Ovarian Cancer Tumor Growth Research Articles

ASK1-dependent endothelial cell activation is critical in ovarian cancer growth and metastasis.

We have recently reported that tumor-associated macrophages (TAMs) promote early transcoelomic metastasis of ovarian cancer by facilitating TAM-ovarian cancer cell spheroid formation. ASK1 is known to be important for macrophage activation and inflammation-mediated tumorigenesis. In the present study, we show that ASK1 deficiency attenuates TAM-spheroid formation and ovarian cancer progression in an orthotopic ovarian cancer model. Interestingly, ASK1 in stroma, but not in TAMs, is critical for peritoneal tumor growth of ovarian cancer. Moreover, overexpression of an ASK1 inhibitory protein (suppressor of cytokine signaling-1; SOCS1) in vascular endothelium attenuates vascular permeability, TAM infiltration, and ovarian cancer growth. Mechanistically, we show that ASK1 mediates degradation of endothelial junction protein VE-cadherin via a lysosomal pathway to promote macrophage transmigration. Importantly, a pharmacological ASK1 inhibitor prevents tumor-induced vascular leakage, macrophage infiltration, and tumor growth in two mouse models. Since transcoelomic metastasis is also associated with many other cancers, such as pancreatic and colon cancers, our study provides ASK1 as a therapeutic target for the treatment of ovarian cancer and other transcoelomic metastasis cancers.

RETRACTED: Amentoflavone suppresses tumor growth in ovarian cancer by modulating Skp2

AimOvarian cancer is one of most common malignancies in women and is associated with high reoccurrence rate and poor prognosis. This study is designed to investigate the anti-tumor effects of amentoflavone (AF), one of the major active ingredients of S. tamariscina, against ovarian cancer. Materials and methodsHuman ovarian cancer cell lines SKOV3 and OVCAR-3 were used in this study. The effect of AF on cell viability was examined by CCK-8 assay. Cell apoptosis and cell cycle distribution was determined by flow cytometry. ROS generation was detected using fluorescent staining. Expression of signaling molecules was determined by western blots. Xenograft model was established to evaluate the therapeutic efficacy of AF in vivo. Key findingsOur results showed that AF could significantly suppress cell proliferation, induce apoptosis and block cell cycle progression. Mechanistically, downregulation of S-phase kinase protein 2 (Skp2) by AF contributed to its anti-tumor effect against ovarian cancer. Furthermore, our results showed that AF repressed the expression of Skp2 through ROS/AMPK/mTOR signaling. The anti-tumor effect of AF against ovarian cancer was also confirmed in a xenograft animal model. SignificanceOverall, our present findings highlighted the potential of AF in the treatment of ovarian cancer. Moreover, our study also provided a new elucidation regarding the anti-tumor mechanisms of AF.

Epigenetic modifiers upregulate MHC II and impede ovarian cancer tumor growth.

Expression of MHC class II pathway proteins in ovarian cancer correlates with prolonged survival. Murine and human ovarian cancer cells were treated with epigenetic modulators – histone deacetylase inhibitors and a DNA methyltransferase inhibitor. mRNA and protein expression of the MHC II pathway were evaluated by qPCR and flow cytometry. Treatment with entinostat and azacytidine of ID8 cells in vitro increased mRNA levels of Cd74, Ciita, and H2-Aa, H2-Eb1. MHC II and CD74 protein expression were increased after treatment with either agent. A dose dependent response in mRNA and protein expression was seen with entinostat. Combination treatment showed higher MHC II protein expression than with single agent treatment. In patient derived xenografts, CIITA, CD74, and MHC II mRNA transcripts were significantly increased after combination treatment. Expression of MHC II on ovarian tumors in MISIIR-Tag mice was increased with both agents relative to control. Combination treatment significantly reduced ID8 tumor growth in immune-competent mice. Epigenetic treatment increases expression of MHC II on ovarian cancer cells and impedes tumor growth. This approach warrants further study in ovarian cancer patients.

