Targeted blockade of HSP90 impairs DNA-damage response proteins and increases the sensitivity of ovarian carcinoma cells to PARP inhibition.

Pharmacological inhibition of PARP is a promising approach in treating high grade serous ovarian carcinoma (HGSOC). PARP inhibitors (PARPi) are most active in patients with defects in DNA damage repair (DDR) mechanisms, such as alterations in expression/function of DNA repair and homologous recombination (HR) genes/proteins, including BRCA1 and BRCA2. Benefit of PARPi could be extended towards HR-proficient patients by combining PARPi with agents that functionally abrogate HR. An attractive molecular target for this purpose is heat shock protein 90 (HSP90), which mediates the maturation and stability of several key proteins required for DDR. Here, we tested the hypothesis that targeted inhibition of HSP90 with a small-molecule inhibitor ganetespib would sensitize non-BRCA mutant ovarian carcinoma (OC) cells to PARP inhibition by talazoparib. We used commercially available cell lines, along with several novel HGSOC OC cell lines established in our laboratory. Ganetespib treatment destabilized HSP90 client proteins involved in DNA damage response and cell cycle checkpoint, and disrupted γ-irradiation-induced DNA repair. The effects of the combination of ganetespib and talazoparib on OC cell viability and survival were also analyzed, and among the non-BRCA mutant cell lines analyzed, the combination was synergistic in several cell lines (OVCAR-3, OC-1, OC-16). Together, our data suggest that ganetespib-mediated inhibition of HSP90 effectively disrupts critical DDR pathway proteins and may sensitize OC cells without 'BRCAness' to PARPi. From a clinical perspective, this suggests that HSP90 inhibition has the potential to sensitize some HGSOC patients without HR pathway alterations to PARPi, and potentially other DNA-damage inducing agents.

NEDD9 promotes oncogenic signaling, a stem/mesenchymal gene signature, and aggressive ovarian cancer growth in mice.

Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) supports oncogenic signaling in a number of solid and hematologic tumors. Little is known about the role of NEDD9 in ovarian carcinoma (OC), but available data suggest elevated mRNA and protein expression in advanced stage high-grade cancers. We used a transgenic MISIIR-TAg mouse OC model combined with genetic ablation of Nedd9 to investigate its action in the development and progression of OC. A Nedd9-/- genotype delayed tumor growth rate, reduced incidence of ascites, and reduced expression and activation of signaling proteins including SRC, STAT3, E-cadherin, and AURKA. Cell lines established from MISIIR-TAg;Nedd9-/- and MISIIR-TAg;Nedd9+/+ mice exhibited altered migration and invasion. Growth of these cells in a syngeneic allograft model indicated that systemic Nedd9 loss in the microenvironment had little impact on tumor allograft growth, but in a Nedd9 wild-type background Nedd9-/- allografts exhibited significantly reduced growth, dissemination, and oncogenic signaling compared to Nedd9+/+ allografts. Gene expression analysis revealed that Nedd9+/+ tumors exhibited more mesenchymal "stem-like" transcriptional program, including increased expression of Aldh1a1 and Aldh1a2. Conversely, loss of Nedd9 resulted in increased expression of differentiation genes, including fallopian tube markers Foxj1, Ovgp1, and Pax8. Collectively, these data suggest that tumor cell-intrinsic Nedd9 expression promotes OC development and progression by broad induction of oncogenic protein signaling and stem/mesenchymal gene expression.

In Vitro and In Vivo evaluation of a novel folate-targeted theranostic nanoemulsion of docetaxel for imaging and improved anticancer activity against ovarian cancers.

Ovarian cancer ranks fifth in cancer related deaths for women in USA. The high mortality rate associated with ovarian cancer is due to diagnosis at later stages of disease and the high recurrence rate of 60-80%. Recurrent ovarian cancers are more likely to present as multidrug resistance (MDR) leading to unfavorable response from 2nd and 3rd line chemotherapy. Nanoemulsions (NEs) are emerging as an attractive drug delivery system to overcome MDR challenges. NEs can also minimize exposure of therapeutic cargo to normal tissues potentially reducing side effects. In >80% of ovarian cancers, Folate Receptor-α (FR-α) is expressed at 10- to 100-fold higher levels than on non-pathological tissues. Therefore, folate (FA) is being evaluated as an active targeting moiety for FR-α+ ovarian cancer. To improve therapeutic outcome with reduced toxicity, we developed NMI-500, a FA targeted gadolinium (Gd) annotated NE loaded with docetaxel (DTX). NMI-500 has been developed as theranostic agents as Gd will enable physician to acquire real time pharmacodynamics data on NE + DTX accumulation in target lesions. In present study, characterization for key translational metrics of NMI-500 showed size distribution in range of 120 to 150 nm and zeta potential around -45 mV. Active targeting of FA was evaluated against FR-α+ KB cells and results demonstrated significant improvement in cell association which was surface ligand density dependent. We found that NMI-500 was able to inhibit tumor growth in a spontaneous transgenic ovarian cancer model with improved safety profile and this growth inhibition could be longitudinally followed by MRI. These results indicate NMI-500 warrants advancement to clinical trials.

