Orthotopic Ovarian Cancer Model Research Articles

Highlights of This Issue

Metronomic chemotherapy, or the frequent administration of low, minimally toxic doses, inhibits the proliferation of endothelial cells. Previs, Armaiz-Pena, Lin, and colleagues tested the efficacy of nab-paclitaxel and topotecan (increases TSP-1 and suppresses carbonic anhydrase IX) at metronomic doses on multiple orthotopic ovarian cancer models and demonstrated decreased angiogenesis and increased apoptosis. Additionally, metronomic treatment suppresses hypoxia in the tumor microenvironment. Treatment with both agents led to a significantly prolonged overall survival compared to vehicle and other monotherapy groups (P < 0.001). These findings support further clinical development of this combination, which has potent antiangiogenic effects.SS1P is a recombinant immunotoxin (RIT) that targets mesothelin. In clinical studies, it has produced dramatic responses in patients with advanced mesothelioma when combined with immunosuppressive therapy so that several treatment cycles could be given. Otherwise its activity is limited by its immunogenicity. To overcome this impediment, a RIT with low immunogenicity was developed by removal of eight T-cell epitopes. LMB-T20 is more active than SS1P in vitro. It also has potent antitumor activity in mice, and has reduced immunogenicity as measured by cytokine secretion assays. LMB-T20 is a favorable candidate for evaluation in clinical trials.The murine double minute 2 (Mdm2) protein is elevated in many therapy-refractive breast cancers, making it an attractive therapeutic target. In triple-negative breast cancer (TNBC) cells, Nutlin-3a modulation of Mdm2 protein–protein interactions sensitized mutant p53 TNBC cells to carboplatin. Combination Nutlin-3a/carboplatin was synergistic in promoting p73a-dependent cell death. This correlated with persistence of DNA damage as assessed by increased γH2AX and association of Mdm2 with chromatin. Moreover, in a human breast-to-lung metastatic TNBC model, combination Nutlin-3a/carboplatin resulted in decreased tumor growth at primary and metastatic sites. This investigation provides rationale for developing novel combination therapies that target Mdm2 in TNBC.Continued refinements in the delivery of ionizing radiation to solid tumors and development of novel radiosensitizing agents have great potential to improve patient treatment. However, identifying reliable, predictive markers of genotoxic stress and radiation response remains elusive. Sandulache, Chen, and colleagues demonstrate that irradiation triggers quantifiable, transient, and reversible perturbations in tumor lactate levels, which correlate with intrinsic and acquired radioresistance. These findings provide a critical biochemical foundation for using noninvasive imaging techniques to assess tumor lactate levels. Incorporation of lactate as a real-time biomarker of radiation effectiveness in the treatment of solid tumors could revolutionize current clinical paradigms.

Elaiophylin, a novel autophagy inhibitor, exerts antitumor activity as a single agent in ovarian cancer cells

Currently, targeting the autophagic pathway is regarded as a promising new strategy for cancer drug discovery. Here, we screened the North China Pharmaceutical Group Corporation's pure compound library of microbial origin using GFP-LC3B-SKOV3 cells and identified elaiophylin as a novel autophagy inhibitor. Elaiophylin promotes autophagosome accumulation but blocks autophagic flux by attenuating lysosomal cathepsin activity, resulting in the accumulation of SQSTM1/p62 in various cell lines. Moreover, elaiophylin destabilizes lysosomes as indicated by LysoTracker Red staining and CTSB/cathepsin B and CTSD/ cathepsin D release from lysosomes into the cytoplasm. Elaiophylin eventually decreases cell viability, especially in combination with cisplatin or under hypoxic conditions. Furthermore, administration of a lower dose (2 mg/kg) of elaiophylin as a single agent achieves a significant antitumor effect without toxicity in an orthotopic ovarian cancer model with metastasis; however, high doses (8 mg/kg) of elaiophylin lead to dysfunction of Paneth cells, which resembles the intestinal phenotype of ATG16L1-deficient mice. Together, these results provide a safe therapeutic window for potential clinical applications of this compound. Our results demonstrate, for the first time, that elaiophylin is a novel autophagy inhibitor, with significant antitumor efficacy as a single agent or in combination in human ovarian cancer cells, establishing the potential treatment of ovarian cancer by this compound.

Photoacoustic Tomography Detects Early Vessel Regression and Normalization During Ovarian Tumor Response to the Antiangiogenic Therapy Trebananib.

The primary aim of this study was to assess the potential of in vivo photoacoustic tomography for direct functional measurement of ovarian tumor response to antiangiogenic therapy. In vivo studies were performed with institutional animal care and use committee approval. We used an orthotopic mouse model of ovarian cancer treated with trebananib (n = 9) or vehicle (n = 9). Tumor-bearing mice were randomized into trebananib or vehicle groups at day 10 and dosed on days 12, 15, and 18 after implantation. Photoacoustic tomography and blood draws were performed at day 10 and then 24 h after each drug dose. Tumors were excised for histopathology after the final studies on day 19. Data analysis to test for statistical significance was performed blinded. Blockade of angiopoietin signaling using trebananib resulted in reduced total hemoglobin-weighted photoacoustic signal (n = 9, P = 0.01) and increased oxyhemoglobin-weighted photoacoustic signal (n = 9, P < 0.01). The latter observation indicated normalization of the residual tumor vessels, which was also implied by low levels of angiopoietin 1 in serum biomarker profiling (0.76 ± 0.12 ng/mL). These noninvasive measures reflected a 30% reduction in microvessel density and increased vessel maturation in ex vivo sections. Photoacoustic tomography is able to evaluate both vessel regression and normalization in response to trebananib. Noninvasive imaging data were supported by modulation of serum markers in vitro and ex vivo histopathology.

