Progression Of Serous Ovarian Cancer Research Articles

The β-arrestin1/endothelin axis bolsters ovarian fibroblast-dependent invadosome activity and cancer cell metastatic potential

Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix (ECM) remodeling, invasion, and metastasis, highlighting the need to investigate the molecular mechanisms driving CAF function. Endothelin-1 (ET-1) regulates the communication between cancer and stroma and facilitates the progression of serous ovarian cancer (SOC). By binding to Endothelin A (ETA) and B (ETB) receptors, ET-1 enables the recruitment of β-arrestin1 (β-arr1) and the formation of signaling complexes that coordinate tumor progression. However, how ET-1 receptors might “educate” human ovarian fibroblasts (HOFs) to produce altered ECM and promote metastasis remains to be elucidated. This study identifies ET-1 as a pivotal factor in the activation of CAFs capable of proteolytic ECM remodeling and the generation of heterotypic spheroids containing cancer cells with a propensity to metastasize. An autocrine/paracrine ET-1/ETA/BR/β-arr1 loop enhances HOF proliferation, upregulates CAF marker expression, secretes pro-inflammatory cytokines, and increases collagen contractility, and cell motility. Furthermore, ET-1 facilitates ECM remodeling by promoting the lytic activity of invadosome and activation of integrin β1. In addition, ET-1 signaling supports the formation of heterotypic HOF/SOC spheroids with enhanced ability to migrate through the mesothelial monolayer, and invade, representing metastatic units. The blockade of ETA/BR or β-arr1 silencing prevents CAF activation, invadosome function, mesothelial clearance, and the invasive ability of heterotypic spheroids. In vivo, therapeutic inhibition of ETA/BR using bosentan (BOS) significantly reduces the metastatic potential of combined HOFs/SOC cells, associated with enhanced apoptotic effects on tumor cells and stromal components. These findings support a model in which ET-1/β-arr1 reinforces tumor/stroma interaction through CAF activation and fosters the survival and metastatic properties of SOC cells, which could be counteracted by ETA/BR antagonists.

Abstract A105: Untangling the effect of ascites in shaping high grade serous ovarian cancer progression

Abstract High Grade Serous Ovarian Cancer (HGSOC) is the eighth cause of cancer-related mortality worldwide. Progress in eradicating this disease has been hampered by several factors, including the uncertainty about its tissues of origin, late diagnosis coupled to early dissemination through accumulation of ascitic fluid and extensive inter-patient variability. While being histopathologically homogeneous, HGSOC is characterized by several layers of heterogeneity that require the development of ad hoc research strategies and to focus on specific scales in order to deconvolve HGSOC pathogenetic mechanisms. In this context, our lab has previously shown that HGSOC can be derived both from the distal tract of the fallopian tube (fallopian tube epithelium, FTE) and the ovarian surface epithelium (OSE), with a prognostic impact for the tissue of origin and origin-specific molecular phenotypes, including an immune-suppressive response in the more aggressive OSE-derived subtype (Lo Riso, Villa et al. 2020), pointing at the role of tumor microenvironment in shaping therapeutical response. Moreover, we have also shown the role of ascitic fluid in sustaining the generation of single cell-derived metastatic ovarian cancer spheroids (sMOCS), that enrich for cancer stem-like cells, and allowing to identify a consistent intra-patient variability in response to carboplatin treatment (Velletri, Villa, Cilli, Barzaghi et al. 2022). To understand the specific impact of the tumor microenvironment in shaping HGSOC complexity, here we show an experimental framework aimed at the identification of the downstream effect of ascites in shaping cellular and molecular features of HGSOC. Here we leverage a newly generated single cell transcriptomic atlas, encompassing 1.5 million cells from 79 patients derived from primary tumors, ascites and metastatic samples, to identify specific expression networks activated during the metastatization process. In parallel, we derive from bulk transcriptomic data the set of genes that are induced upon ascitic fluid supplementation in 2D and 3D in vitro models, that are coupled to model-specific phenotypes, including cell proliferation, stemness and invasiveness. These genes are then integrated in the single-cell derived metastasis-specific expression networks to identify the upstream regulators imputable to ascitic fluid exposure. The linked regulators and downstream effectors are then used to identify in the atlas cell populations whose expression landscape is directly affected by ascitic fluid. The identification of such cell populations will allow to highlight cell-to-cell interactions existing between i) different tumor subpopulations; ii) tumor and tumor-associated compartments, in both primary and metastatic samples, that can be ascribed to the presence of ascitic fluid. Moreover, the molecular characterization of these interacting populations will allow to identify novel targets, paving the way to the identification of novel therapeutical avenues for HGSOC patients. Citation Format: Pietro Lo Riso, Carlo Emanuele Villa, Bianca Barzaghi, Marta R. Sallese, Giulia Picco, Alessia Gatto, Ugo Cavallaro, Giuseppe Testa. Untangling the effect of ascites in shaping high grade serous ovarian cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A105.

