Squamous Cell Carcinoma Related Oncogene/DCUN1D1 Is Highly Conserved and Activated by Amplification in Squamous Cell Carcinomas

Research Article

Inderpal Sarkaria,1,3 Pornchai O-charoenrat,1,2 Simon G. Talbot,1,2 Pabbathi G. Reddy,1 Ivan Ngai,1 Ellie Maghami,1,2 Kepal N. Patel,1,2 Benjamin Lee,1,3 Yoshihiro Yonekawa,1 Maria Dudas,4 Andrew Kaufman,1,3 Russell Ryan,1 Ronald Ghossein,4 Pulivarthi H. Rao,6 Archontoula Stoffel,5 Y. Ramanathan,1 and Bhuvanesh Singh1,2

1Laboratory of Epithelial Cancer Biology, 2Head and Neck Service, 3Thoracic Surgery Service, and 4Department of Pathology, Memorial Sloan-Kettering Cancer Center; 5Laboratory of Cancer Biology, Rockefeller University, New York, New York; and 6Children’s Cancer Center, Baylor College of Medicine, Houston, Texas

Abstract genes are present in the minimal common region of 3q f Chromosomal amplification at 3q is common to multiple amplification ( 40 Mb), defined by comparative genomic hybrid- human cancers, but has a specific predilection for squamous ization. Given that several genes can be overexpressed in a single cell carcinomas (SCC) of mucosal origin. We identified and amplification locus, it is not surprising that multiple putative characterized a novel oncogene, SCC-related oncogene targets that drive selection for 3q amplification have been identified. Despite functional assessments of some candidate genes (SCCRO), which is amplified along the 3q26.3 region in human i SCC. Amplification and overexpression of SCCRO in these (including PIK3CA, PKC , LAMP3, and eIF-5A2), the precise target(s) tumors correlate with poor clinical outcome. The importance of 3q amplification remains ill defined (12–14). of SCCRO amplification in malignant transformation is Most studies suggest that the 3q25-27 region likely harbors the established by the apoptotic response to short hairpin RNA important candidate genes. Fine-resolution mapping show that against SCCRO, exclusively in cancer cell lines carrying SCCRO subpeaks of amplification exist within the 3q25-27 region, and that amplification. The oncogenic potential of SCCRO is under- the gene(s) driving selection for the amplified locus is present within scored by its ability to transform fibroblasts (NIH-3T3 cells) these subpeaks. Lower-resolution positional cloning analyses (using in vitro and in vivo. We show that SCCRO regulates Gli1—a key yeast artificial chromosome clones) show overlapping results with regulator of the hedgehog (HH) pathway. Collectively, these an amplification subpeak centered around 3q26.3 (4, 10, 15). Fine- data suggest that SCCRO is a novel component of the HH resolution mapping has been less homogenous in defining signaling pathway involved in the malignant transformation amplification subpeaks. Our group identified two amplification peaks, one in the region of f180 Mb of chromosome 3 and the of squamous cell lineage. (Cancer Res 2006; 66(19): 9437-44) second at f184Mb in lung and head and neck cancers (9). Snijders et al. (16) also identified two amplification subpeaks at 3q in Introduction fallopian tube carcinomas (map positions f165-175 and f181-185 Several studies suggest that amplified DNA is unstable. Accord- Mb). Interestingly, these authors excluded PIK3CA (180.4Mb) as ingly, increased prevalence in human cancers implies specific a candidate gene, as it was outside their amplification boundaries. selection for the amplified region (1–3). Amplification at 3q is seen Conversely, Or et al. (17) found the amplification apex around the in multiple tumor types but shows a higher predilection for region covered by bacterial artificial chromosome (BAC) clone squamous cell carcinoma (SCC) of mucosal origin, including those RE11-510K16 at f181.6 Mb, suggesting that PIK3CA may be the of the lung, head and neck, esophagus, vagina, vulva, and cervix, target. Jiang et al. (18) did comparative genomic hybridization on where it has been associated with tumor progression and an cDNA arrays in nasopharyngeal carcinomas showing three ampli- aggressive clinical course (4–11). The high prevalence, combined fied loci at 3q, including the region around TP73L (f192 Mb) and with biological implications and clinical associations, suggests that SOX2 (f182 Mb) and AP2M1 (185.3 Mb). In contrast, Snijders et al. amplification at 3q may play a role in squamous cell carcinogenesis. (19) did not identify any recurrent amplifications at 3q26-27 in their Identification of gene(s) driving selection for 3q amplification analysis of primary oral cancers. Combined, the fine-resolution has been a significant research focus. Unlike deletions, trans- mapping suggests that multiple amplification subpeaks may exist, locations, and mutations, there is no pathognomonic attribute that with frequent overlap in the 183 to 186 Mb position at 3q26.3. Here, confirms a gene as a target of amplification. Over 100 candidate we report the identification and functional validation of SCCRO/ RP42/DCUN1D1 (184.1 Mb) as one potential target driving selection for 3q amplification in SCC of mucosal origin. Moreover, our findings suggest that the oncogenic activity of SCCRO results at least Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). partly from activation of hedgehog (HH) signaling. I. Sarkaria, P. O-charoenrat, and S.G. Talbot contributed equally to this work. This work is dedicated to the memory of Dr. P.G. Reddy, who passed away unexpectedly during the preparation of this article. Requests for reprints: Bhuvanesh Singh, Laboratory of Epithelial Cancer Biology, Materials and Methods Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Genomic sequence analysis and prediction. Genomic annotation, Phone: 212-639-2024; Fax: 212-717-3302; E-mail: [email protected]. I2006 American Association for Cancer Research. computational analyses, and homology searches were done using internet doi:10.1158/0008-5472.CAN-06-2074 accessible software (see Supplementary Methods). www.aacrjournals.org 9437 Cancer Res 2006; 66: (19). October 1, 2006