Abstract POSTER-THER-1440: Targeted delivery of doxorubicin loaded nanobins to ovarian cancer cells through the urokinase plasminogen activator system

Abstract The therapeutic efficacy of antitumor drugs is often limited by non-specific, systematic delivery. Here we report a novel targeted delivery platform designed by coupling liposomal nanobins (NB) with the urokinase plasminogen activator receptor (u-PAR) antibody (ATN-658). The urokinase system is overexpressed in epithelial ovarian cancer (OvCa) cells and is expressed only at low levels in normal cells. Doxorubicin (Dox), a FDA approved antitumor drug, had been loaded into the nanobins as a payload. The size and surface charge of the nanobins were optimized to facilitate specific binding to u-PAR expressing OvCa cells. Confocal and transmission electron microscopy showed that ATN-658-NB(Dox) was internalized in OvCa cells in a receptor-dependent manner and was released from endosomes as a function of time. This uptake could be blocked by stably down-regulating u-PAR expression in the OvCa cells using shRNA. In an orthotopic ovarian cancer model, athymic mice treated with ATN-658-NB(Dox) had a significantly greater reduction in tumor burden (0.06±0.01g versus 0.1±0.01g; p&amp;lt;0.001) than mice treated with NB(Dox), which was corroborated by in vivo fluorescence imaging results. A survival study showed that targeted nanobins prolonged the survival of mice by 3 days (43 days versus 40 days, median survival) as compared to untargeted nanobins. Immunoblot analysis of tumor sections showed that targeted nanobins induced apoptosis (cleaved caspase 3) and inhibited proliferation (Ki-67) more efficiently when compared to untargeted nanobins. Furthermore, tumor-targeted delivery of ATN-658-ND(Dox) was confirmed by ex vivo fluorescence imaging, FACS analysis, and confocal microscopy. Taken together, our findings show that receptor-mediated delivery of targeted nanobins to cancer cells could serve as a novel approach to ovarian cancer treatment. Citation Format: Yilin Zhang, Elden P. Swindell, Patrick L. Hankins, Hilary A. Kenny, Thomas V. O’Halloran, Andrew P. Mazar, Ernst Lengyel. Targeted delivery of doxorubicin loaded nanobins to ovarian cancer cells through the urokinase plasminogen activator system [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1440.

Abstract 142: SiRNA therapy against novel lncRNA NRCP: shutting down the fuel for cancer cells

Abstract Long noncoding RNAs (lncRNAs) significantly influence the development and genomic regulation of cells. Even though several lncRNAs have been discovered using model systems such as yeast, few have been proven to be involved in cancer-specific phenotypes. In this study, we report the role of lncRNA NRCP in altering cancer cell metabolism and provide a strategy to target cancer metabolism using siRNAs. Methodology and Results: Using genomic profiling of ovarian tumors (n = 29) and normal ovarian tissues (n = 11) and comparative analyses, we found that NRCP was highly upregulated in ovarian tumors (&amp;gt;100 fold, p&amp;lt;0.05). In another cohort of 218 patients with high-grade serous ovarian cancer, NRCP expression in tumor tissues was negatively correlated with overall survival (p&amp;lt;0.01), and the tumor tissues had significantly higher NRCP expression than the normal ovarian tissues or fallopian tube derived epithelial cells (&amp;gt;6 fold, p&amp;lt;0.01). Using siRNA mediated knockdown of NRCP in cancer cells and mRNA microarray, we carried out functional analyses. Upon performing Ingenuity Pathway Analysis of the significantly altered genes, we found that the cellular pathways most altered by siNRCP included glycolysis, cellular respiration, and glucose metabolism pathways. Significant decreases in glucose uptake and decreases in amount of lactate were observed in the cells treated with siNRCP compared with the control-treated cells. There was significant increase in basal respiration and ATP synthase dependent respiration in siNRCP- compared to control-treated cells. Analysis of global metabolite changes upon silencing NRCP, showed lactate as the key metabolite decreased compared to control siRNA treated cells (&amp;gt;40%). Additionally, metabolites such as glutamate and acetate were also decreased significantly in NRCP silenced cells compared to siControl treated cells. NRCP knockdown in SKOV3 or A2780 ovarian cancer cells showed significantly increased apoptosis and decreased cell proliferation compared with control cancer cells.The cancer cells were more sensitive to cisplatin-mediated apoptosis in vitro after treatment with siNRCP plus cisplatin. In an orthotopic mouse model of ovarian cancer, siNRCP delivered via a liposomal carrier DOPC, significantly reduced tumor weight and number of distant metastatic nodules compared with control treatment (&amp;gt;80%, p&amp;lt;0.01). The mice treated with siNRCP showed enhanced cisplatin sensitivity in tumors when compared to mice treated with the control siRNA. More importantly, metabolite analysis of the in vivo samples revealed significant reduction in lactate levels in the tumors treated with siNRCP. Significance: We report an unrecognized role of the lncRNA NRCP in modulating metabolism in ovarian cancer. DOPC nanoparticle-incorporated siRNA-mediated silencing of this lncRNA combined with cisplatin in vivo demonstrates the first known preclinical model indicating therapeutic potential of modulating lncRNAs in cancer. Citation Format: Rajesha Rupaimole, Rebecca Previs, Jaehyuk Lee, Sunila Pradeep, Sherry Y. Wu, Cristina Ivan, Manuela Ferracin, Jennifer Dennison, Cristian Rodriguez-Aguayo, George A. Calin, Gordon Mills, Wei Zhang, Gabriel Lopez-Berestein, Pratip Bhattacharya, Anil K. Sood. SiRNA therapy against novel lncRNA NRCP: shutting down the fuel for cancer cells. [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 142. doi:10.1158/1538-7445.AM2015-142

