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Ubiquitin E3 ligase WWP1 as an oncogenic factor in human prostate cancer

Abstract

The gene for E3 ubiquitin ligase WWP1 is located at 8q21, a region frequently amplified in human cancers, including prostate cancer. Recent studies have shown that WWP1 negatively regulates the TGFβ tumor suppressor pathway by inactivating its molecular components, including Smad2, Smad4 and TβR1. These findings suggest an oncogenic role of WWP1 in carcinogenesis, but direct supporting evidence has been lacking. In this study, we examined WWP1 for gene dosage, mRNA expression, mutation and functions in a number of human prostate cancer samples. We found that the WWP1 gene had copy number gain in 15 of 34 (44%) xenografts and cell lines from prostate cancer and 15 of 49 (31%) clinical prostate cancer samples. Consistently, WWP1 was overexpressed in 60% of xenografts and cell lines from prostate cancer. Mutation of WWP1 occurred infrequently in prostate cancer. Functionally, WWP1 overexpression promoted colony formation in the 22Rv1 prostate cancer cell line. In PC-3 prostate cancer cells, WWP1 knockdown significantly suppressed cell proliferation and enhanced TGFβ-mediated growth inhibition. These findings suggest that WWP1 is an oncogene that undergoes genomic amplification at 8q21 in human prostate cancer, and WWP1 overexpression is a common mechanism involved in the inactivation of TGFβ function in human cancer.

Introduction

Prostate cancer is one of the most common solid tumors in men. The molecular basis of prostate cancer is not well understood, although it has been recognized that identifying molecular alterations underlying the development and progression of prostate cancer will be necessary for improving its detection and treatment. Copy number gain or loss is a common genetic alteration in solid tumors. Many chromosomal regions have been identified for such changes in cancer by comparative genomic hybridization (CGH) and other approaches, yet the underlying target genes remain to be identified and characterized for most cancers. The q21 band of chromosome 8 (8q21) shows frequent copy number gain in prostate cancer, and the gain at 8q21 is often associated with aggressive behaviors of prostate cancer. For example, in men with clinically localized prostate cancer, gain of 8q21 is associated with poor outcome and metastasis (Nupponen et al., 1998; van Dekken et al., 2003; Rubin et al., 2004). Therefore, 8q21 is believed to harbor an important oncogene for prostate cancer.

Many important molecules involved in cell proliferation, differentiation and carcinogenesis are tightly controlled by different mechanisms, including ubiquitin proteasome pathway (UPP)-mediated protein degradation. Many E3 ubiquitin ligases in the UPP have been implicated in cell cycle control and uncontrolled cell proliferation. Some E3 ligases such as MDM2 and 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 including regulation of epithelial sodium channels, viral budding, receptor trafficking and transcription (Malbert-Colas et al., 2003; Ingham et al., 2004; Zhang et al., 2004). With regard to a role in cell proliferation and carcinogenesis, it has been demonstrated that WWP1 negatively regulates the TGFβ tumor suppressor pathway by mediating the ubiquitination and degradation of multiple components of the pathway, including Smad2 (Seo et al., 2004), Smad4 (Moren et al., 2005), and TGFβ receptor 1 (TβR1) proteins (Komuro et al., 2004). WWP1 appears to be regulated by androgen, and is highly expressed in androgen-independent prostate cancer cells in the LAPC-9 model (Gu et al., 2005). In our recent study, we found that WWP1 also mediates the ubiquitination and degradation of the KLF5 transcription factor, which was identified as a candidate tumor suppressor gene in prostate and breast cancers (Chen et al., 2002, 2003a, 2005a). Consistent with a previous study showing upregulation of WWP1 in multiple carcinoma cell lines (Komuro et al., 2004), we found that WWP1 is overexpressed in some prostate and breast cancer cell lines. Furthermore, WWP1 overexpression leads to excessive degradation of KLF5 in these cancer samples (Chen et al., 2005a, 2005b). These findings suggest that WWP1 could have an oncogenic role in prostate cancer.

