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Advanced ovarian cancer usually spreads to the omentum. However, the omental cell-derived molecular determinants modulating its progression have not been thoroughly characterized. Here, we show that circulating ITLN1 has prognostic significance in patients with advanced ovarian cancer. Further studies demonstrate that ITLN1 suppresses lactotransferrin's effect on ovarian cancer cell invasion potential and proliferation by decreasing MMP1 expression and inducing a metabolic shift in metastatic ovarian cancer cells. Additionally, ovarian cancer-bearing mice treated with ITLN1 demonstrate marked decrease in tumor growth rates. These data suggest that downregulation of mesothelial cell-derived ITLN1 in the omental tumor microenvironment facilitates ovarian cancer progression.

Purpose: Recent studies demonstrate the role of the tumor microenvironment in tumor progression. However, strategies used to overcome the malignant phenotypes of cancer cells modulated by the microenvironment have not been thoroughly explored. In this study, we evaluated the therapeutic efficacy of a newly developed mAb targeting microfibril-associated protein 5 (MFAP5), which is secreted predominately by cancer-associated fibroblast (CAF), in ovarian and pancreatic cancer models. Experimental Design: MAbs were developed using human MFAP5 recombinant protein as an antigen in mice, and antibodies from hybridoma clones were evaluated for their specificity to human and murine MFAP5. An Octet RED384 system was used to determine the kinetics of binding affinity and the specificity of the antibody clones, which were followed by epitope mapping and functional characterization by in vitro assays. The therapeutic efficacy of a lead anti-MFAP5 antibody clone 130A in tumor suppression was evaluated by ovarian tumor- and pancreatic tumor-bearing mouse models., Results: Three hybridoma clones, which produced antibodies with high affinity and specificity to MFAP5, were selected for functional studies. Antibody clone 130A, which recognizes a common epitope shared between human and murine MFAP5 protein, was further selected for in vivo studies. Results showed that clone 130A downregulated MFAP5-induced collagen production in CAFs, suppressed intratumoral microvessel leakiness, and enhanced paclitaxel bioavailability in both ovarian and pancreatic cancer mouse models., Conclusions: These data suggest that MFAP5 blockade using an immunologic approach inhibits fibrosis, induces tumor vessel normalization, and enhances chemosensitivity in ovarian and pancreatic cancer, and can be used as a novel therapeutic agent., (©2019 American Association for Cancer Research.)

Objective: To investigate the prognostic and biologic significance of immune-related gene expression in high grade serous ovarian cancer (HGSOC)., Methods: Gene expression dependent survival analyses for a panel of immune related genes were evaluated in HGSOC utilizing The Cancer Genome Atlas (TCGA). Prognostic value of LCK was validated using IHC in an independent set of 72 HGSOC. Prognostic performance of LCK was compared to cytolytic score (CYT) using RNAseq across multiple tumor types. Differentially expressed genes in LCK high samples and gene ontology enrichment were analyzed., Results: High pre-treatment LCK mRNA expression was found to be a strong predictor of survival in a set of 535 ovarian cancers. Patients with high LCK mRNA expression had a longer median progression free survival (PFS) of 29.4 months compared to 16.9 months in those without LCK high expression (p = 0.003), and longer median overall survival (OS) of 95.1 months versus 44.5 months (p = 0.001), which was confirmed in an independent cohort by IHC (p = 0.04). LCK expression was compared to CYT across tumor types available in the TCGA and was a significant predictor of prognosis in HGSOC where CYT was not predictive. Unexpectedly, LCK high samples also were enriched in numerous immunoglobulin-related and other B cell transcripts., Conclusions: LCK is a better prognostic factor than CYT in ovarian cancer. In HGSOC, LCK high samples were characterized by higher expression of immunoglobulin and B-cell related genes suggesting that a cooperative interaction between tumor infiltrating T and B cells may correlate with better survival in this disease.

