Research ArticleExperimental Studies

Anti-tumor Effect of Hedgehog Signaling Inhibitor, Vismodegib, on Castration-resistant Prostate Cancer

AYA ISHII, KATSUMI SHIGEMURA, KOICHI KITAGAWA, SHIAN-YING SUNG, KUAN-CHOU CHEN, CHIANG YI-TE, MING-CHE LIU and MASATO FUJISAWA
Anticancer Research September 2020, 40 (9) 5107-5114; DOI: https://doi.org/10.21873/anticanres.14514

Abstract

Background/Aim: Epithelial-mesenchymal transition (EMT) via Sonic Hedgehog (Shh) signaling may be one of the mechanisms of progression of castration-resistant prostate cancer (CRPC). In this study, we investigated the possible therapeutic effect of vismodegib, a new Shh inhibitor, in a mouse CRPC model. Materials and Methods: We determined cell proliferation, apoptosis and the expression of EMT-related genes for three prostate cancer cell lines; androgen-dependent LNCaP and independent C4-2B and PC-3 in the presence of vismodegib in vitro. Fifty mg/kg of vismodegib were orally administered into mice bearing C4-2B and PC-3 tumors, respectively every other week for 3 weeks. Results: Vismodegib significantly inhibited cell proliferation and induced cell apoptosis in all cell lines in vitro (p<0.05). Vismodegib significantly inhibited EMT in CRPC cells and tumor growth in C4-2B-bearing mice compared to controls in vivo (p<0.05). Higher expression of caspase-3 and lower expression of vimentin in PC-3 and C4-2B tumors were induced by vismodegib in immunohistochemical analysis. Conclusion: Vismodegib inhibited cell proliferation via apoptosis and also suppressed EMT, showing anti-tumor effects in mice. Further mechanistic studies are needed to investigate the feasibility of vismodegib for CRPC treatment.

Prostate cancer (PC) is the second most prevalent cancer in men worldwide and the sixth leading cause of death in men (1, 2). Androgen-deprivation therapy (ADT) using analogs of gonadotropin-releasing hormone and/or nonsteroidal anti-androgens like hydroxyflutamide or bicalutamide is usually effective in patients with non-organ-confined PC (2), but many patients eventually develop lethal castration-resistant PC (CRPC) for which long-term treatment options are limited (3). Approximately 10-20% of PC patients develop CRPC within 5 years of follow-up (4). Due to intolerable side-effects, over 60% of CRPC patients are ineligible for further chemotherapy, and receive only steroids and supportive care (4). New therapeutic strategies that go beyond current anticancer drugs and androgen deprivation therapy are needed.

Hedgehog signaling is one of the pathways seen in animal development, and has been implicated in tumor growth (5). Hedgehog signaling is activated in various cancers including brain, lung, breast, prostate, and basal cell carcinima (5, 6). Activation of Hedgehog signaling causes angiogenesis and weakens the adhesion of epithelial cells, facilitating both tumor cell proliferation and metastasis in cancers. One of the major proteins in Hedgehog signaling is Sonic Hedgehog (Shh). Vismodegib, a Shh signaling inhibitor induced cell apoptosis via blockade of Smoothened (SMO) (7, 8). Therefore, we hypothesized that vismodegib could be useful for PC treatment as a molecular targeted drug.

Loss of cell adhesion and epithelial-mesenchymal transition (EMT) due to Hedgehog signaling activation has been reported (9). In EMT, epithelial cells lose their cell polarity and cell adhesion, and acquire migration and invasive ability. Since EMT leads to tumor growth, invasion and metastasis in PC, suppressing EMT in CRPC may reduce the risk of metastasis and improve patient prognosis.

In this study, we determined the possible therapeutic effect of vismodegib via inhibition of Shh signaling pathway on cell proliferation, apoptosis, and EMT in PC cells. And we investigated the antitumor effects of vismodegib in CRPC mice model.

Materials and Methods

Cells lines and reagents. Three human PC cell lines, androgen-dependent LNCaP and androgen-independent C4-2B and PC-3, were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin at 37°C and 5% CO2. Vismodegib, a Hedgehog signaling inhibitor, was dissolved and diluted with dimethyl sulfoxide (DMSO).