Serine hydroxymethyl transferase 1 stimulates pro-oncogenic cytokine expression through sialic acid to promote ovarian cancer tumor growth and progression

High-grade serous (HGS) ovarian cancer accounts for 90% of all ovarian cancer-related deaths. However, factors that drive HGS ovarian cancer tumor growth have not been fully elucidated. In particular, comprehensive analysis of the metabolic requirements of ovarian cancer tumor growth has not been performed. By analyzing The Cancer Genome Atlas mRNA expression data for HGS ovarian cancer patient samples, we observed that six enzymes of the folic acid metabolic pathway were overexpressed in HGS ovarian cancer samples compared with normal ovary samples. Systematic knockdown of all six genes using short hairpin RNAs (shRNAs) and follow-up functional studies demonstrated that serine hydroxymethyl transferase 1 (SHMT1) was necessary for ovarian cancer tumor growth and cell migration in culture and tumor formation in mice. SHMT1 promoter analysis identified transcription factor Wilms tumor 1 (WT1) binding sites, and WT1 knockdown resulted in reduced SHMT1 transcription in ovarian cancer cells. Unbiased large-scale metabolomic analysis and transcriptome-wide mRNA expression profiling identified reduced levels of several metabolites of the amino sugar and nucleotide sugar metabolic pathways, including sialic acid N-acetylneuraminic acid (Neu5Ac), and downregulation of pro-oncogenic cytokines interleukin-6 and 8 (IL-6 and IL-8) as unexpected outcomes of SHMT1 loss. Overexpression of either IL-6 or IL-8 partially rescued SHMT1 loss-induced tumor growth inhibition and migration. Supplementation of culture medium with Neu5Ac stimulated expression of IL-6 and IL-8 and rescued the tumor growth and migratory phenotypes of ovarian cancer cells expressing SHMT1 shRNAs. In agreement with the ovarian tumor-promoting role of Neu5Ac, treatment with Neu5Ac-targeting glycomimetic P-3Fax-Neu5Ac blocked ovarian cancer growth and migration. Collectively, these results demonstrate that SHMT1 controls the expression of pro-oncogenic inflammatory cytokines by regulating sialic acid Neu5Ac to promote ovarian cancer tumor growth and migration. Thus, targeting of SHMT1 and Neu5Ac represents a precision therapy opportunity for effective HGS ovarian cancer treatment.

CDDO-Me reveals USP7 as a novel target in ovarian cancer cells.

Deubiquitinating enzyme USP7 has been involved in the pathogenesis and progression of several cancers. Targeting USP7 is becoming an attractive strategy for cancer therapy. In this study, we identified synthetic triterpenoid C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) as a novel inhibitor of USP7 but not of other cysteine proteases such as cathepsin B and cathepsin D. CDDO-Me inhibits USP7 activity via a mechanism that is independent of the presence of α, β-unsaturated ketones. Molecular docking studies showed that CDDO-Me fits well in the ubiquitin carboxyl terminus-binding pocket on USP7. Given that CDDO-Me is known to be effective against ovarian cancer cells, we speculated that CDDO-Me may target USP7 in ovarian cancer cells. We demonstrated that ovarian cancer cells have higher USP7 expression than their normal counterparts. Knockdown of USP7 inhibits the proliferation of ovarian cancer cells both in vitro and in vivo. Using the cellular thermal shift assay and the drug affinity responsive target stability assay, we further demonstrated that CDDO-Me directly binds to USP7 in cells, which leads to the decrease of its substrates such as MDM2, MDMX and UHRF1. CDDO-Me suppresses ovarian cancer tumor growth in an xenograft model. In conclusion, we demonstrate that USP7 is a novel target of ovarian cancer cells; targeting USP7 may contribute to the anti-cancer effect of CDDO-Me. The development of novel USP7 selective compounds based on the CDDO-Me-scaffold warrants further investigation.

Synergistic suppression effect on tumor growth of ovarian cancer by combining cisplatin with a manganese superoxide dismutase-armed oncolytic adenovirus.

Gene therapy on the basis of oncolytic adenovirus is a novel approach for human cancer therapeutics. We aim to investigate whether it will synergistically reinforce their antiovarian cancer activities when the combined use of ZD55-manganese superoxide dismutase (MnSOD) and cisplatin was performed. The experiments in vitro showed that ZD55-MnSOD enhances cisplatin-induced apoptosis and causes remarkable ovarian cancer cell death. Apoptosis induction by treatment with ZD55-MnSOD and/or cisplatin was detected in SKOV-3 by apoptotic cell staining, flow cytometry, and western blot analysis. In addition, the cytotoxicity caused by ZD55-MnSOD to normal cells was examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay and western blot analysis. Animal experiment further confirmed that combination of ZD55-MnSOD and cisplatin achieved significant inhibition of SKOV-3 ovarian tumor xenografted growth. In summary, we have demonstrated that ZD55-MnSOD can sensitize human ovarian cancer cells to cisplatin-induced cell death and apoptosis in vitro and in vivo. These findings indicate that the combined treatment with ZD55-MnSOD and cisplatin could represent a rational approach for antiovarian cancer therapy.