cDNA cloning, expression, and transfection. The human SCCRO cDNA pSUPER-SCCRO (5¶-GTTCAGAGCAGCAACACAG-3¶), as well as a scrambled was generated by reverse transcription-PCR (RT-PCR) of total RNA isolated shRNA construct as control (pSUPER-control;5¶-CGTCTACCTA- from head and neck cancer cell line MDA1386 and subcloned into pGEM-T CACTCCCTC-3¶) were used. Transfection was done by lipofection as (Promega, Madison, WI), pUSEamp (Upstate Biotechnology, Lake Placid, previously described (9). NY), pCMV-HA (BD Biosciences, San Jose, CA), and pGEX-4T-3 (GE Statistical analysis. All analyses were done using JMP4statistical Healthcare Life Sciences, Piscataway, NJ) vectors (see Supplementary software (SAS Institute, Inc., Cary, NC; see Supplementary Methods). Methods). 5¶ Rapid amplification of cDNA ends. The sequences of the 5¶ end of Results cDNA were derived using SMART RACE cDNA Amplification kit (BD Biosciences Clontech, Palo Alto, CA) according to protocols of the Molecular cloning and analysis of SCCRO. We applied a manufacturer and sequenced (see Supplementary Methods). positional cloning approach to systematically refine the minimal Cell lines, tumor tissue, and chemical reagents. The derivation and common region of 3q amplification. Analysis of five cancer cell growth conditions for cell lines were published or are available through the lines with 3q amplification identified two recurrent amplification American Type Culture Collection (Manassas, VA; ref. 10). Primary tumors subpeaks contained within BAC clones, 202B22 and 386M7 (4, 5, 9). from head and neck and lung cancers were collected from patients PIK3CA, previously identified as amplified in ovarian and cervix undergoing surgical resection, after obtaining informed consent and cancers, was an obvious candidate within one of these peaks following institutional guidelines (see Supplementary Methods). (386M7; ref. 25). Analysis of the genomic insert in BAC clone Cell growth and colony formation assays. 3-[4,5-Dimethylthiazol-2-yl]- 202B22 representing the second subpeak showed no known genes. 2,5-diphenyl-tetrazolium bromide (MTT)–based colorimetric assay for cell growth (Sigma-Aldrich, St. Louis, MO) was done as described previously (9). Annotation using the GENSCAN and Genie prediction programs Soft agar colony formation assay was done by plating f2,000 cells on 0.9% identified several potential genes. RT-PCR done on total RNA from agarose-coated plates and incubated for 1 week in DMEM containing 250 two SCC cell lines (MDA886 and MDA1186) and normal human Ag/mL neomycin, essentially as described previously (see Supplementary placenta resulted in a cDNA product for only one of these genes, Methods; ref. 20). which upon sequencing revealed a 780 bp open reading frame Flow cytometry and fluorescence-activated cell sorting analysis. (ORF; Supplementary Fig. S1A and B). This gene was designated Cells were collected by trypsinization and resuspended in PBS containing as SCCRO. 2 mmol/L EDTA. The cells were then fixed in 70% ethanol, digested with Sequence analysis of SCCRO revealed a single ATG at the 5¶ end A A A RNase A (0.02 g/ L), stained with 50 g/mL ethidium bromide, and in the context of a sequence that can support translation initiation analyzed using FACScan and CellQuest software (Becton Dickinson, and a polyadenylation signal at the 3¶ end (Supplementary Fig. S1A; Franklin Lakes, NJ). Apoptosis assessment. Apoptosis was quantified using Annexin/7- ref. 26). In vitro translation of this cDNA yielded a protein of f amino-actinomycin D (7AAD) staining according to protocol of the predicted size ( 30 kDa; data not shown). A multi-tissue Northern manufacturer (R&D Systems, Minneapolis, MN). blot was probed with full-length ORF for SCCRO. Transcripts of Quantified in vitro migration and invasion. The migratory potential different sizes were detected (f2.0, 4.1, and 4.3 kb). The expression and invasive capacity of cultured cells were assayed by a modified Boyden level and relative ratios of these transcripts varied from tissue to chamber method as described previously (21). tissue (Supplementary Fig. S1C). Gene expression was reconfirmed Athymic mouse tumorigenicity. pUSEamp-SCCRO-3T3 or pUSEamp- by real-time RT-PCR (data not shown) in adult RNA with an 3T3 cells were injected s.c. into each flank of BALB/C athymic nude mice. expression pattern similar to that detected by Northern blot Autopsy studies were done on all animals at the end of the experiment analysis. These observations suggest that the expression and the following institutional guidelines (see Supplementary Methods). relative levels of SCCRO transcripts are regulated in a tissue- Fluorescence in situ hybridization. Dual-color fluorescence in situ hybridization (FISH) was done essentially as described earlier using DNA specific manner. A search of the genome database shows signi- from BAC clone 202B22 and chromosome 3 centromeric probes (10). ficant evolutionary conservation, suggesting SCCRO has important 8 Southern and Northern blot analyses. Southern and Northern blot biological functions (Supplementary Fig. S2). No functional data analyses were done according to standard protocols using probes is available for SCCRO orthologues, with the exception of DCN1, corresponding to the entire coding region of SCCRO, generated by PCR which has been shown to play a role in cullin neddylation (27). (see Supplementary Methods; ref. 9). SCCRO is amplified and highly expressed in human tumor. Western blot analysis. Western blots were done using rabbit polyclonal In agreement with the reported frequency of 3q amplification, anti-SCCRO antibody at a concentration of 1:5,000 (see Supplementary SCCRO mRNA overexpression was detected in 21 of 44 (48%) Methods). Antibody against Gli1 was obtained from Chemicon International primary lung, 16 of 45 (36%) head and neck, and 4 of 9 (44%) (Temecula, CA) and used at a concentration of 1:5,000. cervical carcinomas relative to matched histologically normal lung, Quantification of mRNA levels using real-time RT-PCR analysis. Real-time PCR was done as described in Supplementary Methods. Melt oral mucosa, or cervical mucosal controls, respectively (levels >2 curve analysis was done following amplification (22). The acquisition SD; refs. 4–8, 28). Moreover, a significant correlation was observed temperature was set 1jCto2jC below the Tm of the specific PCR product. between SCCRO copy number, the corresponding mRNA, and The relative quantification of a target gene compared with a reference gene protein levels in cell lines and primary tumors derived from lung (18S rRNA) was done as described (23, 24). PCR primers and conditions are and head and neck cancer (Fig. 1A and B; r = 0.81; P < 0.001; see detailed in Supplementary Table S2. Supplementary Figs. S3 and S4for anti- SCCRO antibody validation). Immunohistochemistry. Immunohistochemistry was done using poly- In addition, non–small cell lung cancers showed SCCRO over- clonal antibody against SCCRO raised in rabbit (antibody 1B) and goat expression in SCC (61%), but not in adenocarcinomas (9%; human Gli1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Details P = 0.004), reflecting the known predilection of 3q amplification for immunohistochemistry are provided in Supplementary Methods. Protein knockdown. pSUPER RNA interference (RNAi) system (Oligo Engine Inc., Seattle, WA) was used to stably direct endogenous synthesis of short hairpin RNA (shRNA) against SCCRO and Gli1. We designed two RNAi 7 sequences using OligoEngine software. A Gli1-specific silencer pSUPER- 7 http://www.cerebre.com. Gli-1502 (5¶-CCTTAGGCTGGATCAGCTG-3¶) and a SCCRO-specific silencer 8 R. Ryan et al., unpublished data.

