The anti-tumor effects of AZD4547 on ovarian cancer cells: differential responses based on c-Met and FGF19/FGFR4 expression.

BACKGROUND: The FGF/FGFR signaling pathway plays a critical role in human cancers. We analyzed the anti-tumor effect of AZD4547, an inhibitor targeting the FGF/FGFR pathway, in epithelial ovarian cancer (EOC) and strategies on overcoming AZD4547 resistance. METHODS: The effect of AZD4547 on cell viability/migration was evaluated and in vivo experiments in intraperitoneal xenografts using EOC cells and a patient-derived xenograft (PDX) model were performed. The effect of the combination of AZD4547 with SU11274, a c-Met-specific inhibitor, FGF19-specific siRNA, or an FGFR4 inhibitor was evaluated by MTT assay. RESULTS: AZD4547 significantly decreased cell survival and migration in drug-sensitive EOC cells but not drug-resistant cells. AZD4547 significantly decreased tumor weight in xenograft models of drug-sensitive A2780 and SKOV3ip1 cells and in a PDX with drug sensitivity but not in models with drug-resistant A2780-CP20 and SKOV3-TR cells. Furthermore, c-Met expression was high in SKOV3-TR and HeyA8-MDR cells, and co-administration of SU11274 and AZD4547 synergistically induced cell death. In addition, expressions of FGF19 and FGFR4 were high in A2780-CP20 cells. Combining AZD4547 with FGF19 siRNA or with a selective FGFR4 inhibitor led to significantly reduced cell proliferation in A2780-CP20 cells. CONCLUSIONS: This study showed that AZD4547 has significant anti-cancer effects in drug-sensitive cells and PDX models but not in drug-resistant EOC cells. In drug-resistant cells, the expression level of c-Met or FGF19/FGFR4 may be a predictive biomarker for AZD4547 treatment response, and a combination strategy of drugs targeting c-Met or FGF19/FGFR4 together with AZD4547 may be an effective therapeutic strategy for EOC.

Ulipristal acetate, a selective progesterone receptor modulator, induces cell death via inhibition of STAT3/CCL2 signaling pathway in uterine sarcoma.

Ulipristal acetate (UPA) is a selective progesterone receptor modulator and is used for the treatment of uterine leiomyoma (a benign tumor). Uterine sarcoma which is highly malignant cancer with a poor prognosis is clinically resembled with uterine leiomyoma. There has been no experimental research on the effect of UPA on uterine sarcoma. In this study, we examined the efficacy of UPA in uterine sarcoma with in vitro and in vivo animal models. Cytotoxicity of UPA was determined in uterine sarcoma cell lines (MES-SA, SK-UT-1, and SK-LMS-1). Apoptotic genes and signaling pathways affected by UPA were analyzed by complementary DNA (cDNA) microarray of uterine sarcoma cell lines and western blot, respectively. An in vivo efficacy of UPA was examined with uterine sarcoma cell line- and patient-derived xenograft (PDX) mice models. UPA inhibited cell growth in uterine sarcoma cell lines and primary culture cells from a PDX mouse (PDX-C). cDNA microarray analysis revealed that CCL2 was highly down-regulated by UPA. Phosphorylation and the total expression of STAT3 were inhibited by UPA. UPA also inhibited CCL2 and STAT3 in PDX-C. The inhibitory effect of UPA had not changed in the overexpression of PR and treatment of progesterone. In vivo efficacy studies with cell line-derived xenografts and a PDX model with leiomyosarcoma, a typical uterine sarcoma, demonstrated that UPA significantly decreased tumor growth. UPA had significant anti-tumor effects in uterine sarcoma through the inhibition of STAT3/CCL2 signaling pathway and might be a potential therapeutic agent to treat this disease.

Anti-cancer effect of fenbendazole-incorporated PLGA nanoparticles in ovarian cancer.