The Clinical Significance of Genetic Variation in Ovarian Cancer.

Genetic variation is a well-known contributor to the onset and progression of cancer. The goal of this study is to provide a comprehensive examination of the nucleotide and chromosomal variation associated with the onset and progression of serous ovarian cancer. Using a variety of computational and statistical methods, we examine the exome sequence profiles of genetic variants present in the primary tumors of 432 ovarian cancer patient samples to compute: (1) the tumor mutational burden for all genes and (2) the chromosomal copy number alterations associated with the onset/progression of ovarian cancer. Tumor mutational burden is reduced in the late vs. early stages, with the highest levels being associated with loss-of-function mutations in DNA-repair genes. Nucleotide variation and copy number alterations associated with known cancer driver genes are selectively favored over ovarian cancer development. The results indicate that genetic variation is a significant contributor to the onset and progression of ovarian cancer. The measurement of the relative levels of genetic variation associated with individual ovarian cancer patient tumors may be a clinically valuable predictor of potential tumor aggressiveness and resistance to chemotherapy. Tumors found to be associated with high levels of genetic variation may help in the clinical identification of high-risk ovarian cancer patients who could benefit from more frequent monitoring.

TEAD4 predicts poor prognosis and transcriptionally targets PLAGL2 in serous ovarian cancer.

The oncogenic function of TEA domain transcription factor 4 (TEAD4) has been confirmed in multiple human malignancies, while its potential role and regulatory mechanism in serous ovarian cancer progression are left unknown. By the gene expression analyses from Gene Expression Profiling Interactive Analysis (GEPIA) database, TEAD4 expression is shown to be up-regulated in serous ovarian cancer samples. Here, we confirmed the high expression of TEAD4 in clinical serous ovarian cancer specimens. In the following functional experiments, we found that TEAD4 overexpression promoted serous ovarian cancer malignant phenotypes, including proliferation, migration and invasion in serous ovarian cancer SK-OV-3 and OVCAR-3 cells, while TEAD4 knockout exerted the opposite function. The tumor growth inhibition of TEAD4 depletion was also affirmed by a Xenograft model in mice. In addition, this phenotypic deterioration induced by TEAD4 overexpression was diminished by PLAG1 like zinc finger 2 (PLAGL2) silencing. More importantly, combined with the results of the dual-luciferase assay, the transcriptional regulation of TEAD4 on PLAGL2 promoter was evidenced. Our results showed that the cancer-promoting gene TEAD4 was involved in serous ovarian cancer progression via targeting PLAGL2 at the transcriptional level.

Linc00261 Inhibited High-Grade Serous Ovarian Cancer Progression through miR-552-ATG10-EMT Axis.

In recent years, long non-coding RNAs (lncRNAs) play an important role in a multitude of pathways across species; however, their functions are still unknown. In this study, we demonstrate that Linc00261 is downregulation in high-grade serous ovarian cancer (HGSOC) and can inhibit cell proliferation and migration of high-grade serous ovarian cancer cells. We further validate the targeting interactions among Linc00261, miR-552, and ATG10. Interestingly, they all play important roles for regulating epithelial-mesenchymal transition (EMT) progression. Collectively, these findings suggest that Linc00261, a mediator of EMT progression, can target oncogenic miR-552, elevating ATG10 expression, to prevent high-grade serous ovarian cancer tumorigenesis and may serve as a potential novel therapeutic target.

