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The standard treatment for high-grade serous ovarian cancer is primary debulking surgery followed by chemotherapy. The extent of metastasis and invasive potential of lesions can influence the outcome of these primary surgeries. Here, we explored the underlying mechanisms that could increase metastatic potential in ovarian cancer. We discovered that FABP4 (fatty acid binding protein) can substantially increase the metastatic potential of cancer cells. We also found that miR-409-3p regulates FABP4 in ovarian cancer cells and that hypoxia decreases miR-409-3p levels. Treatment with DOPC nanoliposomes containing either miR-409-3p mimic or FABP4 siRNA inhibited tumor progression in mouse models. With RPPA and metabolite arrays, we found that FABP4 regulates pathways associated with metastasis and affects metabolic pathways in ovarian cancer cells. Collectively, these findings demonstrate that FABP4 is functionally responsible for aggressive patterns of disease that likely contribute to poor prognosis in ovarian cancer., In ovarian cancer, metastatic phenotype may impact surgical outcomes. Here, the authors show miR-409-3p regulates FABP4 which can increase metastatic potential of ovarian cancer, and treatment with DOPC nanoliposomes containing either miR-409--3p mimic or FABP4 siRNA inhibits tumor progression in mice.

Primary debulking surgery followed by adjuvant chemotherapy is the standard treatment for ovarian cancer. Residual disease after primary surgery is associated with poor patient outcome. Previously, we discovered ADH1B to be a molecular biomarker of residual disease. In the current study, we investigated the functional role of ADH1B in promoting ovarian cancer cell invasiveness and contributing to residual disease. We discovered that ADH1B overexpression leads to a more infiltrative cancer cell phenotype, promotes metastasis, increases the adhesion of cancer cells to mesothelial cells, and increases extracellular matrix degradation. Live cell imaging revealed that ADH1B-overexpressing cancer cells efficiently cleared the mesothelial cell layer compared to control cells. Moreover, gene array analysis revealed that ADH1B affects several pathways related to the migration and invasion of cancer cells. We also discovered that hypoxia increases ADH1B expression in ovarian cancer cells. Collectively, these findings indicate that ADH1B plays an important role in the pathways that promote ovarian cancer cell infiltration and may increase the likelihood of residual disease following surgery.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

For mucinous ovarian cancer (MOC), standard platinum-based therapy is largely ineffective. We sought to identify possible mechanisms of oxaliplatin resistance of MOC and develop strategies to overcome this resistance. A kinome-based siRNA library screen was carried out using human MOC cells to identify novel targets to enhance the efficacy of chemotherapy. In vitro and in vivo validations of antitumor effects were performed using mouse MOC models. Specifically, the role of PRKRA/PACT in oxaliplatin resistance was interrogated. We focused on PRKRA, a known activator of PKR kinase, and its encoded protein PACT because it was one of the five most significantly downregulated genes in the siRNA screen. In orthotopic mouse models of MOC, we observed a significant antitumor effect of PRKRA siRNA plus oxaliplatin. In addition, expression of miR-515-3p was regulated by PACT–Dicer interaction, and miR-515-3p increased the sensitivity of MOC to oxaliplatin. Mechanistically, miR-515-3p regulated chemosensitivity, in part, by targeting AXL. The PRKRA/PACT axis represents an important therapeutic target in MOC to enhance sensitivity to oxaliplatin.

Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessels and starving tumor cells. This strategy has shown transient and modest efficacy. Here, we report a better approach to target cancer-associated endothelial cells (ECs), reverse permeability and leakiness of tumor blood vessels, and improve delivery of chemotherapeutic agents to the tumor. First, we identified deregulated microRNAs (miRs) from patient-derived cancer-associated ECs. Silencing these miRs led to decreased vascular permeability and increased maturation of blood vessels. Next, we screened a thioaptamer (TA) library to identify TAs selective for tumor-associated ECs. An annexin A2-targeted TA was identified and used for delivery of miR106b-5p and miR30c-5p inhibitors, resulting in vascular maturation and antitumor effects without inducing hypoxia. These findings could have implications for improving vascular-targeted therapy.

Mounting clinical and preclinical evidence supports a key role for sustained adrenergic signaling in the tumor microenvironment as a driver of tumor growth and progression. However, the mechanisms by which adrenergic neurotransmitters are delivered to the tumor microenvironment are not well understood. Here we present evidence for a feed-forward loop whereby adrenergic signaling leads to increased tumoral innervation. In response to catecholamines, tumor cells produced brain-derived neurotrophic factor (BDNF) in an ADRB3/cAMP/Epac/JNK-dependent manner. Elevated BDNF levels in the tumor microenvironment increased innervation by signaling through host neurotrophic receptor tyrosine kinase 2 receptors. In patients with cancer, high tumor nerve counts were significantly associated with increased BDNF and norepinephrine levels and decreased overall survival. Collectively, these data describe a novel pathway for tumor innervation, with resultant biological and clinical implications. Significance: Sustained adrenergic signaling promotes tumor growth and metastasis through BDNF-mediated tumoral innervation. Cancer Res; 78(12); 3233–42. ©2018 AACR.