In this study, we performed genetic and functional analyses to evaluate the role of WWP1 in human prostate cancer. Both copy number gain and overexpression were frequently detected in human prostate cancer samples, although mutations were rare. Functionally, WWP1 overexpression appeared to promote cell proliferation by antagonizing TGFβ's inhibitory effect on cell proliferation.

Results

Frequent copy number gain

The WWP1 gene is located in the q21 band of chromosome 8 (8q21), which is frequently amplified in human prostate cancer, including the PC-3 prostate cancer cell line (Porkka et al., 2002; Wang et al., 2004). To evaluate if WWP1 undergoes genomic amplification in prostate cancer, we first examined WWP1's DNA copy number in 34 prostate cancer cell lines/xenografts by using real-time polymerase chain reaction (PCR) assay, with normal human DNA as a normal control and PC-3 prostate cancer cell line as a control with doubled WWP1 genome. Fifteen of the 34 cases (44%) showed an increased copy number by at least one fold (Figure 1a). Among the samples examined, LuCaP 23.1, LuCaP 23.12 and LuCaP 23.8 were from different metastases of one patient. LuCap35 and LuCaP35V were also from one patient. Cell line 22Rv1 was derived from the CWR22 xenograft. As all these samples were in the non-amplified group, the actual amplification rate for WWP1 could be higher than described in this study. Conversely, none of the four un-transformed cell lines (PZ-HPV-7, PWR-1E, RWPE-1 and BPH55T) showed a copy number change at WWP1. In agreement with previous findings, the PC-3 prostate cancer cell line had a doubled copy number for WWP1 when compared to normal samples. Increased copy number at WWP1 was also validated in six of the 34 samples by duplex PCR combined with gel electrophoresis (Figure 1a, inset), with normal human DNA and BPH55T untransformed cell line as negative controls.

Figure 1
figure1

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.

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-transformed 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 cancer cell line (Figure 2a).

Figure 2
figure2

Increased expression of WWP1 RNA in prostate cancer. (a) Expression of WWP1 mRNA in prostate cell lines and xenografts detected by real time PCR. GAPDH was used as a control to normalize the cDNA input. Black bars identify the samples with WWP1 overexpression, while empty bars indicate non-transformed prostate samples and gray bars indicate tumor samples without WWP1 overexpression. (b) Expression of WWP1 RNA in prostate cancer cell lines detected by Northern blot analysis. Lanes 1–6 are non-transformed prostate samples, and the rest are prostate cancer cell lines. β-Actin serves as a RNA loading control. (c) Protein expression of WWP1, TβR1 and Smad4 in cell lines from the prostate, as detected by western blot analysis. β-Actin serves as a loading control.

Northern blot analysis was performed to confirm WWP1 overexpression in prostate cancer. Whereas a normal prostate tissue and five non-transformed prostate epithelial cell lines expressed little WWP1 RNA, four of six prostate cancer cell lines, including DU145, PC-3, BRF-41T and LNCaP/C4-2, showed an increased expression of WWP1 (Figure 2b). Except for BRF-41T, expression levels of WWP1 in these samples were consistent between real-time PCR assay and northern blot analysis. BRF-41T showed strong signals for both WWP1 and β-actin in northern blot analysis, but its WWP1 expression did not show a significant increase in real-time PCR analysis in which GAPDH was used as the normalizing control. In addition, a smaller isoform of WWP1, shown as a band below the wild-type WWP1 mRNA at 4.2 Kb, was clearly detected in some samples. However, the isoform was not associated with cancer, because it was present in both cancer samples and non-tumor controls (Figure 2b).

