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Keywords:

  • WWP1;
  • breast cancer;
  • gene amplification;
  • overexpression;
  • apoptosis

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The amplification of the q21 band of chromosome 8 (8q21) occurs in a large percentage of breast cancers. WWP1, an HECT domain-containing ubiquitin E3 ligase located in the 8q21 region, negatively regulates the TGF-β tumor suppressor pathway. To characterize the role of WWP1 in breast cancer, we analyzed WWP1 gene dosage and expression level as well as WWP1's function. A copy number gain of WWP1 was found in 51% (18/35) of breast cancer cell lines and in 41% (17/41) of primary breast tumors. Expression of WWP1 mRNA was analyzed with real-time RT-PCR, Northern blot, and Western blot. WWP1 mRNA is up-regulated in 58% (19/33) of breast cancer cell lines, and overexpression of WWP1 is significantly correlated with a gene copy number gain. In a panel of cDNA from primary breast tumors and normal tissues, expression of WWP1 in tumors is significantly higher than that in normal tissues. Functionally, RNAi-mediated WWP1 knockdown significantly induced cell growth arrest and apoptosis in the MCF7 and HCC1500 breast cancer cell lines. Consistently, WWP1 inhibition activated caspases. Forced overexpression of WWP1 by the lentiviral system in 2 immortalized breast epithelial cell lines MCF10A and 184B5 promoted cell proliferation. These results suggest that genomic aberrations of WWP1 may contribute to the pathogenesis of breast cancer. © 2007 Wiley-Liss, Inc.

A copy number gain or loss is a frequent genetic alteration in solid tumors including breast cancer. A copy number gain of 8q21 was detected in a high percentage of familial and sporadic breast tumors.1–3 Although several 8q21 genes, such as TPD52 and E2F5, have been reported to be amplified and overexpressed in breast cancer,4, 5 their roles have not been firmly established in breast tumorigenesis, and the underlying target genes of 8q21 amplification in breast cancer remain to be elucidated.

Ubiquitin proteasome pathway (UPP)-mediated protein degradation plays an important role in breast cancer cell proliferation, apoptosis and carcinogenesis.6 Several oncogenic ubiquitin E3 ligases have been identified as diagnosis markers or potential drug targets in human breast cancer.7 For example, Skp2, an F-box protein in the SCF ubiquitin ligase complex, targets the CDK inhibitor p27kip for degradation and is overexpressed in a subset of breast carcinomas.8 The ring finger E3 ligase EFP mediates estrogen-induced cell growth and facilitates 14-3-3σ tumor suppressor ubiquitination and proteolysis.9EFP mRNA and protein were reported to be overexpressed and significantly correlated with poor prognosis of breast cancer patients.10

WW domain-containing protein 1 (WWP1) is an HECT (homologous to the E6-associated protein carboxyl terminus) domain-containing E3 ubiquitin ligase. Growing evidence suggests that WWP1 negatively regulates the TGF-β tumor suppressor pathway by mediating the ubiquitination and degradation of TGF-β receptor 1 (TβR1),11 Smad212 and Smad4.13 In addition, WWP1 has recently been demonstrated as an ubiquitin E3 ligase for several transcription factors, Runx2,14, 15 KLF216 and KLF5.17 In our previous study, we found that WWP1 is amplified and overexpressed in prostate cancer.18 Most recently, WWP1 was demonstrated to promote p53 ubiquitination and target p53 for nuclear exportation.19

Here we performed extensive genetic and functional analyses in a large number of human breast cancer samples to evaluate the role of WWP1 in breast cancer. Both a copy number gain and overexpression were frequently detected in human breast cancer samples including primary tumors. Functionally, inhibition of WWP1 expression by RNAi significantly suppressed cell proliferation and induced apoptosis of breast cancer cells. In contrast, forced overexpression of WWP1 promoted immortalized breast epithelial cell proliferation. Our findings suggest that WWP1 could be a potential molecular target for breast cancer diagnosis and therapy.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Tumor specimens and cell lines