Abstract 2705: A computational algorithm to predict tumor growth and cancer stem cell proportion in-vitro and in-vivo from single-cell observations

Abstract We have developed a new mathematical algorithm and corresponding computer software that uses observations of single cell behavior to predict the growth of the cancer stem-like cell proportion in larger cancer cell groups. Cancer stem-like cells (CSCs) have been implicated in ovarian cancer tumor growth, chemotherapy resistance, and disease recurrence. Aldehyde dehydrogenase (ALDH) is a primary discriminator of CSCs in ovarian cancer as well as in other cancer types. Unfortunately, the rarity and ability of CSCs to rapidly differentiate makes them difficult to study in-vitro and in-vivo. Microfluidic capture devices now allow us to grow and evaluate single ovarian cancer cells in isolated culture. We deployed microfluidic devices to evaluate the different self-renewal and asymmetric division patterns of ALDH+ and ALDH(-) ovarian CSCs. We analyzed data gathered from arrays of single cell chamber observations and found that purified ALDH+ cells were more proliferative than ALDH(-) cells in both cell-line (n = 112, p < 0.001) and primary (n = 41, p = 0.008) ovarian cancer specimens. Importantly, ALDH+ cells could produce both ALDH+ and ALDH(-) cells, whereas ALDH- cells were only able to produce ALDH- cells. Based on this hierarchy-defining data, we developed an easy to use computer algorithm on the freely-available software R to predict cancer cell population growth in-vitro and in-vivo. In our algorithm, size changes of cell populations are simulated by drawing from observed events. We compared the predictions from our hybrid microfluidics chip and computational algorithm with validation experiments in-vivo and in-vitro for both cell line and primary ovarian cancer cells. We found a superb correlation between observed and predicted in-vitro total and CSC counts for both cell line and primary ovarian cancer cells (correlation r = 0.98, p < 0.0001). Furthermore, this approach appropriately predicted changes in cell growth in the presence of the CSC-promoting growth factor EGFL6 both in vitro and in vivo, over time frames of up to 28 days. The single cell division based sampling algorithm developed here allows for rapid and inexpensive means to predict in-vivo ovarian tumor growth based on microfluidic chip culture and can easily be adapted to evaluate new therapeutic options across other cancer subtypes. Citation Format: Alexander T. Pearson, Patrick Ingram, Shoumei Bai, Euisik Yoon, Trachette Jackson, Ronald J. Buckanovich. A computational algorithm to predict tumor growth and cancer stem cell proportion in-vitro and in-vivo from single-cell observations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2705.

Pharmacological focal adhesion kinase (FAK) inhibition suppresses cisplatin-resistant human ovarian carcinoma growth

Objectives: FAK is a cytoplasmic protein tyrosine kinase promoting ovarian cancer tumor growth and metastasis. Phase I and II clinical trials are testing small molecule inhibitors to FAK in different cancers. Determination of cancer cell susceptibility to FAK inhibition is an important yet undefined parameter. We test the hypothesis that elevated FAK activity contributes to the growth of aggressive cisplatin (CP)–resistant ovarian cancer. Methods: Growth of paired CP-sensitive (A2780 and OVCAR10) and CP-resistant (A2780-CP and OVCAR10-CP) human ovarian cells was evaluated in adherent, nonadherent, and methylcellulose spheroid culture in the presence or absence of 100 nM concentration of FAK inhibitor (FAKi). Quantification of cancer stemlike cells (CSCs) and cell cycle analyses was performed using flow cytometry for aldehyde dehydrogenase (ALDH) activity and propidium iodide DNA staining, respectively. Cell apoptosis was evaluated with Annexin-V and (7-aminoactinomycin D) 7-AAD staining. Changes in cellular protein expression were determined with quantitative immunoblotting. Intraperitoneal tumor growth in mice was evaluated for combined inhibitory effects of CP and FAKi administration. Results: FAK tyrosine phosphorylation and CSC protein markers were elevated in CP-resistant cells, which was reversed by FAKi addition. On immunoblotting, the levels of the transcription factor OCT4, ALDH, and the cell surface protein N-cadherin were increased in CP-resistant cells. FAKi addition selectively prevented CP-resistant cell growth as spheroids. The combination of FAKi with CP exhibited a combined effect on the CP-resistant cells, with significant decreases in spheroid and tumor growth compared with either agent alone. When comparing the percentage of CSCs with flow cytometry, FAKi addition dramatically reduced the ALDH-positive cell population in both parental and CP-resistant cells. This was associated with a G1 cell cycle growth phase arrest, decreased cyclin D protein expression, but not increased cell apoptosis. (See Fig. 1.) Conclusions: CSCs are implicated in the molecular mechanisms of cisplatin-resistance, and we find that FAK inhibition prevents CP-resistant CSC growth. Because FAKi therapy is well tolerated with limited adverse events, our results support further testing of FAKi in either primary or recurrent settings of CP-resistant ovarian cancer.