We also measured WWP1 protein levels, along with TβRI and Smad4, in one untransformed and five prostate cancer cell lines, using Western blot analysis. Consistent with mRNA expression results (Figure 2b), WWP1 protein level is higher in prostate cancer cell lines PC-3, LNCaP and DU 145, but lower in the untransformed PZ-HPV7 cell line and 22Rv1 prostate cancer cell line (Figure 2c). An extra band below normal WWP1 band was detected in PC-3 and LAPC-4 prostate cancer cells but not in the rest of the cell lines. Protein expression of TβR1 and Smad4 was also analysed in these cell lines, because they have been demonstrated to be negatively regulated by WWP1 (Komuro et al., 2004; Moren et al., 2005). In all but two (PZ-HPV-7 and LNCaP) of the six cell lines analysed, an inverse correlation was noted between WWP1 expression and the level of both TβRI and Smad4 (Figure 2c).

In combination with the results of copy number gain at WWP1, 11 of 15 cancer cell lines with an increased WWP1 copy number showed WWP1 overexpression, whereas only four of 18 samples without WWP1 copy number gain showed WWP1 overexpression (Figures 1 and 2) (Fisher exact test, P=0.004).

Mutation of WWP1 is rare in prostate cancer

To determine if WWP1 is mutated in prostate cancer, the coding region of WWP1 was amplified by PCR with two pairs of primers, and the overlapping PCR products were sequenced in 30 cell lines and xenografts from prostate cancer. Two sequence alterations were detected in xenografts. One was 2393A → T (Glu798Val) in CWR91 and the other was 721A → T (Thr241Ser) in LuCaP35. Both changes were heterozygous and had not been reported in the SNP database at NCBI.

WWP1 promotes cell proliferation

To test whether WWP1 affects cell proliferation, we transfected both wild-type WWP1 and a dominant-negative mutant WWP1 (WWP1C886S) (Chen et al., 2005a) into 22Rv1 prostate cancer cells, which express a lower level of WWP1 (Figure 2). Ectopic expression of wild-type and mutant WWP1 in 22Rv1 cells was verified by Western blot analysis (Figure 3a). As expected, wild-type WWP1 increased but mutant WWP1 suppressed colony formation in 22Rv1 cells when compared to empty vector control (Figure 3a). Measurement of cell numbers by SRB staining in different groups showed significant differences between both vector control and wild-type WWP1 (P=0.02) and between vector control and mutant WWP1 (P=0.02) (Figure 3a). These findings suggest that WWP1 promotes cell proliferation in 22Rv1 prostate cancer cells.

Figure 3
figure3

WWP1 promotes cell proliferation in different prostate cancer cell lines. (a) Compared to vector control (V), ectopic expressions of wild-type and mutant WWP1 respectively promoted or suppressed cell growth in 22Rv1 prostate cancer cells. Expression of WWP1 was verified by western blot analysis (lower panel). The P-values for the difference between vector control and wild-type WWP1 and that between vector control and mutant WWP1 were both 0.02. (b) Knockdown of WWP1 by RNAi (WWP1siRNA) slowed cell proliferation in PC-3 prostate cancer cells, as determined by growth curve analysis. The siRNA for luciferase (LucsiRNA) was used as a control. The P-value for the difference at day 6 is 0.009 (Student's t-test). (c) Verification of RNAi-reduced WWP1 expression by real-time PCR assay. SiRNA treatments were in duplicate.

In our previous study, a siRNA for WWP1 was shown to efficiently knockdown WWP1 expression in the PC-3 prostate cancer cell line (Chen et al., 2005a). To further evaluate the effect of WWP1 on cell growth, we treated PC-3 cells with siRNA for either WWP1 or the luciferase gene (Figure 3b and c). The latter served as a negative control. Cell growth was analysed also by the SRB staining method. Real-time PCR assay showed that the siRNA silenced WWP1 expression by up to 80% (Figure 3c). As shown in Figure 3b, cells with reduced WWP1 expression grew significantly slower at day 6 when compared to the control group (P=0.009). These results also suggest that WWP1 plays a growth-promoting role in prostate cancer cells.