Antibody-mediated targeting of iron oxide nanoparticles to the folate receptor alpha increases tumor cell association in vitro and in vivo.

Active molecular targeting has become an important aspect of nanoparticle development for oncology indications. Here, we describe molecular targeting of iron oxide nanoparticles (IONPs) to the folate receptor alpha (FOLRα) using an engineered antibody fragment (Ffab). Compared to control nanoparticles targeting the non-relevant botulinum toxin, the Ffab-IONP constructs selectively accumulated on FOLRα-overexpressing cancer cells in vitro, where they exhibited the capacity to internalize into intracellular vesicles. Similarly, Ffab-IONPs homed to FOLRα-positive tumors upon intraperitoneal administration in an orthotopic murine xenograft model of ovarian cancer, whereas negative control particles showed no detectable tumor accumulation. Interestingly, Ffab-IONPs built with custom 120 nm nanoparticles exhibited lower in vitro targeting efficiency when compared to those built with commercially sourced 180 nm nanoparticles. In vivo, however, the two Ffab-IONP platforms achieved equivalent tumor homing, although the smaller 120 nm IONPs were more prone to liver sequestration. Overall, the results show that Ffab-mediated targeting of IONPs yields specific, high-level accumulation within cancer cells, and this fact suggests that Ffab-IONPs could have future utility in ovarian cancer diagnostics and therapy.

Differential platelet levels affect response to taxane-based therapy in ovarian cancer.

We hypothesized that platelet levels during therapy could serve as a biomarker for response to therapy and that manipulation of platelet levels could impact responsiveness to chemotherapy. The medical records of patients with recurrent or progressive ovarian cancer were retrospectively queried for changes in platelet and CA-125 levels during primary therapy. In vitro coculture experiments and in vivo orthotopic models of human ovarian cancer in mice were used to test the effect of modulating platelet levels on tumor growth and responsiveness to docetaxel. Thrombocytosis at the diagnosis of ovarian cancer was correlated with decreased interval to progression (P = 0.05) and median overall survival (P = 0.007). Mean platelet levels corrected during primary therapy and rose at recurrence. Contrary to treatment-responsive patients, in a cohort of patients refractory to primary therapy, platelet levels did not normalize during therapy. In A2780, HeyA8, and SKOV3-ip1 ovarian cancer cell lines, platelet coculture protected against apoptosis (P < 0.05). In orthotopic models of human ovarian cancer, platelet depletion resulted in 70% reduced mean tumor weight (P < 0.05). Compared with mice treated with docetaxel, mice treated with both docetaxel and platelet-depleting antibody had a 62% decrease in mean tumor weight (P = 0.04). Platelet transfusion increased mean aggregate tumor weight 2.4-fold (P < 0.05), blocked the effect of docetaxel on tumor growth (P = 0.55) and decreased tumor cell apoptosis. Pretransfusion aspirinization of the platelets blocked the growth-promoting effects of transfusion. Platelet-driven effects of chemotherapy response may explain clinical observations.

Immunotherapy targeting folate receptor induces cell death associated with autophagy in ovarian cancer.

Cancer cells are highly dependent on folate metabolism, making them susceptible to drugs that inhibit folate receptor activities. Targeting overexpressed folate receptor alpha (FRα) in cancer cells offers a therapeutic opportunity. We investigated the functional mechanisms of MORAB-003 (farletuzumab), a humanized mAb against FRα, in ovarian cancer models. We first examined FRα expression in an array of human ovarian cancer cell lines and then assessed the in vivo effect of MORAB-003 on tumor growth and progression in several orthotopic mouse models of ovarian cancer derived from these cell lines. Molecular mechanisms of tumor cell death induced by MORAB-003 were investigated by cDNA and protein expression profiling analysis. Mechanistic studies were performed to determine the role of autophagy in MORAB-003-induced cell death. MORAB-003 significantly decreased tumor growth in the high-FRα IGROV1 and SKOV3ip1 models but not in the low-FRα A2780 model. MORAB-003 reduced proliferation, but had no significant effect on apoptosis. Protein expression and cDNA microarray analyses showed that MORAB-003 regulated an array of autophagy-related genes. It also significantly increased expression of LC3 isoform II and enriched autophagic vacuolization. Blocking autophagy with hydroxychloroquine or bafilomycin A1 reversed the growth inhibition induced by MORAB-003. In addition, alteration of FOLR1 gene copy number significantly correlated with shorter disease-free survival in patients with ovarian serous cancer. MORAB-003 displays prominent antitumor activity in ovarian cancer models expressing FRα at high levels. Blockade of folate receptor by MORAB-003 induced sustained autophagy and suppressed cell proliferation.