Cancer Res 2006; 66: (19). October 1, 2006 9438 www.aacrjournals.org

Figure 1. Copy number and expression level of SCCRO in different primary tumors and cell lines. A, Western blot analysis showing SCCRO protein levels in the indicated cell lines and primary tumors. The corresponding SCCRO mRNA levels and DNA copy number (assessed by real-time RT-PCR and FISH, respectively) are shown. B, correlation between SCCRO amplification (assessed by FISH using BAC 202B22 as a probe) and mRNA expression (assessed by real-time PCR) in human cancer cell lines (n = 15) derived from lung and head and neck SCC (r = 0.81; P < 0.001). C, scatter plot of SCCRO mRNA levels, determined by real-time PCR in 23 lung SCCs, 12 adenocarcinomas, and 45 matched histologically normal lung tissues (lines, median levels; P = 0.01). D, Kaplan- Meier survival curves showing cause- specific survival based on SCCRO mRNA expression status in primary lung carcinomas (P = 0.05).

for SCC (Fig. 1C; ref. 29). Survival analysis was done in a larger 8.7% invasive fraction for clones 14and 28, respectively), compared cohort of previously untreated non–small cell lung cancer cases with the vector-transfected 3T3 cells (18.5 F 6.7% invasive fraction) that underwent primary surgical treatment (n = 79). Although as determined by the modified Boyden chamber invasion assay there were no differences in tumor-node-metastasis (TNM) stage, (P < 0.001). In contrast to vector-transfected NIH-3T3 cells, both histology, treatment, or follow-up (median 44 months) based on SCCRO-transformed clones also displayed anchorage-independent SCCRO expression status, SCCRO expression negatively correlated growth, as tested on soft agar colony formation assay, indicating with cause-specific survival (P = 0.05), indicating that SCCRO a transformed phenotype (Fig. 2C). Finally, in vivo xenograft assay overexpression imparts an aggressive phenotype in these tumors in NIH BALB/c nude mice showed that both SCCRO-transfected (Fig. 1D). The effects of SCCRO overexpression on cause-specific clones were oncogenic, resulting in tumor formation in six of six survival remained significant even after controlling for the effects mice within 8 weeks. In contrast, no tumors developed in six mice of confounding TNM stage by multivariate analysis (relative risk, injected with vector-transfected 3T3 cells even after 12 weeks 4.38; P = 0.04). These findings are similar to our prior analysis of (Fig. 2D). Autopsy and histopathologic analyses revealed the pre- head and neck SCC (11). To exclude the possibility of an oncogenic sence of poorly differentiated tumors (Supplementary Fig. S5B) mutation in SCCRO, we did RT-PCR and sequenced the coding with metastases to pelvic lymph nodes identified in three of six region of SCCRO from 120 cases of SCC of lung or head and neck tumors. origin. No mutations were identified. Combined, these observa- Because SCCRO is purported to play a role in keratinocyte tions suggest that amplification of SCCRO results in its over- transformation, we did transformation experiments in cells of expression, which is associated with aggressive behavior in epithelial derivation (HaCaT). For indeterminate reasons, stable mucosal SCC. colonies could not be established with adequate SCCRO expression SCCRO is oncogenic. To further validate SCCRO as an levels. Accordingly, we assessed colony-forming ability in HaCaT amplification target in cancer, we tested its oncogenic potential cells transiently transfected with SCCRO. SCCRO-transfected in transformation assays. We used NIH-3T3 cells for the initial HaCaT cells formed 352 F 97 colonies per 12-well plate on the experiments, as this cell system is well established for assessment soft agar forced suspension culture assay, compared with z50 of transforming activity. We stably transfected NIH-3T3 cells with a colonies in wild-type and empty vector–transfected cells (P < 0.01; plasmid expressing SCCRO under the control of a cytomegalovirus see Supplementary Fig. S6 for basal expression). These results show (CMV) promoter. Two independent clones (clones 14and 28) were that SCCRO induces transformation in two distinct mammalian cell specifically selected, as they expressed SCCRO protein at levels lines. comparable with human cancers cell lines carrying SCCRO SCCRO is required for survival of cancer cell lines carrying amplification (Fig. 2A). SCCRO-transfected NIH-3T3 cells acquired amplification. To more firmly establish a role for SCCRO a dedifferentiated morphology, with increased nuclear size and amplification/overexpression in the pathogenesis of SCC, we higher nuclear-to-cytoplasmic ratio. Moreover, focus formation was assessed the requirement of SCCRO expression for tumor seen in both SCCRO-transfected clones, but not in the one maintenance. Transfection of shRNA against SCCRO into cancer transfected with vector (data not shown). Cell growth, as cell lines resulted in a 3- to 5-fold decrease in SCCRO protein levels determined by MTT cell viability assay, was significantly higher in relative to empty vector and scrambled shRNA controls (Fig. 3A). SCCRO-transformed cells (Fig. 2B). In addition, SCCRO-trans- Significant cell death was observed following SCCRO knockdown in formed cells did not senesce in absence of growth signals (serum representative cancer cell lines carrying SCCRO amplification/ deficient conditions) in contrast to vector-transfected cells overexpression, but not in those without it (Fig. 3B). Fluorescence- (Supplementary Fig. S5A). SCCRO-transfected cells showed an activated cell sorting (FACS) analysis showed a significant increase increase in in vitro invasive potential (53 F 12.3% and 69.5 F in the sub-G1 fraction, and Annexin/7AAD staining suggested that www.aacrjournals.org 9439 Cancer Res 2006; 66: (19). October 1, 2006

