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

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 analysis experiments were done in triplicate. of the 43 glioblastoma multiforme cases and did not observe an Colony formation in soft-agar assay. Twenty-four hours after siRNA association between SEC61γ amplification and a patient's survival transfection, H80 and HeLa were seeded at 500 cells in 10 cm plates and (Supplementary Fig. S2), which indicates that chromosome 7p11.2 cultured for 14 days. The colonies were identified by crystal violet staining amplification alone may not have a prognostic value to predict for 4 h and then counted. The experiments were done at least twice. t glioblastoma multiforme patients' survival. Statistical analysis was done using Student's test. γ Antibodies. Rabbit anti-SEC61γ polyclonal antibody (pAb) was purchased SEC61 is overexpressed in glioblastoma multiforme. We from Proteintech Group; anti–glyceraldehyde-3-phosphate dehydrogenase also examined the expression levels of genes within the chromo- monoclonal antibody was from Santa Cruz Biotechnology; anti-AKT pAb, some 7p11 amplicon using data derived from the human GeneChip anti–phospho-Ser473-AKT pAb, anti-extracellular signal-regulated kinase 1/2 array U133A (Affymetrix) of 4 normal adult brain tissues and the 43 pAb, anti–phospho-Thr202/Tyr204 extracellular signal-regulated kinase 1/2 primary glioblastoma multiforme samples that we analyzed by Q- pAb, anti-EGFR pAb, and anti–phospho-EGFR were from Cell Signaling Tech- PCR for amplification. Relative to its neighboring genes, SEC61γ nology; the antibody for detection of the active caspase-3 was from Abcam; displayed more prominent overexpression. Among the 43 glioblas- and anti-GRP78/Bip pAb was from Cell Signaling Technology. toma multiforme, 33 (77%) tumors expressed SEC61γ at significantly higher levels than normal brain tissues, and genes in the vicinity of SEC61γ, that is, EGFR, LANCL2, and ECOP, displayed significant Results overexpression in 25 (58%), 10 (23%), and 27 (63%) of the 43 glio- SEC61γ is frequently amplified in glioblastoma multiforme. blastoma multiforme, respectively (Fig. 2A). Although in most cases To identify cancer-specific genetic changes in glioblastoma multi- the level of EGFR expression correlated with that of SEC61γ,intwo forme, we applied two genomic approaches, digital karyotyping and glioblastoma multiforme samples, SEC61γ was expressed at a rela- the Illumina HumanHap550 Genotyping BeadChip array, to tively higher level, whereas EGFR was expressed at the same level as interrogate the glioblastoma multiforme genome for gene copy-num- the control sample (Fig. 2A, samples labeled with asterisk). Addi- ber variations in 59 human patient glioblastoma multiforme samples. tionally, 7 samples in clade I (Fig. 2A) have overexpression of Digital karyotyping allows precise, quantitative delineation of chro- SEC61γ and EGFR but not LANCL2 and ECOP. Furthermore, mosomal amplifications at a resolution of 100 kb (13). Of the 16 cases SEC61γ is overexpressed in every sample with SEC61γ amplifica- of primary tumors and xenografts examined by digital karyotyping, 10 tion (Supplementary Table S1). Consistent with the RNA data, im- samples displayed dramatic amplification (8- to 103-fold) at chromo- munoblotting analysis with anti-SEC61γ antibody detected high some 7p11.2 (Fig. 1A), which is the most frequently amplified region in levels of SEC61γ expression in 6 of 8 (75%) randomly selected glio- the samples analyzed. However, none of the 10 glioblastoma multi- blastoma multiforme xenografts (Supplementary Fig. S3). Further- forme cell lines contained chromosome 7p11.2 amplification. In all more, we examined 3 secondary glioblastoma multiforme but did the samples that displayed chromosome 7p11.2 amplification, two not detect SEC61γ overexpression (Supplementary Fig. S4). genes, SEC61γ and EGFR, were encompassed in the minimal, over- We also compared the differential expression profiles of the three ER lapped regions of amplification (Fig. 1A). In an additional 33 primary translocon subunit genes, SEC61α,SEC61β,andSEC61γ, in glioblasto- tumors and xenografts, the Illumina HumanHap550 single nucleotide ma multiforme samples versus control samples. We found that al- polymorphism data revealed chromosome 7p11.2 focal, high gain of though the expression of all three gene subunits tends to be elevated copy number in 12 cases and gain of copy number in 14 cases. In in glioblastoma multiforme samples, the expression of SEC61γ is often agreement with the digital karyotyping results, the Illumina data also at greater levels than the α and β subunits (Supplementary Fig. S5). displayed minimal overlapped region containing only SEC61γ and To determine whether SEC61γ overexpression is associated with EGFR (Supplementary Fig. S1). stage of astrocytoma progression, we analyzed the SEC61γ gene EGFR amplification is a well-known genetic event occurring in expression in different grades of astrocytomas using the Serial Anal- 40% to 60% of primary glioblastoma multiforme (17, 18). In addi- ysis of Gene Expression data from the National Cancer Institute

