Glioblastoma Proto-Oncogene Sec61γ Is Required for Tumor Cell Survival and Response to Endoplasmic Reticulum Stress

Published OnlineFirst November 17, 2009; DOI: 10.1158/0008-5472.CAN-09-2775 Molecular Biology, Pathobiology, and Genetics Glioblastoma Proto-oncogene SEC61γ Is Required for Tumor Cell Survival and Response to Endoplasmic Reticulum Stress Zheming Lu,1,3 Lei Zhou,4 Patrick Killela,1 Ahmed B. Rasheed,1 Chunhui Di,1 William E. Poe,1 Roger E. McLendon,1 Darell D. Bigner,1 Christopher Nicchitta,2 and Hai Yan1 1Department of Pathology, The Pediatric Brain Tumor Foundation Institute, and The Preston Robert Tisch Brain Tumor Center at Duke and 2Department of Cell Biology, Duke University Medical Center, Durham, North Carolina; 3Department of Etiology, Peking University School of Oncology, Beijing Cancer Hospital and Institute, Beijing, People's Republic of China; and 4Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida Abstract cells to apoptosis (7). These findings may be of particular relevance Glioblastoma multiforme is the most prevalent type of adult to glioblastoma multiforme, which, because they are highly prolifer- brain tumor and one of the deadliest tumors known to man- ative tumors, display substantial microheterogeneity in glucose and kind. The genetic understanding of glioblastoma multiforme oxygen delivery. Recent studies show that UPR genes are upregu- is, however, limited, and the molecular mechanisms that facil- lated in gliomas (8), suggesting that the genetic alteration of the itate glioblastoma multiforme cell survival and growth within ER-based, protein folding machinery, a prominent metabolic feature the tumor microenvironment are largely unknown. We applied of glioblastoma multiforme, confers a growth advantage. γ digital karyotyping and single nucleotide polymorphism ar- SEC61 is a member of the SEC61 translocon, a heterotrimeric α β γ rays to screen for copy-number changes in glioblastoma multi- protein channel comprising three subunits, SEC61 , , and (9). forme samples and found that the most frequently amplified The SEC61 complex forms a transmembrane pore for the translo- region is at chromosome 7p11.2. The high resolution of digital cation of nascent polypeptides into the ER lumen as well as the karyotyping and single nucleotide polymorphism arrays per- integration of transmembrane proteins into the ER bilayer (10). mits the precise delineation of amplicon boundaries and has In addition to the SEC61 complex, other proteins, such as ERj1, enabled identification of the minimal region of amplification SEC62, and SEC63, form the complete protein translocase involved at chromosome 7p11.2, which contains two genes, EGFR and in protein folding, modification, and translocation (11). Mutations SEC61γ. SEC61γ encodes a subunit of a heterotrimeric protein of ER-resident chaperones and the translocon have been identified channel located in the endoplasmic reticulum (ER). In addi- in cancer cells, indicating that ER proteins play an important role tion to its high frequency of gene amplification in glioblastoma in tumor pathogenesis. Of particular note, SEC62 and SEC63 are multiforme, SEC61γ is also remarkably overexpressed in 77% among the most frequently mutated and/or overexpressed genes of glioblastoma multiforme but not in lower-grade gliomas. in prostate, gastric, and colorectal cancers (12). γ The small interfering RNA–mediated knockdown of SEC61γ In the study described here, we found that SEC61 is not only expression in tumor cells led to growth suppression and apo- always coamplified with epidermal growth factor receptor (EGFR) ptosis. Furthermore, we showed that pharmacologic ER stress in 47% of glioblastoma multiforme but also overexpressed in 77% γ agents induce SEC61γ expression in glioblastoma multiforme of glioblastoma multiforme. In addition, SEC61 is required for tu- cells. Together, these results indicate that aberrant expression mor cell survival and response to ER stress reagents. of SEC61γ serves significant roles in glioblastoma multiforme cell survival likely via a mechanism that is involved in the cy- Materials and Methods toprotective ER stress–adaptive response to the tumor microenvironment. [Cancer Res 2009;69(23):9105–11] Tissue samples. Glioblastoma multiforme cell lines and frozen xeno- graft tumor samples were obtained from the tissue bank of the Preston Robert Tisch Brain Tumor Center at Duke. Acquisition of tissue specimens Introduction was approved by the Duke University Health System Institutional Review In tumors, hypoxia and nutrient deprivation of the solid-tumor Board. microenvironment inevitably subject tumor cells to ongoing cell Digital karyotyping. Digital karyotyping library construction and data stress (1–3). Under conditions of reduced oxygen and/or glucose