In total, we analyzed 88 breast cancer samples, including 53 clinical tumor specimens (only for gene dosage analysis) and 35 cancer cell lines. Among the 53 clinical tumor specimens were 41 cases with matched nonneoplastic cells including 19 primary tumors and 22 metastases. According to the tumor grade, 17 were Grade 1, 9 were Grade 2, 10 were Grade 3, and 5 were not available. The patient ages at diagnosis ranged from 27 to 74 years (average 54.2). 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.20 For WWP1 expression analysis, 24 cDNA samples prepared from normal (12) and tumor (12) breast tissues were purchased from OriGene Technologies, Rockville, MD. All breast epithelial cell lines were described in our previous study.21

Detection of the gene copy number and expression for WWP1

The gene copy number in cell lines was detected by SYBR real time PCR.18 Duplex PCR was used to detect the copy number gain for WWP1 for all clinical specimens.18 Real time RT-PCR, Northern blot and Western blot for WWP1 were performed as described in our previous study.18 For real time RT-PCR results, the average ratio of WWP1 to GAPDH control in MCF10A 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 equal or greater than two. Antibodies for Caspase 9, Caspase 7, Caspase 3 and PARP were purchased from cell signaling.

Knockdown of WWP1 by siRNA

MCF7 cells were cultured in DMEM media with 5% FBS, 0.01 mg/ml Insulin, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids and 1.5 g/l sodium bicarbonate. HCC1500 cells were cultured in RPMI1640 media with 5% FBS, 4.5 g/l glucose, 1 mM sodium pyruvate, 10 mM HEPES and 1.5 g/l sodium bicarbonate. MCF10A and 184B5 cells were cultured in F12/DMEM media with 5% horse serum, 20 ng/ml EGF, 100 ng/ml cholera toxin, 0.01 mg/ml insulin and 500 ng/ml hydrocortisone. The target sequences for WWP1 siRNA#2 and luciferase control siRNA were described in our previous study.17 WWP1 siRNA#1 and a negative control siRNA were purchased from Dharmacon. HCC1500, MCF7 and MCF10A cells were plated into 6-well plates at 1.2 × 106, 6 × 105 or 2 × 105 cells/well respectively. Cells were transfected with 100 nM of chemically synthesized siRNA (Dharmacon, IL) using Lipofectamine 2000 (Invitrogen). RNA, protein or cells were collected at 48 hr for different types of analyses after siRNA transfection if not specifically indicated.

Expression of WWP1 by lentivirus

The hWWP1 gene was amplified from the IMAGE clone 5296005 with the pfu enzymes by PCR using primers 5′-ATGGCCACTGCTTCACCAAG-3′ and 5′-TTCCGCGGTCATTCTTGTCCAAATCCCTCT-3′. The PCR product was cloned into the pLenti6/V5-D-TOPO vector and verified by sequencing. A mutant hWWP1C890A was also amplified and cloned into the same vector based on the FLAG-Tiul1C890A construct (kindly provided by Dr. Atfi12). The pLenti6/V5-GW/LacZ vector (Invitrogen) was used as a negative control. All plasmids were transfected into 293FT packing cells using lipofectamine 2000 following the standard protocol. Lentiviruses were collected at 72 hr after transfection and used to transduce MCF10A and 184B5 cells in a 6-well plate. Forty eight hour after transduction, the cells were trypsinized and seeded into 60-mm dishes. Antibiotic blasticidin (10 μg/ml) was added to select drug resistant cell populations until the control cells were completely killed. The stable cell populations were used for cell growth analysis.

