Prostasin may contribute to chemoresistance, repress cancer cells in ovarian cancer, and is involved in the signaling pathways of CASP/PAK2-p34/actin

Abstract

Ovarian cancer is the deadliest of gynecologic cancers, largely due to the development of drug resistance in chemotherapy. Prostasin may have an essential role in the oncogenesis. In this study, we show that prostasin is decreased in an ovarian cancer drug-resistant cell line and in ovarian cancer patients with high levels of excision repair cross-complementing 1, a marker for chemoresistance. Our cell cultural model investigation demonstrates prostasin has important roles in the development of drug resistance and cancer cell survival. Forced overexpression of prostasin in ovarian cancer cells greatly induces cell death (resulting in 99% cell death in a drug-resistant cell line and 100% cell death in other tested cell lines). In addition, the surviving cells grow at a much lower rate compared with non-overexpressed cells. In vivo studies indicate that forced overexpression of prostasin in drug-resistant cells greatly inhibits the growth of tumors and may partially reverse drug resistance. Our investigation of the molecular mechanisms suggests that prostasin may repress cancer cells and/or contribute to chemoresistance by modulating the CASP/P21-activated protein kinase (PAK2)-p34 pathway, and thereafter PAK2-p34/JNK/c-jun and PAK2-p34/mlck/actin signaling pathways. Thus, we introduce prostain as a potential target for treating/repressing some ovarian tumors and have begun to identify their relevant molecular targets in specific signaling pathways.

Figure 1

Reduced prostasin expression in patients with high expression of ERCC1, a marker for chemoresistance in ovarian cancer. (a) Prostasin mRNA levels were compared in tumors with high ERCC1 levels (ERCC1 average=1.54, n=18) representing the chemoresistant phenotype, and in tumors with low ERCC1 levels (ERCC1 average=0.44, n=31) representing the chemosensitive phenotype^, by real-time qPCR analysis. Relative levels of prostasin in chemoresistant versus chemosensitive tumors are shown, respectively. **P<0.01. Mean±s.d. are given, and P values were calculated using the two-sided Student's t-test. (b) Expression of prostasin by fluorescent immunohistochemistry in (patient-derived) ovarian tumor samples. Prostasin is decreased in ovarian ERCC1-high tumors compared with the ERCC1-low tumors

Figure 2

Prostasin has important roles in chemoresistance and cell death in cell culture model. (a) Prostasin decreased in paclitaxel-resistance cancer cell line. O432: ovarian cancer cell line Ovca432 (sensitive to paclitaxel); O432-RP: paclitaxel resistance cell line generated from Ovca432. The prostasin protein levels in O432 and O432-RP cells are shown in immunoblots with specific antibodies. (b) Prostasin siRNAs transfection reduced prostasin. O432-pro-D cells (transfected with prostasin siRNA) express lower prostasin, compared with control cells of O432 (transfected with reagent only) and O432-Cs (transfected with no-targeting siRNA). The prostasin protein levels are shown in immunoblots with specific antibodies. (c) Downregulation of prostasin in O432 cells resulted in increase of chemoresistant activity. Cells were treated with paclitaxel at different concentrations for 24 h (starting 48 h after siRNAs transfection) and cultured with normal medium for an additional 7 to 10 days before cell survival was assayed. Relative cell survival rates of each cell line are shown. (d) Overexpression of prostasin greatly induces cell death in ovarian cancer cells. The cell survival rates are shown after forced overexpression of prostasin in several cell lines from day-0 to day-8, respectively. (e) Prostasin cDNA transfection resulted in overexpression of prostasin in chemoresistant O432-RP cells. O432-RP-pro-O cells (transfected with prostasin cDNA) express higher prostasin compared with control cells O432-RP and O432-RP-C (transfected with control vector). The prostasin protein levels are shown in immunoblots with specific antibodies. (f) Forced overexpression of prostasin represses growth of chemoresistant cells. Relative cell growth rates are shown for O432-RP-pro-O and control cells O432-RP and O432-RP-C. (g) Forced overexpression of prostasin in O432-RP cells re-sensitizes chemoresistant cells. Cells were plated at about 10–20% confluence and treated with paclitaxel at different concentrations for 24 h, cultured with normal medium for additional 7 to 10 days, then assayed for cell survival. Relative survival rates of cell lines are shown

Figure 3

Forced restoration of prostasin represses and re-sensitizes chemoresistant tumors. (a) Overexpression of prostasin represses and re-sensitizes chemoresistant tumors. O432-RP-pro-O cells (stably transfected with prostasin cDNA) or control cells O432-RP-C (stably transfected with control pCI-neo vector) were injected into the left and right flanks of mice, respectively. The animals were treated with paclitaxel (15 mg/kg/week) or control vehicle PBS for 2 weeks after the tumors reached about 100 or 50 mm³. Tumor volumes before and after treatment are shown. O432-RP-psp-O-c and O432-RP-psp-O-t: PBS- and paclitaxel-treated O432-RP-psp-O tumors; O432-RP-C-c and O432-RP-C-t: PBS- and paclitaxel-treated O432-RP-C tumors. n=6 per group, **P<0.01. Mean±s.d. are given, and the P-values were calculated using the two-sided Student's t-test. (b) Tumors in mice before and after treatments. Scale bar=5 mm

Figure 4

Prostasin regulates CASPs-PAK2-p34 axis and thereafter downstream signaling. (a) Immunoblot of mlck, β-actin, PAK2-p34, JNK, C-Jun in O432-RP-pro-O (O432-RP cells transfected with prostasin cDNA which express higher levels of prostasin) control O432-RP-C cells (O432-RP cells transfected with pCI-neo vector). Mlck, PAK2-p34, JNK, C-Jun expressions increase and β-actin decreases in O432-RP-pro-O cells when compared with control O432-RP-C. (b) Comparison of actin, mlck, and pak1 expression between O432-RP-pro-O and control O432-RP-C cells by real-time qPCR. mRNA levels of actin decreases and mlck and pak1 increase in O432-RP-pro-O cells compared with control O432-RP-C cells. (c) mRNA levels of CASPs detection by real-time qPCR in O432-RP-pro-O cells and control O432-RP-C cells. Several CASP expression increase in O432-RP-pro-O cells compared with the controls. (d) Immunoblot of CASP3, 9, and 10 in O432-RP-pro-O and O432-RP-C cells. CASP3, 9, and 10 protein levels increase in O432-RP-pro-O cells compared with control O432-RP-C cells. (e) CASP inhibitors block CASP activity and thus PAK2 cleavage. PAK2-p34 protein was examined by immunoblot after CASP inhibitors were added to the medium. PAK2-p34 was seen decreased or lost when CASPs were inhibited at several time points. (f) Immunoblot of mlck, β-actin, PAK2-p34, JNK, C-Jun, CASP3, 9, and 10 in prostasin knockdown O432-pro-D cells and controls O432 (mock transfected with reagent only) and O432-Cs (transfected with no-targeting siRNA). Reverse expression patterns for these genes are revealed in O432-pro-D cells and controls compared with O432-RP-pro-O cells and controls

Figure 5

Proposed model of prostasin-regulated signaling network affecting cell survival and/or chemoresistance. Prostasin appears to regulate cell survival and/or chemoresistance may be through CASPs/Pak2-p34 axis and thereafter PAK2-p34/JNK/c-jun and PAK2-p34/mlck/actin signaling pathways