analysis based on 25 glioblastoma multiforme samples were done as described previously (13, 14). Digital karyotyping protocols and software for availability, tumors exploit cellular stress response pathways to sup- 5 port tumor cell proliferation (4). The unfolded protein response extraction and analysis of genomic tags are available online. Analysis of copy-number variation. Single nucleotide polymorphism (UPR) is a set of cytoprotective signaling pathways that expands genotyping on genomic DNA from 32 glioblastoma multiforme samples, in- the endoplasmic reticulum (ER) processing capacity for misfolded cluding 7 pediatric glioblastoma multiforme, was done using the Illumina proteins and activates adaptive, antiapoptotic pathways (5, 6). How- HumanHap550 Genotyping BeadChip array. Raw data from the single nu- ever, if the survival mechanisms are exhausted, the UPR can commit cleotide polymorphism chips were collected and subjected to copy-number analysis using Nexus Copy Number Professional software (BioDiscovery). Quantitative real-time PCR analysis. Differences between glioblasto- Note: Supplementary data for this article are available at Cancer Research Online ma multiforme and normal brain cells in genomic DNA content of EGFR (http://cancerres.aacrjournals.org/). or SEC61γ were determined by quantitative real-time PCR (Q-PCR) as Requests for reprints: Hai Yan, Duke University Medical Center, Box 3156, Durham, NC 27710. Phone: 919-668-7850; Fax: 919-684-8756; E-mail: yan00002@mc. described previously (15). Relative gene expression levels of ATF4, Bip, duke.edu. ©2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-09-2775 5 http://www.digitalkaryotyping.org www.aacrjournals.org 9105 Cancer Res 2009; 69: (23). December 1, 2009 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst November 17, 2009; DOI: 10.1158/0008-5472.CAN-09-2775 Cancer Research CHOP, ATF6, Xbp1, EGFR, cyclin D1, and SEC61 α, β, and γ were measured tion, amplification of the EGFR is often associated with structural before and after treating the cells with 1 μg/mL of the ER stress inducer alterations in the gene, with the most common being variant III tunicamycin, and cDNA content was normalized to that of glyceraldehyde- (EGFR vIII), a 801-bp in-frame deletion of exons 2 to 7 of EGFR 3-phosphate dehydrogenase. present in 20% to 50% of glioblastoma multiforme with EGFR am- GeneChip analysis. Human U133A GeneChips (Affymetrix) were used plification (19). Here, the high resolution of genomic approaches for analysis of gene expression in 43 primary glioblastoma multiforme samples and 4 normal brain tissues. The experimental protocols and data pro- revealed both focal amplification and intragenetic deletions of EGFR A cessing were described in a previous report (16). A hierarchical clustering (Fig. 1 ; Supplementary Fig. S1). tree and heat map were generated with dChip. For both cancer and normal It is of note that the genomic analyses revealed that SEC61γ,adja- tissues, an average difference value was normalized against the average dif- cent to EGFR, is always coamplified with EGFR in glioblastoma multi- ference of β-actin to obtain the internally normalized expression value. The forme. To verify the results obtained from genome-wide studies, we data were inputted into dChip, normalized against a normal tissue sample, performed Q-PCR analysis of EGFR and SEC61γ copy numbers on ge- and divided by the SD for each gene. nomic DNA extracted from an additional 43 tumors of primary glio- γ Small interfering RNA. SEC61 small interfering RNA (siRNA) se- blastoma multiforme patients (age >50 years). Of the 43 samples, 20 ′ ′ ′ quences 5 -GCCAAGUCGGCAGUUUGUAAAGGAC-3 and 5 -GUCCUUUA- samples (47%) displayed SEC61γ high copy-number gain (>4-fold) and CAAACUGCCGACUUGGC-3′ and a control scrambled GC siRNA were 17 samples (40%) displayed coincident EGFR high copy-number gain purchased from Invitrogen. B EGFR MTT assay. (>4-fold; Fig. 1 ). Interestingly, in no case was high copy-num- Cell viability assays were done as described previously (14). SEC61γ Briefly, assays were done consecutively from days 1 to 6 after siRNA trans- ber gain present without a corresponding high copy-number fection. A 10% solution of 5 mg/mL MTT diluted in PBS was added to the gain in the Q-PCR analysis (Fig. 1B;SupplementaryTableS1). cells and incubated for 30 min, and 600 μL isopropanol was then added to A previous population-based study indicated that the presence the plate and incubated for 5 min. Next, 200 μL of the mixed solution were of EGFR amplification does not affect survival of glioblastoma mul- transferred to a 96-well plate, and absorbance was measured at 570 nm. All tiforme patients at any age (20). We performed a survival

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