Cell proliferation

For DNA synthesis assay, HCC1500, MCF7 and MCF10A cells were seeded into 24-well plates and labeled with 14C-thymidine (0.025 μCi/ml) overnight. SiRNAs for WWP1 and control were transfected into the cells for 48 hr. The cells were then labeled with 3H-thymidine (1 μCi/ml) for 4 hr. Upon the completion of treatment, the cells were washed with PBS twice and treated with 10% TCA. Radiolabeled DNA was solubilized by 100 μl of 0.3 M NaOH and transferred to glass fiber filter membranes, and radioactivity for 3H and 14C was measured using a Beckman-Coulter LS6500 multipurpose scintillation counter. The DNA synthesis rate was defined by the ratio of incorporated 3H to 14C. Only 3H-thymidine was counted for stable MCF10A and 184B5 cell populations.

For growth curve analysis, HCC1500 and MCF7 cells were seeded into 12-well plates. Transfection was conducted 24 hr later if necessary. Two to four days after transfection, the cells were fixed with 10% TCA, stained with 0.4% Sulforhodamine-B (SRB) and washed by 1% acetic acid. The cell densities were measured with a spectrometer at a 500 nm wavelength.

Apoptosis analysis

Cell apoptosis was analyzed by 2 different assays. One is DNA content assay by propidium iodide (PI) staining as described in our previous study.22 The subG1 cell populations were compared between the control siRNA and the WWP1 siRNA#1 transfected cells. The other apoptotic assay is Annexin V staining as performed following the standard protocol in the manual (BD Pharmingen, San Diego, CA). Briefly, siRNA transfected MCF7 and HCC1500 cells were washed once with cold PBS and then trypsinized and resuspended in a1× binding buffer (0.01 mM HEPES, pH 7.4, 0.14 M NaCl, 2.5 mM CaCl2) at a concentration of ∼2 × 106 cells/ml. One hundred microliter of the cell solution was transfered into a 5-ml culture tube. Five microliter Annexin V and 7-AAD were added respectively. The cells were mixed and incubated for 15 min at RT in the dark. After adding 400 μl of the 1× binding buffer, the cells were analyzed by flow cytometry within 1 hr.

Statistics Analysis

Fisher's exact test was used to analyze the correlation between the gene copy change and the gene expression change for cell lines, the age at diagnosis, or tumor grades for breast tumors. The student t-test was used to determine statistical differences between the experimental and control groups for DNA synthesis, cell viability and apoptosis. The result was considered statistically significant if a p-value is smaller than 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

WWP1 is frequently amplified in breast cancer

The WWP1 gene is located at the 8q21, a region frequently amplified in human breast cancer.3, 23 To evaluate if WWP1 undergoes genomic amplification in breast cancer, we first examined WWP1's DNA copy number in 35 breast cancer cell lines and 4 immortalized breast cell lines by using real-time PCR assay, with normal human DNA as a control. Eighteen of the 35 cases (51%) showed an increased copy number by at least 2-fold (Fig. 1a). The WWP1 gene may be highly amplified (∼10-fold) in the MCF7 cell line. Conversely, none of the 4 nontransformed cell lines (MCF10A, 184A1, 184B5 and 97T) showed a copy number change at WWP1. Interestingly, 1 cancer cell line MDA-MB-468 showed hemizygous deletion at the WWP1 locus (Fig. 1a).

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Figure 1. Frequent gene amplification for WWP1 was detected in breast cancer. (a) WWP1 copy numbers in breast cancer and immortalized breast epithelial cell lines, as detected by SYBR real time PCR. The KAI1 gene serves as a control for normalizing input DNA. The ratio between WWP1/KAI1 in the normal human DNA was defined as 1. Accordingly, 18 cancer cell lines with the ratio of more than 2 were considered as samples with WWP1 amplification. The empty bars indicate normal control and nontransformed breast samples. The normal human DNA is indicated by an arrow. (b) Examples of WWP1 copy number gain in clinical breast cancer specimens detected by duplex PCR assay. The KAI1 gene serves as a loading control. Case numbers are shown at the top. N is normal; and T is tumor.