An immunocompetent, orthotopic mouse model of epithelial ovarian cancer utilizing tissue engineered tumor cell sheets.

Despite the development of a myriad of anticancer drugs that appeared promising in preclinical ovarian cancer animal models, they failed to predict efficacy in clinical testing. To improve the accuracy of preclinical testing of efficacy and toxicity, including pharmacokinetic and pharmacodynamic evaluations, a novel animal model was developed and characterized. In this study, murine ID8 (epithelial ovarian cancer [EOC]) cells as injected cell suspensions (ICS) and as intact cultured monolayer cell sheets (CS) were injected or surgically grafted, respectively, into the left ovarian bursa of 6-8 week-old, female C57BL/6 black mice and evaluated at 8 and 12 weeks after engraftment. Tumor volumes at 8 weeks were as follows: 30.712 ± 18.800 mm(3) versus 55.837 ± 10.711 mm(3) for ICS and CS, respectively, p = 0.0990 (n = 5). At 12 weeks, tumor volumes were 128.129 ± 44.018 mm(3) versus 283.953 ± 71.676 mm(3) for ICS and CS, respectively, p = 0.0112 (n = 5). The ovarian weights at 8 and 12 weeks were 0.02138 ± 0.01038 g versus 0.04954 ± 0.00667 g for ICS and CS, respectively (8 weeks), p = 0.00602 (n = 5); and 0.10594 ± 0.03043 g versus 0.39264 ± 0.09271 g for ICS and CS, respectively (12 weeks), p = 0.0008 (n = 5). These results confirm a significant accelerated tumorigenesis in CS-derived tumors compared with ICS-derived tumors when measured by tumor volume/time and ovarian weight/time. Furthermore, the CS-derived tumors closely replicated the metastatic spread found in human EOC and histopathological identity with the primary tumor of origin.

158 The development of a series of orthotopic solid tumour models of prostate, lung and ovarian cancer using optical and X ray imaging

158 The development of a series of orthotopic solid tumour models of prostate, lung and ovarian cancer using optical and X ray imaging

PP242 synergizes with suberoylanilide hydroxamic acid to inhibit growth of ovarian cancer cells.

Overexpression of histone deacetylases and activation of the phosphatidylinositol 3-kinase/mammalian target of rapamycin pathway are common aberrations in ovarian cancer. For this reason, simultaneous inhibition of such targets is a rational therapeutic strategy to treat patients with ovarian cancer. This study aimed to investigate the biological effect of the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), in combination with the dual mTOR complex 1 and mTOR complex 2 inhibitor, PP242, against ovarian cancer cells. The effects of SAHA and PP242 on the growth of SKOV3 and A2780 cells were examined using Cell Counting Kit-8. The apoptosis was analyzed through flow cytometry, and the expression of apoptosis-related proteins was investigated through Western blotting. Induction of autophagy was determined through fluorescence microscopy using a stably transfected green fluorescent protein/microtubule-associated protein light chain 3 construct to visualize autophagosome formation. The expression of autophagy-related proteins was determined through Western blot analysis. The effect of SAHA and PP242 on the growth of ovarian cancer was also examined in an orthotopic ovarian cancer model. The combination of SAHA and PP242 significantly inhibited cell proliferation and synergistically increased apoptosis and autophagy compared with each agent alone in vitro. In vivo, this combination exhibited greater inhibition on tumor growth than monotreatments did and it significantly prolonged the survival time of the mice. These results suggest that the combination of SAHA and PP242 may lead to a novel strategy in treating patients with ovarian cancer.

Abstract 666: AB-3A4: A novel KAAG1-targeting antibody-drug conjugate is active in models of ovarian carcinoma, triple-negative breast cancer and castration-resistant prostate cancer

Abstract Starting from total RNA prepared from late-stage serous ovarian tumors and low-malignancy potential tumors, a sensitive subtractive cloning technology called STAR was used to identify differentially expressed sequences that were up-regulated in the malignant tumors. This analysis led to the identification of KAAG1, an antigen that is specifically expressed in the vast majority of the ovarian tumors. Monoclonal antibodies (mAbs) that bind to human KAAG1 were generated and one of these, AB-3A4, interacted with KAAG1 expressed on the surface of several ovarian cancer, triple-negative breast cancer (TNBC) and prostate cancer cell lines. Further studies indicated that the AB-3A4/KAAG1 complex was rapidly internalized and co-localized with LAMP1, a marker of lysosomes, indicating that AB-3A4 has potential as an antibody-drug conjugate (ADC). Here we describe preclinical results of AB-3A4 conjugated to the cytotoxic agent monomethyl auristatin E (AB-3A4-vcMMAE). Binding studies with KAAG1-positive cell lines, such as the SKOV-3 (ovarian), MDA-MB-231 (TNBC) and PC-3 (prostate) showed that AB-3A4-vcMMAE interacted with its antigen in a similar manner to the unconjugated AB-3A4. In cytotoxicity assays, AB-3A4-vcMMAE killed cancer cell lines expressing KAAG1 with an IC50 value of 1 - 2 nM, whereas it displayed no specific potency in KAAG1-low or -negative cells. SKOV-3 cells were subcutaneously implanted in SCID mice and allowed to grow to a volume of 150 - 200 mm3 after which four injections of AB-3A4-vcMMAE at 5 mg/kg were administered over two weeks. Rapid and sustained tumor regression was observed for several weeks post-injection. In PC-3 xenografts, a single injection of AB-3A4-vcMMAE at 3 mg/kg was sufficient to cause significant tumor regression and this efficacy was found to be dose-dependent between doses of 1 and 10 mg/kg. In parallel treatment groups, no significant tumor shrinkage was observed with either a control IgG-vcMMAE or a mixture of unconjugated AB-3A4 and free MMAE at a drug-antibody ratio corresponding to that of AB-3A4-vcMMAE. Furthermore, xenografts of TNBC MDA-MB-231 cells were significantly regressed following a single injection of AB-3A4-vcMMAE with no recurrence after several weeks post-treatment. Finally, using an intraperitoneal orthotopic model of ovarian cancer, treatment with AB-3A4-vcMMAE significantly increased the overall survival of mice compared to mice treated with the control ADC. Collectively, our results demonstrate the potent efficacy of AB-3A4-vcMMAE in several disease models and underscore the potential of this novel agent in ovarian cancer, TNBC and castration-resistant prostate cancer, three areas for which very few targeted therapies exist. Citation Format: Gilles B. Tremblay, Anna Moraitis, Dominique Bédard, Adriana Orimoto, Martine Pagé, Anne-Marie Mes-Masson, Mario Filion. AB-3A4: A novel KAAG1-targeting antibody-drug conjugate is active in models of ovarian carcinoma, triple-negative breast cancer and castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 666. doi:10.1158/1538-7445.AM2014-666