Background: Bulk tumor tissue samples are used for generating gene expression profiles in most research studies, making it difficult to decipher the stroma-cancer crosstalk networks. In the present study, we describe the use of microdissected transcriptome profiles for the identification of cancer-stroma crosstalk networks with prognostic value, which presents a unique opportunity for developing new treatment strategies for ovarian cancer., Methods: Transcriptome profiles from microdissected ovarian cancer-associated fibroblasts (CAFs) and ovarian cancer cells from patients with high-grade serous ovarian cancer (n = 70) were used as input data for the computational systems biology program CCCExplorer to uncover crosstalk networks between various cell types within the tumor microenvironment. The crosstalk analysis results were subsequently used for discovery of new indications for old drugs in ovarian cancer by computational ranking of candidate agents. Survival analysis was performed on ovarian tumor-bearing Dicer/Pten double-knockout mice treated with calcitriol, a US Food and Drug Administration-approved agent that suppresses the Smad signaling cascade, or vehicle control (9-11 mice per group). All statistical tests were two-sided., Results: Activation of TGF-β-dependent and TGF-β-independent Smad signaling was identified in a particular subtype of CAFs and was associated with poor patient survival (patients with higher levels of Smad-regulated gene expression by CAFs: median overall survival = 15 months, 95% confidence interval [CI] = 12.7 to 17.3 months; vs patients with lower levels of Smad-regulated gene expression: median overall survival = 26 months, 95% CI = 15.9 to 36.1 months, P = .02). In addition, the activated Smad signaling identified in CAFs was found to be targeted by repositioning calcitriol. Calcitriol suppressed Smad signaling in CAFs, inhibited tumor progression in mice, and prolonged the median survival duration of ovarian cancer-bearing mice from 36 to 48 weeks (P = .04)., Conclusions: Our findings suggest the feasibility of using novel multicellular systems biology modeling to identify and repurpose known drugs targeting cancer-stroma crosstalk networks, potentially leading to faster and more effective cures for cancers., (© The Author(s) 2018. Published by Oxford University Press.)

The molecular mechanism by which cancer-associated fibroblasts (CAFs) confer chemoresistance in ovarian cancer is poorly understood. The purpose of the present study was to evaluate the roles of CAFs in modulating tumor vasculature, chemoresistance, and disease progression. Here, we found that CAFs upregulated the lipoma-preferred partner (LPP) gene in microvascular endothelial cells (MECs) and that LPP expression levels in intratumoral MECs correlated with survival and chemoresistance in patients with ovarian cancer. Mechanistically, LPP increased focal adhesion and stress fiber formation to promote endothelial cell motility and permeability. siRNA-mediated LPP silencing in ovarian tumor-bearing mice improved paclitaxel delivery to cancer cells by decreasing intratumoral microvessel leakiness. Further studies showed that CAFs regulate endothelial LPP via a calcium-dependent signaling pathway involving microfibrillar-associated protein 5 (MFAP5), focal adhesion kinase (FAK), ERK, and LPP. Thus, our findings suggest that targeting endothelial LPP enhances the efficacy of chemotherapy in ovarian cancer. Our data highlight the importance of CAF-endothelial cell crosstalk signaling in cancer chemoresistance and demonstrate the improved efficacy of using LPP-targeting siRNA in combination with cytotoxic drugs.

Peritoneal dissemination is the primary metastatic route of ovarian cancer (OvCa), and is often accompanied by the accumulation of ascitic fluid. The peritoneal cavity is lined by mesothelial cells (MCs), which can be converted into carcinoma-associated fibroblasts (CAFs) through mesothelial-to-mesenchymal transition (MMT). Here, we demonstrate that MCs isolated from ascitic fluid (AFMCs) of OvCa patients with peritoneal implants also undergo MMT and promote subcutaneous tumour growth in mice. RNA sequencing of AFMCs revealed that MMT-related pathways - including transforming growth factor (TGF)-β signalling - are differentially regulated, and a gene signature was verified in peritoneal implants from OvCa patients. In a mouse model, pre-induction of MMT resulted in increased peritoneal tumour growth, whereas interfering with the TGF-β receptor reduced metastasis. MC-derived CAFs showed activation of Smad-dependent TGF-β signalling, which was disrupted in OvCa cells, despite their elevated TGF-β production. Accordingly, targeting Smad-dependent signalling in the peritoneal pre-metastatic niche in mice reduced tumour colonization, suggesting that Smad-dependent MMT could be crucial in peritoneal carcinomatosis. Together, these results indicate that bidirectional communication between OvCa cells and MC-derived CAFs, via TGF-β-mediated MMT, seems to be crucial to form a suitable metastatic niche. We suggest MMT as a possible target for therapeutic intervention and a potential source of biomarkers for improving OvCa diagnosis and/or prognosis. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Transcription factors are master switches for various biochemical pathways. However, transcription factors involved in the pathogenesis of ovarian cancer have yet to be explored thoroughly. Therefore, in the present study, we assessed the prognostic value of the transcription factor E74-like factor 3 (ELF3) identified via transcriptome profiling of the epithelial components of microdissected ovarian tumor samples isolated from long- and short-term survivors and determined its roles in ovarian cancer pathogenesis. Immunohistochemical analysis of ELF3 in tumor tissue sections suggested that ELF3 was exclusively expressed by epithelial ovarian cancer cells. Furthermore, using 112 high-grade ovarian cancer samples isolated from patients and The Cancer Genome Atlas (TCGA) data, we found that downregulation of ELF3 expression was markedly associated with reduced survival. Functional studies demonstrated that overexpression of ELF3 in ovarian cancer cells suppressed proliferation and anchorage-dependent growth of the cells and that ELF3 silencing increased cell proliferation. Furthermore, upregulation of ELF3 increased expression of epithelial markers, decreased expression of mesenchymal markers, and mediated translocation of epithelial-mesenchymal transition (EMT) signaling molecules in ovarian cancer cells. Finally, we validated the tumor-inhibitory roles of ELF3 using animal models. In conclusion, ELF3 is a favorable prognostic marker for ovarian cancer. As a negative regulator of EMT, ELF3-modulated reversal of EMT may be a new effective modality in the treatment of ovarian cancer.