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To determine if WWP1 also has copy number gains in clinical breast cancer samples, we examined 41 cases of breast cancer and their matched normal cells by performing duplex PCR and gel electrophoresis, which were developed and used to detect gene dosage in our previous study.18 As shown in Figure 1b, the ratios between WWP1 signals and control KAI1 signals in 17 (41%) tumors were significantly greater (>2) than that in the normal DNA. No breast tumors showed gene deletion at the WWP1 locus compared with the matched normal. No significant association was found between the copy number gain for WWP1 and age at the diagnosis, stage, tumor grade or overall survival.

Expression of WWP1 is frequently up-regulated in breast cancer

A copy number gain typically leads to overexpression for a gene. To test whether WWP1 is overexpressed in breast cancer, we first examined WWP1 mRNA expression by real time RT-PCR in 33 breast cancer cell lines and 5 normal or immortalized breast cell lines. Compared with the expression level of WWP1 in the MCF10A immortalized breast cell line, the expression of WWP1 was increased by at least 2-fold in 19 of the 33 (58%) breast cancer samples (Fig. 2a). The HCC1500 cell line showed the highest level (10.7-fold) of expression for WWP1. The immortalized cell line 97T also showed more than a twofold increase in WWP1 mRNA expression. Four other nontransformed breast cell lines, including MCF10A, 184A1, 184B5 and Human mammary epithelial cell (HMEC), showed low expression levels for WWP1.

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Figure 2. Expression of WWP1 is up-regulated in breast cancer. (a) Expression of WWP1 mRNA in breast cell lines detected by SYBR real time RT-PCR. GAPDH was used as a control to normalize the cDNA input. Empty bars indicate nontransformed breast samples. MCF10A control is indicated by an arrow. (b) The average level of WWP1 mRNA in 12 normal and 12 tumor breast samples was detected by real time RT-PCR. Normal and tumor samples are not matched in pairs. (c) Expression of WWP1 mRNA in breast cell lines as detected by Northern blot analysis. Lanes 1–4 are non-transformed breast samples, and the rest are breast cancer cell lines. β-actin serves as a RNA loading control. (d) WWP1 protein expression in breast cancer cell lines as detected by Western blot.

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In combination with the results of the copy number gain for WWP1, 13 of the 17 cancer cell lines with an increased WWP1 copy number showed WWP1 overexpression, whereas only 6 of the 16 samples without a WWP1 copy number gain showed WWP1 overexpression (Figs. 1 and 2) (Fisher's exact test, p = 0.03). These results suggest that the overexpression of WWP1 is significantly correlated with its DNA copy number gain in breast cancer.

To test whether WWP1 is overexpressed in primary breast tumors, we examined WWP1 expression in 12 normal and 12 tumor breast specimens by real-time RT-PCR. Because these normal and tumor samples are not matched in pairs, we compared the average mRNA level for WWP1. As shown in Figure 2b, the average level of WWP1 mRNA in the tumor samples is about three times of that in normal breast tissue. In other words, WWP1 mRNA is significantly upregulated in breast tumors (p < 0.001, t-test).

Northern blot analysis was performed to confirm WWP1 overexpression in breast cancer. Whereas a normal breast tissue and 2 nontransformed breast epithelial cell lines (MCF10A and 184A1) expressed little WWP1 mRNA, 7 of the 9 breast cancer cell lines, including HCC1937, MDA-MB-361, HCC70, BT474, BT549, MDA-MB-134 and HCC1500, showed an increased expression of WWP1 (Fig. 2c). Consistent with the real-time RT-PCR results aforementioned, the immortalized cell line 97T showed a slight increase of the expression of WWP1 mRNA.

It has been reported that WWP1 has multiple isoforms resulting from alternative splicing based on a study of the breast cancer cell line T-47D.24 Our Northern blot analysis showed that a smaller isoform of WWP1, indicated as a band below the wild type WWP1 mRNA at 4.2 Kb, was detected in some samples. However, the isoform was not associated with breast cancer because it was present in both cancer samples and nontumor controls (Fig. 2b).