WWP1 antagonizes TGFβ in suppressing cell proliferation in PC-3 cells

Previous studies showed that WWP1 mediates the degradation of three components of the TGFβ pathway, including Smad2, Smad4 and TβR1 (Komuro et al., 2004; Seo et al., 2004; Moren et al., 2005). It is thus possible that WWP1 inactivates TGFβ in epithelial cells. To test this hypothesis, we analysed DNA synthesis rates in PC-3 cells treated with different concentrations of TGFβ in combination with siRNA for WWP1 or luciferase control. Consistent with colony formation results, knockdown of WWP1 by siRNA transfection significantly inhibited the DNA synthesis rate, which indicates cell proliferation, in the presence or absence of TGFβ (Figure 4a). Furthermore, whereas PC-3 cells treated with control siRNA did not show an obvious response to TGFβ‘s inhibitory effect on cell proliferation, siRNA-mediated WWP1 knockdown significantly enhanced such an effect at both TGFβ concentrations tested (P<0.05, Student's t-test). These findings suggest that knockdown of WWP1 sensitizes cells to TGFβ's effect.

Figure 4
figure4

WWP1 antagonizes TGFβ function in PC-3 cells. (a) RNAi-mediated WWP1 knockdown (WWP1si) sensitized PC-3 to TGFβ's inhibitory effect on cell proliferation when compared to the negative control (Lucsi), as determined by DNA synthesis assay. (b) Knockdown of WWP1 increased protein levels of Smad2, Smad4, TβR1 and p15 in PC-3 cells. All TGFβ treatments were for 24 h. β-Actin served as a loading control.

To explore the mechanisms responsible for WWP1's effect on TGFβ activity, we examined protein expression of several components of the TGFβ signaling pathway, including Smad2, Smad3, Smad4 and TβR1, by Western blot analysis (Figure 4b). A downstream target molecule of TGFβ, the CDK inhibitor p15, was also examined for expression. Without exogenous TGFβ, knockdown of WWP1 by siRNA transfection increased expression levels of Smad2, Smad4 and TβR1 in PC-3 cells, which is consistent with published studies implicating WWP1 in the degradation of these molecules. As expected, knockdown of WWP1 also increased the expression of p15 but not the expression of Smad3 in the same cells. On the other hand, addition of exogenous TGFβ to the culture medium increased expression of Smad2, Smad4 and TβRI, but the effect of WWP1 knockdown on these molecules' expression was not detectable when exogenous TGFβ was present (Figure 4b). In the induction of p15 expression, however, the effects of TGFβ and WWP1 knockdown were additive, which is consistent with an additive inhibitory effect on cell proliferation (Figures 3b and 4a).

Discussion

In this study, we present multiple lines of evidence for an oncogenic role of the WWP1 E3 ubiquitin ligase in human prostate epithelial cells. Genetically, WWP1 is located at 8q21, which contains a frequently amplified locus in human prostate cancer, and showed frequent copy number gain in cell lines, xenografts and clinical samples from prostate cancer. WWP1 is also frequently overexpressed in prostate cancer samples, and its overexpression is significantly associated with its copy number gain. Functionally, ectopic expression of WWP1 enhanced and knockdown of WWP1 suppressed cell proliferation. Furthermore, WWP1 antagonizes TGFβ's inhibitory effect on cell proliferation, likely by mediating the degradation of key components of TGFβ pathway including Smad2, Smad4 and TβRI as indicated in previous studies (Komuro et al., 2004; Seo et al., 2004; Moren et al., 2005). Therefore, WWP1 is a potential oncogene in prostate cancer. Because copy number gain at 8q21 has been detected in prostatic intraepithelial neoplasia (PIN) of both low and high grades (Zitzelsberger et al., 1998), which is considered a precursor of prostate cancer, it is likely that WWP1 overexpression could also play a role in early stages of prostate cancer development.