OBJECTIVE: Fenbendazole (FZ) has potential anti-cancer effects, but its poor water solubility limits its use for cancer therapy. In this study, we investigated the anti-cancer effect of FZ with different drug delivery methods on epithelial ovarian cancer (EOC) in both in vitro and in vivo models. METHODS: EOC cell lines were treated with FZ and cell proliferation was assessed. The effect of FZ on tumor growth in cell line xenograft mouse model of EOC was examined according to the delivery route, including oral and intraperitoneal administration. To improve the systemic delivery of FZ by converting fat-soluble drugs to hydrophilic, we prepared FZ-encapsulated poly(D,L-lactide-co-glycolide) acid (PLGA) nanoparticles (FZ-PLGA-NPs). We investigated the preclinical efficacy of FZ-PLGA-NPs by analyzing cell proliferation, apoptosis, and in vivo models including cell lines and patient-derived xenograft (PDX) of EOC. RESULTS: FZ significantly decreased cell proliferation of both chemosensitive and chemoresistant EOC cells. However, in cell line xenograft mouse models, there was no effect of oral FZ treatment on tumor reduction. When administered intraperitoneally, FZ was not absorbed but aggregated in the intraperitoneal space. We synthesized FZ-PLGA-NPs to obtain water solubility and enhance drug absorption. FZ-PLGA-NPs significantly decreased cell proliferation in EOC cell lines. Intravenous injection of FZ-PLGA-NP in xenograft mouse models with HeyA8 and HeyA8-MDR significantly reduced tumor weight compared to the control group. FZ-PLGA-NPs showed anti-cancer effects in PDX model as well. CONCLUSION: FZ-incorporated PLGA nanoparticles exerted significant anti-cancer effects in EOC cells and xenograft models including PDX. These results warrant further investigation in clinical trials.

High-throughput organo-on-pillar (high-TOP) array system for three-dimensional ex vivo drug testing.

The development of organoid culture technologies has triggered industrial interest in ex vivo drug test-guided clinical response prediction for precision cancer therapy. The three-dimensional culture encapsulated with basement membrane (BM) components is extremely important in establishing ex vivo organoids and drug sensitivity tests because the BM components confer essential structures resembling tumor histopathology. Although numerous studies have demonstrated three-dimensional culture-based drug screening methods, establishing a large-scale drug-screening platform with matrix-encapsulated tumor cells is challenging because the arrangement of microspots of a matrix-cell droplet onto each well of a microwell plate is inconsistent and difficult to standardize. In addition, relatively low scales and lack of reproducibility discourage the application of three-dimensional organoid-based drug screening data for precision treatment or drug discovery. To overcome these limitations, we manufactured an automated organospotter-integrated high-throughput organo-on-pillar (high-TOP) drug-screening platform. Our system is compatible with various extracellular matrices, including BM extract, Matrigel, collagen, and hydrogel. In addition, it can be readily utilized for high-content analyses by simply exchanging the bottom plates without disrupting the domes. Our system demonstrated considerable robustness, consistency, reproducibility, and biological relevancy in three-dimensional drug sensitivity analyses using Matrigel-encapsulated ovarian cancer cell lines. We also demonstrated proof-of-concept cases representing the clinical feasibility of high-TOP-assisted ex vivo drug tests linked to clinical chemo-response in ovarian cancer patients. In conclusion, our platform provides an automated and standardized method for ex vivo drug-sensitivity-guided clinical response prediction, suggesting effective chemotherapy regimens for patients with cancer.

Changes in subcellular localization of Lysyl-tRNA synthetase and the 67-kDa laminin receptor in epithelial ovarian cancer metastases.

