WWP2 is overexpressed in human oral cancer, determining tumor size and poor prognosis in patients: downregulation of WWP2 inhibits the AKT signaling and tumor growth in mice

Abstract

The WW domain containing E3 ubiquitin protein ligase 2 (WWP2) encodes a member of the Nedd4 family of E3 ligases, which catalyzes the final step of the ubiquitination cascade. WWP2 is involved in tumoral growth with degradation of the tumor suppressor phosphatase and tensin homologue deleted on chromosome TEN (PTEN). However, little is known about the mechanisms and roles of WWP2 in human malignancies including oral squamous cell carcinomas (OSCCs). We found frequent WWP2 overexpression in all OSCC-derived cell lines examined that was associated with cellular growth by accelerating the cell cycle in the G1 phase via degradation of PTEN and activation of the PI3K/AKT signaling pathway. Our in vivo data of WWP2 silencing showed dramatic inhibition of tumoral growth with increased expression of PTEN. Our 104 primary OSCCs had significantly higher expression of WWP2 than their normal counterparts. Moreover, among the clinical variables analyzed, enhanced WWP2 expression was correlated with primary tumoral size and poor prognosis. These data suggested that WWP2 overexpression contributes to neoplastic promotion via the PTEN/PI3K/AKT pathway in OSCCs. WWP2 is likely to be a biomarker of tumoral progression and prognosis and a potential therapeutic target for development of anticancer drugs in OSCCs.

Keywords: PTEN/PI3K/AKT pathway; WWP2; cell cycle; oral squamous carcinoma; tumoral growth.

Figure 1. Overexpression of WWP2 in OSCC-derived cell lines

(A) Quantification of WWP2 mRNA expression in OSCC-derived cell lines by qRT-PCR analysis. Eight OSCC-derived cell lines have significant (P < 0.05, Student's t-test) up-regulation of WWP2 mRNA compared with the HNOKs. (B) Western blotting of WWP2 protein in the OSCC-derived cell lines and HNOKs. WWP2 protein expression is up-regulated in the OSCC-derived cell lines compared with that in the HNOKs. Densitometric WWP2 protein data are normalized to α-tubulin protein levels. The values are expressed as a percentage of the HNOKs.

Figure 2. shWWP2 transfection and reduced cellular growth in shWWP2 cells

(A) WWP2 mRNA levels in shWWP2-transfected cells. qRT-PCR shows that WWP2 is down-regulated significantly (P < 0.05, Student's t-test) in shWWP2 cells compared with shMock cells. (B) Representative Western blotting analysis and densitometric data of WWP2 protein levels in shWWP2 cells and shMock cells. WWP2 protein is decreased markedly (P < 0.05, Student's t-test) in shWWP2-transfected cells compared with shMock cells. Densitometric WWP2 protein data are normalized to α-tubulin protein levels. (C) Proliferation assay of the shWWP2 cells. In all cell lines, the cellular growth of shWWP2 cells is inhibited significantly (P < 0.05, Student's t-test) compared with shMock cells after 7 days. The results are expressed as the means ± SEM of values from six assays.

Figure 3. shWWP2 increases PTEN and promotes G2/M arrest via the PI3K-AKT pathway

(A) Western blot analyses relevant to the PTEN/PI3K/AKT pathway in shWWP2 and shMock cells. shWWP2 cells have overexpression of PTEN and down-expression of p-AKT. Densitometric PTEN, AKT, and p-AKT protein data are normalized to α-tubulin protein levels. (B) Western blot analyses of cell-cycle-related genes in shWWP2 and shMock cells. shWWP2 cells indicate overexpression of p21^Cip1 and p27^Kip1 as CDK inhibitors and down-regulation of cyclin D1, cyclin E, CDK2, CDK4, and CDK6.

Figure 4. shWWP2 increases PTEN and promotes G2/M arrest via the PI3K-AKT pathway

Cell-cycle analysis by flow cytometry. The percentage of the G1/S phase in shKIF4A cells is significantly (P < 0.05, Student's t-test) higher than in shMock cells. The data are expressed as the means ± SEM of values from three assays.

Figure 5. shWWP2 inhibits tumoral growth in vivo

(A) To define the effect of WWP2 on tumoral growth in vivo, shWWP2 and shMock-transfected cells of two cell lines, KOSC-2 and HSC-3, were injected subcutaneously into the backs of female nude mice, respectively (3or 4 mice in each group). The in vitro findings showed that the mean tumoral volume of the shWWP2-transfected cells is significantly (P < 0.05, Student's t-test) smaller compared with the shMock-transfected. (B) Original magnification, ×200 (hematoxylin and eosin) and ×400 (WWP2 and PTEN). Scale bars, 50 μm. IHC clearly shows decreased immunostaining for WWP2 and increased immunostaining for PTEN in the shWWP2-derived tumors than in the shMock-injected mice. Hematoxylin and eosin staining confirms the presence of tumoral cells. H&E, hematoxylin and eosin.

Figure 6. Evaluation of WWP2 protein expression in primary OSCCs and correlation with poor prognosis

(A) Representative IHC results for WWP2 protein in normal oral tissue and primary OSCC. Original magnification, ×200. Scale bars, 50 μm. WWP2 is highly overexpressed in OSCCs compared to normal oral tissues. The state of WWP2 protein expression in primary OSCCs (n=104) and the normal counterparts. The WWP2 IHC scores of normal oral tissues range from 10 to 105 (median, 45.0) and OSCCs range from 50 to 225 (median, 122.5). WWP2 protein expression levels in OSCCs are significantly (P< 0.01, Student's t-test) higher than in normal oral tissues. (B) Five-year survival rate. The left graph shows survival rates with WWP2 expression. The survival rates in the WWP2-positive group are lower than in WWP2-negative group, but the difference does not reach significance. The right graph shows the survival rates based on the degree of primary tumoral progression, T1+T2 or T3 +T4, with the WWP2 expression. In the T1+T2 group. there is no significant difference in the survival rates between the WWP2-positive and -negative groups. In the T3+T4 group, the survival rates in the WWP2-positive group are significantly (P < 0.05, log-rank test) lower than in the WWP2-negative group.