CircSETDB1 knockdown inhibits the malignant progression of serous ovarian cancer through miR-129-3p-dependent regulation of MAP3K3

BackgroundCircular RNA (circRNA) is recently found to participate in the regulation of tumor progression, including ovarian cancer. However, the application of circRNA SET domain bifurcated histone lysine methyltransferase 1 (circSETDB1) as a therapeutic target in serous ovarian cancer (SOC) remains to be elucidated. Herein, circSETDB1 role in SOC malignant progression and underlying mechanism are revealed.MethodsThe expression of circSETDB1, microRNA-129-3p (miR-129-3p) and mitogen-activated protein kinase kinase kinase 3 (MAP3K3) messenger RNA (mRNA) was detected by quantitative real-time polymerase chain reaction. Protein abundance was determined by western blot analysis. Cell proliferation, apoptosis, invasion and migration were demonstrated by cell counting kit-8 and 5-Ethynyl-29-deoxyuridine assays, flow cytometry analysis, transwell invasion assay and wound-healing assay, respectively. The interaction between miR-129-3p and circSETDB1 or MAP3K3 was predicted by online database, and identified by mechanism assays. The effect of circSETDB1 knockdown on tumor formation in vivo was unveiled by mouse model experiment.ResultsCircSETDB1 and MAP3K3 expression were apparently upregulated, whereas miR-129-3p expression was downregulated in SOC tissues and cells in comparison with normal fallopian tube tissues or normal ovarian epithelial cells. CircSETDB1 knockdown inhibited cell proliferation, invasion and migration, but induced cell apoptosis in SOC cells. Additionally, miR-129-3p inhibitor impaired circSETDB1 silencing-mediated SOC malignant progression. MiR-129-3p repressed SOC cell processes via binding to MAP3K3. Furthermore, circSETDB1 knockdown suppressed tumor growth in vivo.ConclusionCircSETDB1 silencing repressed SOC malignant progression through miR-129-3p/MAP3K3 pathway. This study supports circSETDB1 as a new therapeutic target for SOC.

Oncogenic Role of miR-200c-3p in High-Grade Serous Ovarian Cancer Progression via Targeting the 3'-Untranslated Region of DLC1.

High-grade serous ovarian cancer (HGSC) is the most common ovarian cancer with highly metastatic properties. A small non-coding RNA, microRNA (miRNA) was discovered to be a major regulator in many types of cancers through binding at the 3′-untranslated region (3′UTR), leading to degradation of the mRNA. In this study, we sought to investigate the underlying mechanisms involved in the dysregulation of miR-200c-3p in HGSC progression and metastasis. We identified the upregulation of miR-200c-3p expression in different stages of HGSC clinical samples and the downregulation of the tumor suppressor gene, Deleted in Liver Cancer 1 (DLC1), expression. Over expression of miR-200c-3p in HGSC cell lines downregulated DLC1 but upregulated the epithelial marker, E-cadherin (CDH1). Based on in silico analysis, two putative binding sites were found within the 3′UTR of DLC1, and we confirmed the direct binding of miR-200c-3p to the target binding motif at position 1488–1495 bp of 3′UTR of DLC1 by luciferase reporter assay in a SKOV3 cell line co-transfected with vectors and miR-200c-3p mimic. These data showed that miR-200c-3p regulated the progression of HGSC by regulating DLC1 expression post-transcription and can be considered as a promising target for therapeutic purposes.