validate the results from the gene array analysis (Supplementary Tables S1 and S2). Of the genes identified by array analysis, up-regulation of Gli1 was of particular interest, as it plays an essential role in the development of lungs, trachea, and esophagus—the same organs where SCCRO is purported to have oncogenic activity (30). The expressions of multiple genes known to be downstream of Gli1 was also altered, including Gli2, cyclin D1, FGFR, IGF, IGFR, and ceruloplasmin (31–33). To validate the findings of the gene expression analysis, we did Western blot (Fig. 4A; Supplementary Fig. S6), real-time RT-PCR (Fig. 4B), and immunohistochemical analyses (Fig. 4C) in SCC cell lines and several primary tumors. Results suggest that there is a positive correlation between SCCRO and Gli1 levels in the analyzed cases (Fig. 4A-C; r = 0.75; P < 0.001). This association was further tested in an independent MEF/3T3 tetracycline-repressible SCCRO expression system (Tet-off). As expected, removal of tetracycline from the medium resulted in an increase in SCCRO expression and a resultant increase in Gli1 levels as detected by Western blot analysis and real-time PCR analyses (5.0-fold; P = 0.03; Fig. 4D). Conversely, no change in SCCRO or Gli1 expression was seen in the presence of tetracycline in the medium. Figure 2. SCCRO induces malignant transformation. A, Western blot analysis In addition, mRNA levels of Gli2 (3.5-fold; P = 0.05) and PTCH showing SCCRO protein levels in NIH-3T3 cells stably transfected with either an expression vector containing SCCRO (clones 14 and 28) or empty (5.4-fold; P = 0.01), genes known to be activated by the HH pathway, vector. B, MTT cell proliferation assay showing increased growth rate in were also up-regulated. In contrast, no significant expression SCCRO-transfected NIH-3T3 cells relative those transfected with empty vector. C, soft agar assay showing increased colony formation in SCCRO-transfected changes were detected in Gli3 (P > 0.05) or SMO (P > 0.05), both of NIH-3T3 cells (clones 14 and 28) compared with vector alone (P < 0.001). which are not targets of the HH pathway. Finally, SCCRO expression D, tumor formation in BALB-c nude mice injected with SCCRO-transfected (left) in the developing mouse embryo was similar to Gli1 (Supplemen- or vector-transfected (right) NIH-3T3 cells (1 cm scale). tary Fig. S7). Taken together, these results strongly suggest that SCCRO is a positive regulator of the HH signaling pathway. SCCRO binds to and activates the Gli1 promoter. To reduction of SCCRO protein levels by shRNA transfection resulted determine if Gli1 is a transcriptional target of SCCRO, we did a in death by apoptosis in MDA1386 cells (Fig. 3C). Conversely, no reporter assay using a construct containing luciferase gene under apoptosis was seen in MDA1386 cells transfected with the control the control of Gli1 promoter. Transfection of the Gli1-luciferase shRNA (Fig. 3C) or in cell lines with normal SCCRO copy number reporter construct into cells overexpressing SCCRO (either stably (data not shown). Soft agar colony formation assay showed a or transiently) resulted in a significant increase in luciferase significant reduction in the number of colonies in shRNA- expression relative to control (Fig. 5A). Moreover, in transient transfected cells compared with the control (Fig. 3D). Because cotransfection experiments, the level of luciferase expression the specificity of RNAi-based knockdown has been questioned, correlated with the amount of SCCRO expression plasmid trans- even when unique sequences are targeted, we repeated the cell fected (Fig. 5A). viability experiments after knocking down SCCRO by transfection To further elucidate the mechanism of SCCRO-mediated effects of antisense in the same cell lines. This produced identical results on Gli1 transcription, we did modified McKay and chromatin in vitro and in vivo in xenograft models,9 strongly suggesting the immunoprecipitation (ChIP) assays to assess SCCRO-DNA inter- observed apoptosis results from SCCRO protein knockdown. These actions. Modified McKay experiment showed SCCRO to interact experiments suggest that SCCRO not only confers survival with a fragment that spans the region +122 to +496 of Gli1 advantages, but is required for the viability of cancer cell lines promoter (Fig. 5B and C). SCCRO-DNA interaction was further carrying amplification/overexpression. Collectively, these experi- confirmed in vivo by ChIP assay. Three primer sets, spanning ments strongly implicate SCCRO as one of the targets of 3q overlapping segments of the Gli1 promoter (À962 and +496), were amplification. used to detect the in vivo binding region (Supplementary Table S2). SCCRO is involved in HH signaling. As a step toward ChIP assay validated the results from the modified McKay assay, understanding the functional role of SCCRO,wedidgene showing that SCCRO binds to a region spanning +122 to +496 of expression analysis using Affymetrix oligonucleotide microarrays Gli1 promoter (Fig. 5D). Taken together, it seems that Gli1 is a on NIH-3T3 cells stably transfected with SCCRO (clones 14and 28). direct transcriptional target of SCCRO. After normalizing for expression changes in vector-transfected Gli1 contributes to SCCRO-induced transformation. To NIH-3T3 cells, the expression of several genes was altered in both determine the role of Gli1 in SCCRO-driven oncogenesis, we SCCRO-transfected clones, including many linked to growth and assessed the effects of Gli1 knockdown on the viability of SCCRO- malignant transformation (Supplementary Table S1). Real-time T1 cells (derived from pUSEamp-SCCRO-3T3 after passage through RT-PCR of eight representative genes was done to internally a mouse). Specific silencing was achieved with transfection of pSUPER-Gli1-1502, whereas there was no change in Gli1 protein levels with transfection of either scrambled RNAi (pSUPER-control) or empty vector (pSUPER) in SCCRO-T1 cells (Fig. 6A). Silencing of 9 I. Ganly et al., unpublished data. Gli1 in SCCRO-T1 cells resulted in a 48% mean reduction in colony