BACKGROUND: Although lysyl-tRNA synthetase (KARS1) is predominantly located in the cytosol, it is also present in the plasma membrane where it stabilizes the 67-kDa laminin receptor (67LR). This physical interaction is strongly increased under metastatic conditions. However, the dynamic interaction of these two proteins and the turnover of KARS1 in the plasma membrane has not previously been investigated. OBJECTIVE: Our objective in this study was to identify the membranous location of KARS1 and 67LR and investigate if this changes with the developmental stage of epithelial ovarian cancer (EOC) and treatment with the inhibitor BC-K01. In addition, we evaluated the therapeutic efficacy of BC-K01 in combination with paclitaxel, as the latter is frequently used to treat patients with EOC. METHODS: Overall survival and prognostic significance were determined in EOC patients according to KARS1 and 67LR expression levels as determined by immunohistochemistry. Changes in the location and expression of KARS1 and 67LR were investigated in vitro after BC-K01 treatment. The effects of this compound on tumor growth and apoptosis were evaluated both in vitro and in vivo. RESULTS: EOC patients with high KARS1 and high 67LR expression had lower progression-free survival rates than those with low expression levels of these two markers. BC-K01 reduced cell viability and increased apoptosis in combination with paclitaxel in EOC cell xenograft mouse models. BC-K01 decreased membranous KARS1 expression, causing a reduction in 67LR membrane expression in EOC cell lines. BC-K01 significantly decreased in vivo tumor weight and number of nodules, especially when used in combination with paclitaxel. CONCLUSIONS: Co-localization of KARS1 and 67LR in the plasma membrane contributes to EOC progression. Inhibition of the KARS1-67LR interaction by BC-K01 suppresses metastasis in EOC.

Anticancer Activity of the Combination of Cabozantinib and Temozolomide in Uterine Sarcoma.

PURPOSE: To evaluate the anticancer effects of cabozantinib, temozolomide, and their combination in uterine sarcoma cell lines and mouse xenograft models. EXPERIMENTAL DESIGN: Human uterine sarcoma cell lines (SK-LMS-1, SK-UT-1, MES-SA, and SKN) were used to evaluate the anticancer activity of cabozantinib, temozolomide, and their combination. The optimal dose of each drug was determined by MTT assay. Cell proliferation and apoptosis were assessed 48 and 72 hours after the drug treatments. The tumor weights were measured in an SK-LMS-1 xenograft mouse model and a patient-derived xenograft (PDX) model of leiomyosarcoma treated with cabozantinib, temozolomide, or both. RESULTS: Given individually, cabozantinib and temozolomide each significantly decreased the growth and viability of cells. This inhibitory effect was more pronounced when cabozantinib (0.50 µmol/L) and temozolomide (0.25 or 0.50 mmol/L) were co-administered (P < 0.05). The combination of the drugs also significantly increased apoptosis in all cells. Moreover, this effect was consistently observed in patient-derived leiomyosarcoma cells. In vivo studies with SK-LMS-1 cell xenografts and the PDX model with leiomyosarcoma demonstrated that combined treatment with cabozantinib (5 mg/kg/d, per os administration) and temozolomide (5 mg/kg/d, per os administration) synergistically decreased tumor growth (both P < 0.05). CONCLUSIONS: The addition of cabozantinib to temozolomide offers synergistic anticancer effects in uterine sarcoma cell lines and xenograft mouse models, including PDX. These results warrant further investigation in a clinical trial.

Combination effect of poly (ADP-ribose) polymerase inhibitor and DNA demethylating agents for treatment of epithelial ovarian cancer.