Lastly, the overexpression of WWP1 in breast cancer cells was confirmed at the protein level. We examined the WWP1 protein expression in seven breast cancer cell lines by Western blot using an anti-WWP1 antibody.18 Consistent with real-time RT-PCR and northern blot results, high levels of WWP1 protein were detected in several breast cancer cell lines, such as MDA-MB-361, MCF7, SKBR-3, MDA-MB-134, and HCC1500 (Fig. 2d). Two immortalized cell lines (184A1 and 184B5) and 2 cancer cell lines (BT20 and SW527) showed WWP1 protein at low levels. In our previous study, a short WWP1 isoform was detected in PC-3 and LAPC-4 prostate cancer cells18; however, this WWP1 isoform was not found in any breast cancer cell lines examined.

Inhibition WWP1 by RNA interference suppresses cell proliferation

In our previous study, an siRNA for WWP1 was shown to efficiently knock down WWP1 expression in the PC-3 prostate and MCF7 breast cancer cell lines.17 This siRNA also reduced PC-3 cell growth.18 To further evaluate the effect of WWP1 on breast cancer cell growth, we treated MCF7 and HCC1500 cells with two WWP1 siRNAs, a negative control siRNA or no siRNA respectively. The high levels of WWP1 mRNA and protein in MCF7 and HCC1500 breast cancer cell lines have been demonstrated in Figure 2. Two different WWP1 siRNAs were used to reduce the concern of off-target effects. The knock down of WWP1 was examined by Western blot. Both WWP1 siRNA#1 and #2 silence endogenous WWP1 protein expression from 50 to 80% in both MCF7 and HCC1500 cell lines. WWP1 siRNA#1 functions slightly better than #2 in both cell lines (Fig. 3a). The negative control siRNA has no significant effect on WWP1 protein expression compared to the mock (no siRNA) group in both cell lines.

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Figure 3. Inhibiting WWP1 by RNAi suppresses MCF7 and HCC1500 breast cancer cell proliferation. (a) RNAi-reduced WWP1 expression in MCF7 and HCC1500 cells as detected by Western blot. Mock, no siRNA; CTRL, control siRNA#1 (Dharmacon); W#1 and W#2 are 2 different WWP1 siRNAs. All siRNA were transfected using Lipofectamine 2000 at 100 nM final concentration. Proteins were collected 48 hr after transfection. (b) WWP1 knockdown significantly suppresses DNA synthesis at 48 hr after siRNA transfection when compared to the negative controls, as determined by DNA synthesis assay as described in materials and methods. (c) Knockdown of WWP1 by RNAi slowed cell growth in the course of 4 days, as determined by SRB assay. All functional experiments were performed in triplicate.

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We then examined the cell proliferation by using 3H/14C-thymidine incorporation assay. HCC1500 shows less DNA synthesis than MCF7. Both WWP1 siRNAs dramatically suppress DNA synthesis compared to the control siRNA in both breast cancer cell lines (Fig. 3b, p < 0.001, t-test). Consistent with the knock down effect, DNA synthesis is more severely inhibited by WWP1siRNA#1 (∼70%) than siRNA#2 (∼45%) in both cell lines. The control siRNA does not affect DNA synthesis compared to the mock group.

Cell growth was further analyzed by the Sulphorhodamine-B (SRB) staining25 at day 2 and day 4 after siRNA transfection. Analogue to DNA synthesis, MCF7 and HCC1500 cells with reduced WWP1 expression grew significantly slower when compared to the control siRNA or mock groups.

WWP1 siRNAs induce breast cancer cell apoptosis

We noticed that WWP1 knock down by siRNA dramatically induces alteration of MCF7 and HCC1500 morphology and cell death compared to the control siRNA (Fig. 4a). Similar phenomena were observed for both WWP1 siRNAs. In DNA synthesis assay as aforementioned, incorporated 14C-thymidine was also significantly reduced in the WWP1 siRNAs transfected cells compared to the control siRNA transfected cells (Data not shown).