The mutation of WWP1 also occurs in prostate cancer, although the frequency could be low. In 30 cancer samples examined, two showed sequence alterations. Although it is not clear if the change of Thr241Ser has any functional consequence, the change of Glu798Val in the CWR91 xenograft should be a function-altering mutation, because the Glu798 codon is located in the HECT domain of WWP1, it is conserved in all HECT domains among different molecules, it participates in intermolecular interaction between N-lobe and C-lobe to maintain protein conformation, and mutation of this codon from Glu to Ala significantly decreases WWP1's ubiquitin transfer activity (Verdecia et al., 2003).

WWP1 has multiple isoforms resulting from alternative splicing (Flasza et al., 2002). Different isoforms have different domain structures and thus may function differently. Although a previous study of the breast cancer cell line T-47D suggests the presence of tumor-specific splicing of WWP1 (Flasza et al., 2002), our northern blot analysis showed that a smaller isoform of WWP1 is present in both normal and cancer cells, and this isoform is not related to cancer (Figure 2b). In addition, wild-type WWP1 is the dominant form that is overexpressed in some cancer samples.

In addition to WWP1, at least two other genes from 8q21 have been shown to undergo copy number gain and overexpression in prostate cancer, including PrLZ/TPD52 (Rubin et al., 2004; Wang et al., 2004) and TCEB1 (Elongin C or transcription elongation factor B) (Porkka et al., 2002). Although neither PrLZ/TPD52 nor TCEB1 has been shown to affect cell proliferation as WWP1, it is possible that each of these molecules plays a role in prostatic carcinogenesis. A more definitive approach such as transgenic overexpression in mice is needed to better define the role of these molecules in prostate cancer. Currently we are testing if prostate-specific overexpression of WWP1 in the prostate can cause prostate cancer in mice. It is also necessary to analyse protein expression of WWP1 in prostate cancer to determine whether WWP1 is also overexpressed in clinical samples and whether WWP1 overexpression is associated with clinicopathologic characteristics of prostate cancer. In addition, the MYC oncogene is located in 8q24, another chromosomal region that undergoes frequent copy number gain in prostate cancer. The role of MYC in prostate cancer has been well documented (Dong, 2006). It is possible that both WWP1 and MYC are overexpressed in some prostate cancers, and thus have a cooperative role in prostatic carcinogenesis.

It is well known that TGFβ is a potent tumor suppressor in the development of cancer, but it becomes a promoter of metastasis during cancer progression (Massague et al., 2000; Roberts and Wakefield, 2003). The TGFβ signaling pathway involves a series of molecules. Among them are the TGFβ receptor 1 (TβR1), Smad2 and Smad4, which are negatively regulated by WWP1 at the protein level (Komuro et al., 2004; Seo et al., 2004; Moren et al., 2005). Consistently, we found that protein levels of TβR1, Smad2 and Smad4 were increased when WWP1 expression was knocked down by siRNA in PC-3 cells. Furthermore, one of the well-established TGFβ targets involved in cell cycle control, the CDK inhibitor p15, was significantly induced at the protein level upon the knockdown of WWP1. When exogenous TGFβ was added, the expression of Smad2, Smad4 and TβRI was increased with or without WWP1 knockdown, but WWP1 knockdown did not further increase their expression. For the expression of p15, however, the effects of TGFβ and WWP1 knockdown are additive. These results further suggest that, in addition to increased PI3K/AKT signaling, androgen receptor overexpression, and reduced TGFβ receptors I and II which all have been shown to inhibit TGFβ signaling in prostate cancer, overexpression of WWP1 is another mechanism that impairs TGFβ's inhibitory effect on cell proliferation.

Escape from TGFβ inhibition is considered an early event in prostate cancer. Several mechanisms have been found to overcome the growth inhibitory effect of TGFβ, including increased PI3K/AKT/mTOR signaling (van der Poel, 2004), overexpression of androgen receptor (Danielpour, 2005), and loss of TGFβ receptor (Kim et al., 1996). Whereas WWP1 overexpression could lead to downregulation of TβRI, it remains to be determined whether WWP1 overexpression also affects PI3K/AKT signaling and AR activity.

