Loss of Connective Tissue Growth Factor Expression Promotes Remodeling of the Extracellular Matrix and Epithelial-to-Mesenchymal Transition in Ovarian Cancer

Background: Ovarian Cancer (OC) is the leading cause of death from gynecologic malignancies in the United States largely due to the advanced stage at the time of diagnosis. Epithelial-to-mesenchymal transition (EMT) is a key biological process implicated in the pathophysiology of the metastatic spread of OC. Discovering the “trigger/s,” its downstream targets, and therapeutic targeting are essential to substantively improve the survival of women with OC. The objective of our study is to evaluate the role of Connective Tissue Growth Factor (CTGF) in EMT in OC. Methods: R182 and R2615 are well-described epithelial OC cell and MR182 and MR2615 are the mesenchymal counterparts. R182/R2615 CTGF knock outs (KO) were derived utilizing a Cas9/CRISPR-Cas9 lentivirus plasmid vector and verified by indel sequencing. Invasion, anoikis resistance, and chemosensitivity assays were performed in wild-type (WT) and KO cells. RNA sequence analysis was performed and analyzed using iPathway guide. Top five upregulated and downregulated genes involved in ECM organization pathway were validated by quantitative PCR (qPCR). Immunofluorescence was performed for F-actin. Results: CTGF was expressed in the epithelial and not in the mesenchymal OC cell lines. Loss of CTGF was associated with anoikis resistance, where KO and WT cells displayed 75% and 10% viability, respectively. KO cells were significantly more invasive than WT cells. Administration of exogeneous CTGF in KO cells decreased invasion in a dose dependent manner. No change was seen in chemosensitivity to Cisplatin in KO cells. RNA seq analysis identified ECM organization as the biologic process most affected by loss of CTGF. Upregulated (FREM2, LAMC2, ITGB4) and downregulated (SPP1, SV2A, RELN, COL6A3, COL4A6) extracellular matrix genes were validated by qPCR. Immunofluorescence staining of F-actin demonstrated increased cytoskeleton expression of F-actin in CTGF KO cells. Conclusion: Our data suggests that CTGF expression maintains the epithelial phenotype in OC. Loss of CTGF may be one of the early triggers of EMT in OC through extracellular matrix remodeling affecting anoikis and adhesion characteristics, thus acquiring a more migratory and invasive phenotype

Immunological modifications following chemotherapy are associated with delayed recurrence of ovarian cancer

IntroductionOvarian cancer recurs in most High Grade Serous Ovarian Cancer (HGSOC) patients, including initial responders, after standard of care. To improve patient survival, we need to identify and understand the factors contributing to early or late recurrence and therapeutically target these mechanisms. We hypothesized that in HGSOC, the response to chemotherapy is associated with a specific gene expression signature determined by the tumor microenvironment. In this study, we sought to determine the differences in gene expression and the tumor immune microenvironment between patients who show early recurrence (within 6 months) compared to those who show late recurrence following chemotherapy.MethodsPaired tumor samples were obtained before and after Carboplatin and Taxol chemotherapy from 24 patients with HGSOC. Bioinformatic transcriptomic analysis was performed on the tumor samples to determine the gene expression signature associated with differences in recurrence pattern. Gene Ontology and Pathway analysis was performed using AdvaitaBio’s iPathwayGuide software. Tumor immune cell fractions were imputed using CIBERSORTx. Results were compared between late recurrence and early recurrence patients, and between paired pre-chemotherapy and post-chemotherapy samples.ResultsThere was no statistically significant difference between early recurrence or late recurrence ovarian tumors pre-chemotherapy. However, chemotherapy induced significant immunological changes in tumors from late recurrence patients but had no impact on tumors from early recurrence patients. The key immunological change induced by chemotherapy in late recurrence patients was the reversal of pro-tumor immune signature.DiscussionWe report for the first time, the association between immunological modifications in response to chemotherapy and the time of recurrence. Our findings provide novel opportunities to ultimately improve ovarian cancer patient survival

The Role of Connective Tissue Growth Factor in Maintaining the Epithelial Phenotype of Ovarian Cancer Cells during Epithelial-to-Mesenchymal Transition