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Figure 4. WWP1 knock down induces MCF7 and HCC1500 breast cancer cell apoptosis. (a) A WWP1 siRNA induces cell death compared to the non-targeting control siRNA, as observed under an inverted phase contrast microscope. (b) WWP1 knockdown increases subG1 cell populations, as determined by propidium iodide (PI) staining. (c) The WWP1 siRNA (arrows) increases Annexin V staining. (d) The WWP1 siRNA induces cleavage of PARP and activation of caspases. CF represents cleaved fragment. Data were collected 48 hr after siRNA (final concentration is 100 nM) transfection in all the above experiments. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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To determine if WWP1 knock down indeed induces cell apoptosis, we first performed DNA content analysis by PI staining. We found that both MCF7 and HCC1500 cell lines have a high background of apoptosis and the WWP1 siRNA significantly increases sub-G1 cell populations by 1.4-fold and 1.7-fold respectively (Fig. 4b). Then, we performed Annexin V staining and found that Annexin V positive cell populations are significantly increased by the WWP1 siRNA in both MCF7 and HCC1500 (Fig. 4c). Further investigation revealed that the WWP1 siRNA induces PARP cleavage, an apoptotic marker, in both MCF7 and HCC1500 cancer cell lines (Fig. 4d).

To understand the mechanism of cell apoptosis induced by WWP1 knock down, we further examined the status of several caspases. We detected a decrease of caspase 9 by the WWP1 siRNA in both cell lines (Fig. 4d). Interestingly, caspase 7 is only activated in MCF7 but not in HCC1500 although caspase 7 is expressed in both cell lines (Fig. 4d). In contrast, caspase 3 is only activated in HCC1500 but not in MCF7 (Fig. 4d). Actually, MCF7 is a caspase 3 deficient cell line.26 These findings suggest that WWP1 ablation may induce apoptosis through activating common or different caspases in MCF7 and HCC1500 breast cancer cell lines.

WWP1 overexpression promotes immortalized breast epithelial cell proliferation

To test if WWP1 overexpression affects normal breast epithelial cell growth, we transduced the human wild type (WT) WWP1, a catalytically inactive mutant WWP1C890A, in which the catalytic cystein was replaced with an alanine,12 or a LacZ control into MCF10A, and 184B5 immortalized breast epithelial cell lines by a lentiviral system. As shown in Figure 5a, both WT and mutant WWP1 showed elevated protein expression compared to the parental or LacZ control cell populations by Western blot.

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Figure 5. WWP1 overexpression promotes MCF10A and 184B5 immortalized breast epithelial cell proliferation. (a) WWP1 expression was detected by Western blot. LacZ, human WT WWP1, and mutant WWP1C890A were transduced into MCF10A and 184B5 by lentivirus. Blasticidin (10 μg/ml) resistant cell populations were used. (b) WWP1 expressing cells increased 3H-thymidine incorporation compared to parental or lacZ control cells (p < 0.05). (c) A WWP1 siRNA knocked down overexpressed WWP1 protein. (d) Inhibition of overexpressed WWP1 reverses the growth rate as determined by DNA synthesis assay (p < 0.05).

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Then, we compared the cell growth of eight populations by DNA synthesis. We found that the cell population with WWP1 overexpression grows significantly faster than the control cell populations (Fig. 5b). Interestingly, both WT and catalytic inactive WWP1 increase DNA synthesis at same efficiency (p < 0.05). We analyzed 2 batches of transduced MCF10A cell populations and obtained the same results. We also confirmed cell proliferation by cell viability assays (Data not shown). These results suggest that WWP1 may promote cell proliferation in a ubiquitination-independent mechanism.