E3 ubiquitin ligases are a group of scaffold proteins that recognize and conjugate ubiquitins to specific substrates. There are hundreds of E3 ligases actively functioning in mammalian cells (Pickart, 2001). Several E3 ligases, including Mdm2, Skp2 and β-TrCP, have been demonstrated to play an oncogenic role in human carcinogenesis; and frequent gene amplification and overexpression of these E3 ligases have been detected in different types of human cancers. Our findings in this study present WWP1 as another oncogenic E3 ligase. These E3 ligases can be therapeutic targets in the treatment of cancer. For example, targeting Mdm2 can restore the apoptotic response of cells to androgen deprivation therapy in prostate cancer (Zhang et al., 2003; Mu et al., 2004). It is possible that inactivating WWP1 can sensitize cells to TGFβ's inhibitory effect on cell proliferation, and thus targeting WWP1 could be a useful therapeutic approach in treating cancers with WWP1 overexpression. We are currently testing this possibility.

In our previous studies, we identified the KLF5 transcription factor as a candidate tumor suppressor gene that is inactivated by genomic deletion and transcriptional downregulation in human prostate and breast cancers (Chen et al., 2002, 2003a). We have recently established WWP1 as an E3 ligase for posttranslational regulation of KLF5 (Chen et al., 2005a). In cancer cell lines, we have shown that overexpression of WWP1 leads to excessive degradation of KLF5 (Chen et al., 2005a, 2005b). Taken together, the finding of copy number gain and overexpression of WWP1 in human prostate cancer in this study provides another mechanism for inactivation of KLF5 in human cancer, that is, excessive protein degradation caused by WWP1 overexpression. Interestingly, our ongoing studies suggest that KLF5 also mediates TGFβ's function in the control of cell proliferation (unpublished data), which provides another line of evidence for WWP1's antagonizing role in regulating the TGFβ signaling pathway.

In summary, we found that the E3 ubiquitin ligase WWP1 undergoes frequent copy number gain and overexpression in human prostate cancer. WWP1 is also mutated in some prostate cancers. Functionally, WWP1 promotes cell proliferation, likely by antagonizing TGFβ's inhibitory function. These results suggest that WWP1 could be a potential biomarker and therapeutic target in the detection and treatment of prostate cancer.

Materials and methods

Tumor specimens, cell lines and xenografts

In total, we analysed 83 prostate cancer samples, including 49 clinical tumor specimens and 34 cell lines and xenografts derived from prostate cancer. Among the 49 tumor specimens with matched non-neoplastic cells, 40 were primary tumors, six were hormone refractory local cancers and three were metastases. Patient age at diagnosis ranged from 50 to 79 years (average 63.3). The Gleason scores for the tumors ranged from five to nine, with 23 having a Gleason score of five or six, 22 having seven, and four having eight or nine. All tumor and matched normal cells were collected from H&E stained sections of formalin-fixed, paraffin-embedded tissues by manual microdissection, as described in our previous study (Dong et al., 2000). All prostatic epithelial cell lines and xenografts were described in our previous studies (Chen et al., 2003a, 2003b; Sun et al., 2005).

Detection of copy number gain for WWP1

Two approaches were used to detect gene copy number changes. One was real-time PCR with the SYBR green dye using the ABI 7000 Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA). Primer sequences for human WWP1 were 5′-IndexTermCAGGAGGTTGACTTGGCAGA-3′ and 5′-IndexTermAAACTGTGTCCAAAAGCAGTCTC-3′; and those for control gene KAI1 were 5′-IndexTermCAGGTG GGCACGGGTTT-3′ and 5′-IndexTermTCCTCGCGACTGCTGTTGTA-3′. Briefly, 10 ng of genomic DNA were amplified in a volume of 25 μl with primers for either WWP1 or KAI1 following the manufacturer's protocols. The KAI1 gene serves as a non-deleting normalizing control. All PCRs were performed in duplicate with 40 cycles using standard program with an annealing and extension temperature of 60°C. Relative DNA copy number was determined by the ΔΔCt method, and WWP1 copy number gains were indicated by the ratio of the WWP1 reading to the KAI1 reading, with normal samples defined as 1. Copy number gain was defined in a sample when the ratio of WWP1 to KAI1 was equal or greater than two, based on the reading for the PC-3 cell line, which has doubled the WWP1 genome.

