analysis Chromatin immunoprecipitation Immunofluorescence and immunohistochemistry (IHC) Chromatin immunoprecipitation (ChIP) analysis was con- were conducted as described previously (23) with primary ducted using an Agarose-Chip Kit (Pierce) according to the antibodies against Grp78, CHOP (Santa Cruz), Ki67, and CD31 manufacturer's instruction. Cells were treated for 18 hours with (BD Pharmingen). Goat antirabbit Alexa Flour 594 (Molecular complete DMEM containing normal glucose (25 mmol/L) or Probes) and 40,6-diamidino-2-phenylindole (DAPI) counter- low glucose (2 mmol/L), respectively. The chromatin solution staining were used for immunofluorescence staining. The was incubated overnight with ATF4 antibody (sc-200, Santa Polink-2 horseradish peroxidase broad kit with 3,30-diami- Cruz), nonimmune rabbit IgG, and anti-RNA polymerase II nobenzidine (DAB) chromogen was used for IHC staining. antibody. Purified complexes and input DNAs were analyzed by Normal human oral mucosa (NHM, from Dr. Hector Rios, PCR. The ATF4-binding sites of VEGF gene (AsnSyn site)