Cancer Res 2006; 66: (19). October 1, 2006 9440 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. SCCRO/DCUN1D1 Is Activated by Amplification formation relative to pSUPER-control–transfected cells (Fig. 6B; primary tumors and cell lines derived from head and neck and lung P = 0.01) These findings suggest that Gli1 partially mediates carcinomas. Because overexpression of an amplified gene in itself is SCCRO-induced transformation, but does not exclude involvement insufficient to support its role in cancer pathogenesis, we did a in other pathways. series of experiments to establish SCCRO as an amplification target. We found that SCCRO overexpression was associated with an aggressive clinical course in primary SCC of the lung. We showed Discussion that SCCRO transforms cells of both fibroblastic and keratinocytic SCC of mucosal origin include tumors arising from the head and lineage, suggesting that it functions as an oncogene. Finally, we neck, lung, esophagus, and cervix, and account for over one third of showed that silencing using shRNA against SCCRO results in all cancers and cancer-related deaths annually in the United States apoptosis only in cancer cell lines carrying SCCRO amplification, (34). Although these tumors are anatomically diverse, they share a suggesting that SCCRO not only induces transformation but is also causal association with tobacco exposure and a common genetic required for maintenance of the malignant phenotype. This composition (35). Amplification of 3q26-27 locus is a common and phenomenon, termed ‘‘oncogene addiction,’’ suggests that the gene crucial event in squamous cell carcinogenesis, associated with both is directly involved in the maintenance of the malignant state, tumor progression and an aggressive clinical course (4, 5, 9–11, strongly suggesting it is an oncogenic signal rather than noise in 25, 36–39). In this study, we identified a novel gene (SCCRO) within malignant progression (41). Although it remains possible that other a recurrent amplification subpeak at 3q26.3 in SCC. Interestingly, important cancer gene(s) may be present, our data strongly suggest at least three other genes, namely LAMP3, AP2M1, and eIF-4c, are that SCCRO is a candidate oncogene that drives selection for 3q in the same relative genomic location as SCCRO, supporting the amplification. presence of an amplification subpeak in the region (14, 18, 40). To To begin to ascertain its function, we screened the effects of validate SCCRO as an amplification target, we first correlated SCCRO transfection on gene expression in NIH-3T3 cells and genomic amplification with mRNA and protein expression in identified multiple targets. Given the magnitude of up-regulation

Figure 3. shRNA-mediated suppression of SCCRO expression results in apoptosis. A, Western blot analysis of SCCRO protein levels following transfection of representative SCC cell lines with shRNA against SCCRO showing specific decrease in SCCRO levels relative to cells transfected with scrambled shRNA. Representative Western blot showing h-actin levels is shown as a loading control. B, MTT cell viability assay done 48 hours posttransfection of shRNA against SCCRO showing a significant decrease in cell viability in cancer cell lines carrying high-level SCCRO amplification (SCC15 and MDA1386) with minimal effect on cell lines without amplification (584 and H157; P < 0.05). FACS analysis and Annexin/7AAD staining (C) and soft agar colony formation assay (D)of MDA1386 cells transfected with SCCRO-shRNA (bottom) shows an increase in apoptotic cell death and decrease in colony formation relative to vector-transfected cells (top).

www.aacrjournals.org 9441 Cancer Res 2006; 66: (19). October 1, 2006

Figure 4. SCCRO and Gli1 are coexpressed in cancer cell lines and primary tumors. A, Western blot analysis of Gli1 protein levels in cell lines derived from head and neck and lung carcinomas. In these cell lines, Gli1 levels show a positive correlation with SCCRO (as in Fig. 1A). B, analysis of SCCRO and Gli1 expression done in 26 non–small cell carcinomas of lung origin and matched histologically normal lung tissue shows a significant correlation between SCCRO and Gli1 expression, relative to matched normal controls (r = 0.75; P < 0.001). C, immunohistochemical analysis shows coexpression of SCCRO (bottom) and Gli1 (top) in three primary lung SCCs and absence of expression in histologically normal lung tissue. D, Western blot showing SCCRO and Gli1 protein levels in MEF/3T3 cells expressing SCCRO under the control of a tetracycline-repressible promoter with (top) and without (bottom) tetracycline withdrawal.