OBJECTIVE: Poly (ADP)-ribose polymerase inhibitors (PARPi) are effective clinical agents for treatment of epithelial ovarian cancer (EOC) harboring BRCA mutations as well as those without BRCA mutations. In this study, we evaluate the efficacy of combined PARPi and DNA methyltransferase inhibitor (DNMTi) in EOCs. METHODS: Expression levels of DNMT1 and PARP1 proteins in EOC cells were assessed using western blot analysis and immunohistochemistry. To evaluate the effects of co-treatment of PARPi (olaparib) and DNMTi (5-azacitidine, 5-AZA), we performed cell proliferation, apoptosis, and wound-healing assays in EOC cells. In addition, we performed in vivo experiments using both cell-line and patient-derived xenograft (PDX) models of EOC. RESULTS: The combination of olaparib and 5-AZA significantly inhibited cell proliferation and migration and induced apoptosis compared with olaparib or 5-AZA alone in EOC cell lines including A2780, HeyA8, A2780-CP20, and HeyA8-MDR. Moreover, in vivo experiments with this combination showed significantly decreased weight and nodule numbers of tumors in cell-line xenograft models with A2780 cells and a PDX model compared with control, olaparib, and 5-AZA groups. As a potential mechanism, the expression of intracellular reactive oxygen species (ROS) and its related proteins, including p-ERK, NRF2, p-p38, HO-1, and γH2AX, was affected in EOC cells. CONCLUSIONS: Co-treatment with PARPi and DNMTi had a significant anti-tumor effect in EOC cells. This combination might be a potential therapeutic strategy for EOCs.

Establishment and preclinical application of a patient-derived xenograft model for uterine cancer.

BACKGROUND: The patient-derived xenograft (PDX) model is a promising translational platform for duplicating the characteristics of primary tumors. Here, we established and characterized PDX models of uterine cancer to demonstrate their utility for preclinical drug testing. MATERIALS AND METHODS: We generated PDX tumors surgically derived from 58 cases of uterine cancer. Subrenal capsule xenografts and primary tumors were compared using microscopic examination, short tandem repeat analyses, and targeted sequencing analyses. A phosphatidylinositol 3-kinase (PI3K) inhibitor was administered to mice whose PDX tumors harbored a PTEN deletion or PIK3CA mutation. We also generated an orthotopic PDX model using uterine horn implantation. RESULTS: Thirty-three (56.9%) PDXs were successfully generated and passaged to maintain tumors. The histological features of the PDX tumors were stable over subsequent passages. By contrast, the proportions of epithelial and mesenchymal components of carcinosarcoma PDX models varied by generation. Targeted sequencing analyses revealed that all mutated cancer-related genes were stable during establishment and subgrafting. Treatment with a PI3K inhibitor cased a significant decrease in tumor weight in the clear cell carcinoma PDX harboring a frameshift PTEN deletion (p = 0.049) and in the serous carcinoma PDX harboring a missense PI3KCA mutation (p = 0.003) compared with matched controls. We also successfully established orthotopic PDX models (3/3; 100.0%). CONCLUSIONS: The histological and genetic features of PDXs were similar to those of primary tumors. This model is a promising translational platform for preclinical testing of new anticancer drugs and will enable the personalized development of therapeutic options for uterine cancer.

Chloroquine reverses chemoresistance via upregulation of p21WAF1/CIP1 and autophagy inhibition in ovarian cancer.

Overcoming drug-resistance is a big challenge to improve the survival of patients with epithelial ovarian cancer (EOC). In this study, we investigated the effect of chloroquine (CQ) and its combination with cisplatin (CDDP) in drug-resistant EOC cells. We used the three EOC cell lines CDDP-resistant A2780-CP20, RMG-1 cells, and CDDP-sensitive A2780 cells. The CQ-CDDP combination significantly decreased cell proliferation and increased apoptosis in all cell lines. The combination induced expression of γH2AX, a DNA damage marker protein, and induced G2/M cell cycle arrest. Although the CQ-CDDP combination decreased protein expression of ATM and ATR, phosphorylation of ATM was increased and expression of p21WAF1/CIP1 was also increased in CQ-CDDP-treated cells. Knockdown of p21WAF1/CIP1 by shRNA reduced the expression of γH2AX and phosphorylated ATM and inhibited caspase-3 activity but induced ATM protein expression. Knockdown of p21WAF1/CIP1 partly inhibited CQ-CDDP-induced G2/M arrest, demonstrating that knockdown of p21WAF1/CIP1 overcame the cytotoxic effect of the CQ-CDDP combination. Ectopic expression of p21WAF1/CIP1 in CDDP-treated ATG5-shRNA/A2780-CP20 cells increased expression of γH2AX and caspase-3 activity, demonstrating increased DNA damage and cell death. The inhibition of autophagy by ATG5-shRNA demonstrated similar results upon CDDP treatment, except p21WAF1/CIP1 expression. In an in vivo efficacy study, the CQ-CDDP combination significantly decreased tumor weight and increased expression of γH2AX and p21WAF1/CIP1 in A2780-CP20 orthotopic xenografts and a drug-resistant patient-derived xenograft model of EOC compared with controls. These results demonstrated that CQ increases cytotoxicity in combination with CDDP by inducing lethal DNA damage by induction of p21WAF1/CIP1 expression and autophagy inhibition in CDDP-resistant EOC.