Ovarian high-grade serous carcinoma (HGSC) results in the highest mortality among gynecological cancers, developing rapidly and aggressively. Dissimilarly, serous borderline ovarian tumors (BOT) can progress into low-grade serous carcinomas and have relatively indolent clinical behavior. The underlying biological differences between HGSC and BOT call for accurate diagnostic methodologies and tailored treatment options, and identification of molecular markers of aggressiveness could provide valuable biochemical insights and improve disease management. Here, we used desorption electrospray ionization (DESI) mass spectrometry (MS) to image and chemically characterize the metabolic profiles of HGSC, BOT, and normal ovarian tissue samples. DESI-MS imaging enabled clear visualization of fine papillary branches in serous BOT and allowed for characterization of spatial features of tumor heterogeneity such as adjacent necrosis and stroma in HGSC. Predictive markers of cancer aggressiveness were identified, including various free fatty acids, metabolites, and complex lipids such as ceramides, glycerophosphoglycerols, cardiolipins, and glycerophosphocholines. Classification models built from a total of 89,826 individual pixels, acquired in positive and negative ion modes from 78 different tissue samples, enabled diagnosis and prediction of HGSC and all tumor samples in comparison with normal tissues, with overall agreements of 96.4% and 96.2%, respectively. HGSC and BOT discrimination was achieved with an overall accuracy of 93.0%. Interestingly, our classification model allowed identification of three BOT samples presenting unusual histologic features that could be associated with the development of low-grade carcinomas. Our results suggest DESI-MS as a powerful approach for rapid serous ovarian cancer diagnosis based on altered metabolic signatures. Cancer Res; 77(11); 2903–13. ©2017 AACR.

Purpose: Chronic adrenergic activation has been shown to associate with adverse clinical outcomes in cancer patients, but the underlying mechanisms are not well understood. The focus of the current study was to determine the functional and biologic effects of adrenergic pathways on response to chemotherapy in the context of ovarian cancer. Experimental Design: Increased DUSP1 production by sympathetic nervous system mediators (e.g., norepinephrine) was analyzed by real-time quantitative RT-PCR and by Western blotting. In vitro chemotherapy-induced cell apoptosis was examined by flow cytometry. For in vivo therapy, a well-characterized model of chronic stress was used. Results: Catecholamines significantly inhibited paclitaxel- and cisplatin-induced apoptosis in ovarian cancer cells. Genomic analyses of cells treated with norepinephrine identified DUSP1 as a potential mediator. DUSP1 overexpression resulted in reduced paclitaxel-induced apoptosis in ovarian cancer cells compared with control; conversely, DUSP1 gene silencing resulted in increased apoptosis compared with control cells. DUSP1 gene silencing in vivo significantly enhanced response to paclitaxel and increased apoptosis. In vitro analyses indicated that norepinephrine-induced DUSP1 gene expression was mediated through ADRB2 activation of cAMP–PLC–PKC–CREB signaling, which inhibits JNK-mediated phosphorylation of c-Jun and protects ovarian cancer cells from apoptosis. Moreover, analysis of The Cancer Genome Atlas data showed that increased DUSP1 expression was associated with decreased overall (P = 0.049) and progression-free (P = 0.0005) survival. Conclusions: These findings provide a new understanding of the mechanisms by which adrenergic pathways can impair response to chemotherapy and have implications for cancer management. Clin Cancer Res; 22(7); 1713–24. ©2015 AACR.

Purpose: XPO1 inhibitors have shown promise for cancer treatment, and yet the underlying mechanisms for the antitumor effects are not well understood. In this study, we explored the usefulness of selective inhibitors of nuclear export (SINE) compounds that are specific inhibitors of XPO1. Experimental Design: We used proteomic analysis in XPO1 inhibitor–treated ovarian cancer cell lines and examined antitumor effects in ovarian and breast cancer mouse models. We also studied the effects of XPO1 inhibitor in combination with chemotherapeutic agents. Results: XPO1 inhibitor treatment substantially increased the percentage of apoptotic cells (60%) after 72 hours of incubation. XPO1 inhibitor promoted the accumulation of eIF5A in mitochondria, leading to cancer cell death. Topotecan showed the greatest synergistic effect with XPO1 inhibitor. XPO1 inhibitors prevented the translocation of IGF2BP1 from the nucleus to the cytoplasm, thereby permitting the localization of eIF5A in the mitochondria. This process was p53, RB, and FOXO independent. Significant antitumor effects were observed with XPO1 inhibitor monotherapy in orthotopic ovarian (P < 0.001) and breast (P < 0.001) cancer mouse models, with a further decrease in tumor burden observed in combination with topotecan or paclitaxel (P < 0.05). This mitochondrial accumulation of eIF5A was highly dependent on the cytoplasmic IGF2BP1 levels. Conclusions: We have unveiled a new understanding of the role of eIF5A and IGF2BP1 in XPO1 inhibitor–mediated cell death and support their clinical development for the treatment of ovarian and other cancers. Our data also ascertain the combinations of XPO1 inhibitors with specific chemotherapy drugs for therapeutic trials. Clin Cancer Res; 21(14); 3286–97. ©2015 AACR.