To test if the phenotype is reversible, we knocked down the overexpressed WT WWP1 in MCF10A by using a WWP1 siRNA. As shown in Figure 5c, the WWP1 siRNA efficiently silenced both endogenous and exogenous WWP1 protein expression. We examined the DNA synthesis. Consequently, WWP1 knock down in WWP1 overexpressed MCF10A cells significantly (p < 0.05) reverses the cell proliferation rate back to the same level of LacZ control cells. However, WWP1 knock down in LacZ overexpressed cells only slightly decreases cell proliferation, if at all. These findings strongly suggest that WWP1 promotes “normal” breast epithelial cell proliferation.

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this study, we present 4 lines of evidence for an oncogenic role of the WWP1 ubiquitin E3 ligase in human breast epithelial cells. First, WWP1 is located at 8q21, which is frequently amplified in human breast cancer. We showed that WWP1 indeed frequently gains DNA copy numbers in breast cell lines and clinical samples from breast cancer. Second, WWP1 is frequently overexpressed in breast cancer cell lines and primary tumors, and the overexpression is significantly associated with its copy number gain in breast cancer cell lines. Additionally, knockdown of WWP1 suppresses MCF7 and HCC1500 breast cancer cell proliferation and induces apoptosis. Lastly, forced WWP1 overexpression promotes the immortalized breast epithelial cell lines MCF10A and 184B5 cell proliferation. Therefore, WWP1 is a potential oncogene in breast cancer.

In addition to WWP1, at least 2 other target genes from 8q21 have been shown to undergo a copy number gain and overexpression in breast cancer, including TPD524 and E2F5.5 Importantly, TPD52 is also amplified and overexpressed in human prostate27, 28 and ovarian cancer.29 Similarly, we found that the WWP1 gene is also amplified and overexpressed in human prostate cancer.17, 18 Thus, it is possible that co-amplification and overexpression of these molecules additively contribute to both breast and prostate carcinogenesis.

Although overexpression of WWP1 is significantly correlated with its DNA copy number gain in breast cancer cell lines, we can not exclude other mechanisms involved in WWP1 overexpression. Six breast cancer cell lines with a high level of WWP1 mRNA, such as HCC1500, BT474 and HCC1937, did not show gene amplification. Additionally, we failed to detect expression up-regulation for 4 breast cancer cell lines with a gene copy gain, such as MDA-MB-453, ZR-75-1, HCC2218 and HCC1599. Therefore, the WWP1 overexpression could be independent of gene amplification at least under certain circumstances. Recently, WWP1 expression has been demonstrated to be suppressed by p53.19 Whether p53 regulates WWP1 transcription in breast cancer remains to be identified.

WWP1 may promote cell survival in breast cancer through a mechanism distinct from inhibition of the TGF-β pathway which has been demonstrated in several other cell models. First, 2 WWP1 overexpressed breast cancer cell lines, MCF7 and HCC1500, are actually sensitive to TGF-β induced growth arrest (data not shown and Ref.30). Second, TGF-β cannot inhibit MCF10A cell proliferation but, instead, can induce cell migration and invasion.31 The expression level of WWP1 in MCF10A is low; furthermore, WWP1 overexpression promotes the MCF10A cell proliferation. It is clear that TGF-β resistance in MCF10A is not mediated by WWP1 overexpression. Lastly, although several different components in the TGF-β pathway, such as TβR1, Smad2, Smad4 and p15, have been shown to be regulated in other cell models,11–13, 18 their expressions were not induced by WWP1 siRNAs in MCF7 and HCC1500 (Data not shown). In MCF10A, WWP1 overexpression does not affect protein levels of Smad4 (Data not shown). Therefore, inhibition of TGF-β signaling by WWP1 cannot explain the observation from WWP1 in these 3 breast epithelial cell lines although we can not completely exclude the possibility that WWP1 promotes other breast cell transformation through inhibiting the TGF-β pathway.