Anti-Cancer Activity of As4O6 and its Efficacy in a Series of Patient-Derived Xenografts for Human Cervical Cancer.

PURPOSE: To investigate the anti-cancer effects of tetraarsenic hexoxide (TAO, As4O6) in cervical cancer cell lines and in a series of patient-derived xenograft (PDX) mouse models. METHODS: Human cervical cancer cell lines, including HeLa, SiHa and CaSki, and human umbilical vein endothelial cells (HUVECs), were used to evaluate the anti-cancer activity of TAO. Cellular proliferation, apoptosis, and enzyme-linked immunosorbent assay (ELISA) for matrix metallopeptidase 2 (MMP-2) and 9 (MMP-9) were assessed. The tumor weights of the PDXs that were given TAO were measured. The PDXs included primary squamous cell carcinoma, primary adenocarcinoma, recurrent squamous cell carcinoma, and recurrent adenocarcinoma. RESULTS: TAO significantly decreased cellular proliferation and increased apoptosis in cervical cancer cell lines and HUVEC. The functional studies on the cytotoxicity of TAO revealed that it inhibited the activation of Akt and vascular endothelial growth factor receptor 2 (VEGFR2). It also decreased the concentrations of MMP-2 in both cervical cancer cell lines and HUVECs. Active caspase-3 and p62 were both increased by the treatment of TAO, indicating increased rates of apoptosis and decreased rates of autophagy, respectively. In vivo studies with PDXs revealed that TAO significantly decreased tumor weight for both primary squamous cell carcinoma and adenocarcinoma of the cervix. However, this anti-cancer effect was not seen in PDXs with recurrent cancers. Nevertheless, the combination of TAO with cisplatin significantly decreased tumor weight in PDX models for both primary and recurrent cancers. CONCLUSIONS: TAO exerted inhibitory effects on angiogenesis, cellular migration, and autophagy, and it showed stimulatory effects on apoptosis. Overall, it demonstrated anti-cancer effects in animal models for human cervical cancer.

Preclinical assessment of the VEGFR inhibitor axitinib as a therapeutic agent for epithelial ovarian cancer.

Axitinib, small molecule tyrosine kinase inhibitor, demonstrates anti-cancer activity for various solid tumors. We investigated anti-cancer effect of axitinib in epithelial ovarian cancer (EOC). We treated EOC cells (A2780, HeyA8, RMG1, and HeyA8-MDR) with axitinib to evaluate its effects on cell viabilty, apoptosis and migration. Western blots were performed to assess VEGFR2, ERK, and AKT levels, and ELISA and FACS to evaluate apoptosis according to axitinib treatment. In addition, in vivo experiments in xenografts using A2780, RMG1, and HeyA8-MDR cell lines were performed. We repeated the experiment with patient-derived xenograft models (PDX) of EOC. Axitinib significantly inhibited cell survival and migration, and increased apoptosis in EOC cells. The expression of VEGFR2 and phosphorylation of AKT and ERK in A2780, RMG1, and HeyA8 were decreased with axitinib treatment in dose-dependent manner, but not in HeyA8-MDR. In in vivo experiments, axitinib significantly decreased tumor weight in xenograft models of drug-sensitive (A2780), and clear cell carcinoma (RMG1) and PDX models for platinum sensitive EOC compared to control, but was not effective in drug-resistant cell line (HeyA8-MDR) or heavily pretreated refractory PDX model. Axitinib showed significant anti-cancer effects in drug-sensitive or clear cell EOC cells via inhibition of VEGFR signals associated with cell proliferation, apoptosis and migration, but not in drug-resistant cells.