Despite being among the most common oncogenes in human cancer, to date, there are no effective clinical options for inhibiting KRAS activity. We investigated whether systemically delivered KRAS siRNAs have therapeutic potential in KRAS-mutated cancer models. We identified KRAS siRNA sequences with notable potency in knocking down KRAS expression. Using lung and colon adenocarcinoma cell lines, we assessed antiproliferative effects of KRAS silencing in vitro. For in vivo experiments, we used a nanoliposomal delivery platform, DOPC, for systemic delivery of siRNAs. Various lung and colon cancer models were used to determine efficacy of systemic KRAS siRNA based on tumor growth, development of metastasis, and downstream signaling. KRAS siRNA sequences induced >90% knockdown of KRAS expression, significantly reducing viability in mutant cell lines. In the lung cancer model, KRAS siRNA treatment demonstrated significant reductions in primary tumor growth and distant metastatic disease, while the addition of CDDP was not additive. Significant reductions in Ki-67 indices were seen in all treatment groups, whereas significant increases in caspase-3 activity were only seen in the CDDP treatment groups. In the colon cancer model, KRAS siRNA reduced tumor KRAS and pERK expression. KRAS siRNAs significantly reduced HCP1 subcutaneous tumor growth, as well as outgrowth of liver metastases. Our studies demonstrate a proof-of-concept approach to therapeutic KRAS targeting using nanoparticle delivery of siRNA. This study highlights the potential translational impact of therapeutic RNA interference, which may have broad applications in oncology, especially for traditional “undruggable” targets. Mol Cancer Ther; 13(12); 2876–85. ©2014 AACR.

Purpose Bisphosphonates have been shown to inhibit and deplete macrophages. The effects of bisphosphonates on other cell types in the tumor microenvironment have been insufficiently studied. Here, we sought to determine the effects of bisphosphonates on ovarian cancer angiogenesis and growth via their effect on the microenvironment, including macrophage, endothelial and tumor cell populations. Experimental Design Using in vitro and in vivo models, we examined the effects of clodronate on angiogenesis and macrophage density, and the overall effect of clodronate on tumor size and metastasis. Results Clodronate inhibited the secretion of pro-angiogenic cytokines by endothelial cells and macrophages, and decreased endothelial migration and capillary tube formation. In treated mice, clodronate significantly decreased tumor size, number of tumor nodules, number of tumor-associated macrophages and tumor capillary density. Conclusions Clodronate is a potent inhibitor of tumor angiogenesis. These results highlight clodronate as a potential therapeutic for cancer.

Cancer-related deregulation of miRNA biogenesis has been suggested, but the underlying mechanisms remain elusive. Here we report a previously unrecognized effect of hypoxia in the downregulation of Drosha and Dicer in cancer cells that leads to dysregulation of miRNA biogenesis and increased tumour progression. We show that hypoxia-mediated downregulation of Drosha is dependent on ETS1/ELK1 transcription factors. Moreover, mature miRNA array and deep sequencing studies reveal altered miRNA maturation in cells under hypoxic conditions. At a functional level, this phenomenon results in increased cancer progression in vitro and in vivo, and data from patient samples are suggestive of miRNA biogenesis downregulation in hypoxic tumours. Rescue of Drosha by siRNAs targeting ETS1/ELK1 in vivo results in significant tumour regression. These findings provide a new link in the mechanistic understanding of global miRNA downregulation in the tumour microenvironment.

Platinum compounds display clinical activity against a wide variety of solid tumors; however, resistance to these agents is a major limitation in cancer therapy. Reduced platinum uptake and increased platinum export are examples of resistance mechanisms that limit the extent of DNA damage. Here, we report the discovery and characterization of the role of ATP11B, a P-type ATPase membrane protein, in cisplatin resistance. We found that ATP11B expression was correlated with higher tumor grade in human ovarian cancer samples and with cisplatin resistance in human ovarian cancer cell lines. ATP11B gene silencing restored the sensitivity of ovarian cancer cell lines to cisplatin in vitro. Combined therapy of cisplatin and ATP11B-targeted siRNA significantly decreased cancer growth in mice bearing ovarian tumors derived from cisplatin-sensitive and -resistant cells. In vitro mechanistic studies on cellular platinum content and cisplatin efflux kinetics indicated that ATP11B enhances the export of cisplatin from cells. The colocalization of ATP11B with fluorescent cisplatin and with vesicular trafficking proteins, such as syntaxin-6 (STX6) and vesicular-associated membrane protein 4 (VAMP4), strongly suggests that ATP11B contributes to secretory vesicular transport of cisplatin from Golgi to plasma membrane. In conclusion, inhibition of ATP11B expression could serve as a therapeutic strategy to overcome cisplatin resistance.