Cell proliferation assay. Fifty thousand LNCaP, C4-2B and PC-3 cells were seeded and incubated for 24 h, then divided into 2 groups. The medium was changed to contain 100 μM vismodegib or DMSO, respectively. The concentration of vismodegib was determined with reference to previous studies (10). Cell proliferation was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Wako, Osaka, Japan) after incubation for 0, 24, 48, and 72 h according to the manufacturer's instructions. All experiments were carried out in triplicate.

Cell immunofluorescence staining. Fifty thousand cancer cells were seeded and incubated for 24 h. Then cells were treated with vismodegib or DMSO as described above and incubated for an additional 48 h. These cells were fixed by 4% paraformaldehyde for 15 min. After washing, cells were treated with 0.5% Triton-X for 15 min. Cells were incubated with anti-caspase-3 and anti-caspase-9 antibodies overnight at 4°C. Cells were washed and incubated with Alexa Fluor 488-conjugated secondary antibodies (Life Technologies, Carlsbad, CA) and DAPI for 1 h. After washing, the cells were mounted and viewed under a fluorescent microscope.

Flow cytometry analysis. Apoptosis was detected to investigate the relationship between Shh signaling inhibition and apoptosis induction. Each cell line was seeded at 1×106 cells/well (n=3) in a 6-well plate and cultured at 37°C under 5% CO2 for 24 h. After the medium was removed, and a serum-free medium containing 50 μM vismodegib and 0.5% DMSO was added to each well, and the cells were cultured for 48 h. Each cell line was collected and washed by centrifugation with PBS. Apoptosis of each cell was detected by flow cytometry using Annexin V-FITC Apoptosis Detection Kit (Nacalai Tesque, Kyoto, Japan).

Reverse transcription quantitative PCR. Fifty thousand cancer cells were seeded and incubated for 24 h. Then cells were treated with vismodegib or DMSO as described above and incubated for an additional 48 h. Total cellular RNA was isolated using NucleoSpin® RNA (MACHEREY-NAGEL, Neumann, Germany) according to the manufacturer's protocol. RT-qPCR reaction was performed with Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) using the StepOne Plus™ Real-time PCR System (Applied Biosystems, Waltham, MA, USA). Gene-specific primers used were: E-cadherin: 5’-ACGTCGTAATCACCACACTGA-3’ and 5’-TTCGTCACTGCTACGTGTAGAA-3’; N-cadherin: 5’-ACAGTGGCCACCTACAAAGG-3’ and 5’-CCGAGATGGGGTTGATAATG-3’; Vimentin: 5’-GAGAACTTTGCCGTTGAAGC-3’ and 5’-GCTTCC TGTAGGTGGCAATC-3’ (Eurofins Genomics, Tokyo, Japan) (11). The levels of mRNAs were represented as relative values normalized to the level of β-actin mRNA.

Figure 1.
Figure 1.

Cell proliferation assay. In vitro cell proliferation assays in LNCaP, PC-3 and C4-2B cell lines treated with DMSO (vehicle control) and vismodegib (n=3). Cell growth ratio over time for each cell line is shown. The number of cells at 24, 48, 72 h, assuming the number of live cells at the start of the experiment as 1, is shown. *p<0.05.

Figure 2.
Figure 2.

Immunocytochemical staining for caspase-3 and caspase-9. LNCaP, PC-3 and C4-2B were treated with vismodegib or DMSO for 48 h and stained with caspase-3 and caspase-9 antibodies, respectively. Nuclei were stained blue by DAPI. Green staining by anti-caspase antibody indicates apoptosis induction.

Animal experiments. Balb/c nu/nu mice 6-8 weeks of age were purchased from CLEA Japan (Tokyo, Japan). Mice were inoculated with 1×106 C4-2B and PC-3 cells (n=10 each). The day when treatment started was designated as day 0. Tumor volume was calculated by the formula: (longest diameter) × (shortest diameter)2 ×0.5 (12). After tumor formation, mice with tumors (C4-2B, PC-3) were randomly divided into treatment groups and control groups. In order to reduce the vismodegib dose, mice were treated orally (50 mg/kg) every other week for 3 weeks by using feeding needles (13). Therapy was performed 7 days on and 7 days off. After 47 days of treatment, mice were sacrificed and tumors removed. Tissue specimens were prepared by fixing tumors and embedding them in paraffin. All aspects of the experimental design and procedure were reviewed and approved by the institutional ethics and animal welfare committees of Kobe University.

