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 underexpressed (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; Ral activation assays and site-directed mutagenesis. Ral activation for RalB amplicon 1 were 5¶-CTGGTCCCTGCTCTTCAGTGG-3¶ forward was assayed and quantitated in indicated bladder cancer cell lines or and 5¶-CCGCTCCTCTAGGTCAGACTTG-3¶ reverse; and for RalB ampli- transfectants using RalBP1-agarose beads from Upstate that bind con 2 were 5¶-CAGCAACTGCCGAATTCAGGG-3¶ forward and activated, GTP-bound Ral GTPase (10). Percentage activation was 5¶-ACAAGCAAGAAGTCAACCATAGC-3¶ reverse. The RalA mutation calculated by densitometric comparison of bands of activated Ral was additionally independently verified by sequencing a genomic A pulled down by beads from 1,000 g of lysate to bands of total Ral in PCR product directly amplified from the UM-UC-6 genome, using A 10 g whole-cell lysate, divided by 100. The wild-type RalA and V23 primers 5¶-TGAGCCTACCAAAGCAGAC-3¶ forward and 5¶-GGAATAAA- constitutively active RalA in pcDNA3.1 expression vectors, as well as CACAATTAGAGACAGA-3¶ reverse. protocol for creation of the E97Q mutant RalA by site-directed Visualization of RalA structure. Visualization of the location of the mutagenesis have been described before (10). These constructs were RalA mutation identified in the RalA protein structure was carried out transiently transfected into HEK293T cells (American Type Culture with INSIGHTII (Molecular Simulations, Inc.) and labeled with Collection) using the FUGENE 6 (Roche Applied Sciences) and assayed SHOWCASE (Silicon Graphics, Inc.). and quantitated for activation after 48 h, as reported (10). Tumor tissue immunoblotting. Samples of adjacent normal bladder Quantitative Ral immunoblotting of human bladder cell lines. For mucosa (N1-5) or muscle invasive T2+ bladder tumor (T1-5) were quantitative immunoblotting, cells from 28 different bladder cancer obtained from cystectomy specimens in compliance with Human cell lines were grown to 80% to 90% confluence, washed with PBS, and Investigation Committee of the University of Virginia and Federal serum-free medium was added. Cells were then incubated for 24 h, guidelines. Ice-cold standard radioimmunoprecipitation assay lysis washed thrice with cold PBS, and lysed in a modified 2 SDS buffer buffer plus protease and phosphatase inhibitors were used in an Omni [0.125 mol/L Tris (pH 6.8), 4% SDS, 10% glycerol] with protease TH tissue homogenizer (Omni) to produce protein extracts. Samples inhibitors. The samples were sonicated and protein concentrations were quantitated using the bicinchoninic acid assay, and 20 Agof were estimated using a bicinchoninic acid protein assay kit (Pierce). each was run on 4% to 20% gradient SDS-PAGE minigels (Invitrogen) DTT was added to a final concentration of 0.2 mol/L, proteins were as described previously (13). Tumor blots were probed for RalA or A boiled, and 10 g of proteins were loaded on gels, as reported before RalB (both 1:1,000, described above); RalBP1 mouse monoclonal, (10). After electroblotting to polyvinylidene difluoride membrane and 1:1,000 (clone 2A1, Abnova); and Filamin A mouse monoclonal, blocking with 4% bovine serum albumin in TBST, RalA was detected 1:2,000 (mAb1680, Chemicon). Blots were stained with Ponceau S to with monoclonal anti-RalA (clone 8, BD PharMingen), as described ensure equal loading and transfer. before (9, 10, 13), whereas RalB was detected using goat polyclonal Databases and statistical analysis. The significance of expression of antibodies raised against RalB (R&D Systems) 1:1,000. Horseradish RalA, RalB, and associated genes with stage in bladder cancer was tested peroxidase–conjugated donkey anti-goat secondary antibodies (Sigma)