Pharmacogenomic analysis of patient-derived tumor cells in gynecologic cancers.

BACKGROUND: Gynecologic malignancy is one of the leading causes of mortality in female adults worldwide. Comprehensive genomic analysis has revealed a list of molecular aberrations that are essential to tumorigenesis, progression, and metastasis of gynecologic tumors. However, targeting such alterations has frequently led to treatment failures due to underlying genomic complexity and simultaneous activation of various tumor cell survival pathway molecules. A compilation of molecular characterization of tumors with pharmacological drug response is the next step toward clinical application of patient-tailored treatment regimens. RESULTS: Toward this goal, we establish a library of 139 gynecologic tumors including epithelial ovarian cancers (EOCs), cervical, endometrial tumors, and uterine sarcomas that are genomically and/or pharmacologically annotated and explore dynamic pharmacogenomic associations against 37 molecularly targeted drugs. We discover lineage-specific drug sensitivities based on subcategorization of gynecologic tumors and identify TP53 mutation as a molecular determinant that elicits therapeutic response to poly (ADP-Ribose) polymerase (PARP) inhibitor. We further identify transcriptome expression of inhibitor of DNA biding 2 (ID2) as a potential predictive biomarker for treatment response to olaparib. CONCLUSIONS: Together, our results demonstrate the potential utility of rapid drug screening combined with genomic profiling for precision treatment of gynecologic cancers.

Anti-Tumor Effects of Wee1 Kinase Inhibitor with Radiotherapy in Human Cervical Cancer.

Although the concurrent use of a chemotherapeutic agent and radiotherapy improves survival in patients with locally advanced or recurrent cervical cancer, severe side effects related to chemotherapy are frequent and may result in a low quality of life for the patients. In this study, we investigated the effects of a combination of Wee1 inhibitor (AZD1775) and irradiation in cervical cancer. In vitro effects of AZD1775 with irradiation in human cervical cancer cells were assessed by clonogenic survival and apoptosis assays. The effects on DNA damage response signaling and the cell cycle were also explored. Tumor growth delay was evaluated to investigate the in vivo effects of AZD1775 with irradiation in cervical cancer mouse models, including xenografts and patient-derived xenografts (PDXs). The co-treatment of AZD1775 and irradiation significantly decreased clonogenic survival and increased apoptosis in cervical cancer cells. These effects were associated with G2 checkpoint abrogation which resulted in persistent DNA damage. Both in the xenografts and the PDXs, the co-treatment significantly decreased tumor growth compared tothe irradiation alone (p < 0.05). These results demonstrate that the Wee1 inhibitor (AZD1775) can be considered as a potential alternative as a radiosensitizer in cervical cancer instead of a chemotherapeutic agent such as cisplatin.

The anti-cancer effects of itraconazole in epithelial ovarian cancer.

We assessed the anti-proliferative activity of itraconazole using an EOC cell line (SKOV3ip1) and endothelial cell lines (HUVEC & SVEC4-10). We also examined angiogenesis (VEGFR2, p-ERK, p-PLCr1/2), hedgehog (Gli1, Ptch1, SMO), and mTOR (pS6K1) signaling pathways to determine the mechanism of action of itraconazole. Furthermore, we evaluated the synergistic effects of itraconazole and paclitaxel using orthotopic mouse models with established EOC cells (SKOV3ip1 or HeyA8) as well as patient-derived xenografts (PDXs). Itraconazole treatment inhibited proliferation of endothelial cells in a dose-dependent manner, but had no effect on EOC cells. The endothelial cell antiproliferative effect was associated with inhibition of hedgehog, and mTOR pathways and angiogenesis. In xenograft models of EOC using SKOV3ip1 or HeyA8, mice treated with the combination of itraconazole and paclitaxel had significantly decreased tumor weight than the control, paclitaxel-alone, or itraconazole-alone groups. Tissue derived from these tumors had significantly lower microvessel density than tissue from the other groups as well as hedgehog and mTOR pathway inhibition. We confirmed those effects in two EOC PDX models. These results suggest that itraconazole selectively inhibits endothelial cells rather than cancer cells by targeting multiple pathways including hedgehog, and mTOR pathways and angiogenesis.