Everolimus exhibits anti-tumorigenic activity in obesity-induced ovarian cancer.

Everolimus inhibits mTOR kinase activity and its downstream targets by acting on mTORC1 and has anti-tumorigenic activity in ovarian cancer. Clinical and epidemiologic data find that obesity is associated with worse outcomes in ovarian cancer. In addition, obesity leads to hyperactivation of the mTOR pathway in epithelial tissues, suggesting that mTOR inhibitors may be a logical choice for treatment in obesity-driven cancers. However, it remains unclear if obesity impacts the effect of everolimus on tumor growth in ovarian cancer. The present study was aimed at evaluating the effects of everolimus on cytotoxicity, cell metabolism, apoptosis, cell cycle, cell stress and invasion in human ovarian cancer cells. A genetically engineered mouse model of serous ovarian cancer fed a high fat diet or low fat diet allowed further investigation into the inter-relationship between everolimus and obesity in vivo. Everolimus significantly inhibited cellular proliferation, induced cell cycle G1 arrest and apoptosis, reduced invasion and caused cellular stress via inhibition of mTOR pathways in vitro. Hypoglycemic conditions enhanced the sensitivity of cells to everolimus through the disruption of glycolysis. Moreover, everolimus was found to inhibit ovarian tumor growth in both obese and lean mice. This reduction coincided with a decrease in expression of Ki-67 and phosphorylated-S6, as well as an increase in cleaved caspase 3 and phosphorylated-AKT. Metabolite profiling revealed that everolimus was able to alter tumor metabolism through different metabolic pathways in the obese and lean mice. Our findings support that everolimus may be a promising therapeutic agent for obesity-driven ovarian cancers.

MicroRNA-873 mediates multidrug resistance in ovarian cancer cells by targeting ABCB1.

Ovarian cancer is commonly treated with cisplatin and paclitaxel combination chemotherapy; however, ovarian cancer cells often develop resistance to these drugs. Increasingly, microRNAs (miRNAs) including miR-873 have been implicated in drug resistance in many cancers, but the role of miR-873 in ovarian cancer remains unknown. MTT cell viability assays revealed that the sensitivities of ovarian cancer lines to cisplatin and paclitaxel increased following transfection with miR-873 (P < 0.05). After predicting the miR-873 binding region in the 3'-untranslated region of ABCB1, dual-luciferase reporter assay confirmed this prediction. RT-PCR and Western blotting revealed that MDR1 expression was significantly downregulated after transfection with miR-873 and upregulated after transfection with anti-miR-873 at both mRNA and protein levels compared to negative controls (P < 0.05). Experiments in a mouse xenograft model confirmed that intratumoral administration of miR-873 could enhance the efficacy of cisplatin in inhibiting tumor growth in ovarian cancer in vivo (P < 0.05). ABCB1 overexpression reduced sensitivities of ovarian cancer lines OVCAR3 and A2780 to cisplatin and paclitaxel, which can be reversed by miR-873 mimic transfection (P < 0.05). In summary, we demonstrated that overexpression of miR-873 increased the sensitivity of ovarian cancer cells to cisplatin and paclitaxel by targeting MDR1 expression. Our findings suggest that combination therapies with chemotherapy agents and miR-873 may suppress drug resistance in ovarian cancer.

Abstract B46: Use of a novel orthotopic ovarian cancer transplant patient derived xenograft model as a preclinical platform for bench to bedside research.