8 http://www.mutationdiscovery.com 7 http://www.dchip.org 9 http://genome.ucsc.edu

Clin Cancer Res 2007;13(13) July 1, 2007 3804 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. RalA and RalB Overexpression in Cancer using a two-tailed Student’s t test, assuming unequal variances. RalA level of GTP-bound E97Q mutant RalA in the UM-UC-6 cell mRNA and protein expression levels for bladder cancer cell lines were line, we decided to directly determine whether this mutation both normalized to h-actin mRNA or protein expression, respectively, changes the activity of the RalA protein. For this purpose, we and their correlation was tested by standard linear regression analysis in used HEK293T cells, derived from normal embryonic kidney Statistica V6 (StatSoft). We used the differential expression function of epithelial cells, to assay the activity of this protein in a cellular the Oncomine10 database (19) to determine whether a statistically background without the complex molecular deregulation significant difference exists in the expression of queried genes for various comparisons. present in cancer cell lines. As shown in Fig. 2E and quantitated in Fig. 2F, this mutant, when transiently overexpressed in HEK293T cells, does not exhibit a higher level of Results GTP-bound activation than wild-type RalA.

Activation state of Ral GTPases in UM-UC-6 and other human Expression of Ral GTPases,their effectors,and activators in bladder cancer cell lines human bladder cancer Given prior reports of overexpression of epidermal growth Expression of RalA, RalB, and Aurora-A mRNAs in bladder factor receptor (20), mutations of fibroblast growth factor cancer. Using Affymetrix HG-U133A oligonucleotide micro- receptor 3 and Ras (21), and overexpression of Aurora-A array data on 65 human bladder tumors and 15 samples of kinase (22) in bladder cancer, we sought to characterize the normal bladder mucosa (16, 17), we evaluated the expression activation state of RalA and RalB in bladder cancer cell lines. of Ral family GTPases as a function of transformation (cancer Activation assays for RalA and RalB, using the Ral binding versus normal or invasive cancer versus normal) as well as domain of RalBP1 that specifically interacts with the GTP- tumor progression (invasive versus superficial cancer; Table 1; bound form of RalA and RalB (10), were carried out on nine Supplementary Fig. S1). We found that RalA mRNA is different bladder cancer cell lines as shown in Fig. 1B. significantly overexpressed in human bladder cancer Interestingly, the activation states of RalA and RalB seem to (P < 0.001). Moreover, RalA overexpression is further correlate with each other, particularly in cells with high levels associated with muscle invasive stage T2+ tumors compared of activation of both GTPases that harbor the G12V HRAS with superficial Ta,Tis (carcinoma in situ), and T1 tumors (P < oncogene (Fig. 1C). 