The conventional approach to assessing cancer invasion is primarily for end-point analysis, which does not provide temporal information on the invasion process or any information on the interactions between invading cells and the underlying adherent cells. To alleviate these limitations, the present study exploited electric cell-substrate impedance sensing (ECIS) to monitor the invasion of ovarian cancer cells (SKOV-3) through an adherent monolayer of human umbilical vein endothelial cells (HUVECs). Impedance was measured at 4 kHz of AC voltage or was measured as a function of AC frequency (25 Hz to 60 kHz). By measuring impedance at 4-kHz AC, we found that the invasion of SKOV-3 cells through the HUVEC monolayer was manifested as a rapid decrease in transendothelial electrical resistance in real time. The invasion was augmented in the presence of hepatocyte growth factor (HGF). The enhancing effect of HGF was attenuated by c-Met inhibitor (SU11274). By measuring the frequency-dependent impedance of SKOV-3 cells over time, we found that HGF-enhanced SKOV-3 cell invasion was accomplished with reduced junctional resistance (R b ), increased average cell-substrate separation (h), and increased micromotion. SU11274 attenuated the effects of HGF on R b , h, and micromotion in the SKOV-3 monolayer. SU11274 also increased the barrier function of the HUVEC monolayer by increasing R b and decreasing h In conclusion, this study demonstrated an improved method for monitoring and studying the interactions between cancer cells and the underlying adherent cells during invasion in real time. Alterations in cellular biophysical properties (R b , h) associated with cancer transendothelial invasion were detected., (Copyright © 2016 the American Physiological Society.)

Advanced ovarian cancer usually spreads to the visceral adipose tissue of the omentum. However, the omental stromal cell-derived molecular determinants that modulate ovarian cancer growth have not been characterized. Here, using next-generation sequencing technology, we identify significantly higher levels of microRNA-21 (miR21) isomiRNAs in exosomes and tissue lysates isolated from cancer-associated adipocytes (CAAs) and fibroblasts (CAFs) than in those from ovarian cancer cells. Functional studies reveal that miR21 is transferred from CAAs or CAFs to the cancer cells, where it suppresses ovarian cancer apoptosis and confers chemoresistance by binding to its direct novel target, APAF1. These data suggest that the malignant phenotype of metastatic ovarian cancer cells can be altered by miR21 delivered by exosomes derived from neighbouring stromal cells in the omental tumour microenvironment, and that inhibiting the transfer of stromal-derived miR21 is an alternative modality in the treatment of metastatic and recurrent ovarian cancer.

Ovarian cancer is a histologically, clinically, and molecularly diverse disease with a five-year survival rate of less than 30%. It has been estimated that approximately 21,980 new cases of epithelial ovarian cancer will be diagnosed and 14,270 deaths will occur in the United States in 2015, making it the most lethal gynecologic malignancy. Ovarian tumor tissue is composed of cancer cells and a collection of different stromal cells. There is increasing evidence that demonstrates that stromal involvement is important in ovarian cancer pathogenesis. Therefore, stroma-specific signaling pathways, stroma-derived factors, and genetic changes in the tumor stroma present unique opportunities for improving the diagnosis and treatment of ovarian cancer. Cancer-associated fibroblasts (CAFs) are one of the major components of the tumor stroma that have demonstrated supportive roles in tumor progression. In this review, we highlight various types of signaling crosstalk between ovarian cancer cells and stromal cells, particularly with CAFs. In addition to evaluating the importance of signaling crosstalk in ovarian cancer progression, we discuss approaches that can be used to target tumor-promoting signaling crosstalk and how these approaches can be translated into potential ovarian cancer treatment.