Abstract Introduction: Treatment for cancer is moving from a “one size fits all” to a personalized and targeted approach addressing the unique biology of each patients' tumor. Preclinical research in ovarian cancer (OVCA) has relied on established cell lines that have recently been shown to have marked differences in molecular profiles when compared to ovarian tumors from the TCGA. Patient derived xenografts (PDXs) are emerging as a reliable preclinical model that can recapitulate the principal characteristics of the patients' original tumor while remaining biologically stable while passaged in mice. We developed an orthotopic model that involves transplanting tumor directly to the fallopian tube/ovary in order to accurately study ovarian cancer tumorigenicity and metastasis in the native environment. Experimental Procedures: Fresh OVCA tumor was transplanted orthotopically to the fallopian tube/ovary of NSG 5-8 week female mice. Tumor growth was followed over time with ultrasound. Tumors were evaluated by IHC, genomic and proteomic analysis. Alu II probe staining was performed to evaluate human stroma content. DNA sequencing analysis was performed using a 153 ovarian cancer gene panel, which includes all genes relevant to OVCA, including homologous recombination and all actionable genes. Reverse Phase Protein Array Analysis (RPPA) was used to evaluate signaling pathway activation. Several primary ovarian tumor cultures were also developed from the patients' tumor for mechanistic studies. Results: To date, we have transplanted tumor from 18 primary, 4 interval, and 6 recurrent ovarian debulking surgeries using an orthotopic ovarian tumor transplant approach with an 90% success rate in generating tumors in mouse passage 1 (MP1) and 100% in generating MP2 and MP3. The mean time for tumors to reach ~1 cm (first passage in mice) was 8 weeks and ~6 weeks when tumors were then passaged again (MP2/MP3). We have generated 12 BRCA1/2 deleterious mutation positive carrier PDXs. Alu staining of PDXs demonstrated human cells in the stroma. We have generated several primary tumor cell lines from the original tumors/PDXs. These primary cells express ovarian epithelial markers CK7, and PAX8. Targeted DNA sequencing analysis showed that P53 and BRCA1/2 pathogenic mutations were highly conserved from the patient tumor, to the PDXs (MP1-3) and primary tumor cell lines. We evaluated 280 phospho/total proteins in our tumor samples by RPPA. Unsupervised cluster analysis of 24 patient tumors and correlating PDX (MP1-3) and 6 cell lines showed that several parent tumors clustered together with their MP1-3 PDXs. WO-2-1 BRCA 2 mutant PDXs were sensitive to platinum treatment demonstrating a response similar to that noted in the original patient after platinum chemotherapy. Conclusions: Although technically more challenging the orthotopic transplantation technique is feasible in generating ovarian cancer PDX models that more closely resemble the natural environment for ovarian cancer tumor growth and metastasis. PDXs maintain consistent gene alterations and signaling pathway activation to the original patient tumor. Given PDXs maintain the characteristics of the patients' original tumor, they are excellent models to study therapeutic response. Citation Format: Erin George, Hyoung Kim, Clemens Krepler, Janos Tanyi, Zhi Wei, Katrin Sproesser, Patricia Brafford, Marilda Beqiri, Adina-Monica Vultur, Rachel Lee, Mark Morgan, Ronny Drapkin, Tan Ince, Meenhard Herlyn, Fiona Simpkins. Use of a novel orthotopic ovarian cancer transplant patient derived xenograft model as a preclinical platform for bench to bedside research. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B46.

Abstract 4382: Anti-human LSR monoclonal antibody inhibits tumor growth of ovarian cancer directly

Abstract Ovarian cancer is the most lethal gynecologic malignancy; thus developing new treatment options is urgently required. Molecular targeted therapies for cancers, which are generally more tolerable than widely used cytotoxic agents, have shown highly specific inhibition of target molecules. We previously identified bone marrow stromal antigen 2 (BST2) as an endometrial cancer antigen using iTRAQ-based quantitative proteomic technology focused on cell surface membrane proteins, and also demonstrated the usefulness of an anti-BST2 monoclonal antibody (mAb) for endometrial cancer. In this study, we aimed to identify a new ovarian cancer antigen. We also aimed to develop a novel monoclonal antibody (mAb) and evaluate its preclinical efficacy against ovarian cancer. To identify a new ovarian cancer antigen, cell surface membrane proteins of normal ovarian epithelial and ovarian cancer cell lines were analyzed by iTRAQ-based proteomic technology. As the new therapeutic target for ovarian cancer, we identified lipolysis-stimulated lipoprotein receptor (LSR) which had one of the largest significant differences in protein level between normal ovarian epithelial and ovarian cancer cell lines. Immunohistochemical analysis showed that the overall survival of ovarian serous carcinoma patients with high LSR expression was significantly shorter than those with low LSR (p = 0.0293). We newly developed anti-LSR mAb and investigated its preclinical efficacy. Anti-LSR mAb showed significant in vivo inhibition of tumor growth against a xenograft model of hLSR-positive ovarian cancer in an antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) independent manner (p = 0.0001). And anti-LSR mAb also induced G0/G1 cell cycle arrest by regulation of MEK and p44/42 MAPK activities and expression levels of cell cycle related proteins in vitro. Furthermore, anti-hLSR mAb, which crossreacts with mouse LSR, had little detectable toxicity in mice. In summary, high expression of LSR in ovarian cancer was the poor prognostic factor. Our newly developed anti-LSR mAb showed significant tumor growth inhibition in ADCC and CDC independent manner in vivo. Anti-human LSR mAb also inhibited LSR function and showed direct tumor growth inhibition inducing G0/G1 cell cycle arrest in vitro. Our preclinical data demonstrated that targeting LSR by mAb is a promising therapy for patients with LSR-positive ovarian cancer. Citation Format: Kosuke Hiramatsu, Satoshi Serada, Takayuki Enomoto, Satoshi Nakagawa, Akiko Morimoto, Minoru Fujimoto, Takuhei Yokoyama, Yusuke Takahashi, Yutaka Ueda, Kiyoshi Yoshino, Eiichi Morii, Tadashi Kimura, Tetsuji Naka. Anti-human LSR monoclonal antibody inhibits tumor growth of ovarian cancer directly. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4382. doi:10.1158/1538-7445.AM2015-4382