0.001; Fig. 3A). Finally, we compared RalA expression between different tumor grades, finding higher expression in RalA but not RalB mutations are present in human bladder grade 3 (n = 45) than grade 2 (n = 14) tumors (P < 0.001) cancer and higher expression in grade 4 (n = 3) than grade 3 tumors Cellular Ral activity may be influenced by its mutational (P = 0.07). Additionally, a slight overexpression of RalB status, including being rendered constitutively active by mRNA in cancer was noted (1.2-fold, P = 0.03; Fig. 3B), but mutation of G23V and Q72L (Ras codons 12 and 61; was not significantly associated with higher tumor grade. ref. 10). Therefore, we evaluated the 20 human bladder cell Table 1 summarizes findings on association of expression of lines for the presence of Ral mutations. We chose to sequence Ral pathway genes with tumor stage and grade. the cell lines because of the relative simplicity compared with Expression of activators of Ral in bladder cancer. Activation tissues and as well due to the observation that only the most of molecules upstream of Ral that would contribute to Ral malignant tumors were able to generate cell lines in bladder activation, including mutations of Ras (21) and both cancer. Thus, we would expect that if present, Ral mutations overexpression (20) or mutation (21) of receptor tyrosine would be enriched in this sample cohort, optimizing the kinases, have been extensively characterized in bladder cancer chance for their discovery. The cell lines used for this analysis (reviewed in ref. 4). In vivo, activation of Ral to the GTP- are summarized in Supplementary Table S1. bound conformation occurs through interaction with Ral- By sequencing the cDNA coding sequence of these cell lines, GEFs, including RalGDS, Rgl1, and Rgl2, which in turn are we found one mutation in the coding sequence of RalA from activated by recruitment to the membrane by their Ras bladder cancer cell line UM-UC-6, a heterozygous G-to-C Binding Domain binding to GTP-bound Ras (1). Because transversion at nucleotide position 589 of the RalA Refseq in vitro expression of these GEFs activates Ral (25), we (NM_005402) resulting in a missense mutation of glutamate evaluated the expression of these proteins in our cohort to 97 to glutamine (E97Q; Fig. 2A). We confirmed this mutation ascertain if differential expression of these factors were by independently sequencing a PCR product containing the associated with bladder cancer. We did not observe signifi- RalA mutation out of the genome of UM-UC-6. The crystal cant levels of overexpression of RalGDS or Rgl1 mRNA in structure of RalA (Pdbcode: 1UAD) has recently been bladder cancer or tumor progression. However, Rgl2, known published (23, 24). Using this structural knowledge, Fig. 2B commonly by the name of its mouse ortholog, Rlf, was shows a stereo view of the crystal structure of RalA, indicating slightly, but statistically significantly overexpressed in bladder the position of the E97Q mutation. tumors (P = 0.01, 1.4-fold). We then sought to evaluate the RalA activation state in the Recent reports have identified Aurora-A kinase (12) and the UM-UC-6 cell line, which harbors the mutation described NF2 gene product merlin, which inhibits RalGDS (5), as above, finding an intermediate level of RalA activation positive and negative regulators, respectively, of Ral activity. between the highest, J82, and lowest, UM-UC-3, of the cell Interestingly, as reported before on the level of immunohis- lines studied (Fig. 2C and D). Because of the relatively high tochemistry on bladder cancer specimens (22), Aurora-A mRNA is overexpressed in bladder tumors both when tumor tissue is compared with normal mucosa (P < 0.001) and when muscle invasive stage tumors are compared with superficial 10 http://www.oncomine.org ones (P = 0.02; Fig. 3C). Moreover, as was the case with RalA,

