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Expression of Ral Gtpases,Their Effectors, and Activators in Human

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Expression of Ral Gtpases,Their Effectors, and Activators in Human Human Cancer Biology Expression of Ral GTPases, Their Effectors, and Activators in Human Bladder Cancer Steven Christopher Smith,1, 2 Gary Oxford,1, 2 Alexander S. Baras,3 Charles Owens,1, 2 Dmytro Havaleshko,1, 2 David L. Brautigan,4 Martin K. Safo,6 and DanTheodorescu1, 2 , 5 Abstract Purpose: The Ral family of small G proteins has been implicated in tumorigenesis, invasion, and metastasis in in vitro and animal model systems; however, a systematic evaluation of the state of activation, mutation, or expression of these GTPases has not been reported in any tumor type. Experimental Design: We determined the activation state of the RalA and RalB paralogs in 10 bladder cancer cell lines with varying Ras mutation status. We sequenced RalA and RalB cDNAs from 20 bladder cancer cell lines and functionally evaluated the mutations found. We determined the expression of Ral, Ral activators, and Ral effectors on the level of mRNA or protein in human bladder cancer cell lines and tissues. Results: We uncovered one E97Q substitution mutation of RalA in 1 of 20 cell lines tested and higher Ral activation in cells harboring mutant HRAS. We found overexpression of mRNAs for RalA and Aurora-A, a mitotic kinase that activates RalA, in bladder cancer (both P < 0.001), and in association with tumors of higher stage and grade. RalBP1,a canonical Ral effector, mRNA and protein was overexpressed in bladder cancer (P < 0.001), whereas Filamin A was underex- pressed (P = 0.004).We determined that RalA mRNA levels correlated significantly with protein levels (P < 0.001) and found protein overexpression of both GTPases in homogenized invasive cancers. Available data sets suggest that RalA mRNA is also overexpressed in seminoma, glioblastoma, and carcinomas of the liver, pancreas, and prostate. Conclusion: These findings of activation and differential expression of RalA and RalB anchor prior workin model systems to human disease and suggest therapeutic strategies targeting both GTPases in this pathway may be beneficial. Within the Ras family of monomeric G proteins, RalA and downstream functions, particularly in transcription, remain to RalB are highly similar paralogs (85% amino acid identity) be determined. Recent results have indicated key roles for Ral that participate in diverse cellular functions. Among the proteins in tumorigenesis and metastasis (2, 3). processes regulated by Ral proteins are endocytosis, exocyto- Ral GTPases may be activated in a Ras-dependent manner, sis, actin cytoskeletal dynamics, and transcription (reviewed via several guanidine nucleotide exchange factors, or RalGEFs, in ref. 1). In some cases, Ral involvement in these processes including RalGDS (4), which is inhibited by the neurofibro- has been shown to be mediated through effectors such as matosis type 2 (NF2) gene product, Merlin (5). Activation of RalBP1, Sec5, Filamin A, and phospholipase D1 (Fig. 1A); in the Ral pathway has been recently shown to be a other cases, particular Ral-effector interactions responsible for requirement for transformation of human cells (6, 7), and Ras-mediated transformation depends on activation of RalA (8). Based on our prior observation of Ral involvement in Authors’ Affiliations: Departments of 1Molecular Physiology and Biological cancer cell motility (9), we recently reported antagonistic Physics, 2Urology, and 3Pathology; 4Center for Cell Signaling and Department of roles for these two GTPases, where RalB depletion by small Microbiology, University ofVirginia; 5Mellon Prostate Cancer Institute, University of interfering RNA or expression of activated mutant RalA both 6 Virginia, Charlottesville, Virginia; and Department of Medicinal Chemistry,Virginia inhibit cancer cell migration (10). Another group recently Commonwealth University, Richmond,Virginia Received 10/4/06; revised 2/20/07; accepted 4/6/07. found a similar role for RalB in motility through the exocyst Grant support: Medical Scientist Training Program training grant T32GM007267 complex in normal cells (11). Interestingly, RalA has been and Cancer Training Grant CA009109-29 (S.C. Smith), P01-CA40042 project 2 identified as a substrate of Aurora-A kinase, and RalA activity (D.L. Brautigan), and CA075115 (D.Theodorescu). may be increased by phosphorylation at serine 194 (12). The costs of publication of this article were defrayed in part by the payment of page advertisement Finally, we recently reported a role for RalA and RalB in the charges. This article must therefore be hereby marked in accordance CD24 with 18 U.S.C. Section 1734 solely to indicate this fact. transcriptional regulation of , a metastasis-associated Note: Supplementary data for this article are available at Clinical Cancer Research gene in bladder and other cancers (13) that is also a stem Online (http://clincancerres.aacrjournals.org/). cell marker (14). Requests for reprints: DanTheodorescu, Department of Molecular Physiology Given these findings, we sought to systematically investigate and Biological Physics, University of Virginia, Box 422, Charlottesville,VA 22908. Phone: 434-924-0042; Fax: 434-982-3652; E-mail: [email protected]. Ral GTPase activation state, mutation status, and expression in F 2007 American Association for Cancer Research. human bladder cancers and cell lines. Our results show that doi:10.1158/1078-0432.CCR-06-2419 GTP-bound, activated RalA and RalB are present in cell lines www.aacrjournals.orgClin Cancer Res 2007;13(13) July 1,3803 2007 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology derived from bladder cancers, and that the activation state is were used for enhanced chemiluminescence detection and quantitation higher in lines harboring the G12V HRAS oncogene. We using an Alpha Innotech Fluorchem 8800 and AlphaEase software. h A identified only one mutation, a missense mutation resulting in Monoclonal anti– -actin clone AC-47 (Sigma) at 0.5 g/mL in 1% substitution of glutamine for glutamate at position 97 of the bovine serum albumin in TBST was used to detect actin expression for normalization of expression of RalA or RalB. sequence of RalA in 1 of 20 lines screened, UM-UC-6. Most Ral sequencing. RalA and RalB coding sequences were examined by importantly, however, we identified overexpression of RalA and Transgenomic Labs. mRNA from 20 human bladder cancer cell lines RalB protein in bladder cancer and overexpression of RalA (15) was extracted; cDNAs were synthesized and sequenced to mRNA in other tumors. These results support the notion that determine if mutations were present in coding sequences. Two Ral GTPases are likely mediators in human bladder cancer and overlapping amplicons per gene were generated from cell line cDNA suggest that targeting the Ral pathway is a rational therapeutic for downstream analysis of variants. Mutation analysis of RalA and approach. RalB cDNA PCR products was conducted using fluorescent denaturing high-performance liquid chromatography technology and Surveyor Nuclease mismatch cleavage analysis both with the WAVE-HS System (Transgenomic). Aliquots of PCR product were scanned for variants by Materials and Methods denaturing high-performance liquid chromatography, confirmed by Surveyor mismatch cleavage, and characterized with bidirectional Cell culture and gene expression profiling of bladder tumor cell lines sequence analysis on an ABI 3100 sequencer using BigDye V3.1 and human bladder cancers. Forty-one human bladder cancer cells terminator chemistry (Applied Biosystems, Inc.). PCR products and were obtained and cultured as described (15). These and primary sequencing reactions were cleaned for cycle sequencing and chroma- human bladder carcinomas and normal bladder tissues were profiled tography using AMPure and CleanSEQ, respectively, as per manufac- on HG-U133A GeneChip arrays (Affymetrix) as described (16). The turer’s standard protocols (Agencourt). For semiquantitative complete cohort of 65 human bladder cancers and 15 normal bladder determination of mutant and normal allele frequencies, relative peak tissues used for this study were all analyzed on the HG-U133A areas of denaturing high-performance liquid chromatography elution GeneChip Array platform and was generated by compiling our data profiles, Surveyor mismatch cleavage products, and sequence chroma- (16) with others (17) available on National Center for Biotechnology tograms were examined after normalization and comparison to Information Gene Expression Omnibus (18). Standardization was reference controls (using the WAVE Navigator software for denaturing achieved by using original cel files for all cases, which were then high-performance liquid chromatography and Surveyor data). 7 processed with the dCHIP 2006 algorithm. The final human bladder Sequencher software (GeneCodes) was used to visualize and align tumor set includes 15 normal mucosae, 30 stage Ta,5Tis,6T1,5T2, sequence chromatograms and Mutation Discovery8 and the University 9T3, and 10 T4 tumors. Affymetrix probes used were RalA 214435_x_at, of California Santa Cruz genome browser9 were used for protein RalB 202100_at, RalBP1 202844_s_at, Rgl1 209568_s_at, Rgl2 annotation. Primers for RalA amplicon 1 were 5¶-GACTGGCTCGCGG- 209110_s_at, RalGDS 209050_s_at, Filamin A 214752_x_at, PLD1 TGCAGATTC-3¶ forward, and 5¶-GTTAACATTCCACTGCTCAGCTCTG- 177_at, Sec5 219349_s_at, Aurora-A 208079_s_at, and Merlin 3¶ reverse; for RalA amplicon 2 were 5¶-CTACAGCTGACTTCAGGGAG- 204991_s_at. CAG-3¶ forward and 5¶-TTCCATTGCAGTTCCAATTCATGC-3¶ reverse;
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