Follicle-Stimulating Hormone Receptor Is Expressed by Most Ovarian Cancer Subtypes and Is a Safe and Effective Immunotherapeutic Target.

PURPOSE: To define the safety and effectiveness of T cells redirected against follicle-stimulating hormone receptor (FSHR)-expressing ovarian cancer cells. EXPERIMENTAL DESIGN: FSHR expression was determined by Western blotting, immunohistochemistry, and qPCR in 77 human ovarian cancer specimens from 6 different histologic subtypes and 20 human healthy tissues. The effectiveness of human T cells targeted with full-length FSH in vivo was determined against a panel of patient-derived xenografts. Safety and effectiveness were confirmed in immunocompetent tumor-bearing mice, using constructs targeting murine FSHR and syngeneic T cells. RESULTS: FSHR is expressed in gynecologic malignancies of different histologic types but not in nonovarian healthy tissues. Accordingly, T cells expressing full-length FSHR-redirected chimeric receptors mediate significant therapeutic effects (including tumor rejection) against a panel of patient-derived tumors in vivo In immunocompetent mice growing syngeneic, orthotopic, and aggressive ovarian tumors, fully murine FSHR-targeted T cells also increased survival without any measurable toxicity. Notably, chimeric receptors enhanced the ability of endogenous tumor-reactive T cells to abrogate malignant progression upon adoptive transfer into naïve recipients subsequently challenged with the same tumor. Interestingly, FSHR-targeted T cells persisted as memory lymphocytes without noticeable PD-1-dependent exhaustion during end-stage disease, in the absence of tumor cell immunoediting. However, exosomes in advanced tumor ascites diverted the effector activity of this and other chimeric receptor-transduced T cells away from targeted tumor cells. CONCLUSIONS: T cells redirected against FSHR+ tumor cells with full-length FSH represent a promising therapeutic alternative against a broad range of ovarian malignancies, with negligible toxicity even in the presence of cognate targets in tumor-free ovaries. Clin Cancer Res; 23(2); 441-53. ©2016 AACR.

The BRCA1-Δ11q Alternative Splice Isoform Bypasses Germline Mutations and Promotes Therapeutic Resistance to PARP Inhibition and Cisplatin.

Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and therapeutic response. Cancer cell lines and tumors harboring mutations in exon 11 of BRCA1 express a BRCA1-Δ11q splice variant lacking the majority of exon 11. The introduction of frameshift mutations to exon 11 resulted in nonsense-mediated mRNA decay of full-length, but not the BRCA1-Δ11q isoform. CRISPR/Cas9 gene editing as well as overexpression experiments revealed that the BRCA1-Δ11q protein was capable of promoting partial PARPi and cisplatin resistance relative to full-length BRCA1, both in vitro and in vivo Furthermore, spliceosome inhibitors reduced BRCA1-Δ11q levels and sensitized cells carrying exon 11 mutations to PARPi treatment. Taken together, our results provided evidence that cancer cells employ a strategy to remove deleterious germline BRCA1 mutations through alternative mRNA splicing, giving rise to isoforms that retain residual activity and contribute to therapeutic resistance. Cancer Res; 76(9); 2778-90. ©2016 AACR.

Targeted Blockade of JAK/STAT3 Signaling Inhibits Ovarian Carcinoma Growth.