For all clinical specimens and some cell lines/xenografts, we also performed duplex PCR to detect copy number gain for WWP1 following our established procedures (Dong et al., 2000; Chen et al., 2003a). Genomic DNA is severely degraded in formalin-fixed clinical tissue specimens, so a pair of primers amplifying a small fragment of WWP1 (103 basepairs) were used. Primer sequences for WWP1 were 5′-IndexTermGTATGGATCCTGTACGGCAGCA-3′ and 5′-IndexTermAACAGAGTTGCTACTTAAATGCAAGTCAG-3′). Again, KAI1 was used as a normalizing control, and the primer sequences were 5′-IndexTermGGTGGGATGGTGCAGAGCGG-3′, and 5′-IndexTermCAGAGACAACCCAGGAGGAC-3′. Gel images were scanned, and band intensities were measured by using the IMAGE J computer program from NIH (http://rsb.info.nih.gov/ij). The ratio of signal intensity for WWP1 to that for the KAI1 control in a tumor sample was compared to the ratio in normal human DNA to define a DNA copy number change.

WWP1 expression analyses

Total RNA was extracted using the Trizol reagent following its standard protocol (Invitrogen). The cDNA was prepared by using the Script kit according to the manufacturer's protocol (Bio-Rad). Real-time RT–PCR with SYBR green dye was performed as described above with different primers. Primer sequences for WWP1 were 5′-IndexTermGTATGGATCCTGTACGGCAGCA-3′ (forward) and 5′-IndexTermGTTGTGGTCTCTCCCATGTGGT-3′ (reverse); and those for GAPDH were 5′-IndexTermGTGGTCCAGGGGTCTT ACTC-3′ and 5′-IndexTermAACGGGAAGCTCACTGGC-3′. All real-time PCRs were performed in duplicate. The average ratio of WWP1 to GAPDH control in PZ-HPV7 was defined as 1, and the ratios for other samples were normalized accordingly. WWP1 overexpression was defined in a sample when the ratio of WWP1 to GAPDH was 2.

For the northern blot analysis, a probe was prepared from the WWP1 coding region by PCR with primers 5′-IndexTermAAGAATGGCATAGCACAAAC-3′ and 5′-IndexTermTTCTGCACTGGTAGAAGGAA-3′. The sequence for this probe was unique to WWP1 among different gene family members. The probe was labeled with α-32P-dCTP using a random primer DNA labeling system (Invitrogen). For each sample, 15 μg of total RNA were loaded into a denaturing gel, which was electrophoresed. Nylon membrane with transferred RNA was hybridized with labeled probe at 65°C for 3 h in the QuikHyb hybridization solution (Stratagene, La Jolla, CA, USA), washed following the manufacturer's instructions, and exposed to X-ray film. WWP1 probe was stripped from the blot and the membrane was re-hybridized with the β-actin probe.

Mutation detection

In 30 cell lines and xenografts from prostate cancer, the WWP1 coding region was amplified by RT–PCR with two pairs of primers. Primers sequences were 5′-IndexTermGAAAGAGGGAATCGTGTCTTAC-3′ and 5′-IndexTermACGATCATCAACTCTTCTTTCCC-3′ for one pair, and 5′-IndexTermGTATGGATCCTGTACGGCAGCA-3′ and 5′-IndexTermTCACTGTACAGAATGAACAGCTT-3′ for the other pair. After purification from gels using Qiagen's Gel Purification Kit, the PCR products were sequenced with each of the PCR primers and an additional primer 5′-IndexTermACCTCCTGCATGCCACACAAC-3′ by Macrogene (Korea).