www.aacrjournals.orgClin Cancer Res 2007;13(13) July 1,3805 2007 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology increased Aurora-A kinase expression is associated with higher bladder cancer cells, we assayed homogenates of tissue tumor grade, as we found higher levels of expression of samples of normal bladder mucosae and muscle invasive Aurora-A in grade 3 tumors compared with grade 2 (P = 0.05) bladder cancers taken from cystectomy specimens for their and in grade 4 tumors compared with grade 3 (P = 0.06; expression of these proteins by immunoblotting. Tissue Table 1). Merlin mRNA expression is not significantly different homogenates were quantitated for total protein, equal between normal bladder mucosa or bladder cancer (data not amounts of protein for each sample were loaded, and blots shown). were probed for either of these GTPases by standard means, Expression of Ral effectors in bladder cancer. mRNAs for the with equivalent protein transfer validated by Ponceau S Ral effectors, RalBP1 (26), and Filamin A (27), were staining (not shown). As shown in Fig. 4C, invasive bladder differentially expressed in bladder tumors compared with cancers overexpress both RalA and RalB proteins. Moreover, normal bladder tissue. Interestingly, RalBP1 was significantly given our findings of differential mRNA expression of RalBP1 overexpressed (P < 0.001), whereas Filamin A was significant- and Filamin A in bladder cancer, we probed blots for these ly underexpressed (P = 0.004; Fig. 3D and E; Supplementary Ral effectors. As the mRNA results suggested, RalBP1 protein Fig. S2). However, neither was further consistently differen- was overexpressed in four of five invasive bladder cancers, tially expressed with increasing tumor stage or grade. Ral has whereas Filamin A protein was underexpressed in all five been reported to interact with EXOC2, the human homologue (Fig. 4C, bottom). of the Sec5 subunit of the exocyst (28), and PLD1, Ral overexpression is associated with cancer and progression in phospholipase D1 (29); however, microarray probes did not other tumor types reliably detect expression of these effectors above background. We used Oncomine (19) to determine if differential RalA,but not RalB,mRNA expression correlates with protein expression of RalA and RalB mRNA exists between cancer and expression corresponding normal tissue in other tumors than bladder Given the results obtained above, we sought to determine if cancer. We found that Ral GTPases were overexpressed in tumor RalA protein expression is significantly associated with tumor types other than bladder cancer. Table 2 shows associations stage in human cancers. Unfortunately, despite persistent between RalA overexpression and cancer in published studies of attempts, the commercially available antibodies for RalA were seminoma, glioblastoma, hepatocellular carcinoma, pancreatic not suitable for immunohistochemistry in either frozen or carcinoma, and prostate cancer, and underexpression in two paraffin sections.11 To overcome this limitation, we reasoned variants of lung cancer. Increased RalA expression is further that if RalA mRNA expression was proportional to RalA pro- associated with metastasis compared with primary prostate tein expression in bladder cancer cell lines, we could infer cancer (P < 0.001), a finding corroborated by another recent that human tumor expression of RalA mRNA was a reason- study, which discovered increased expression of RalA protein as able surrogate of the RalA protein levels in patient tumor a marker for metastatic prostate cancer compared with clinically material. Using a mouse monoclonal anti-RalA extensively localized and benign disease (31). Interestingly, this study also published in immunoblotting (clone 8 from BD PharMingen; found that Aurora-A kinase mRNA and protein levels parallel refs. 10, 27), we immunoblotted whole-cell extracts of 28 those of RalA and are overexpressed in metastatic prostate bladder cancer cell lines (data not shown). RalA bands were cancer compared with localized disease. For RalB, we found quantitated and then normalized to h-actin expression reduced levels of expression in cancer, including in hepatocel- reprobed on the same stripped blot. Using microarray lular carcinoma, seminoma, ovarian carcinoma, and meningi- expression data for 28 human bladder cancer cell lines (15), oma (Table 2). we then normalized RalA mRNA expression to h-actin mRNA expression, and correlated normalized mRNA expression of RalA to normalized protein expression of RalA. As shown in Discussion Fig. 4A, we found a significant positive correlation between Recent reports have determined that activation of Ral GTPase RalA mRNA and protein expression (R = 0.87, R2 = 0.76, P < is one of the most basic and distinct requirements for Ras- 0.001). This very high degree of correspondence is as good as mediated transformation of human cells (6, 7) and that specific the best found in a recent report using proteomic analysis of loss of function of either RalA or RalB gene product may the NCI-60 cancer cell lines, which reported a correlation abrogate important aspects of the cancer phenotype in different between oligonucleotide microarray expression data and cells (9–11). However, to date, no report has systematically protein expression level of R = 0.40 (range 0.15-0.88) across examined Ral GTPase expression or mutations in any single 19 gene products (30). In contrast, when we applied similar tumor type. Here, we integrate evaluation of Ral GTPase analysis to RalB expression in the same 28 cell lines, we found signaling, mutation, and expression data on human cancer that there was essentially no correlation between RalB mRNA samples to establish the clinical relevance of prior model and protein expression (Fig. 4B). system results. Overexpression of Ral pathway proteins in bladder tumor blots Our survey of the activation state of 10 different bladder Because of the intriguing difference between RalA and RalB cancer cell lines provides both interesting clues to the as regards correlation of mRNA and protein expression in activation state of Ral GTPases in human tumors as well as makes the case for more careful determination of the specificities of signaling of Ras paralogs, RalGEFs, and Ral paralogs. The highest levels of activation of RalA and RalB are 11 Henry F. Frierson, University of Virginia Department of Pathology, personal exhibited in J82, T24, and a metastatic variant of T24, T24T communication. (32, 33), all of which harbor G12V mutant HRAS, providing

