Oncogene (2007) 26, 2386–2394 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ONCOGENOMICS Ubiquitin E3 ligase WWP1 as an oncogenic factor in human prostate cancer C Chen1,2, X Sun1, P Guo1, X-Y Dong1, P Sethi1, W Zhou1, Z Zhou2, J Petros1,3,4, HF Frierson Jr5, RL Vessella6, A Atfi7 and J-T Dong1,3,8 1Winship Cancer Institute and Department of Hematology and Oncology, Emory University School of Medicine, Atlanta, GA, USA; 2Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY, USA; 3Department of Urology, Emory University School of Medicine, Atlanta, GA, USA; 4Atlanta VA Medical Center, Decatur, GA, USA; 5Department of Pathology, University of Virginia Health System, Charlottesville, VA, USA; 6Department of Urology, University of Washington, Seattle, WA, USA; 7INSERM U482, Hoˆpital St-Antoine, Paris, France and 8Department of Genetics and Cell Biology, Nankai University College of Life Sciences, Tianjin, China The gene for E3 ubiquitin ligase WWP1 is located at Introduction 8q21, a region frequently amplified in human cancers, including prostate cancer. Recent studies have shown that Prostate cancer is one of the most common solid tumors WWP1 negatively regulates the TGFb tumor suppressor in men. The molecular basis of prostate cancer is not pathway by inactivating its molecular components, well understood, although it has been recognized that including Smad2, Smad4 and TbR1. These findings identifying molecular alterations underlying the deve- suggest an oncogenic role of WWP1 in carcinogenesis, lopment and progression of prostate cancer will be but direct supporting evidence has been lacking. In this necessary for improving its detection and treatment. study, we examined WWP1 for gene dosage, mRNA Copy number gain or loss is a common genetic expression, mutation and functions in a number of human alteration in solid tumors. Many chromosomal regions prostate cancer samples. We found that the WWP1 gene have been identified for such changes in cancer by had copy number gain in 15 of 34 (44%) xenografts and comparative genomic hybridization (CGH) and other cell lines from prostate cancer and 15 of 49 (31%) clinical approaches, yet the underlying target genes remain to be prostate cancer samples. Consistently, WWP1 was over- identified and characterized for most cancers. The q21 expressed in 60% of xenografts and cell lines from band of chromosome 8 (8q21) shows frequent copy prostate cancer. Mutation of WWP1 occurred infre- number gain in prostate cancer, and the gain at 8q21 is quently in prostate cancer. Functionally, WWP1 over- often associated with aggressive behaviors of prostate expression promoted colony formation in the 22Rv1 cancer. For example, in men with clinically localized prostate cancer cell line. In PC-3 prostate cancer cells, prostate cancer, gain of 8q21 is associated with poor WWP1 knockdown significantly suppressed cell prolifera- outcome and metastasis (Nupponen et al., 1998; van tion and enhanced TGFb-mediated growth inhibition. Dekken et al., 2003; Rubin et al., 2004). Therefore, 8q21 These findings suggest that WWP1 is an oncogene that is believed to harbor an important oncogene for prostate undergoes genomic amplification at 8q21 in human cancer. prostate cancer, and WWP1 overexpression is a common Many important molecules involved in cell proli- mechanism involved in the inactivation of TGFb function feration, differentiation and carcinogenesis are tightly in human cancer. controlled by different mechanisms, including ubiquitin Oncogene (2007) 26, 2386–2394. doi:10.1038/sj.onc.1210021; proteasome pathway (UPP)-mediated protein degrada- published online 2 October 2006 tion. Many E3 ubiquitin ligases in the UPP have been implicated in cell cycle control and uncontrolled cell ONCOGENOMICS Keywords: WWP1; amplification; overexpression; prostate proliferation. Some E3 ligases such as MDM2 and cancer; KLF5; TGFb SKP2, which target p53 and CDKN1A/CDKN1B, are considered oncoproteins (Leite et al., 2001; Lu et al., 2002; Drobnjak et al., 2003), while some other E3 ligases are considered tumor suppressors. WWP1 is an E3 ubiquitin ligase that contains four tandem WW domains and a HECT domain. Published studies suggest that WWP1 plays a role in different biological processes Correspondence: Dr JT Dong, Winship Cancer Institute, Emory including regulation of epithelial sodium channels, University School of Medicine, 1365 Clifton Road, Room C4080, viral budding, receptor trafficking and transcription Atlanta, GA 30322, USA. E-mail: [email protected] (Malbert-Colas et al., 2003; Ingham et al., 2004; Received 27 April 2006; revised 26 June 2006; accepted 30 June 2006; Zhang et al., 2004). With regard to a role in published online 2 October 2006 cell proliferation and carcinogenesis, it has been WWP1 in prostate cancer C Chen et al 2387 demonstrated that WWP1 negatively regulates the Results TGFb tumor suppressor pathway by mediating the ubiquitination