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Figure 1. SEC61γ and EGFR coamplification in glioblastoma multiforme. A, identification of consensus SEC61γ and EGFR coamplification in glioblastoma multiforme by digital karyotyping. Each line stands for the amplified region in each glioblastoma multiforme sample. The two vertical lines illustrate the consensus region of amplification reveled from the 10 glioblastoma multiforme samples. The fold of amplification is shown on the right of each amplicon. The arrow points to the actual digital karyotyping data generated from the corresponding sample, which displays 60-fold amplification on chromosome 7 and an interstitial deletion within the amplified region. B, verification of SEC61γ and EGFR coamplification by Q-PCR in glioblastoma multiforme. Q-PCR was done on genomic DNA from 43 primary glioblastoma multiforme samples. Relative genomic amplification levels are indicated as copies per diploid genome and were generated by Q-PCR at each gene. SEC61γ displayed high copy-number gain (>4 fold) with EGFR in 16 of 43 cases. Note that, in samples 17, 33, 39, and 43, SEC61γ displayed a larger scale of amplification than EGFR.

Cancer Genome Anatomy Project Web site.6 As shown in Fig. 2B, SEC61γ is required for tumor cell growth. Based on our none of the 8 WHO grade I and II samples exhibited overexpression genetic studies, our hypothesis is that overexpression of SEC61γ of SEC61γ, whereas 2 of 11 (18%) grade III samples and 14 of 19 is needed for glioblastoma multiforme tumor cell progression. (82%) glioblastoma multiforme samples overexpressed SEC61γ. To assess the requirement for SEC61γ in malignant cells, we investigated the effect of siRNA-mediated knockdown of SEC61γ on tumor cell growth. First, SEC61γ siRNA-transfected tumor cells were examined for viability by a MTT assay. In glioblasto- 6 http://cgap.nci.nih.gov/SAGE ma multiforme cell line H80, significantly reduced cell viability www.aacrjournals.org 9107 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 was observable 48 h post-transfection with siRNA against the UPR, a cytoprotective signaling network that enables cells to SEC61γ (Fig. 3A). The growth-inhibitory effects of SEC61γ process misfolded proteins and activate adaptive, antiapoptotic knockdown were observed under complete medium conditions pathways. Given that the SEC61 complex forms the core of the ER and more prominently under serum starvation. The siRNA also protein translocation apparatus, we postulated that SEC61γ may play markedly repressed anchorage-independent tumor cell growth a role in the ER stress response in glioblastoma multiforme cells. in soft agar (Fig. 3B). To determine if the SEC61γ knockdown- To assess the involvement of SEC61γ in the cellular response induced decrease in cell viability was due to increased apopto- to ER stress, we exposed H80 and HeLa cells to the pharmacologic sis, we treated H80 cells with SEC61γ siRNA and assayed them ER stress inducer tunicamycin and examined the expression for cleaved caspase-3, a protein that plays a key role in the ex- of SEC61γ and GRP78, an ER chaperone protein and central regu- ecution of the late-stage apoptotic program. SEC61γ siRNA- lator of ER homeostasis, the upregulation of which is widely used mediated knockdown of SEC61γ led to significantly elevated as a sentinel marker for ER stress under pathologic conditions caspase-3 activity (Fig. 3C). To verify the results, we repeated (21). We observed upregulation of both SEC61γ and GRP78 pro- the above experiments using the highly proliferative cervical teins 3 h after tunicamycin treatment, with peak levels at cancer cell line HeLa. We found that siRNA-mediated knock- 24 h (Fig. 4). To further evaluate expression of the SEC61 translo- down of SEC61γ also resulted in deceased cell growth and in- con subunits under ER stress, the expression of SEC61 α, β, and γ creased caspase-3 activity in HeLa cells (Fig. 3A-C). and of five UPR activation markers was examined by Q-PCR after SEC61γ and ER stress. Aggressive solid tumors like glioblastoma tunicamycin treatment in H80 cells. EGFR and cyclin D were multiforme typically outgrow their blood supply, which leads to included as controls. Consistent with previous reports, cyclin D chronic hypoxia and nutrient deprivation. This physiologic stress expression was downregulated in response to tunicamycin (22), usually results in disruption of ER homeostasis and leads to ER whereas EGFR transcription was not upregulated in the cells. stress, a condition in which the protein-folding capacity of the ER is We found significant upregulation of the ER stress response overwhelmed and misfolded proteins accumulate in the ER lumen. genes GRP78, Xbp1, ATF4, ATF6, and CHOP by tunicamycin treat- To survive in this condition, solid tumors cope with ER stress through ment (Supplementary Fig. S6). Following a short exposure to