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evidence in bladder cancer cells for association of activating line tested. Intriguingly, UM-UC-3, which is the only cell line mutations of HRAS with higher activation states of both Ral studied that is known to harbor a KRAS2 mutation (21), has GTPases. Conversely, RT4, which possesses wild-type HRAS a low level of RalA activation and an even lower level of (34), exhibited the lowest Ral activation levels of any cell RalB activation, consistent with prior reports showing a lack

Fig. 1. Activation of the Ral pathway. A, schematic representation of the Ral pathway. Activated transmembrane receptor tyrosine kinases signal to Ras, resulting in Ras-GTP ^ dependent recruitment of RalGEFs to the membrane, where they can potentiate exchange of GDP for GTP in Ral. Then, activated Ral may signal through various known effectors and mediate diverse cellular processes (1). B, Ral activation assays were done on nine bladder cancer cell lines. Lanes 1to 9, RT4, 5637, J82,T24,T24T, UM-UC-3, TCCSUP, HT1197, and HT1376, respectively. C, activation of Ral GTPases was quantitated (see Materials and Methods) and percentage GTP-bound RalA (white columns)andRalB(black columns) was plotted for each cell line. *, cell lines known to harbor G12VHRAS include J82, T24, and T24T; c,UM-UC-3,whichharbors G12CKRAS2.

www.aacrjournals.orgClin Cancer Res 2007;13(13) July 1,3807 2007 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology of RalA activation downstream from KRAS2 transformed addition of a membrane targeting ‘‘CAAX box’’ for lipid cells (35). modification have been used extensively in in vitro experi- Recent reports have focused on differential outputs of HRAS ments to activate Ral (25), their relevance to cancer in vivo has versus KRAS2 as regards canonical Ras-effector pathways (36), not been characterized. One recent report provides an but, surprisingly, given the importance of the Ral pathway to excellent paradigm for ascertaining in vivo function of RalGEFs transformation and tumorigenesis, the relative specificities of in tumorigenesis, by studying the effect of RalGDS knockout Ras GTPases to RalGEFs to RalA versus RalB has not been in an animal model of skin tumorigenesis (38). Such systematically determined, much less in cells derived from approaches will be necessary to determine the importance of different tissues. Given reports of Ras, Ras-effector class, and the GEFs to tumorigenesis in vivo, and mouse models of Ral paralog–specific signaling and phenotypes, these experi- bladder cancer, particularly driven by the activated HRAS ments will be essential to understanding consequences of oncogene (39), seem to be a relevant platform to test for pathway output, especially as pharmaceutical methods to dependency on GEF function. target specific Ras-effector pathways are developed (37). And We identified only one mutation in the coding sequence of although RalGEFs, or GEFs rendered ‘‘constitutively active’’ by RalA in the UM-UC-6 bladder cancer line, and no mutations of

Fig. 2. Mutation of RalA. A, a heterozygous G-to-C transversion mutation at nucleotide position 589 of the RalA Refseq resulting in a missense mutation of glutamate 97 to glutamine. B, location of the E97Q mutation in the structure of RalA (Pdb code: 1UAD). Stereo view of the GNP (GTP) binding site of RalA with respect to E97.The protein is colored yellow in ribbons with backbone atoms. Magenta sticks, E97; yellow sticks, K128.Blue, GNP; red spheres, water molecules. For clarity, not all secondary structural elements or residue side chains are shown. C, RalA activation compared in J82, UM-UC-3, and UM-UC-6, which harbors the RalA E97Q mutation. D, quantitation (see Materials and Methods) of the percent GTP-bound RalA in these three cell lines from (C). E, activation assays were carried out on HEK293Tcells transiently transfected with pcDNA3.1 expression constructs for wild-type, G23V constitutively active, or E97Q RalA. F, quantitation of the % GTP-bound RalA for the transfected cells in (E).