Immunohistochemical staining. Paraffin-embedded tissue sections were deparaffinized and rehydrated. Antigen retrieval was performed in citrate buffer (pH 6.0) at 120°C for 5 min. Immunohistochemical staining was performed in an automatic tissue processor (BondMax, Leica Microsystems, Wetzlar, Germany) following the standard protocol. Briefly, tissue sections were incubated for 60 min with the anti-E-cadherin, anti-N-cadherin, anti-vimentin, and anti-caspase-3 primary antibodies. After washing, sections were exposed to dextran polymer backbone-conjugated secondary antibodies with HRP for 12 min, according to the instrument's standard protocols. Tissue sections were incubated with diaminobenzidine for 10 min and counterstained with hematoxylin. The resulting tissue slides were observed under a microscope.

Immunohistochemical analysis. Immunohistochemical (IHC) staining was scored by the percentage of positive cell area and the staining intensity of the cells. The staining intensity was scored as 0 (negative), 1+, (weak), 2+ (medium) or 3+ (strong). The total IHC score was determined by the following formula: (percentage of positive cell area) × (intensity scores). This method of calculating the score was based on a previous study (14). Immunohistochemical images were evaluated as normal-power (200×) images.

Ethical approval. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors.

Statistical analysis. We used the Student's t-tests to compare controls to targets in the proliferation assay, flow cytometry analysis, RT-qPCR, and animal experiment. The same statistical method was also used in immunohistochemical analysis. For all tests, statistical significance was defined as p<0.05. We used EZR (Jichi Medical University Saitama Medical Center, Tochigi, Japan) for statistical analysis.

Figure 3.
Figure 3.

Detection of apoptotic cells by Annexin-V in vitro. LNCaP, C4-2B and PC-3 were treated with vismodegib or DMSO for 48 h and stained with Annexin-V-FITC and propidium iodide (PI) in vitro to detect apoptotic cells. The positive ratio of apoptotic cells which were positive for both Annexin-V and PI are shown. *p<0.05, n=3, ctrl: Control group.

Results

Cell proliferation assay. Vismodegib significantly inhibited LNCaP and C4-2B cell growth compared to the control group after 24 h of culture (p<0.05). PC-3 cell growth was significantly inhibited by vismodegib compared to the control group after 48 h of culture (p<0.05) (Figure 1). These results indicated that vismodegib inhibited cell proliferation by blocking Shh signaling in both androgen-dependent and androgen-independent cell lines.

Immunofluorescence cell staining for apoptosis detection. In the LNCaP, PC-3, and C4-2B cell lines in the vismodegib treatment group, the cytoplasm was stained green by the anti-caspase antibodies compared to the control groups (Figure 2). There was no difference in staining between the two antibodies, caspase-3 and caspase-9 as an indicator of the intrinsic apoptosis pathway.

Flow cytometry analysis. In LNCaP, C4-2B and PC-3, the proportion of apoptotic cells significantly increased in the group treated with vismodegib as compared to the control group, respectively (Figure 3) (p<0.05).

Reverse transcription quantitative PCR. The expression of vimentin was significantly reduced in the vismodegib-treatment group compared to the control group (p<0.05) (Figure 4). The expression of N-cadherin was significantly reduced in the vismodegib-treatment groups compared to the control groups (p<0.05). The expression of E-cadherin was significantly higher in vismodegib-treatment groups than in the control groups (p<0.05). In C4-2B, the relative expression level of E-cadherin was highest among 3 cell lines, and that was about 10 times as large as the control group.

Mice tumor growth inhibition by vismodegib. We examined the in vivo effect of vismodegib in mice. After 47 days of treatment with every other week dosing, vismodegib significantly inhibited C4-2B (p<0.05) mice tumor growth compared with control, but not PC-3 (p>0.05) (Figure 5).