Lentivirus-mediated knockdown of Krüppel-like factor 9 inhibits the growth of ovarian cancer.

Ovarian cancer is among the top diseases in the list of malignant gynaecologic tumors. In the present study, we aim to investigate the effect of lentivirus-mediated knockdown of Krüppel-like factor 9 (KLF9) on cell viability and tumor growth in ovarian cancer. Firstly, the expression of KLF9 was determined by real-time PCR and western blot in human ovarian cancer tissues. Then, endogenous KLF9 expression was silenced by lentivirus in SKOV3 and OVCAR3 ovarian cancer cells, and followed by MTT and BrdU incorporation assays, cell cycle analysis and tumor xenografts in nude mice. Our results found that the expression of KLF9 is up-regulated in human ovarian cancer. As expected, KLF9 knockdown significantly inhibited cell proliferation and resulted in cell cycle arrest in the G0/G1 phase. Besides, KLF9 deficiency significantly inhibited tumor growth in nude mice. Therefore, our data reveal that lentivirus-mediated KLF9 silencing might be promising in the treatment of human ovarian cancer.

AAV-mediated persistent bevacizumab therapy suppresses tumor growth of ovarian cancer

RationaleAnti-angiogenesis therapies such as bevacizumab, the monoclonal antibody to vascular endothelial growth factor (VEGF), have been used against ovarian cancer, but transient and low peritoneal drug levels are likely a factor in treatment failure. We hypothesized that a single administration of adeno-associated virus (AAV)-mediated intraperitoneal expression of bevacizumab would direct persistent expression and suppress growth and metastasis of ovarian cancer. MethodsAAVrh.10BevMab, a rhesus serotype 10 adeno-associated viral vector coding for bevacizumab, was evaluated for the capacity of a single intraperitoneal administration to persistently suppress peritoneal tumor growth in an intraperitoneal model of ovarian carcinomatosis with human ovarian cancer cells in nude immunodeficient mice. ResultsThe data demonstrates that AAVrh10.BevMab mediates persistent and high levels of bevacizumab in the peritoneal cavity following a single intraperitoneal administration in mice. In AAVrh10.BevMab treated A2780 human ovarian cancer-bearing mice, tumor growth was significantly suppressed (p<0.05) and the area of blood vessels in the tumor was decreased (p<0.04). Survival of mice with A2780 xenografts or SK-OV3 xenografts was greatly prolonged in the presence of AAVrh10.BevMab (p<0.001). Administration of AAVrh10.BevMab 4days after A2780-luciferase cell implantation reduced tumor growth (p<0.01) and increased mouse survival (p<0.0001). Combination of AAVrh10.BevMab with cytotoxic reagents paclitaxel or topotecan proved to be more effective in increasing survival than treatment with cytotoxic reagent alone. ConclusionA single administration of AAVrh10.BevMab provides sustained and high local expression of bevacizumab in the peritoneal cavity, and significantly suppresses peritoneal carcinomatosis and increases survival in an ovarian cancer murine model.