Colony formation assay

The 22Rv1 prostate cancer cell line was maintained in RPMI-1640 medium supplemented with 5% fetal bovine serum, HEPES (0.1 M), sodium pyruvate (1 mM), sodium bicarbonate (0.15%), glucose (0.45%) and penicillin and streptomycin (1%). Myc-tagged wild-type and mutant WWP1 constructs, that is, myc-WWP1 and myc-WWP1C886S, have been described in our previous study (Chen et al., 2005a). In six-well tissue culture plates, 2 × 105 cells were seeded into each well. After 1 day, WWP1 plasmids were transfected into cells using the Lipofectamine 2000 reagent following the manufacturer's manual (Invitrogen). Selection medium containing 1 mg/ml G418 was replaced 24 h later. At 2 weeks after growth in the selection medium, cells were fixed, stained with 0.4% SRB (Sulphorhodamine-B), and washed by 1% acetic acid. Cell densities were measured with a spectrometer at a 500 nm wavelength, following a published procedure (Sun et al., 1997).

Western blot

Western blot analysis was performed as described in our previous study (Chen et al., 2005b). Antibodies for Smad2 and Smad3 were purchased from Zymed Laboratories, and antibodies for Smad4, TβRI and p15 were purchased from Cell Signaling. Anti-WWP1 antibody was described in a previous study (Seo et al., 2004).

Knockdown of WWP1 and related cell assays

A siRNA with the sequence of 5′-IndexTermGAGTTGATGATCGTAGAAG-3′ was used to target WWP1. A siRNA for the luciferase gene (5′-IndexTermCTTACGCTGAGTACTTCGA-3′) was used as a negative control. PC-3 cells were transfected with 200 nM of chemically synthesized siRNA (Dharmacon, Chicago, IL, USA) using Oligofectamine (Invitrogen) in 12-well plates. RNA was collected 48 h after transfection.

For growth curve analysis, PC-3 cells were plated into 12-well plates at 4 × 104 cells per well. Transfection was conducted 24 h later. At 2, 4 and 6 days after transfection, cells were fixed with 10% TCA and stained with SRB as described above.

For DNA synthesis assay, PC-3 cells were seeded into 12-well plates at 4 × 104 cells per well, and labeled with (14C)-thymidine (0.02 μCi/ml) overnight. SiRNAs for WWP1 and control were transfected into cells for 24 h. Cells were then treated with TGFβ overnight, and labeled with 3H-thymidine (1 μCi/ml) for 4 h. Upon the completion of treatment, cells were washed with PBS and treated with 10% TCA to release genomic DNA. Radiolabeled DNA was solubilized by 120 μl of 0.3 N NaOH and transferred to glass fiber filter membranes, and radioactivity for 3H and 14C was measured using a scintillation counter. The DNA synthesis rate was defined by the ratio of incorporated 3H to 14C.

Statistics analysis

Fisher's exact test was used to analyse the correlation between copy number or expression change with age at diagnosis, tumor grade or tumor stage in all prostate cancer samples. Two-tail Student's t-test was used to determine statistical significance between experimental and control groups in cell proliferation assay, and a P-value smaller than 0.05 was considered statistically significant.

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Acknowledgements

C Chen is an AFUD/AUA Research Scholar. This work was supported in part by grants from the National Cancer Institute (Grant # CA87921), from the Department of Defense Prostate Cancer Research Program (Grant # DAMD17-03-2-0033), from the Georgia Cancer Coalition, and from the Susan G Komen Breast Cancer Foundation.

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Correspondence to J-T Dong.

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Chen, C., Sun, X., Guo, P. et al. Ubiquitin E3 ligase WWP1 as an oncogenic factor in human prostate cancer. Oncogene 26, 2386–2394 (2007). https://doi.org/10.1038/sj.onc.1210021

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Keywords

  • WWP1
  • amplification
  • overexpression
  • prostate cancer
  • KLF5
  • TGFβ

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