Immunohistochemical analysis of mouse tumor samples. We investigated mice tumors to examine the in vivo inhibition of EMT by vismodegib. The expression of caspase-3 was significantly increased by vismodegib compared to controls for both tumors (p<0.05) (Figure 6). In addition, vismodegib significantly suppressed the expression of vimentin in both tumors compared with control (p<0.05) (Figure 6).

Discussion

In this study, we demonstrated that vismodegib inhibited tumor growth of PC compared to controls in vivo. Apoptosis induction by vismodegib probably played a role in suppressing cell proliferation. Analysis of EMT-related markers suggested EMT suppression, which is expected to reduce the risk of metastasis by reducing cell motility. Recent studies demonstrated that the Shh-Gli pathway plays an important role in the growth and survival of PC cells, and gene expression analysis downstream from SMO has been performed (7). We, thus, compared castration-resistant and non-castration-resistant PC cell lines to explore the effects of vismodegib on invasion, which had not been done previously, as well as cell proliferation and apoptosis.

Figure 4.
Figure 4.

Quantification of EMT-related genes by real-time PCR analysis. LNCaP, PC-3 and C4-2B were treated with vismodegib or DMSO for 48 h and total mRNA was extracted and purified. Reverse Transcription RT-PCR was performed to determine the gene expression-related EMT. Relative gene expressions of vimentin, N-cadherin and E-cadherin in cells that were treated with vismodegib were normalized to that of cells with control treatment. *p<0.05, ctrl: Control group, vis: vismodegib treatment group.

Vismodegib has been reported to suppress cell proliferation in colon cancer, basal cell carcinoma and breast cancer (5, 6). Cell growth was reportedly also suppressed in PC (7, 15), but we revealed that the effect of vismodegib on cell growth was obtained after 24 h and was also particularly effective against CRPC cell lines (C4-2B).

Previous studies have reported that vismodegib induces apoptosis in several kinds of cancers (7, 8, 16) including PC (7). However, it is not clear whether induction of apoptosis occurs by an intrinsic or extrinsic pathway. In this study, staining for caspase-3 and caspase-9 revealed that the intrinsic pathway is activated, supporting its apoptosis-inducing effect for possible new therapeutic strategies in the future. Vismodegib treatment was reported to up-regulate E-cadherin, a marker for epithelial property, in lung cancer (17). However, there were few reports on the analysis of EMT marker expression in PC (18). Our RT-qPCR results showed that treatment with vismodegib suppressed EMT and induced the mesenchymal-to-epithelial transition (MET). In particular, up-regulation of E-cadherin in CRPC cell line C4-2B was remarkable. This result suggested the possibility of lowering the risk of PC invasion and metastasis.

In the development of metastasis of CRPC, the interplay among signal transduction pathways in response to external stimuli is a critical mechanism. Signaling pathways such as phosphoinositide 3-kinase (PI3K)-Akt signaling pathway involved in the initiation of EMT often lead to suppression of E-cadherin, resulting in enhanced cell proliferation, motility, and metastasis (19). Vogelmann et al. showed that TGF-β could initiate EMT by dissociation of E-cadherin/catenin complexes from the actin cytoskeleton via PI3K/Akt signaling (20). Several transcriptional repressors such as Snail, Slug, Twist or ZEB1 also initiate EMT involving loss of E-cadherin, up-regulation of N-cadherin and vimentin, then enhancing the cell migration motility and metastasis in PC (19). In this study, we demonstrated that vismodegib significantly increased the expression of E-cadherin and decreased the expression of N-cadherin and vimentin in androgen-independent PC cell lines such as PC-3 and C4-2B as well as androgen-dependent LNCaP in vitro. Our data suggested that vismodegib could inhibit EMT and may suppress the motility and metastasis in PCs.

Figure 5.
Figure 5.

In vivo tumor inhibitory effects of vismodegib. PC-3 and C4-2B were subcutaneously inoculated into right flanks of Balb/c nu/nu mice. After tumor growth was confirmed, mice were treated with oral administration of 50 mg/kg vismodegib or vehicle control (DMSO) every other week for 47 days. The change of relative tumor volume normalized with the tumor volume at the start of treatment is shown. *p<0.05.