PSP94 contributes to chemoresistance and its peptide derivative PCK3145 represses tumor growth in ovarian cancer

Tumor drug resistance remains a major challenge in the treatment of cancer. Here, we show that Prostatic secretory protein 94 (PSP94) levels are reduced in ovarian cancer patients with high levels of excision repair cross-complementing 1 (ERCC1), a marker for chemoresistance. We find that PSP94 is decreased in an ovarian cancer drug-resistant cell line, and plays an important role in the development of drug resistance in vitro. Our studies indicate that PSP94 can partially reverse drug resistance in mouse tumor models in vivo and that a PSP94 peptide derivative PCK3145 suppresses chemoresistant cancer cell and tumor growth in vitro and in vivo. Our investigation of the involved molecular mechanisms suggests that PSP94 may confer drug resistance by modulating the Lin28b/Let-7 signaling pathway. We introduce PSP94 and its peptide derivative PCK3145 as potential target to reverse chemoresistance in ovarian cancer and have begun to identify their relevant molecular targets in specific signaling pathways.

Huaier Aqueous Extract Inhibits Ovarian Cancer Cell Motility via the AKT/GSK3β/β-Catenin Pathway

Traditional Chinese medicine has gained popularity due to its ability to kill tumor cells. Recently, the apoptotic and anti-angiogenic effects of Trametes robiniophila murr (Huaier) have been investigated. The aim of this study was to investigate its effect on cell mobility and tumor growth in ovarian cancer. Cell viability and motility were measured using SRB, scratch and migration assays. Cell apoptosis was analysed by annexin V/PI staining. Using a reverse-phase protein array (RPPA) assay, we analyzed the levels of 153 proteins and/or phosphorylations in Huaier-treated and untreated cells. Huaier inhibited cell viability and induced both early and late apoptosis in SKOV3, SKOV3.ip1 and Hey cells in a time- and dose-dependent manner. Cell invasiveness and migration were also suppressed significantly. The RPPA results showed significant differences (of at least 30%; P <0.05) in the levels of 7 molecules in SKOV3 cells and 10 in SKOV3.ip1 cells between the untreated and treated cells. Most of the molecules identified play roles in cell proliferation, apoptosis or cell adhesion/invasion. Western blot analysis further validated that Huaier treatment resulted in decreased AKT phosphorylation, enhanced expression of total GSK3β, inhibition of the phosphorylation of GSK3β on S9, reduction of both cytoplasmic β-catenin expression and nuclear β-catenin translocation, and transcriptional repression of several Wnt/β-catenin target genes (DIXDC1, LRP6, WNT5A, and cyclin D1). After knocking down GSK3β, β-catenin expression could not be inhibited by Huaier. Finally, Huaier inhibited the growth of ovarian tumor xenografts in vivo. These studies indicate that Huaier inhibits tumor cell mobility in ovarian cancer via the AKT/GSK3β/β-catenin signaling pathway.

Selective Blockade of Matrix Metalloprotease-14 with a Monoclonal Antibody Abrogates Invasion, Angiogenesis, and Tumor Growth in Ovarian Cancer

Most patients with ovarian cancer are diagnosed late in progression and often experience tumor recurrence and relapses due to drug resistance. Surface expression of matrix metalloprotease (MMP)-14 on ovarian cancer cells stimulates a tumor-stromal signaling pathway that promotes angiogenesis and tumor growth. In a cohort of 92 patients, we found that MMP-14 was increased in the serum of women with malignant ovarian tumors. Therefore, we investigated the preclinical efficacy of a MMP-14 monoclonal antibody that could inhibit the migratory and invasive properties of aggressive ovarian cancer cells in vitro. MMP-14 antibody disrupted ovarian tumor-stromal communication and was equivalent to Avastin in suppressing blood vessel growth in mice harboring Matrigel plugs. These effects on angiogenesis correlated with downregulation of several important angiogenic factors. Furthermore, mice with ovarian cancer tumors treated with anti-MMP-14 monotherapy showed a marked and sustained regression in tumor growth with decreased angiogenesis compared with immunoglobulin G (IgG)-treated controls. In a model of advanced peritoneal ovarian cancer, MMP-14-dependent invasion and metastasis was effectively inhibited by intraperitoneal administration of monoclonal MMP-14 antibody. Together, these studies provide a preclinical proof-of-concept for MMP-14 targeting as an adjuvant treatment strategy for advanced ovarian cancer.

Abstract 4990: Complement promotes growth of ovarian cancer.