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Table 1. Ral pathway genes differentially expressed in cancer and invasive cancer

Probe set* Symbol Gene name Cancer vs normal T2-4 cancer vs normal Expression with stage Pc Fold Pc Fold 214435_x_at RALAb RalA 1.0e-10 1.9 2.0e-07 2.6 202845_s_at RALBP1 RalA binding protein 1 2.0e-062.5 0.0018 2.3 x 208079_s_at AURKA Serine/threonine kinase 6, Aurora-A 1.3e-05 1.8 7.2e-06 2.2 214752_x_at FLNA Filamin A, a 0.0037 -4.3 0.00060 -3.5

Probe set* Symbol Gene name Grade 3 vs grade 2 Grade 4 vs grade 3

Expression with grade Pc Fold Pc Fold

214435_x_at RALA RalA 0.00051 1.5 0.070 1.7 208079_s_at AURKA Serine/threonine kinase 6, Aurora-A 0.054 1.3 0.064 1.7

*Probe set on the Affymetrix HG-U133A oligonucleotide microarray. cP values for expression differences tested with a two-tailed Student’s t test. b RalA overexpression is additionally associated with invasive stage T2-4 cancer compared with superficial Ta,Tis, and T1 cancer (P = 9.2e-5, f1.7-fold). x Aurora overexpression is additionally associated with invasive stage T2-4 cancer compared with superficial Ta,Tis, and T1 cancer (P = 0.020, f1.33-fold).

RalB. The E97Q mutation occurs in a region of the structure of bally et al. (31) in their recent integrative genomic and this GTPase that is essential for nucleotide binding. Figure 2D proteomic analysis of prostate cancer progression. Seminoma, shows the location of E97 with respect to the GNP binding site glioblastoma, hepatocellular carcinoma, and pancreatic carci- in the recently determined RalA crystal structure (Pdbcode: noma all also overexpress RalA mRNA, suggesting that over- 1UAD), with GNP tightly sandwiched between the residue expression may be relevant in other tumor types as well. The K128, Loop 1, Turn 1, which provide extensive hydrogen bond overexpression of RalA in bladder cancer is especially interest- and hydrophobic interactions to the GNP. Disturbance of K128 ing because it coincides with overexpression of an activator of and any of these secondary structural elements could affect RalA, Aurora-A kinase (12), a phenomenon also observed in GNP binding, either positively or negatively depending on the prostate cancer (31). structural perturbation. In summary, this mutation seems Aurora-A kinase is overexpressed in many tumor types, and located in an area of RalA structure known to mediate essential pharmacologic inhibitors of Aurora kinase are available (40). functions, and the UM-UC-6 cell line exhibits a high level of One group recently reported that RalA (but not RalB) is a RalA activation compared with other cell lines without mutant substrate for phosphorylation by Aurora-A kinase on serine HRAS. 194. This phosphorylation potentiates RalA activation, Nevertheless, upon transient transfection of this mutant anchorage-independent growth, and collagen I–induced cell into normal cells, this E97Q RalA mutant behaved no migration (12). This association of overexpression of both differently than wild-type RalA. Although this result does kinase and GTPase target is intriguing, although, to our not exclude that the mutant may have some gain of function knowledge, the phosphorylation state of S194 in RalA has in the context of a bladder cancer cell, it suggests that the not been characterized in any tumor samples and will be the intrinsic ability of RalA to bind and hydrolyze GTP is not subject of future reports. Nevertheless, as recent reports have altered by this mutation. In any case, having only found one shown that RalA activation is essential for Ras-mediated RalA mutation in 20 bladder tumor cell lines begs the transformation (6, 7), tumorigenesis (8), and associated question of whether this mutation really occurs with any with tumor maintenance in vivo (41), the potential use of frequency in human cancer or whether it is but a cell culture– Aurora kinase inhibitors to modulate RalA activity merits induced epiphenomenon. Given the findings here that Ral investigation. overexpression correlates with tumor stage and aggressiveness, We also identified overexpression of RalBP1 (26) in bladder and that most human bladder cancer cell lines are derived cancer. A molecule of growing interest in cancer biology, from locally advanced or metastatic tumors, we believe there RalBP1 has been shown to be an ATP-dependent transporter is a low likelihood of Ral mutations in human bladder cancer localized to the plasma membrane by noncanonical mem- tissues. brane-anchoring domains (42). Recently, in lung cancer cells, Our finding of significant RalA mRNA overexpression in RalBP1 has been shown to mediate survival and drug bladder and other cancer types is intriguing and points to Ral resistance (43). Although a function for RalBP1 in drug efflux having a role in the transformed phenotype in human cancers. has not been shown in bladder cancer cells, the potential The results are even more compelling because we found implications of co-overexpression of this target with RalA and increased expression of RalA in muscle invasive versus RalB in bladder cancer are significant. RalBP1 was discovered superficial disease, suggesting a further role in cancer progres- in a yeast two-hybrid system as a binding partner specific for sion. Similarly, we found overexpression of RalA mRNA in active, GTP-bound Ral (44), and we are currently testing if Ral metastatic prostate cancer, a finding corroborated by Varam- has any role in potentiating RalBP1-mediated drug efflux. In