of Gli1 and several of the Gli1 transcriptional targets in SCCRO- have suggested that the HH signal is activated in head and neck transformed cells, we focused on assessing the role of SCCRO in cancer (19). This activation is not induced by receptor-ligand HH signaling. The HH signaling pathway is known to play an activity, as is present in small cell lung cancers, but rather essential role in both embryonic development and cancer involves other mechanisms. In a small percentage of cases, the pathogenesis (42, 43). Gli1 is also an important primary target activation is related to amplification of Gli2 (19). The association in carcinogenesis, as evidenced by its activation by amplification between SCCRO and Gli1 expression and activation of the HH in gliomas (44). It is important to note that prior studies have pathway in both constitutive (stable) and regulated (tetracycline shown a prominent absence of aberrant HH signaling in SCC repressible) SCCRO expression systems suggests that SCCRO (45). However, all of these studies have focused on tumors of amplification may play a role in activating HH signaling in SCC. cutaneous origin, which are etiologically and pathogenetically Moreover, SCCRO and Gli1 mRNA and protein are coexpressed in distinct from SCC of mucosal origin. Interestingly, recent studies primary tumors derived from head and neck and lung

Figure 5. Gli1 is a transcriptional target of SCCRO. A, cotransfection of NIH-3T3 cells stably or transiently transfected with SCCRO-expressing plasmid under the control of a CMV promoter and a luciferase reporter plasmid under the control of the Gli1 promoter. B, modified McKay assay using the human Gli1 promoter (+122 to +496) incubated with whole cell lysate from human SCC cancer cell lines MDA1386 and immunoprecipitated using anti-SCCRO antibody. C, modified McKay assay using the human Gli1 promoter (+122 to +496) incubated with whole cell lysate form hemagglutinin (HA)-SCCRO–transfected HeLa cells and immunoprecipitated using an anti-HA antibody. D, results from ChIP assay with PCR using primer set 7, 8, and 10 are shown. IP, immunoprecipitation.

Cancer Res 2006; 66: (19). October 1, 2006 9442 www.aacrjournals.org

Figure 6. A, Western blot analysis of Gli1 protein levels following transfection of NIH-3T3-SCCRO-T1 cells (SCCRO-transformed NIH-3T3 cells passaged through a mouse) with shRNA against Gli1 showing a specific decrease in expression levels. B, soft agar assay showing a significant decrease in colony formation in shRNA-Gli1 (pSUPER1502)–transfected NIH-3T3-SCCRO-T1 cells relative to scrambled RNAi (pSUPERscram) or vector-transfected cells.

carcinomas. We also found that the expression of known Gli1 of Gli1 expression in mammalian systems is likely to be targets, including Gli2 and PTCH, is increased in response to multifactorial. We showed that SCCRO binds the Gli1 promoter to SCCRO overexpression, whereas the expression of HH pathway activate transcription, establishing a new mechanism for activation components known not to be effected by Gli1 remains of the HH signaling pathway. Kurz et al. (27) recently cloned the unchanged. Analysis of mouse embryo containing lacZ cassette SCCRO homologue of both Caenorhabditis elegans and Saccharo- inserted into SCCRO gene (see Supplementary Fig. S8)10 suggests myces cerevisiae (dcn-1/Dcn1p), and identified it to be a key regulator SCCRO to be highly expressed in the forebrain and midbrain as of cullin neddylation, and, in turn, activation of the SCF complex well as other sites, a pattern similar to Gli1 expression (46). and ubiquitin-targeted protein degradation. Interestingly, prior work Combined with the observation that shRNA-mediated suppression has established the role of cullin neddylation in the regulation of of Gli1 activity resulted in decreased tumorigenicity in SCCRO- HH signaling in Drosophila (50). Whether SCCRO serves as a link transformed cells, we suggest that activation of HH signaling between cullin neddylation and the regulation of HH signaling plays a significant, but not exclusive, role in SCCRO-mediated remains to be established. transformation. Unlike Drosophila, where the HH control of Ci transcription (Gli homologue) is fairly well defined, the precise Acknowledgments mechanisms of Gli regulation in mammalian systems remain Received 6/8/2006; revised 7/13/2006; accepted 8/8/2006. obscure (43). Although the COOH terminus of Gli2 and Gli3 (but Grant support: This work was supported in part by grants from George H.A. not Gli1) may function as transcriptional repressors after Clowes, Jr., MD, FACS, Memorial Research Career Development Award from the American College of Surgeons; Falcone Fund; Clinical Innovator Award, Flight proteolysis, a key mechanism for HH-associated Ci transcriptional Attendant Medical Research Institute (to B. Singh). regulation in Drosophila, most investigators agree that this is The costs of publication of this article were defrayed in part by the payment of page not a major mechanism for HH-associated regulation of Gli charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734solely to indicate this fact. transcription in vertebrate systems (43). Analysis of Gli1 knockout We thank Dr. Lorenz Studer for assistance with the immunofluorescence analysis, mice suggests that HH and Gli have overlapping as well as Dr. Agnes Viale for assistance with gene expression profiling, Dr. Nicholas Socci for help with analysis of gene expression data, Dr. Susan Naylor (Department of Cellular independent functions (43, 47–49). It thus seems that the regulation and Structural Biology, University of Texas Health Science Center, San Antonio, TX) for providing the BACs used in these experiments, Dr. Shunsuke Ishii (Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Ibaraki, Japan) for providing the Gli1 promoter constructs, Drs. Chris Sander and Boris Reva for bioinformatics analyses, Swarna Gogneni and Michael Wyler for technical assistance, Drs. Joan Massague and Andrew Simpson for the critical review of the manuscript, and Nancy Bennett for 10 A. Kaufman et al., in preparation. excellent editorial assistance.