Figure 6.
Figure 6.

Immunohistochemical analysis of PC-3 and C4-2B mouse tumors for EMT markers. After the treatment with vismodegib, tumor tissues were resected and stained with Caspase-3, E-cadherin, vimentin and N-cadherin to evaluate the inhibition of EMT by vismodegib treatment in vivo. These expressions were evaluated by staining score. Vis: vismodegib, *p<0.05.

Animal studies were performed using PC-3 and C4-2B to further clarify the antitumor effects of vismodegib in CRPC. Previously, animal experiments using vismodegib have been performed to suppress tumor growth in pancreatic cancer (21). There were some studies that showed that vismodegib in combination with other treatments such as gemcitabine significantly suppressed tumor growth in mice (22). In our study, vismodegib as a single agent was able to significantly suppress tumor growth even in every other weekly dosing to reduce treatment-related side effects.

Immunohistochemical staining of tumor tissues also showed that apoptosis was induced by vismodegib in vivo. It is suggested that EMT was suppressed, due to a decrease in vimentin, a mesenchymal marker. C4-2B tended to be inhibited cell growth both in vitro and in vivo more than PC-3. It is possible that the difference in the results between PC-3 and C4-2B was related to the presence of androgen receptor (AR). PC-3 and C4-2B are both CRPC cell lines, but PC-3 has no ARs. In adult solid tumors, Hedgehog signaling is composed of a variety of factors including numerous signaling pathways such as Wnt/β-catenin, FGF, Notch, TGF-β/BMP, and ARs (23). In this study, we focused on the Hedgehog signaling pathway alone, but other factors such as AR may also influence outcomes. Future studies of other Hedgehog-related factors may be the key to further antitumor effects.

In this study, the effects of vismodegib on PC cell growth suppression, apoptosis induction, and EMT suppression were demonstrated both in vitro and in vivo. For possible future clinical application, it is necessary to analyze the expression of downstream genes of SMO to achieve more effective treatment. There is a possibility that drug resistance can be delayed by combination with docetaxel (24), which is being studied, or combination with a PARP inhibitor (25), which has been clinically applied in recent years. Such studies will lead to the establishment of a treatment strategy for CRPC.

We would like to emphasize the study limitations. First, a mechanistic study should be performed to investigate how vismodegib affects Hedgehog signaling and suppresses tumor growth in CRPC. Next, the in vivo study using clinical samples are also demanding. Third, we need to determine the dose dependency of vismodegib in the antitumor effect. These limitations should be more assessed in our future study.

In summary, vismodegib inhibited cell proliferation and EMT and induced apoptosis, and showed anti-tumor effects in an animal study with vismodegib. Further mechanistic studies of Hedgehog signaling in PC are needed to pursue promising therapies for CRPC.

Acknowledgements

The Authors appreciate Mrs Naoki Yamada and Kento Nishimoto for their technical support.

Footnotes

  • Authors' Contributions

    Conception and design of the study: Katsumi Shigemura and Shian-Ying Sung. Experiment, acquisition of data and laboratory analysis: Aya Ishii and Koichi Kitagawa. Analysis and interpretation of data: all authors. Drafting the article: Katsumi Shigemura, Aya Ishii and Koichi Kitagawa. Critical revision for important intellectual content: all authors. Supervision: Kuan-Chou Chen, Chiang Ti-Te, Ming-Che Liu and Masato Fujisawa. Final approval of the submitted version: all Authors.

  • Conflicts of Interest

    The Authors declare they have no conflicts of interest.

  • Received June 14, 2020.
  • Revision received July 4, 2020.
  • Accepted July 6, 2020.

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Anti-tumor Effect of Hedgehog Signaling Inhibitor, Vismodegib, on Castration-resistant Prostate Cancer
AYA ISHII, KATSUMI SHIGEMURA, KOICHI KITAGAWA, SHIAN-YING SUNG, KUAN-CHOU CHEN, CHIANG YI-TE, MING-CHE LIU, MASATO FUJISAWA
Anticancer Research Sep 2020, 40 (9) 5107-5114; DOI: 10.21873/anticanres.14514
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