Abstract Objective: The complement system is a component of innate immunity against invading pathogens, but also mediates the cell-cell communication in organ regeneration, angiogenesis, epithelial-mesenchymal transition (EMT) and cell migration. We studied whether the complement system affect tumor growth in ovarian cancer. Materials &amp; Methods: Complement C3 mRNA level was quantified using qRT-PCR in ovarian cancer cell lines and in tumor specimens from patients with ovarian cancer. Small interfering RNA (siRNA) was used to investigate the consequences of C3 gene silencing in vitro and in vivo. Results: C3 was expressed in ovarian cancer cell lines and tumor tissues from patients. Silencing of the C3 gene decreased ovarian cancer cell proliferation, migration and invasion in vitro. In orthotopic murine models of ovarian cancer, C3 siRNA reduced growth of implanted tumors by 60% as was evident by lower number and smaller size of tumor nodules in C3-siRNA treated mice in comparison to those in scrambled siRNA treated control mice (average tumor weight of 0.19 g in C3 siRNA vs. 0.61 g in scrambled siRNA, n=8 in each group, p=0.017; average nodule number of 4.4 in C3 siRNA vs. 11.4 in scrambled siRNA, n=8 in each group, p=0.05; t-test). Consistent with our in vitro and animal studies, ovarian cancer patients with a higher C3 mRNA level in their tumors had a worse prognosis with overall survival rates compared to those with a lower C3 mRNA level (n=75, p=0.03). Conclusion: Our studies revealed a novel role for complement in cancer biology. Cancer cell-derived C3 promotes tumor growth and progression. Our studies might identify the complement system as a new target for the development of new anti-tumor reagents. Citation Format: Min Soon Cho, Hernan G. Vasquez, Hee Dong Han, Sunila Pradeep, Rajesh Rupaimoole, Behrouz Zand, Michael Kroll, Anil K. Sood, Vahid Afshar-Kharghan. Complement promotes growth of ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4990. doi:10.1158/1538-7445.AM2013-4990

Interleukin-1β Promotes Ovarian Tumorigenesis through a p53/NF-κB-Mediated Inflammatory Response in Stromal Fibroblasts

Cancer has long been considered a disease that mimics an “unhealed wound,” with oncogene-induced secretory activation signals from epithelial cancer cells facilitating stromal fibroblast, endothelial, and inflammatory cell participation in tumor progression. However, the underlying mechanisms that orchestrate cooperative interaction between malignant epithelium and the stroma remain largely unknown. Here, we identified interleukin-1β (IL-1β) as a stromal-acting chemokine secreted by ovarian cancer cells, which suppresses p53 protein expression in cancer-associated fibroblasts (CAFs). Elevated expression of IL-1β and cognate receptor IL-1R1 in ovarian cancer epithelial cells and CAFs independently predicted reduced overall patient survival, as did repressed nuclear p53 in ovarian CAFs. Knockdown of p53 expression in ovarian fibroblasts significantly enhanced the expression and secretion of chemokines IL-8, growth regulated oncogene-alpha (GRO-α), IL-6, IL-1β, and vascular endothelial growth factor (VEGF), significantly increased in vivo mouse xenograft ovarian cancer tumor growth, and was entirely dependent on interaction with, and transcriptional up-regulation of, nuclear factor-kappaB (NF-κB) p65. Our results have uncovered a previously unrecognized circuit whereby epithelial cancer cells use IL-1β as a communication factor instructing stromal fibroblasts through p53 to generate a protumorigenic inflammatory microenvironment. Attenuation of p53 protein expression in stromal fibroblasts generates critical protumorigenic functionality, reminiscent of the role that oncogenic p53 mutations play in cancer cells. These findings implicate CAFs as an important target for blocking inflammation in the tumor microenvironment and reducing tumor growth.

Knockdown of mediator complex subunit 19 inhibits the growth of ovarian cancer

Ovarian cancer causes more deaths than any other type of female reproductive cancer. The development of new therapeutic approaches is required due to the low survival rate using routine methods. The goal of this study was to investigate the effect of the gene silencing of mediator complex subunit19 (MED19) on cell viability and tumor growth in ovarian cancer. Immunohistochemistry was used to characterize the expression of MED19 in human ovarian cancer tissues. Lentivirus-mediated RNAi was employed to downregulate endogenous MED19 expression in SKOV-3 and HEY ovarian cancer cells. MTT assay, BrdU incorporation assay, colony formation assay, cell cycle analysis and tumor xenografts in nude mice were performed to determine the effects of MED19 silencing on cell viability and tumor growth invitro and invivo. The data showed that the expression of MED19 in human ovarian cancer tissues correlated with the level of tumor malignancy. The downregulation of MED19 in ovarian cancer cells significantly inhibited cell proliferation and colony formation invitro and led to cell cycle arrest in the G0/G1 phase. MED19 RNAi significantly inhibited ovarian cancer tumor growth in engrafted nude mice. Our findings reveal that the knockdown of MED19 by lentivirus-mediated RNAi may be useful in the treatment of human ovarian cancer.