Besides blocking the TGF-β signal pathway, the WWP1 E3 ligase may also function through targeting other substrates for proteolysis. WWP1 was reported to target the epithelial Na+ channel (ENaC),32 Notch,33 Runx2,14, 15 KLF2,16 and KLF517 for ubiquitin-mediated proteolysis. There is growing evidence that KLF transcription factors play critical roles in the growth and metastasis of many tumor types including breast tumors.34, 35 We previously demonstrated that KLF5 is frequently deleted and down-regulated in breast cancer cell lines.21 Most recently, WWP1 was reported to promote p53 ubiquitination and export p53 from the nucleus.19 It is still to be determined whether WWP1 promotes breast cancer development through regulating these known proteins. Interestingly, WWP1 may promote cell proliferation through a ubiquitin-independent mechanism because the overexpression of a catalytically inactive mutant WWP1 also increased DNA synthesis in MCF10A and 184B5. In line with this, WWP1 was shown to have ligase activity independent function.36

Frequent genetic and expression aberrations of E3s have been documented in human breast cancer.7 E3 ubiquitin ligases have become promising drug targets for cancer therapy. Several E3 ligases, including Mdm2,37 EFP,9 Skp2,8 and β-TrCP,38 have been demonstrated to play an oncogenic role in human breast carcinogenesis. Our findings in this study present WWP1 as another oncogenic E3 ligase in human breast cancer, and these E3 ligases can be therapeutic targets in the treatment of breast cancer. For example, targeting β-TrCP can suppress growth and survival of human breast cancer cells and sensitize the killing effects of anticancer drugs.38 Recently, the potent and selective small-molecule antagonists of MDM2 have been developed to treat human cancer.39 Here we show that the inhibition of WWP1 by the RNAi approach dramatically induces the growth arrest and apoptosis of breast cancer cells. Thus targeting WWP1 by small molecular drugs could be a useful therapeutic approach in treating breast cancers with WWP1 overexpression.

It is worthwhile to point out that WWP1 siRNA does not induce significant growth reduction in SKBR3 and T-47D although WWP1 expression levels are at high levels similar to MCF7 and HCC1500 (Fig. 2) and are effectively knocked down by WWP1 siRNAs (data not shown). Our data suggest that WWP1 siRNAs could suppress a subset of breast cancer cell proliferation and induce apoptosis. More experiments are required to understand the different effects of WWP1 ablation in breast cancer.

We did not observe a significant correlation between WWP1 amplification and breast cancer age at diagnosis, grade, stage or overall survival probably because of the small number of tumor samples. The overexpression of WWP1 is not associated with known p53 status in breast cancer cell lines.40 We noticed that the expression level of WWP1 appears to be higher in ER positive cell lines (9/12, 75%) than in ER negative cell lines (6/13, 46%). However, there is no significant correlation (p = 0.14, Fisher's exact test). In ER positive MCF7 cells, estrogen (0.2 μM E2) treatment for 24 hr slightly increases WWP1 protein expression (Data not shown). Additionally, we can not completely exclude that WWP1 is mutated in breast cancer although the WWP1 mutation rarely occurs in prostate cancer.18 The verification of WWP1 mRNA or protein overexpression in more clinical tumor specimens will be required in the future to assess the value of WWP1 overexpression in breast cancer diagnosis. Additional functional analysis of WWP1 in animal models will be essential to validate the potential of WWP1 inhibition in breast cancer therapy.

In summary, we found that the WWP1 gene undergoes a frequent copy number gain and overexpression in human breast cancer. Functionally, the inhibition of WWP1 by RNAi suppresses breast cell proliferation and induces apoptosis through activating caspases in MCF7 and HCC1500. WWP1 overexpression promotes MCF10A and 184B5 immortalized breast epithelial cell proliferation. These results indicate that WWP1 could be a potential biomarker and therapeutic target in the detection and treatment of breast cancer.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Dr. Dorina Avram's help for apoptosis analysis and Dr. Jihe Zhao for a critical reading of the manuscript. This work was supported in part by a grant (BCTR0503705) from the Susan G. Komen Breast Cancer Foundation (Chen, C), and a grant (CA087921) from the National Cancer Institute (Dong, JT).

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References