References invasive cancer and is a negative prognostic factor in 8. Bockmuhl U, Schluns K, Kuchler I, et al. Genetic head and neck squamous cell carcinomas. Am J Pathol imbalances with impact on survival in head and neck 1. Miele M, Bonatti S, Menichini P, et al. The presence of 2002;161:365–71. cancer patients. Am J Pathol 2000;157:369–75. amplified regions affects the stability of chromosomes 5. Singh B, Wreesmann VB, Pfister D, et al. Chromosomal 9. Singh B, Reddy PG, Goberdhan A, et al. p53 regulates in drug-resistant Chinese hamster cells. Mutat Res 1989; aberrations in patients with head and neck squamous cell survival by inhibiting PIK3CA in squamous cell 219:171–8. cell carcinoma do not vary based on severity of tobacco/ carcinomas. Genes Dev 2002;16:984–93. 2. Bonatti S, Miele M, Menichini P, et al. Preferential loss alcohol exposure. BMC Genet 2002;3:22. 10. Singh B, Gogineni SK, Sacks PG, et al. Molecular of chromosomes containing amplified DNA regions in 6. Heselmeyer K, Macville M, Schrock E, et al. Advanced- cytogenetic characterization of head and neck squa- cultured cells. Prog Clin Biol Res 1989;318:271–6. stage cervical carcinomas are defined by a recurrent mous cell carcinoma and refinement of 3q amplifica- 3. Roth JR, Andersson DI. Amplification-mutagenesis— pattern of chromosomal aberrations revealing high tion. Cancer Res 2001;61:4506–13. how growth under selection contributes to the genetic instability and a consistent gain of chromosome 11. Estilo CL, O-charoenrat P, Ngai I, et al. The role of origin of genetic diversity and explains the phenom- arm 3q. Genes Chromosomes Cancer 1997;19:233–40. novel oncogenes squamous cell carcinoma-related enon of adaptive mutation. Res Microbiol 2004;155: 7. Petersen I, Bujard M, Petersen S, et al. Patterns of oncogene and phosphatidylinositol 3-kinase p110a in 342–51. chromosomal imbalances in adenocarcinoma and squamous cell carcinoma of the oral tongue. Clin 4. Singh B, Stoffel A, Gogineni S, et al. Amplification of squamous cell carcinoma of the lung. Cancer Res Cancer Res 2003;9:2300–6. the 3q26.3 locus is associated with progression to 1997;57:2331–5. 12. Eder AM, Sui X, Rosen DG, et al. Atypical PKCL