Ovarian carcinoma is the fifth leading cause of death among women in the United States. Persistent activation of STAT3 is frequently detected in ovarian carcinoma. STAT3 is activated by Janus family kinases (JAK) via cytokine receptors, growth factor receptor, and non-growth factor receptor tyrosine kinases. Activation of STAT3 mediates tumor cell proliferation, survival, motility, invasion, and angiogenesis, and recent work demonstrates that STAT3 activation suppresses antitumor immune responses and supports tumor-promoting inflammation. We hypothesized that therapeutic targeting of the JAK/STAT3 pathway would inhibit tumor growth by direct effects on ovarian carcinoma cells and by inhibition of cells in the tumor microenvironment (TME). To test this, we evaluated the effects of a small-molecule JAK inhibitor, AZD1480, on cell viability, apoptosis, proliferation, migration, and adhesion of ovarian carcinoma cells in vitro. We then evaluated the effects of AZD1480 on in vivo tumor growth and progression, gene expression, tumor-associated matrix metalloproteinase (MMP) activity, and immune cell populations in a transgenic mouse model of ovarian carcinoma. AZD1480 treatment inhibited STAT3 phosphorylation and DNA binding, and migration and adhesion of cultured ovarian carcinoma cells and ovarian tumor growth rate, volume, and ascites production in mice. In addition, drug treatment led to altered gene expression, decreased tumor-associated MMP activity, and fewer suppressor T cells in the peritoneal TME of tumor-bearing mice than control mice. Taken together, our results show pharmacologic inhibition of the JAK2/STAT3 pathway leads to disruption of functions essential for ovarian tumor growth and progression and represents a promising therapeutic strategy.

Network analysis identifies an HSP90-central hub susceptible in ovarian cancer.

PURPOSE: Epithelial ovarian cancer (EOC) is usually detected at an advanced stage and is frequently lethal. Although many patients respond to initial surgery and standard chemotherapy consisting of a platinum-based agent and a taxane, most experience recurrence and eventually treatment-resistant disease. Although there have been numerous efforts to apply protein-targeted agents in EOC, these studies have so far documented little efficacy. Our goal was to identify broadly susceptible signaling proteins or pathways in EOC. EXPERIMENTAL DESIGN: As a new approach, we conducted data-mining meta-analyses integrating results from multiple siRNA screens to identify gene targets that showed significant inhibition of cell growth. On the basis of this meta-analysis, we established that many genes with such activity were clients of the protein chaperone HSP90. We therefore assessed ganetespib, a clinically promising second-generation small-molecule HSP90 inhibitor, for activity against EOC, both as a single agent and in combination with cytotoxic and targeted therapeutic agents. RESULTS: Ganetespib significantly reduced cell growth, induced cell-cycle arrest and apoptosis in vitro, inhibited growth of orthotopic xenografts and spontaneous ovarian tumors in transgenic mice in vivo, and inhibited expression and activation of numerous proteins linked to EOC progression. Importantly, paclitaxel significantly potentiated ganetespib activity in cultured cells and tumors. Moreover, combined treatment of cells with ganetespib and siRNAs or small molecules inhibiting genes identified in the meta-analysis in several cases resulted in enhanced activity. CONCLUSION: These results strongly support investigation of ganetespib, a single-targeted agent with effects on numerous proteins and pathways, in augmenting standard EOC therapies.

Combined in vivo molecular and anatomic imaging for detection of ovarian carcinoma-associated protease activity and integrin expression in mice.

Most patients with epithelial ovarian cancer (EOC) experience drug-resistant disease recurrence. Identification of new treatments is a high priority, and preclinical studies in mouse models of EOC may expedite this goal. We previously developed methods for magnetic resonance imaging (MRI) for tumor detection and quantification in a transgenic mouse model of EOC. The goal of this study was to determine whether three-dimensional (3D) fluorescence molecular tomography (FMT) and fluorescent molecular imaging probes could be effectively used for in vivo detection of ovarian tumors and response to therapy. Ovarian tumor-bearing TgMISIIR-TAg mice injected with fluorescent probes were subjected to MRI and FMT. Tumor-specific probe retention was identified in vivo by alignment of the 3D data sets, confirmed by ex vivo fluorescent imaging and correlated with histopathologic findings. Mice were treated with standard chemotherapy, and changes in fluorescent probe binding were detected by MRI and FMT. Ovarian tumors were detected using probes specific for cathepsin proteases, matrix metalloproteinases (MMPs), and integrin α(v)ß(3). Cathepsin and integrin α(v)ß(3) probe activation and retention correlated strongly with tumor volume. MMP probe activation was readily detected in tumors but correlated less strongly with tumor volume. Tumor regression associated with response to therapy was detected and quantified by serial MRI and FMT. These results demonstrate the feasibility and sensitivity of FMT for detection and quantification of tumor-associated biologic targets in ovarian tumors and support the translational utility of molecular imaging to assess functional response to therapy in mouse models of EOC.