Abstract 2982: Macrophages modulate adaptive resistance to anti-angiogenic therapy

Abstract Targeted anti-angiogenic therapies were a highly anticipated addition to cancer treatment, but their clinical use has been tempered by the development of resistance. To understand the role of immune cells in potentially mediating such resistance, we carried out systematic immune cell profiling in a syngeneic ovarian cancer mouse model following treatment with the B20 anti-VEGF antibody. Following injection of luciferase-labeled cancer cells and initiation of treatment, mice (n=8/group) were divided into B20-sensitive and resistant groups based on longitudinal bioluminescence imaging of tumor burden. Immune cell profiling confirmed a dramatic increase in macrophages with emergence of resistance to B20. Using the syngeneic model, resistance to B20 was documented with bioluminescence imaging as an increase in tumor burden in mice with previously stable disease. Zoledronic acid, a macrophage-depleting bisphosphonate, was then added to B20 treatment. Restoration of sensitivity to B20 with the addition of zoledronic acid was demonstrated by a 68% prolongation of survival in this treatment group as compared to those receiving B20 alone (p&amp;lt;0.0001). Zoledronic acid in combination with the VEGF antibody, bevacizumab, significantly reduced both tumor size and nodules when compared to either drug alone in additional orthotopic models of ovarian cancer. CD68 staining confirmed significantly reduced macrophage numbers in tumor samples following treatment with zoledronic acid. In vitro immunofluorescence imaging of macrophages following prolonged exposure to anti-VEGF therapy showed downregulation of VEGF receptors compared to short-term exposure, suggesting a possible mechanism for their persistence in an anti-VEGF resistant microenvironment. Collectively, these data demonstrate a novel role for macrophages in resistance to anti-VEGF therapy and offer a new therapeutic strategy to overcome this problem with important translational implications. Citation Format: Heather J. Dalton, Sunila Pradeep, Guillermo N. Armaiz-Pena, Rebecca Previs, Ashley Davis, Rajesha Rupaimoole, Behrouz Zand, Yared Hailemichael, Willem W. Overwijk, Gabriel Lopez-Berestein, Anil K. Sood. Macrophages modulate adaptive resistance to anti-angiogenic therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2982. doi:10.1158/1538-7445.AM2014-2982

Abstract LB-28: Suppression of ovarian tumor growth and metastasis with a STAT3 inhibitor, HO-3867, in preclinical orthotopic models and ex vivo cultures of ovarian cancer

Abstract Objective: We previously studied a novel anticancer compound, HO-3867, in in vitro culture and xenograft ovarian tumor models. In our current study, we tested our compound in more clinically relevant preclinical models of ovarian cancer. Our goal is to employ an orthotopic mouse model of ovarian cancer by injecting tumor cells directly into intrabursal space and allow the tumor to grow in its native microenvironment. This model allows the investigator to not only evaluate the efficacy of new drugs in treating the primary tumor mass, but also permits investigation into the potential for prevention and/or treatment of metastatic tumor growth. Methods: Development of an orthotopic model of ovarian tumor. In vivo antitumor activity of STAT3 inhibitor, body weight measurements, feed intake and tumor weight assessment. Assessment of tumor volume and hypoxia using PET/CT. Tumor excision assays with clonogenic survival endpoints. In vivo analysis was performed on mice administered oral HO-3867 using histopathological analysis and TUNEL assays. In vivo bio-absorption of DAP compounds in tumors and in vivo toxicity evaluation. Bio-absorption analysis by EPR and LCMS. Results: Treatment with HO-3867 significantly suppressed ovarian tumor growth and metastasis compared with Cisplatin. Markers specific to cell proliferation (Ki-67, Cyclin D1), angiogenesis (VEGF and Kinase array) and apoptosis (caspase-3 activity) were demonstrated to be affected by treatment with HO-3867. In vivo histopathological evaluation of internal organs collected from treated tumor mice revealed no evidence of toxicity specific to HO-3867. Normal and malignant tissues were collected and TUNEL/8-OHdG staining revealed selective apoptotic induction limited to neoplastic cells and concomitant increase in reactive oxygen species within the orthotopic tumor. Suppression of STAT3 and its downstream target proteins (cell proliferative, anti-apoptotic and angiogenic) was confirmed with proteomic array. HO-3867 was also found to have cytotoxic effects on human tumors resulting from pharmacologic intervention in ex vivo culture of freshly collected tumor sections Conclusion: Our results highlight the clinical anti-cancer potential of HO-3867 using a relevant preclinical model, and provide a rationale for the inclusion of ex vivo tumor slice culture in oncologic drug development processes. This pre-clinical screening of HO-3867 may have a significant impact on patient selection for clinical trials and in predicting response to small-molecule STAT3 inhibitor therapy for ovarian cancer. Citation Format: Shan K. Naidu, Uksha Saini, Adam C. ElNaggar, Bid K. Hemant, Kellie S. Rath, Ross Wanner, Adrian Suarez, John Hays, Peter Houghton, David E. Cohn, Karuppaiyah Selvendiran. Suppression of ovarian tumor growth and metastasis with a STAT3 inhibitor, HO-3867, in preclinical orthotopic models and ex vivo cultures of ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-28. doi:10.1158/1538-7445.AM2014-LB-28