Patient-Derived Xenograft Models of Epithelial Ovarian Cancer for Preclinical Studies.

PURPOSE: Patient-derived tumor xenografts (PDXs) can provide more reliable information about tumor biology than cell line models. We developed PDXs for epithelial ovarian cancer (EOC) that have histopathologic and genetic similarities to the primary patient tissues and evaluated their potential for use as a platform for translational EOC research. MATERIALS AND METHODS: We successfully established PDXs by subrenal capsule implantation of primary EOC tissues into female BALB/C-nude mice. The rate of successful PDX engraftment was 48.8% (22/45 cases). Hematoxylin and eosin staining and short tandem repeat analysis showed histopathological and genetic similarity between the PDX and primary patient tissues. RESULTS: Patients whose tumors were successfully engrafted in mice had significantly inferior overall survival when compared with those whose tumors failed to engraft (p=0.040). In preclinical tests of this model, we found that paclitaxel-carboplatin combination chemotherapy significantly deceased tumor weight in PDXs compared with the control treatment (p=0.013). Moreover, erlotinib treatment significantly decreased tumor weight in epidermal growth factor receptor-overexpressing PDX with clear cell histology (p=0.023). CONCLUSION: PDXs for EOC with histopathological and genetic stability can be efficiently developed by subrenal capsule implantation and have the potential to provide a promising platform for future translational research and precision medicine for EOC.

Proton Pump Inhibition Enhances the Cytotoxicity of Paclitaxel in Cervical Cancer.

PURPOSE: This study was conducted to investigate whether a proton pump inhibitor (PPI) could enhance chemosensitivity via the inhibition of vacuolar-type H+ ATPase (V-ATPase) in cervical cancer. MATERIALS AND METHODS: The expression of V-ATPase was evaluated in 351 formalin-fixed, paraffin-embedded human cervical cancer tissues using immunohistochemistry and compared with clinicopathologic risk factors for disease prognosis. The influence of cell proliferation and apoptosis following V-ATPase siRNA transfection or esomeprazole pretreatment was assessed in cervical cancer cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and enzyme-linked immunosorbent assay, respectively. RESULTS: Immunohistochemical analysis revealed that V-ATPase was expressed in about 60% of cervical cancer tissue samples (211/351), and the expression was predominantly found in adenocarcinoma histology (p=0.016). Among patients with initially bulky cervical cancer (n=89), those with V-ATPase expression had shorter disease-free survival (p=0.005) and overall survival (p=0.023). Co-treatment with V-ATPase siRNA or esomeprazole with paclitaxel significantly decreased the cell proliferation of cervical cancer cell lines, including HeLa and INT407, compared to cell lines treated with paclitaxel alone (p < 0.01). Moreover, V-ATPase siRNA or esomeprazole followed by paclitaxel significantly increased the expression of active caspase-3 in these cells compared to cells treated with paclitaxel alone (both, p < 0.05). CONCLUSION: V-ATPase was predominantly expressed in cervical adenocarcinoma, and the expression of V-ATPases was associated with poor prognosis. The inhibition of V-ATPase via siRNA or PPI (esomeprazole) might enhance the chemosensitivity of paclitaxel in cervical cancer cells.

c-MET as a Potential Therapeutic Target in Ovarian Clear Cell Carcinoma.