www.aacrjournals.orgClin Cancer Res 2007;13(13) July 1,3809 2007 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology the end, RalBP1 may prove to be an important therapeutic experiments will determine if such a mechanism exists in target in bladder cancer, as one recent report found that bladder cancer cells. RalBP1 was part of a defining gene expression signature of the Our finding of co-overexpression of RalA and RalB proteins disease (45). in invasive bladder tumors is most interesting, however, RalA has been shown to target Filamin A to induce taken in context of prior findings on Ral function. Also, the filopodia (27). Interestingly, we found that expression of lack of correlation between mRNA and protein in RalB while another Ral effector, Filamin A, is significantly down-regulated a striking correlation exits between those variables in RalA is in bladder cancer—to our knowledge, the first reported intriguing and suggests that these two paralogs may be association of loss of Filamin A expression with any tumor regulated in different ways. Although a body of work has type. Although the significance of coincident loss of Filamin A now implicated RalA in tumorigenesis of human cells, our expression and gain of RalBP1 expression in bladder tumor understanding of the role of RalB in human cancer remains cells remains unknown, reports have shown that Exo84 and much more limited. We and other groups (10, 11) have Sec5, two other Ral effectors, are competitive effectors of RalA discovered a role for RalB in cellular migration, one function. It is intriguing to speculate that loss of one effector component of invasion and metastasis. Although such a might potentiate signaling through another, and future model for RalA and RalB function—in tumorigenesis and

Fig. 3. Expression of Ral pathway mRNAs. A, plot of HG-U133A expression level of RalA as a function of normal tissue and tumor stage. RalA is overexpressed in bladder cancer compared with normal mucosa (P = 1.0e-10) and in muscle invasive (tumor stageT2-4)cancer compared with superficial cancer (Ta,Tis,T1; P = 9.2e-05). Columns, individual tumor samples at the stage indicated. B to E, expression of indicated Ral pathway mRNAs in normal bladder mucosa (n =15); superficial Ta,Tis,andT1bladder tumors (n = 41); and muscle invasive T2,T3,and T4 bladder tumors (n =24).Columns, average expression for each category of tissue from the HG-U133A oligonucleotide microarray platform; bars, SE of measured expression across each category. For fold changes and P values, see Table 1.