and degradation of multiple components Frequent copy number gain of the pathway, including Smad2 (Seo et al., 2004), The WWP1 gene is located in the q21 band of Smad4 (Moren et al., 2005), and TGFb receptor 1 chromosome 8 (8q21), which is frequently amplified in (TbR1) proteins (Komuro et al., 2004). WWP1 appears human prostate cancer, including the PC-3 prostate to be regulated by androgen, and is highly expressed cancer cell line (Porkka et al., 2002; Wang et al., 2004). in androgen-independent prostate cancer cells in the To evaluate if WWP1 undergoes genomic amplification LAPC-9 model (Gu et al., 2005). In our recent in prostate cancer, we first examined WWP1’s DNA study, we found that WWP1 also mediates the ubi- copy number in 34 prostate cancer cell lines/xenografts quitination and degradation of the KLF5 transcription by using real-time polymerase chain reaction (PCR) factor, which was identified as a candidate tumor assay, with normal human DNA as a normal control suppressor gene in prostate and breast cancers (Chen and PC-3 prostate cancer cell line as a control with et al., 2002, 2003a, 2005a). Consistent with a previous doubled WWP1 genome. Fifteen of the 34 cases (44%) study showing upregulation of WWP1 in multiple showed an increased copy number by at least one fold carcinoma cell lines (Komuro et al., 2004), we found (Figure 1a). Among the samples examined, LuCaP 23.1, that WWP1 is overexpressed in some prostate and LuCaP 23.12 and LuCaP 23.8 were from different breast cancer cell lines. Furthermore, WWP1 over- metastases of one patient. LuCap35 and LuCaP35V expression leads to excessive degradation of KLF5 in were also from one patient. Cell line 22Rv1 was derived these cancer samples (Chen et al., 2005a, b). These from the CWR22 xenograft. As all these samples were in findings suggest that WWP1 could have an oncogenic the non-amplified group, the actual amplification rate role in prostate cancer. for WWP1 could be higher than described in this study. In this study, we performed genetic and functional Conversely, none of the four un-transformed cell lines analyses to evaluate the role of WWP1 in human (PZ-HPV-7, PWR-1E, RWPE-1 and BPH55T) showed prostate cancer. Both copy number gain and over- a copy number change at WWP1. In agreement with expression were frequently detected in human prostate previous findings, the PC-3 prostate cancer cell line had cancer samples, although mutations were rare. Func- a doubled copy number for WWP1 when compared to tionally, WWP1 overexpression appeared to promote normal samples. Increased copy number at WWP1 was cell proliferation by antagonizing TGFb’s inhibitory also validated in six of the 34 samples by duplex PCR effect on cell proliferation. combined with gel electrophoresis (Figure 1a, inset), Figure 1 Frequent copy number gain at WWP1 in prostate cancer. (a) WWP1 copy numbers in cell lines and xenografts from prostate cancer, as detected by real-time PCR. The KAI1 gene serves as a control for normalizing input DNA. Black bars indicate samples with WWP1 copy number gain. The empty bars indicate non-transformed prostate samples, while gray bars indicate tumor samples without obvious copy number gains. The inset shows WWP1 copy number gain in six prostate cancer samples detected by duplex PCR and gel electrophoresis. (b) Examples of WWP1 copy number gain in clinical prostate cancer specimens detected by duplex PCR assay. Case numbers are shown at the top. N, normal; T, tumor. Oncogene WWP1 in prostate cancer C Chen et al 2388 with normal human DNA and BPH55T untransformed cell line as negative controls. To determine if WWP1 also has copy number gains in clinical prostate cancer samples, we examined 49 cases of prostate cancer and their matched normal cells by performing duplex PCR and gel electrophoresis, which was developed and used to detect gene dosage in our previous studies (Dong et al., 2000; Chen et al., 2003a). As shown in Figure 1b, the ratios between WWP1 signals and control KAI1 signals were significantly greater (>2) than that in the normal DNA in 15 of 49 (31%) tumors examined. It is worth noting that eight of 49 (16%) tumors appeared to have copy number losses at WWP1. No significant association was found between copy number change at WWP1 and age at diagnosis, tumor grade, or tumor stage, which could be due to the smaller sample size. WWP1 expression is frequently upregulated in prostate cancer Copy number gain often causes overexpression for a gene. To test whether WWP1 is overexpressed in prostate cancer, we first examined WWP1 mRNA expression by real time PCR in 30 cell lines and xenografts from prostate cancer. Compared to the average level of WWP1 expression in six non-trans- formed prostate samples, the level of WWP1 expression was increased by at least onefold in 18 of 30 (60%) prostate cancer samples, including the PC-3 prostate Figure 2 Increased expression of WWP1 RNA in prostate cancer.