Abstract 1406: Evoking potent RNAi response using novel 2′-OMe-phosphorodithioated modified siRNAs

Abstract Improving small interfering RNA (siRNA) efficacy in target cell populations remains a critical challenge to bringing siRNA therapy into the clinic. There is currently an unmet need to develop a reliable strategy to globally enhance siRNA stability and potency. Here, we report a novel chemical modification, consisting of phosphorodithioate (PS2) and 2′-O-Methyl (2′-OMe) MePS2 on a single nucleotide, that significantly enhances potency and resistance to nuclease degradation for a variety of siRNA sequences. We show a 3.5-fold improvement in gene silencing in tumors following systemic delivery of MePS2-modified siRNAs using DOPC nanoliposomes compared to unmodified counterparts. We found that this enhanced potency stems from an unforeseen increase in loading of siRNAs to the RNA-induced silencing complex (RISC), likely due to the unique interaction between 2′-OMe and PS2 moieties. We subsequently demonstrate the therapeutic utility of MePS2 siRNAs in orthotopic mouse models of chemoresistant ovarian cancer, focusing on targeting GRAM domain containing 1B (GRAMD1B), a protein whose role in taxane resistance is first reported here. Efficient silencing of GRAMD1B (&amp;gt;80%) was achieved in tumors following systemic delivery of these MePS2-modified siRNAs and a synergistic anti-tumor effect was observed when combined with paclitaxel treatment. Given that limited success has been achieved thus far toward broadly enhancing siRNA potency with chemically modified siRNAs, our finding represents an important step forward in bringing siRNA therapeutics into the clinic. Citation Format: Sherry Wu, Xianbin Yang, Martin Egli, Kshipra Ghaupure, Hiroto Hatakeyama, Michael McGuire, Rajesha Rupaimoole, Takahito Miyake, Morgan Taylor, Sunila Pradeep, Archana Nagaraja, Malgorzata Sierant, Richa Singhania, Cristian Rodriguez-Aguayo, Nigel McMillan, Gabriel Lopez-Berestein, Prahlad Ram, Barbara Nawrot, Anil K. Sood. Evoking potent RNAi response using novel 2′-OMe-phosphorodithioated modified siRNAs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1406. doi:10.1158/1538-7445.AM2014-1406

Abstract 2993: Dll4 inhibition plus aflibercept markedly reduces ovarian tumor burden and ascites

Abstract Purpose: To determine the effects of Dll4 blockade with and without aflibercept on ovarian tumor burden and ascites in preclinical models. Methods: Using Dll4-Fc and anti-Dll4 antibodies, we evaluated the biological effects of Dll4 inhibition combined with aflibercept in vivo (orthotopic mouse models of ovarian cancer). Reverse phase protein array (RPPA) analysis was used to identify potential biomarkers of treatment response. Results: To address the biological significance of Dll4 expression in tumor cells versus endothelial cells, we used human and mouse anti-Dll4 antibodies in vivo. In the A2780 model, treatment with mouse (REGN1035) or human (REGN421) anti-Dll4 antibodies reduced tumor weight by 61.5% and 82.4%, respectively, compared to the controls (P &amp;lt; 0.05 for both). Aflibercept alone reduced tumor weight by 90% (p &amp;lt; 0.05). The combinations of REGN1035 plus aflibercept, and REGN421 plus aflibercept resulted in the greatest inhibition of tumor growth, reducing respective tumor weight by 95.8% and 93.9%, compared to the controls (P &amp;lt; 0.05). The anti-tumor effects were related to decreased angiogenesis and increased apoptosis. RPPA analysis revealed that caspase3 and GATA3 were significantly increased; cyclin D1, Bcl-2 and pS6-s240_s244 were decreased in response to Dll4 Inhibition plus aflibercept. Among these, GATA3, a transcription factor for E-cadherin, was significantly induced by Dll4 Inhibition plus aflibercept, resulting in the up-regulation of E-cadherin expression in human cancer cells. Furthermore, GATA3 and E-cadherin expression might be useful as potential biomarkers of response. Conclusions: Dual targeting of Dll4 and VEGF shows promise for inhibiting ovarian tumor growth and may have important clinical implications. Citation Format: Jie Huang, Wei Hu, Heather J. Dalton, Justin Bottsford-Miller, Robert L. Coleman, Robert B. Jaffe, Anil K. Sood. Dll4 inhibition plus aflibercept markedly reduces ovarian tumor burden and ascites. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2993. doi:10.1158/1538-7445.AM2014-2993