www.aacrjournals.org 9443 Cancer Res 2006; 66: (19). October 1, 2006

contributes to poor prognosis through loss of apical- 24. Pfaffl MW. A new mathematical model for relative as a candidate oncogene in head and neck cancer. basal polarity and cyclin E overexpression in ovarian quantification in real-time RT- PCR. Nucleic Acids Res Cancer Res 2002;62:6211–7. cancer. Proc Natl Acad Sci U S A 2005;102:12519–24. 2001;29:E45. 38. Hibi K, Trink B, Patturajan M, et al. AIS is an 13. Regala RP, Weems C, Jamieson L, et al. Atypical 25. Shayesteh L, Lu Y, Kuo WL, et al. PIK3CA is oncogene amplified in squamous cell carcinoma. Proc protein kinase CL is an oncogene in human non-small implicated as an oncogene in ovarian cancer. Nat Genet Natl Acad Sci U S A 2000;97:5462–7. cell lung cancer. Cancer Res 2005;65:8905–11. 1999;21:99–102. 39. Imoto I, Pimkhaokham A, Fukuda Y, et al. SNO is a 14. Kanao H, Enomoto T, Kimura T, et al. Overexpression 26. Kozak M. Initiation of translation in prokaryotes and probable target for gene amplification at 3q26 in of LAMP3/TSC403/DC-LAMP promotes metastasis in eukaryotes. Gene 1999;234:187–208. squamous-cell carcinomas of the esophagus. Biochem uterine cervical cancer. Cancer Res 2005;65:8640–5. 27. Kurz T, Ozlu N, Rudolf F, et al. The conserved protein Biophys Res Commun 2001;286:559–65. 15. Riazimand SH, Welkoborsky HJ, Bernauer HS, et al. DCN-1/Dcn1p is required for cullin neddylation in C. 40. Brass N, Heckel D, Sahin U, et al. Translation Investigations for fine mapping of amplifications in elegans and S. cerevisiae. Nature 2005;435:1257–61. initiation factor eIF-4g is encoded by an amplified gene chromosome 3q26.3-28 frequently occurring in squa- 28. Shinomiya T, Mori T, Ariyama Y, et al. Comparative and induces an immune response in squamous cell lung mous cell carcinomas of the head and neck. Oncology genomic hybridization of squamous cell carcinoma of carcinoma. Hum Mol Genet 1997;6:33–9. 2002;63:385–92. the esophagus: the possible involvement of the DPI gene 41. Weinstein IB. Cancer. Addiction to oncogenes—the 16. Snijders AM, Nowee ME, Fridlyand J, et al. Genome- in the 13q34amplicon. Genes Chromosomes Cancer Achilles heal of cancer. Science 2002;297:63–4. wide-array-based comparative genomic hybridization 1999;24:337–44. 42. Weiner HL, Bakst R, Hurlbert MS, et al. Induction of reveals genetic homogeneity and frequent copy number 29. Bjorkqvist AM, Husgafvel-Pursiainen K, Anttila S, medulloblastomas in mice by sonic hedgehog, indepen- increases encompassing CCNE1 in fallopian tube et al. DNA gains in 3q occur frequently in squamous cell dent of Gli1. Cancer Res 2002;62:6385–9. carcinoma. Oncogene 2003;22:4281–6. carcinoma of the lung, but not in adenocarcinoma. 43. Ruiz i Altaba A, Sanchez P, Dahmane N. Gli and 17. Or YY, Hui AB, Tam KY, et al. Characterization of Genes Chromosomes Cancer 1998;22:79–82. hedgehog in cancer: tumours, embryos and stem cells. chromosome 3q and 12q amplicons in nasopharyngeal 30. Motoyama J, Liu J, Mo R, et al. Essential function of Nat Rev Cancer 2002;2:361–72. carcinoma cell lines. Int J Oncol 2005;26:49–56. Gli2 and Gli3 in the formation of lung, trachea and 44. Kinzler KW, Bigner SH, Bigner DD, et al. Identifica- 18. Jiang F, Yin Z, Caraway NP, et al. Genomic profiles in oesophagus. Nat Genet 1998;20:54–7. tion of an amplified, highly expressed gene in a human stage I primary non small cell lung cancer using 31. Yoon JW, Kita Y, Frank DJ, et al. Gene expression glioma. Science 1987;236:70–3. comparative genomic hybridization analysis of cDNA profiling leads to identification of GLI1-binding ele- 45. Dahmane N, Lee J, Robins P, et al. Activation of the microarrays. Neoplasia 2004;6:623–35. ments in target genes and a role for multiple transcription factor Gli1 and the Sonic hedgehog signal- 19. Snijders AM, Schmidt BL, Fridlyand J, et al. Rare downstream pathways in GLI1-induced cell transforma- ling pathway in skin tumours. Nature 1997;389:876–81. amplicons implicate frequent deregulation of cell fate tion. J Biol Chem 2002;277:5548–55. 46. Hui CC, Slusarski D, Platt KA, et al. Expression of specification pathways in oral squamous cell carcinoma. 32. Kato M, Seki N, Sugano S, et al. Identification of sonic three mouse homologs of the Drosophila segment Oncogene 2005;24:4232–42. hedgehog-responsive genes using cDNA microarray. polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, 20. Mahoney MG, Simpson A, Jost M, et al. Metastasis- Biochem Biophys Res Commun 2001;289:472–8. in ectoderm- and mesoderm-derived tissues suggests associated protein (MTA)1 enhances migration, invasion, 33. Clark JC, Tichelaar JW, Wert SE, et al. FGF-10 disrupts multiple roles during postimplantation development. and anchorage-independent survival of immortalized lung morphogenesis and causes pulmonary adenomas Dev Biol 1994;162:402–13. human keratinocytes. Oncogene 2002;21:2161–70. in vivo. Am J Physiol Lung Cell Mol Physiol 2001;280: 47. Park HL, Bai C, Platt KA, et al. Mouse Gli1 mutants are 21. O-charoenrat P, Modjtahedi H, Rhys-Evans P, et al. L705–15. viable but have defects in SHH signaling in combination Epidermal growth factor-like ligands differentially up- 34. Greenlee RT, Murray T, Bolden S, et al. Cancer with a Gli2 mutation. Development 2000;127:1593–605. regulate matrix metalloproteinase 9 in head and neck statistics, 2000. CA Cancer J Clin 2000;50:7–33. 48. Chiang C, Litingtung Y, Lee E, et al. Cyclopia and squamous carcinoma cells. Cancer Res 2000;60:1121–8. 35. Decker J, Goldstein JC. Risk factors in head and neck defective axial patterning in mice lacking Sonic 22. Morrison TB, Weis JJ, Wittwer CT. Quantification of cancer. N Engl J Med 1982;306:1151–5. hedgehog gene function. Nature 1996;383:407–13. low-copy transcripts by continuous SYBR green I 36. Redon R, Muller D, Caulee K, et al. A simple specific 49. Lee J, Platt KA, Censullo P, et al. Gli1 is a target of monitoring during amplification. Biotechniques 1998; pattern of chromosomal aberrations at early stages of Sonic hedgehog that induces ventral neural tube 24:954–62. head and neck squamous cell carcinomas: PIK3CA but development. Development 1997;124:2537–52. 23. Schmittgen TD, Zakrajsek BA. Effect of experimental not p63 gene as a likely target of 3q26-qter gains. Cancer 50. Ou CY, Lin YF, Chen YJ, et al. Distinct protein treatment on housekeeping gene expression: validation Res 2001;61:4122–9. degradation mechanisms mediated by Cul1 and Cul3 by real-time, quantitative RT-PCR. J Biochem Biophys 37. Redon R, Hussenet T, Bour G, et al. Amplicon controlling Ci stability in Drosophila eye development. Methods 2000;46:69–81. mapping and transcriptional analysis pinpoint cyclin L Genes Dev 2002;16:2403–14.

Cancer Res 2006; 66: (19). October 1, 2006 9444 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. Squamous Cell Carcinoma Related Oncogene/DCUN1D1 Is Highly Conserved and Activated by Amplification in Squamous Cell Carcinomas

Inderpal Sarkaria, Pornchai O-charoenrat, Simon G. Talbot, et al.

Cancer Res 2006;66:9437-9444.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/66/19/9437

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2007/10/10/66.19.9437.DC1

Cited articles This article cites 49 articles, 18 of which you can access for free at: http://cancerres.aacrjournals.org/content/66/19/9437.full#ref-list-1

Citing articles This article has been cited by 18 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/66/19/9437.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 Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/66/19/9437. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.