Figure 2. SEC61γ is overexpressed in glioblastoma multiforme. A, human GeneChip array U133A heat map shows SEC61γ, EGFR, LANCL2, and ECOP expression in normal brain tissue and glioblastoma multiforme. The expression values of SEC61γ, EGFR, LANCL2, and ECOP in all samples were normalized by subtracting the values of control sample N780 and then dividing by the SD of each row (gene). The relative levels of expression of SEC61γ, EGFR, LANCL2, and ECOP, referenced with normal tissue, are hierarchically clustered. Samples 987 and 943 have significant overexpression of SEC61γ but not EGFR (asterisk). Samples in clade I display overexpression of SEC61γ and EGFR but not LANCL2 and ECOP. Samples in clade II had strong overexpression of all four genes. Samples in clade III have little change in the expression of the four genes in comparison with the control normal tissues. B, SEC61γ overexpression is associated with grade IV astrocytomas (GBM). Serial Analysis of Gene Expression (SAGE) analysis shows normal expression (≤20 tags) of SEC61γ in grades I and II astrocytomas but high expression (>20 tags) in 2 of 11 grade III astrocytomas and 14 of 19 grade IV astrocytomas. The SEC61γ tag numbers from each of the normal brain tissues, the grades I to III astrocytomas, and the grade IV astrocytomas were obtained from the Serial Analysis of Gene Expression Web site and normalized to 200,000 tags per library.

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Figure 3. SEC61γ is required for tumor cell growth. A, knockdown of SEC61γ by siRNA inhibited cell proliferation assessed in plastic culture plates using medium containing either 10% or 0.25% serum. H80 glioblastoma multiforme cells and HeLa cells were transfected with SEC61γ or scrambled siRNAs. Average cell number was measured by determining DNA content in three replicate wells using the MTT assay. B, knockdown of SEC61γ by siRNA inhibited tumor cell anchorage-independent proliferation assessed by measuring colony growth in soft agar. Graphs indicate number of colonies >2 mm in diameter observed after 2 wk of growth. C, knockdown of SEC61γ by SEC61γ siRNA induced cleaved caspase-3 in H80 and HeLa cells by Western blot. tunicamycin, the expression of CHOP mRNA was first observed to 47% of glioblastoma multiforme but also overexpressed in 77% of increase in H80 cultures and started to decrease 3 h post-treat- glioblastoma multiforme. In addition, we found that SEC61γ is re- ment. At later intervals, ATF4 and Xbp1 were induced and reached quired for tumor cell survival and for the cellular response to ER their peak levels at 6 h post-treatment. Transcription levels of stress. Our findings suggest that SEC61γ exists as a glioblastoma SEC61 α, β, and γ, ATF6, and GRP78 reached a peak at 12 h after multiforme–specific proto-oncogene, the product of which may fa- tunicamycin treatment (Supplementary Fig. S6). cilitate tumor cells in coping with cellular stress in the tumor microenvironment to support glioblastoma multiforme cell proliferation. Chromosome 7p11 is the most frequently amplified genomic re- Discussion gion in glioblastoma multiforme. Among the genes within the Chromosome 7p11.2 amplification has long been identified as chromosome 7p11 amplicon, our results indicate that, in addition the most prominent genetic lesion for the carcinogenesis of glio- to EGFR, SEC61γ is frequently amplified and overexpressed. Previ- blastoma multiforme. Using high-resolution digital karyotyping ous studies have shown that the size of a specific amplicon varies and single nucleotide polymorphism arrays, we found that the among tumors, ranging from a few hundred to a few thousand minimal amplicon of chromosome 7p11.2 contains two genes, EGFR kilobases (23–28). In many tumor samples, an amplicon may and SEC61γ. SEC61γ is not only always coamplified with EGFR in harbor a cancer-specific gene and its adjacent bystander genes www.aacrjournals.org 9109 Cancer Res 2009; 69: (23). December 1, 2009