Abstract 2992: Zoledronic acid inhibits angiogenesis through Rac1 inactivation in ovarian cancer

Abstract Background Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a complex and crucial process for tumor growth and development of metastasis. Rac1, a member of the Rho GTPase family, plays a central role in angiogenesis through regulation of endothelial cell migration, tube formation, adhesion, invasion, and proliferation. In this study we hypothesized that zoledronic acid (ZA) inhibits ovarian cancer angiogenesis through the inactivation of Rac1. Methods We treated HeyA8 MDR and Ovcar-5 cells with ZA to assess its effects on cell invasion, proliferation, expression of matrix metalloproteinases (MMPs; Milliplex assay) and angiogenic factors. We also determined Rac1 activation by pull-down assay. HeyA8 MDR and Ovcar-5 orthotopic murine models of ovarian cancer were assessed for expression of CD31 and Ki-67, Rac1 activation, and apoptosis by immunohistochemistry after treatment with ZA, nanoparticle, albumin-bound paclitaxel (nab-paclitaxel), or a combination. Results In vitro, we observed inhibition of HeyA8 MDR and Ovcar-5 cell invasion and proliferation by ZA. Moreover, ZA decreased the secretion of MMP-7 and vascular endothelial growth factor C (VEGF-C) in HeyA8 MDR cells and MMP-9, MMP-10, VEGF-C, granulocyte-colony stimulating factor, endothelin-1, follistatin, interleukin-8, placenta growth factor, fibroblast growth factor-1, and hepatocyte growth factor in Ovcar-5 cells. Furthermore, ZA inactivated Rac1 in both cells lines. In vivo, ZA decreased the number of nodules and tumor weight, inhibited angiogenesis and cell proliferation, and increased apoptosis in both HeyA8 MDR and Ovcar-5 animal models. These in vivo antitumor effects were enhanced in both models when ZA was combined with nab-paclitaxel. Conclusion ZA inhibits ovarian cancer angiogenesis through the inactivation of Rac1, suggesting that ZA combined with paclitaxel could be a novel therapeutic approach to ovarian cancer angiogenesis. Citation Format: Viany Gonzalez-Villasana, Cristian Rodriguez-Aguayo, Enrique Fuentes-Mattei, Sunila Pradeep, Cristina Ivan, Anil K. Sood, Gabriel Lopez-Berestein. Zoledronic acid inhibits angiogenesis through Rac1 inactivation in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2992. doi:10.1158/1538-7445.AM2014-2992

Abstract 2995: Metronomic albumin-bound paclitaxel and topotecan has potent antitumor activity in ovarian cancer

Abstract Objective: There is growing recognition of the important role of metronomic chemotherapy for cancer treatment. Based on their unique anti-angiogenic effects, we tested the efficacy of dual albumin-bound paclitaxel (ABP), which stimulates Thrombospondin-1, and topotecan, which inhibits Hypoxia-inducible factor 1-α, chemotherapy at metronomic dosing in ovarian carcinoma. Methods: We examined the effects of ABP and topotecan in vitro (MTT) and in vivo using SKOV3ip1, HeyA8 and HeyA8-MDR orthotopic mouse models of ovarian cancer. We also examined effects on proliferation (Ki67), apoptosis (TUNEL), and angiogenesis (MVD) in tumor samples obtained at necropsy. Results: In vitro cytotoxicity assays revealed similar effects (IC50=20-60 nM) of maximum tolerated dosing (MTD) and metronomic dosing of ABP on HeyA8 tumor cells (p&amp;lt;0.05). In vivo therapy experiments using the SKOV3ip1 and HeyA8 tumor models demonstrated that treatment with metronomic ABP alone and in combination with metronomic topotecan resulted in significant reductions in tumor weight, 62% and 96% respectively as compared to controls (p&amp;lt;0.01). Combination treatment with metronomically dosed ABP and topotecan further reduced tumor growth by 82% compared to topotecan alone (p&amp;lt;0.01). In the chemoresistant tumor model, HeyA8-MDR, metronomically dosed monotherapy with either cytotoxic agent had a modest effect on tumor growth however, combination therapy decreased tumor burden by 74% when compared to controls (p&amp;lt;0.01). Metronomic ABP monotherapy resulted in reduced cell proliferation (p&amp;lt;0.001), reduced tumor angiogenesis (p≤0.01), and increased apoptosis (p&amp;lt;0.01). Conclusions: Metronomically-dosed albumin-bound paclitaxel and topotecan offer a highly effective and novel therapeutic approach in ovarian carcinoma that merits further clinical development. Citation Format: Ashley N. Davis, Yvonne G. Lin-Liu, Rebecca A. Previs, Heather J. Dalton, Behrouz Zand, Justin Bottsford-Miller, Robert Coleman, Anil K. Sood. Metronomic albumin-bound paclitaxel and topotecan has potent antitumor activity in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2995. doi:10.1158/1538-7445.AM2014-2995