In this study, we investigated the therapeutic effects of c-MET inhibition in ovarian clear cell carcinoma (OCCC). Expression levels of c-MET in the epithelial ovarian cancers (EOCs) and normal ovarian tissues were evaluated using real-time PCR. To test the effects of c-MET inhibitors in OCCC cell lines, we performed MTT and apoptosis assays. We used Western blots to evaluate the expression of c-MET and its down-stream pathway. In vivo experiments were performed to test the effects of c-MET inhibitor on tumor growth in orthotopic mouse xenografts of OCCC cell line RMG1 and a patient-derived tumor xenograft (PDX) model of OCCC. c-MET expression was significantly greater in OCCCs compared with serous carcinomas and normal ovarian tissues (p < 0.001). In in vitro study, inhibition of c-MET using c-MET inhibitors (SU11274 or crizotinib) significantly decreased the proliferation, and increased the apoptosis of OCCC cells. SU11274 decreased expression of the p-c-MET proteins and blocked the phosphorylation of down-stream proteins Akt and Erk. Furthermore, SU11274 treatment significantly decreased the in vivo tumor weight in xenograft models of RMG1 cell and a PDX model for OCCC compared to control (p = 0.004 and p = 0.009, respectively).

HER2 as a novel therapeutic target for cervical cancer.

Surgery and radiation are the current standard treatments for cervical cancer. However, there is no effective therapy for metastatic or recurrent cases, necessitating the identification of therapeutic targets. In order to create preclinical models for screening potential therapeutic targets, we established 14 patient-derived xenograft (PDX) models of cervical cancers using subrenal implantation methods. Serially passaged PDX tumors retained the histopathologic and genomic features of the original tumors. Among the 9 molecularly profiled cervical cancer patient samples, a HER2-amplified tumor was detected by array comparative genomic hybridization and targeted next-generation sequencing. We confirmed HER2 overexpression in the tumor and serially passaged PDX. Co-administration of trastuzumab and lapatinib in the HER2-overexpressed PDX significantly inhibited tumor growth compared to the control. Thus, we established histopathologically and genomically homologous PDX models of cervical cancer using subrenal implantation. Furthermore, we propose HER2 inhibitor-based therapy for HER2-amplified cervical cancer refractory to conventional therapy.

Proton pump inhibitors enhance the effects of cytotoxic agents in chemoresistant epithelial ovarian carcinoma.

This study was designed to investigate whether proton pump inhibitors (PPI, V-ATPase blocker) could increase the effect of cytotoxic agents in chemoresistant epithelial ovarian cancer (EOC). Expression of V-ATPase protein was evaluated in patients with EOC using immunohistochemistry, and patient survival was compared based on expression of V-ATPase mRNA from a TCGA data set. In vitro, EOC cell lines were treated with chemotherapeutic agents with or without V-ATPase siRNA or PPI (omeprazole) pretreatment. Cell survival and apoptosis was assessed using MTT assay and ELISA, respectively. In vivo experiments were performed to confirm the synergistic effect with omeprazole and paclitaxel on tumor growth in orthotopic and patient-derived xenograft (PDX) mouse models. Expression of V-ATPase protein in ovarian cancer tissues was observed in 44 patients (44/59, 74.6%). Higher expression of V-ATPase mRNA was associated with poorer overall survival in TCGA data. Inhibition of V-ATPase by siRNA or omeprazole significantly increased cytotoxicity or apoptosis to paclitaxel in chemoresistant (HeyA8-MDR, SKOV3-TR) and clear cell carcinoma cells (ES-2, RMG-1), but not in chemosensitive cells (HeyA8, SKOV3ip1). Moreover, the combination of omeprazole and paclitaxel significantly decreased the total tumor weight compared with paclitaxel alone in a chemoresistant EOC animal model and a PDX model of clear cell carcinoma. However, this finding was not observed in chemosensitive EOC animal models. These results show that omeprazole pretreatment can increase the effect of chemotherapeutic agents in chemoresistant EOC and clear cell carcinoma via reduction of the acidic tumor microenvironment.