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Fig. 4. Protein expression of Ral pathway genes. A, scatter plot and linear regression analysis on 28 human bladder cancer cell lines of normalized RalA mRNA level (RalA HG-U133A expression level multiplied by 10 then divided by h-actin HG-U133A expression level) against normalized RalA protein expression (RalA protein expression level divided by h-actin protein expression level from quantitative analysis of blots as described in Materials and Methods). Our microarray analysis of the cell lines has been reported before (15). B, a similar analysis to (A) done on RalB, finding a lackof correlation between normalized mRNA and protein expression. C, five tissue samples of normal bladder mucosa or muscle invasiveT2+ bladder tumors (see Materials and Methods) were homogenized for protein extraction and equal protein amounts of each were Western blotted for RalA, RalB, RalBP1, and Filamin-A. Blots stained with Ponceau S confirmed equivalent protein transfer (not shown). IB, immunoblot. metastasis, respectively—is attractive, much work remains to harboring mutant Ras. In tumors, a severalfold overexpres- determine the relative contributions of each to the metastatic sion multiplied by severalfold increased activation state process and if differences in effector specificity (largely might yield a dramatic increase in total signaling output, unknown) explain them. and we propose to address this question in future reports Moreover, overexpression of RalA and RalB could represent using Ral activation assays on frozen tumor extracts. only part of a mechanism for increasing signaling through Alternatively, recent techniques using antibody phage display this pathway in cancer. As molecular lesioning of upstream- have yielded recombinant antibodies that detect specifi- activating pathways of Ral is common in bladder cancer cally the GTP-bound conformation of the related Rab6 (4, 20, 21), there exists the potential that these overexpressed small GTPase (46), and such technology could be applied GTPases are additionally hyperactivated with supraphysio- to Ral. logic levels of GTP binding in tumors compared with normal In summary, our findings make a strong case for the tissue. Indeed, we observed higher levels of Ral activation pathologic relevance of both Ral GTPases, their regulators, and in bladder cancer cell lines compared with normal HEK293T their effectors in human bladder cancer and other cancers and epithelial cells and particularly high levels in cell lines fill a current void in this area in the literature. Although our

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Table 2. Ral GTPase expression in other tumor types

Probe set* Type Pc Overexpression of RalA compared with corresponding normal tissues IMAGE:260768 Seminoma 3.1e-8 39253_s_at Glioblastoma 5.4e-5 IMAGE:230261 Hepatocellular carcinoma 7.8e-5 IMAGE:260768 Pancreatic adenocarcinoma 0.00016 IMAGE:230261 Prostate adenocarcinomab 0.00050 Underexpression of RalA compared with corresponding normal tissues IMAGE:260768 Lung squamous cell carcinoma 0.00012 IMAGE:260768 Lung adenocarcinoma 0.001 Underexpression of RalB compared with corresponding normal tissues IMAGE:322617 Hepatocellular carcinoma 3.1e-8 IMAGE:322617 Seminoma 5.4e-5 M35416_at Ovarian adenocarcinoma 7.8e-5 228_at Meningioma 0.00016 IMAGE:322617 Prostate cancer 0.00050

*Probe sets used to detect RalA or RalB on various study platforms available on Oncomine. cP value in ascending order, obtained from Oncomine for ‘‘cancer versus normal’’ comparison. bRalA is significantly further overexpressed in metastatic prostate cancer, using the ‘‘cancer versus cancer’’ comparison in Oncomine to compare the ‘‘metastatic prostate cancer’’ with ‘‘prostate carcinoma’’ classes (31, 48).

findings here focus on bladder cancer, the fifth most Acknowledgments commonly diagnosed cancer in the United States (47), overexpression of RalA seems to be a common phenomenon We thankDr. Henry Frierson, University of Virginia Department of Pathology, for in other tumor types as well. As such, we propose Ral GTPases his ongoing collaboration on immunohistochemistry and members of the Theodor- as targets for future therapeutic strategies. escu Laboratory for their suggestions and assistance.

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