Abstract A30: Claudin-4-dependent mechanisms of high-grade serous ovarian cancer progression

Abstract In The Cancer Genome Atlas, Claudins-3, -4, and -7 are highly expressed in high-grade serous ovarian cancer (HGSOC). While claudins-3 and -7 are often expressed at tight junctions, claudin-4 (CLDN4) appears to be cytoplasmic, both in normal cells where it is expressed rarely and in a model of HGSOC, often used to study cellular interactions in this disease, OVCAR3 cells. CLDN4 expression reduces response to paclitaxel and conveys worse overall survival. We, therefore, wanted to further elucidate CLDN4-dependent mechanisms of cancer progression and therapeutic response. To address CLDN4 biology, we utilized immunohistochemical (IHC) analysis, loss-of-function studies, and several nonbiased approaches, including RNA-sequencing and proteomics. The IHC showed CLDN4 protein localized in the Golgi compartment with GM130 and around the cells at sites of cell-cell contact. Knocking down CLDN4 by 80% in OVCAR3 cells led them to undergo a transition from a more epithelial morphology to a mesenchymal phenotype (EMT). RNA-Seq analysis supports this morphologic interpretation by showing an increase in mRNA for genes coding for mesenchymal proteins such as fibronectin-1 (FN1), CD44, VIM, TNC, MMP2, and SNAI2 and a decrease in epithelial genes such as E-cadherin (CDH1), CD24, KRT18, and KRT19. RNA-Seq analysis of non-membrane protein kinases showed a 2-fold increase in PLAU, MSN, CAV1, CAV2, and EPHA2 and a 2-fold decrease in IGFBP2 and ERBB2, results that correlate well with the sensitivity of cancer cells to the BCR-ABL inhibitor, dasatinib. To determine the proteins that associate with CLDN4, we performed biotin ligase analysis (Bio-ID) fusing biotin protein ligase to CLDN4 and introducing the gene for the fusion protein into both OVCAR3 and OVCAR8 cells. Of the 36 proteins repeatedly more highly biotinylated in the presence of CLDN4-biotin ligase, 20 were identified as plasma membrane proteins and eight as Golgi localized by Gene Ontology analysis. Notably, we discovered a tyrosine protein kinase, YES1, to be biotinylated in both cell lines. YES1 is a poor prognostic indicator in HGSOC and promotes signal propagation following receptor tyrosine kinase activation, which contributes to a plethora of downstream outputs, including cell-cell adhesion and cell survival. These findings imply that CLDN4 expression is related to EMT and knockdown of CLDN4 could increase the sensitivity HGSOC to inhibitors such as dasatinib (BCR-ABL) or saracatinib (c-Yes). Citation Format: Patricia Webb, Margaret C. Neville, Heidi Baumgartner-Wilson, Miriam D. Post, Andrew Goodspeed, Benjamin G. Bitler. Claudin-4-dependent mechanisms of high-grade serous ovarian cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A30.

A graph-based multi-sample test for identifying pathways associated with cancer progression

Cancer is in general not a result of an abnormality of a single gene but a consequence of changes in many genes, it is therefore of great importance to understand the roles of different oncogenic and tumor suppressor pathways in tumorigenesis. In recent years, there have been many computational models developed to study the genetic alterations of different pathways in the evolutionary process of cancer. However, most of the methods are knowledge-based enrichment analyses and inflexible to analyze user-defined pathways or gene sets. In this paper, we develop a nonparametric and data-driven approach to testing for the dynamic changes of pathways over the cancer progression. Our method is based on an expansion and refinement of the pathway being studied, followed by a graph-based multivariate test, which is very easy to implement in practice. The new test is applied to the rich Cancer Genome Atlas data to study the (epi)genetic alterations of 186 KEGG pathways in the development of serous ovarian cancer. To make use of the comprehensive data, we incorporate three data types in the analysis representing gene expression level, copy number and DNA methylation level. Our analysis suggests a list of nine pathways that are closely associated with serous ovarian cancer progression, including cell cycle, ERBB, JAK-STAT signaling and p53 signaling pathways. By pairwise tests, we found that most of the identified pathways contribute only to a particular transition step. For instance, the cell cycle and ERBB pathways play key roles in the early-stage transition, while the ECM receptor and apoptosis pathways contribute to the progression from stage III to stage IV. The proposed computational pipeline is powerful in detecting important pathways and gene sets that drive cancers at certain stage(s). It offers new insights into the understanding of molecular mechanism of cancer initiation and progression.