Objective: The transition from epithelial to mesenchymal is essential for the process of ovarian cancer (OC) metastasis. The aim of our study is to evaluate the role and mechanism of Connective Tissue Growth Factor (CTGF) in epithelial to mesenchymal transition (EMT). Methods: R182 is an epithelial OC cell line. CTGF expression +/- Transforming Growth Factor -b (TGF-b) was determined via Western blot and ELISA. R182 CTGF knock out (KO) were derived utilizing a Cas9/CRISPR-Cas9 lentivirus plasmid vector. Anoikis resistance and invasion assays were performed to characterized phenotypes of R182 wild type (WT) and KO cells. For anoikis resistance, cells were plated in triplicate in an ultra-low adhesive (ULA) cell plate with complete culture media. Using Promega CellTiter assay, cell viability was quantified by absorbance at 450 nm at 0, 24, 48, and 72 hr time points. For invasion assay, 3000 cells were suspended in 50% reduced growth factor Basement Membrane Extract. Human recombinant CTGF is added at 50 and 100 ng/mL concentrations. Cells were plated in tissue culture plate and placed in Cytation 5/Biospa. Cells were imaged at 4-hour interval for up to 6 days. Lastly, expression of mesenchymal markers including SNAIL was probed via Western blot analysis in both WT and KO cells. All experiments are being repeated in another epithelial OC cell line, R2615. Results: CTGF is constitutively expressed in R182 with detectable levels as early as 24 hrs of culture. Expression peaked at 2 – 6 hr with TGF-b and return to baseline by 24 hrs. Loss of CTGF promoted anoikis resistance. At 72 hr in ULA plate, R182 CTGF KO cells displayed 75% viability while R182 KO only have 10% viability. Further, we demonstrated that loss of CTGF allows for OC cells to invade the matrix whereas R182 WT does not display invasion. Administration of exogeneous CTGF in KO cells suppresses invasion in a dose dependent manner where 100 ng/mL returns the phenotype similar to R182 WT level. Lastly, loss of CTGF induces SNAIL expression. Conclusion: CTGF plays a role in maintaining the epithelial phenotype of OC cells during EMT. Loss of CTGF promotes anoikis resistance and invasion which are vital characteristics in the metastatic nature of OC. We suggest that loss of CTGF leads to increase in SNAIL expression permitting OC cells to transition to mesenchymal phenotype. Thus, loss of CTGF expression in OC cells could be a potential early target to prevent metastases in OC

Malignant Brenner tumor: Two case reports

Malignant Brenner tumor (MBT) is a rare ovarian tumor that, given the infrequency of the disease, has not been well documented in the literature. Diagnosing MBT both radiographically and histologically remains a challenge. We report two cases of ovarian MBT, detailing the clinical presentation, radiographic characteristics, and histologic findings with supplementary imaging. Our cases demonstrate the pathologic challenge of histologically diagnosing MBT versus other Brenner tumors and transitional cell carcinoma (TCC) of the ovary. Keywords: Malignant brenner tumor, Transitional cell carcinoma, Ovarian cance

A rare case of ovarian hemangioma in a 30-year-old nulligravid woman

A 30-year-old nulligravid woman with a history of polycystic ovarian syndrome presented for evaluation of left adnexal mass. The patient was referred to the gynecologic oncology clinic after endorsing signs of abdominal pain for a month and the pelvis ultrasound demonstrated hypoechoic solid mass in the left ovary. Magnetic resonance imaging with T1- and T2-weighted images demonstrated progressive centripetal “filling-in” of the mass suggesting a unique variation of malignant ovarian mass, similar to what is seen in hepatic hemangioma. Upon resection of the ovarian mass, pathology reported that the mass was filled with numerous small blood vessels with single later of endothelial cells confirming the diagnosis of ovarian hemangioma, capillary-type—a rare finding

Regulatory Role of the Adipose Microenvironment on Ovarian Cancer Progression

The tumor microenvironment of ovarian cancer is the peritoneal cavity wherein adipose tissue is a major component. The role of the adipose tissue in support of ovarian cancer progression has been elucidated in several studies from the past decades. The adipocytes, in particular, are a major source of factors, which regulate all facets of ovarian cancer progression such as acquisition of chemoresistance, enhanced metastatic potential, and metabolic reprogramming. In this review, we summarize the relevant studies, which highlight the role of adipocytes in ovarian cancer progression and offer insights into unanswered questions and possible future directions of research

Generation of Stable Epithelial–Mesenchymal Hybrid Cancer Cells with Tumorigenic Potential

Purpose: Cancer progression, invasiveness, and metastatic potential have been associated with the activation of the cellular development program known as epithelial-to-mesenchymal transition (EMT). This process is known to yield not only mesenchymal cells, but instead an array of cells with different degrees of epithelial and mesenchymal phenotypes with high plasticity, usually referred to as E/M hybrid cells. The characteristics of E/M hybrid cells, their importance in tumor progression, and the key regulators in the tumor microenvironment that support this phenotype are still poorly understood. Methods: In this study, we established an in vitro model of EMT and characterized the different stages of differentiation, allowing us to identify the main genomic signature associated with the E/M hybrid state. Results: We report that once the cells enter the E/M hybrid state, they acquire stable anoikis resistance, invasive capacity, and tumorigenic potential. We identified the hepatocyte growth factor (HGF)/c-MET pathway as a major driver that pushes cells in the E/M hybrid state. Conclusions: Herein, we provide a detailed characterization of the signaling pathway(s) promoting and the genes associated with the E/M hybrid state