Abstract 2: Paxillin enhances angiogenesis through transcriptional regulation of Src in ovarian cancer

Abstract Purpose: Paxillin (PXN) expression is increased in various cancer types, but its biological and clinical impact is not well understood. Here, we examined the biological effects of paxillin in ovarian cancer. Methods: The in vivo (HeyA8 and HeyA8-MDR orthotopic mouse models of ovarian cancer) and in vitro (tube formation assays, reverse phase protein array (RPPA) analysis, RT-PCR chromatin-immunoprecipitation (ChIP) assay) effects of paxillin were examined in ovarian cancer. Results: Paxillin silencing using siRNA incorporated in DOPC nanoliposomes resulted in 55% reduction in tumor growth in a HeyA8 orthotopic ovarian cancer mouse model (p&amp;lt;0.05). There was an unexpected effect of marked reduction in vascular density (67%, p&amp;lt;0.01). In addition, tumor proliferation was decreased by 60% (p&amp;lt;0.01) in PXN-siRNA treatment group. This anti-proliferative effect of siPXN was not observed in vitro. In contrast, a significant anti-angiogenic effect was observed in vitro using a tube formation assay, where condition media collected from ovarian cancer cells treated with siPXN compared to siControl resulted in a prominent (60%,p&amp;lt;0.01) reduction in tube formation ability of endothelial cells. RPPA analyses pointed to Src protein expression being significantly decreased following PXN silencing in HeyA8 cells. Using RT-PCR and western blotting techniques, we observed that Src mRNA and protein levels were significantly decreased in siPXN-treated cells. Moreover, p-Src protein level was also found to be decreased following PXN silencing. ChIP analysis showed direct PXN binding to the Src promoter. Conclusion PXN exerts indirect effects on tumor angiogenesis, mediated through Src. These findings provide a new understanding of the role of PXN in malignant biology. Citation Format: Hyun Jin Choi, Seung-Wook Kim, Sunila Pradeep, Heather J. Dalton, Rajesha Rupaimoole, Selanere Mangala, Gabriel Lopez, Sood K. Anil. Paxillin enhances angiogenesis through transcriptional regulation of Src in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2. doi:10.1158/1538-7445.AM2014-2

Abstract 5403: Metronomic docetaxel in PRINT nanoparticles and EZH2 silencing have synergistic antitumor effect in ovarian cancer

Abstract Purpose: The purpose of this study was to investigate the combined antitumor effects of metronomic docetaxel-containing PLGA-PRINT nanoparticles and antiangiogenic mEZH2 siRNA incorporated into chitosan nanoparticles (CH-mEZH2 siRNA). Method: In vivo dose-finding studies and therapeutic experiments with PLGA-PRINT-docetaxel and CH-mEZH2 siRNA were conducted in well-established orthotopic mouse models of ovarian cancer (HeyA8 and SKOV3ip1). Antitumor effects were quantified through mean tumor weight and tumor nodule counts. Tumor tissues from these studies were stained to evaluate the proliferation index (Ki67), apoptosis index (cleaved caspase-3), and microvessel density (CD31). Result: Dose-finding studies for PLGA-PRINT-docetaxel revealed all doses (0.5, 1.5, 2 mg/kg for metronomic and 20mg/kg for MTD) significantly reduced tumor burden (p&amp;lt;0.05 for MTD; p&amp;lt;0.01 for metronomic doses). The lowest dose (0.5 mg/kg) reduced tumor weight by ∼80% and was selected for subsequent therapeutic studies. We then tested whether combining PLGA-PRINT-docetaxel particles with CH-mEZH2 siRNA would enhance antitumor effect. CH-mEZH2 siRNA alone reduced tumor weight by ∼70% in both models (p&amp;lt;0.01 for HeyA8; p&amp;lt;0.05 for SKOV3ip1). PLGA-PRINT-docetaxel treatment reduced tumor weight by ∼80% (p&amp;lt;0.01). Combination therapy showed the greatest therapeutic benefit with ∼95% reduction in tumor weight in both models (p&amp;lt;0.001 in HeyA8; p&amp;lt;0.01 in SKOV3ip1). In both the models, the combination therapy resulted in the greatest reduction of tumor nodules. Individual as well as combination therapies showed a significant reduction in proliferation (p&amp;lt;0.001). Metronomic PLGA-PRINT-docetaxel and CH-mEZH2 siRNA monotherapy increased the apoptotic index significantly (p&amp;lt;0.05), whereas the combination therapy had an additive effect (p&amp;lt;0.001). CH-mEZH2 siRNA as well as metronomic PLGA-PRINT-docetaxel therapy resulted in significant reduction in MVD (p&amp;lt;0.01). Conclusion: Metronomic delivery of PLGA-PRINT-docetaxel combined with CH-mEZH2 siRNA is an attractive therapeutic strategy. Citation Format: Kshipra M. Gharpure, Kevin S. Chu, Charles Bowerman, Takahito Miyake, Sunila Pradeep, Lingegowda S. Mangala, Hee-Dong Han, Rajesha Rupaimoole, Sherry Y. Wu, Heather J. Dalton, Mary E. Napier, Gabriel Lopez-Berestein, Joseph M. DeSimone, Anil K. Sood. Metronomic docetaxel in PRINT nanoparticles and EZH2 silencing have synergistic antitumor effect in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5403. doi:10.1158/1538-7445.AM2014-5403