MUS81 Participates in the Progression of Serous Ovarian Cancer Associated With Dysfunctional DNA Repair System.

Objective: Methyl methanesulfonate ultraviolet sensitive gene clone 81 (MUS81) is a structure-specific endonuclease that plays a pivotal role in the DNA repair system of cancer cells. In this study, we aim to elucidate the potential association between the dysfunction of MUS81 and the progression of Serous Ovarian Cancer (SOC).Methods: To investigate the association between MUS81 and prognosis of SOC, immunohistochemistry technology and qPCR were used to analyze the level of MUS81 expression, and transcriptional profile analysis and protein interaction screening chip were used to explore the MUS81 related signal pathways. Random amplified polymorphic DNA (RAPD) analysis, immunofluorescence and comet assays were further performed to evaluate genomic instability and DNA damage status of transduced SOC cells. Experiments both in vitro and in vivo were conducted to verify the impact of MUS81 silencing on chemotherapeutic drug sensitivity of SOC.Results: The overexpression of MUS81 in SOC tissues was related to poor clinical outcomes. The transcriptional chip data showed that MUS81 was involved in multiple pathways associated with DNA repair. Deficiency of MUS81 intensified the genome instability of SOC cells, promoted the emergence of DSBs and restrained the formation of RAD51 foci in SOC cells with exposure to UV. Furthermore, downregulation of MUS81 enhanced the sensitivity to Camptothecin and Olaparib in SOC cell lines and xenograft model.Conclusions: MUS81 is involved in the progression of SOC and inhibition of MUS81 could augment the susceptibility to chemotherapeutic agents. MUS81 might represent a novel molecular target for SOC chemotherapy.

Keratin 5 overexpression is associated with serous ovarian cancer recurrence and chemotherapy resistance

This study investigated the clinical significance of keratin 5 and 6 expression in serous ovarian cancer progression and chemotherapy resistance. KRT5 and KRT6 (KRT6A, KRT6B & KRT6C) gene expression was assessed in publically available serous ovarian cancer data sets, ovarian cancer cell lines and primary serous ovarian cancer cells. Monoclonal antibodies which detect both K5/6 or only K5 were used to assess protein expression in ovarian cancer cell lines and a cohort of high grade serous ovarian carcinomas at surgery (n = 117) and after neoadjuvant chemotherapy (n = 21). Survival analyses showed that high KRT5 mRNA in stage III/IV serous ovarian cancers was significantly associated with reduced progression-free (HR 1.38, P < 0.0001) and overall survival (HR 1.28, P = 0.013) whilst high KRT6 mRNA was only associated with reduced progression-free survival (HR 1.2, P = 0.031). Both high K5/6 (≥ 10%, HR 1.78 95% CI; 1.03−2.65, P = 0.017) and high K5 (≥ 10%, HR 1.90, 95% CI; 1.12−3.19, P = 0.017) were associated with an increased risk of disease recurrence. KRT5 but not KRT6C mRNA expression was increased in chemotherapy resistant primary serous ovarian cancer cells compared to chemotherapy sensitive cells. The proportion of serous ovarian carcinomas with high K5/6 or high K5 immunostaining was significantly increased following neoadjuvant chemotherapy. K5 can be used to predict serous ovarian cancer prognosis and identify cancer cells that are resistant to chemotherapy. Developing strategies to target K5 may therefore improve serous ovarian cancer survival.

Modeling the Dynamics of High-Grade Serous Ovarian Cancer Progression for Transvaginal Ultrasound-Based Screening and Early Detection.