Ovarian cancer is the most lethal gynecological malignancy. It is usually diagnosed at a late stage, with a 5-yr survival rate of <30%. The majority of ovarian cancer cases are diagnosed after tumors have widely spread within the peritoneal cavity, limiting the effectiveness of debulking surgery and chemotherapy. Owing to a substantially lower survival rate at late stages of disease than at earlier stages, the major cause of ovarian cancer deaths is believed to be therapy-resistant metastasis. Although metastasis plays a crucial role in promoting ovarian tumor progression and decreasing patient survival rates, the underlying mechanisms of ovarian cancer spread have yet to be thoroughly explored. For many years, researchers have believed that ovarian cancer metastasizes via a passive mechanism by which ovarian cancer cells are shed from the primary tumor and carried by the physiological movement of peritoneal fluid to the peritoneum and omentum. However, the recent discovery of hematogenous metastasis of ovarian cancer to the omentum via circulating tumor cells instigated rethinking of the mode of ovarian cancer metastasis and the importance of the "seed-and-soil" hypothesis for ovarian cancer metastasis. In this review we discuss the possible mechanisms by which ovarian cancer cells metastasize from the primary tumor to the omentum, the cross-talk signaling events between ovarian cancer cells and various stromal cells that play crucial roles in ovarian cancer metastasis, and the possible clinical implications of these findings in the management of this deadly, highly metastatic disease.

Ovarian cancer is the most lethal gynaecologic malignancy in the United States, and advanced serous ovarian adenocarcinoma is responsible for most ovarian cancer deaths. However, the stroma-derived molecular determinants that modulate patient survival are yet to be characterized. Here we identify a stromal gene signature for advanced high-grade serous ovarian cancer using microdissected stromal ovarian tumour samples and find that stromal microfibrillar-associated protein 5 (MFAP5) is a prognostic marker for poor survival. Further functional studies reveal that FAK/CREB/TNNC1 signalling pathways mediate the effect of MFAP5 on ovarian cancer cell motility and invasion potential. Targeting stromal MFAP5 using MFAP5-specific siRNA encapsulated in chitosan nanoparticles significantly decreases ovarian tumour growth and metastasis in vivo, suggesting that it may be a new modality of ovarian cancer treatment.

TGF-β has limited effects on ovarian cancer cells, but its contributions to ovarian tumor growth might be mediated through elements of the tumor microenvironment. In the present study, we tested the hypothesis that TGF modulates ovarian cancer progression by modulating the contribution of cancer-associated fibroblasts (CAF) that are present in the microenvironment. Transcriptome profiling of microdissected stromal and epithelial components of high-grade serous ovarian tumors and TGF-β-treated normal ovarian fibroblasts identified versican (VCAN) as a key upregulated target gene in CAFs. Functional evaluations in coculture experiments showed that TGF-β enhanced the aggressiveness of ovarian cancer cells by upregulating VCAN in CAFs. VCAN expression was regulated in CAFs through TGF-β receptor type II and SMAD signaling. Upregulated VCAN promoted the motility and invasion of ovarian cancer cells by activating the NF-κB signaling pathway and by upregulating expression of CD44, matrix metalloproteinase-9, and the hyaluronan-mediated motility receptor. Our work identified a TGF-β-inducible gene signature specific to CAFs in advanced high-grade serous ovarian tumors, and showed how TGF-β stimulates ovarian cancer cell motility and invasion by upregulating the CAF-specific gene VCAN. These findings suggest insights to develop or refine strategies for TGF-β-targeted therapy of ovarian cancer.

Purpose: To evaluate the prognostic value of fibroblast growth factor receptor 4 (FGFR4) protein expression in patients with advanced-stage, high-grade serous ovarian cancer, delineate the functional role of FGFR4 in ovarian cancer progression, and evaluate the feasibility of targeting FGFR4 in serous ovarian cancer treatment., Experimental Design: Immunolocalization of FGFR4 was conducted on 183 ovarian tumor samples. The collected FGFR4 expression data were correlated with overall survival using Kaplan-Meier and Cox regression analyses. The effects of FGFR4 silencing on ovarian cancer cell growth, survival, invasiveness, apoptosis, and FGF1-mediated signaling pathway activation were evaluated by transfecting cells with FGFR4-specific siRNAs. An orthotopic mouse model was used to evaluate the effect of injection of FGFR4-specific siRNAs and FGFR4 trap protein encapsulated in nanoliposomes on ovarian tumor growth in vivo., Results: Overexpression of FGFR4 protein was significantly associated with decreased overall survival durations. FGFR4 silencing significantly decreased the proliferation, survival, and invasiveness and increased apoptosis of ovarian cancer cells. Also, downregulation of FGFR4 significantly abrogated the mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and WNT signaling pathways, which are activated by FGF1. Targeting FGFR4 with the FGFR4-specific siRNAs and FGFR4 trap protein significantly decreased ovarian tumor growth in vivo., Conclusions: FGFR4 is a prognostic marker for advanced-stage, high-grade serous ovarian carcinoma. Silencing FGFR4 and inhibiting ligand-receptor binding significantly decrease ovarian tumor growth both in vitro and in vivo, suggesting that targeting ovarian cancer cells with high levels of FGFR4 protein expression is a new therapeutic modality for this disease and will improve survival of it., (©2012 AACR.)