Recently, SEC61β has been implicated in EGFR trafficking and EGFR-mediated activation of the phosphoinositide 3-kinase/AKT pathway (41–43), which is known for promoting cell survival and inhibiting apoptosis in most cell types (44, 45), including glioblastoma multiforme (46, 47). The phosphoinositide 3-kinase/AKT pathway may counteract ER stress–induced apoptotic signaling (48, 49). To determine if the growth-inhibitory effects of SEC61γ knockdown might be mediated through the EGFR/AKT signaling pathway, we evaluated the consequence of SEC61γ knockdown on the EGFR/AKT pathway activity and found that EGF-mediated activation of EGFR and AKT was significantly inhibited in SEC61γ siRNA-transfected cells (Supplementary Fig. S7). These results suggest that inhibition of SEC61γ could be an alternative way to inhibit various arms of the AKT signaling network and reverse the fate of tumor cells from survival to cell death. However, the molecular mechanism underlying the observation needs to be illuminated by further investigation of the role of SEC61γ in the EGFR-AKT signaling pathway. In the present study, we show that the expression of SEC61γ is positively correlated with astrocytoma grade and the primary glioblastoma multiforme subtype. Moreover, we show that SEC61γ is Figure 4. SEC61γ is induced by ER stress. H80 (A) and HeLa (B) cells were treated with tunicamycin for the indicated times, and SEC61γ, GRP78, and upregulated in glioblastoma multiforme cell lines in response to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured by the pharmacologic stress agent tunicamycin. The SEC61γ amplifi- Western blotting. cation and increased expression in glioblastoma multiforme in proliferative situations probably reflects increased synthesis and (29–32). However, it is also possible that more than one gene can translocation of proteins to the lumen of the ER and increased ac- exist as a tumor-specific gene in the same amplicon. For example, tivity of the quality-control process of proteins destined for mem- PDGFRA has been found to be coamplified with KIT, along with the branes and for secretion. Knocking down SEC61γ expression vascular endothelial growth factor receptor gene KDR, at 4q12, and resulted in apoptosis and abrogation of EGFR/AKT survival signal- DDX1 and N-Myc are coamplified at 2p24 (33). The coamplification ing. These results suggest that SEC61γ confers a selective growth of multiple genes within a genomic region may have synergistic advantage under physiologic conditions by facilitating a cytopro- effects on neoplastic pathogenesis. Within the chromosome tective response to ER stress. 7p11.2 amplicon, EGFR is amplified and overexpressed in 46% of Further studies are needed to investigate the molecular mechan- glioblastoma multiforme (34), and EGFR truncated mutations have isms through which ER stress stimulates the expression of SEC61γ been identified to be oncogenic in glioblastoma multiforme (34, and mediates its prosurvival effects. A detailed understanding of the 35). Given that receptor tyrosine kinases and the signaling path- signaling networks underlying SEC61γ involvement in ER stress re- ways they control constitute potential therapeutic targets, EGFR sponse, along with those underlying its high overexpression in malig- has been regarded as a major focus of research in glioblastoma nant cells and near absence in normal cells, will make SEC61γ as an multiforme. Two genes adjacent to EGFR, LANCL2 (36) and ECOP attractive viable therapeutic target for pharmaceutical intervention. (37–39), have been reported to coamplify with EGFR in 50% and 33% of glioblastoma multiforme, respectively. However, the role of SEC61γ in glioblastoma multiforme pathogenesis has not been fully characterized. Disclosure of Potential Conflicts of Interest Due to inadequate perfusion, the microenvironment of highly pro- No potential conflicts of interest were disclosed. liferative solid tumors is characterized by chronic hypoxia and a lack of nutrients. These conditions induce ER stress and activate both cytoprotective and cytodestructive branches of the UPR (40). Recent Acknowledgments work has linked various types of cancer to genetic alterations in ER- Received 7/25/09; revised 9/23/09; accepted 9/23/09; published OnlineFirst 11/17/09. resident chaperones (1–3). The ER translocon proteins SEC62 and Grant support: The Pediatric Brain Tumor Foundation Institute at Duke and NIH SEC63 have been identified as the most frequently mutated and over- grants: National Cancer Institute grant R01CA118822, National Institute of Neurolog- ical Disorders and Stroke grant 5P50 NS20023, National Cancer Institute Specialized expressed genes in prostate, gastric, and colorectal cancers (12). An Program of Research Excellence grant 5P50 CA108786, and National Cancer Institute exacerbated proliferation is a hallmark of glioblastoma multiforme merit award R37 CA 011898. The costs of publication of this article were defrayed in part by the payment of page cells as further indicated by the significant overexpression of the charges. This article must therefore be hereby marked advertisement in accordance UPR target genes, including GRP78, in glioblastoma multiforme (8). with 18 U.S.C. Section 1734 solely to indicate this fact.

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www.aacrjournals.org 9111 Cancer Res 2009; 69: (23). December 1, 2009

Glioblastoma Proto-oncogene SEC61γ Is Required for Tumor Cell Survival and Response to Endoplasmic Reticulum Stress

Zheming Lu, Lei Zhou, Patrick Killela, et al.

Cancer Res 2009;69:9105-9111. Published OnlineFirst November 17, 2009.

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