Smoothened antagonists reverse taxane resistance in ovarian cancer

Abstract

The hedgehog pathway has been implicated in the formation and maintenance of a variety of malignancies, including ovarian cancer; however, it is unknown whether hedgehog signaling is involved in ovarian cancer chemoresistance. The goal of this study was to determine the effects of antagonizing the hedgehog receptor, Smoothened (Smo), on chemotherapy response in ovarian cancer. Expression of hedgehog pathway members was assessed in three pairs of parental and chemotherapy-resistant ovarian cancer cell lines (A2780ip2/A2780cp20, SKOV3ip1/SKOV3TRip2, HeyA8/HeyA8MDR) using quantitative PCR and Western blot analysis. Cell lines were exposed to increasing concentrations of two different Smo antagonists (cyclopamine, LDE225) alone and in combination with carboplatin or paclitaxel. Selective knockdown of Smo, Gli1, or Gli2 was achieved using siRNA constructs. Cell viability was assessed by MTT assay. A2780cp20 and SKOV3TRip2 orthotopic xenografts were treated with vehicle, LDE225, paclitaxel, or combination therapy. Chemoresistant cell lines showed higher expression (>2-fold, P < 0.05) of hedgehog signaling components compared with their respective parental lines. Smo antagonists sensitized chemotherapy-resistant cell lines to paclitaxel, but not to carboplatin. LDE225 treatment also increased sensitivity of ALDH-positive cells to paclitaxel. A2780cp20 and SKOV3TRip2 xenografts treated with combined LDE225 and paclitaxel had significantly less tumor burden than those treated with vehicle or either agent alone. Increased taxane sensitivity seems to be mediated by a decrease in P-glycoprotein (MDR1) expression. Selective knockdown of Smo, Gli1, or Gli2 all increased taxane sensitivity. Smo antagonists reverse taxane resistance in chemoresistant ovarian cancer models, suggesting combined anti-hedgehog and chemotherapies could provide a useful therapeutic strategy for ovarian cancer.

Figure 1. Expression of HH signaling comoponents in chemosensitive and chemoresistant ovarian cancer cell lines

Gene expression was calculated relative to the sample/cell line with the highest expression of a particular gene. A) mRNA expression of HH ligands, Sonic (SHH), Indian (IHH) and Desert (DHH). B) mRNA expression of HH receptors, PTCH1 and SMO. C) Protein expression of Smo was also measured using Western blot analysis. β-actin was used as a loading control. D) mRNA expression of HH transcription factors, GLI1 and GLI2. Data are representative of 3 independent experiments. *P < 0.05, compared to parental chemosensitive cell line.

Figure 2. LDE225 reduces HH pathway activity in chemoresistant ovarian cancer cells

A) Chemical structures of NVP-LDE225 and paclitaxel. B) Gene expression of PTCH1, GLI1 and GLI2 was examined in A2780cp20 cells following exposure to increasing concentrations of LDE225 using qPCR. *P < 0.05, compared to DMSO vehicle control. C) Protein expression of Gli1 in A2780cp20 cells following exposure to increasing concentrations of LDE225 was measured using Western blot analysis to confirm mRNA results. β-actin was used as a loading control. Data are representative of 3 independent experiments.

Figure 3. Smo antagonism reverses taxane resistance in chemoresistant ovarian cancer cell lines both in vitro and in vivo

A) A2780cp20 cells were exposed to either 95% ethanol (EtOH, vehicle control) or cyclopamine (5 µM) in combination with increasing concentrations of paclitaxel. Cell viability was determined by MTT assay. B) A2780cp20 cells were exposed to either DMSO (vehicle control) or LDE225 (1 and 5 µM) in combination with increasing concentrations of paclitaxel. Cell viability was determined by MTT assay. C) Cell cycle analysis was performed on A2780cp20 cells treated with DMSO alone, paclitaxel alone, LDE225 alone or combined paclitaxel and LDE225 using propidium iodide (PI) staining. Representative histograms of DMSO- and combination-treated cells are shown on the right. Data are representative of 3 independent experiments. D) Mice injected intraperitoneally with A2780cp20 cells were treated with either vehicle alone, paclitaxel alone, LDE225 alone or combined paclitaxel + LDE225. E) Mice injected intraperitoneally with SKOV3TRip2 cells were treated with either vehicle alone, paclitaxel alone, LDE225 alone or combined paclitaxel + LDE225. For both xenograft models, mice treated with the combination paclitaxel + LDE225 showed a significant reduction in tumor weight compared to treatment with vehicle alone. Mean tumor weights with standard error are presented. *P < 0.05, compared to vehicle control.

Figure 4. LDE225 sensitizes chemoresistant ovarian cancer cells to paclitaxel by downregulating MDR1 expression and sensitizes both ALDH-negative and -positive ovarian cancer cells to paclitaxel

A) A2780cp20 cells were exposed to DMSO, LDE225 (1 or 5 µM), paclitaxel (Tax, 30 nM) or combined LDE225 + paclitaxel for 72 hours and examined for MDR1 gene expression. *P < 0.05, compared to DMSO; †P < 0.05, compared to paclitaxel alone. B) SKOV3TRip2 cells were exposed to DMSO, LDE225 (1 or 5 µM), paclitaxel (Tax, 200 nM) or combined LDE225 + paclitaxel for 72 hours and examined for MDR1 gene expression. *P < 0.05, compared to DMSO; †P < 0.05, compared to paclitaxel alone. Data are representative of 3 independent experiments. C) A2780cp20 xenografts (n = 5 per group) treated with vehicle alone, paclitaxel alone, LDE225 alone or combined LDE225 + paclitaxel were resected after 4 weeks of therapy and examined for MDR1 gene expression. Mean expression with standard error are presented. *P < 0.05, compared to vehicle; †P < 0.05, compared to paclitaxel alone. D) SKOV3TRip2 cells were sorted into aldehyde dehydrogenase-negative (ALDH−) and -positive (ALDH+) populations, using the ALDEFLUOR assay, and then exposed to either DMSO or 5 µM LDE225, both alone and in combination with increasing concentrations of paclitaxel. Cell viability was determined by MTT assay.

Figure 5. Knockdown of Smo diminishes HH pathway activity, reduces viability and reverses taxane resistance in ovarian cancer cells

A) A2780cp20 cells were exposed to either control or Smo siRNA for 72 hours and examined for mRNA expression of HH pathway mediators SMO, PTCH1, GLI1 and GLI2. *P < 0.01, compared to control siRNA. Protein expression of Smo and Gli (inset) was also measured using Western blot analysis to confirm mRNA results. β-actin was used as a loading control. A2780cp20 cells were transfected with either control siRNA or 2 distinct siRNA constructs designed against Smo (B), Gli1 (C) or Gli2 (D) and exposed to increasing concentrations of paclitaxel. Cell viability was determined by MTT assay. Data are representative of 3 independent experiments.