High-grade serous ovarian cancer (HGSOC) represents the majority of ovarian cancers and accounts for the largest proportion of deaths from the disease. A timely detection of low volume HGSOC should be the goal of any screening studies. However, numerous transvaginal ultrasound (TVU) detection-based population studies aimed at detecting low-volume disease have not yielded reduced mortality rates. A quantitative invalidation of TVU as an effective HGSOC screening strategy is a necessary next step. Herein, we propose a mathematical model for a quantitative explanation on the reported failure of TVU-based screening to improve HGSOC low-volume detectability and overall survival.We develop a novel in silico mathematical assessment of the efficacy of a unimodal TVU monitoring regimen as a strategy aimed at detecting low-volume HGSOC in cancer-positive cases, defined as cases for which the inception of the first malignant cell has already occurred. Our findings show that the median window of opportunity interval length for TVU monitoring and HGSOC detection is approximately 1.76 years. This does not translate into reduced mortality levels or improved detection accuracy in an in silico cohort across multiple TVU monitoring frequencies or detection sensitivities. We demonstrate that even a semiannual, unimodal TVU monitoring protocol is expected to miss detectable HGSOC. Lastly, we find that circa 50% of the simulated HGSOC growth curves never reach the baseline detectability threshold, and that on average, 5–7 infrequent, rate-limiting stochastic changes in the growth parameters are associated with reaching HGSOC detectability and mortality thresholds respectively. Focusing on a malignancy poorly studied in the mathematical oncology community, our model captures the dynamic, temporal evolution of HGSOC progression. Our mathematical model is consistent with recent case reports and prospective TVU screening population studies, and provides support to the empirical recommendation against frequent HGSOC screening.

Abstract B61: Leptin induces a pro-inflammatory macrophage-cancer cell reinforcement loop that favors high-grade serous ovarian cancer progression among overweight/obese women.

Abstract Introduction: High-grade serous ovarian cancer remains as the most lethal gynecologic cancer. Despite achieving optimal debunking using ultra-radical surgery followed by platinum-based chemotherapy, in the long term, most of the patient will die from their disease, mainly due to extensive carcinomatosis, bowel obstruction and cachexia. Recently our group has provided biological evidence supporting the role of obesity in impairing ovarian cancer prognosis. Obesity is considered as a chronic low-grade inflammation state where leptin can play a major role in modulating cancer behavior. In the present work we explore the effects of leptin levels found in ascites, comparing lean and obese population, in cytokine profile released from macrophages and cancer cells and its impact in macrophage behavior and invasiveness of ovarian cancer cells. Methods: Ascites from patients undergoing primary cytoreductive surgery for advanced stage high-grade serous ovarian cancer were collected to establish primary tissue cultures and measuring leptin levels by ELISA kit. A database was built up for clinical variables, and patients were stratified by body mass index (BMI) using the WHO classification. Three ovarian cancer cells (e.g. HEY, UCI 101 and SKOV3) were used to establish in vitro spheroids and assessing the effects of leptin alone or conditioned media (CM) collected from leptin-treated macrophages in migration/invasion of cancer cells in matrigel Boyden chamber assays. IL-6, IL-10 and IL-12 release were measured in both cancer cells and macrophages upon leptin treatment, at similar concentrations than those found in ascites collected from lean, overweight and obese patients. Monocyte THP-1 cells were PMA-transformed into macrophages and the IL-10/IL-12 release ratio was used to identify M1 or M2 polarization induced either by leptin alone or CM collected from leptin-treated cancer cells. Results: Ascites collected from obese patients express significantly higher leptin levels compared with ascites from lean patients (13 ±4 ng/ml vs. 4±2 ng/ml; p&amp;lt;0,05). In addition, supplementation with ascites containing higher leptin levels significantly speeds up spheroid formation and increases the spheroid number. Moreover, ascites from obese patients also fosters formation of denser spheroids compared with ascites from lean patients. Adding leptin to the media at concentrations found in obese patients results in significantly increase in cytokine release from both ovarian cancer cells and macrophages. In terms of ovarian cancer cells, leptin fosters the faster formation of spheroids and increase the spheroid number. These cancer cells tend also to migrate and invade more than those not supplemented. In relation to macrophages, leptin induce an increase in the IL-10/IL-12 release ratio, reflecting preferred polarization to a M2 phenotype. Adding CM from macrophages, treated with leptin, induces epithelial mesenchymal transition (EMT) and significantly increases migration and invasion of ovarian cancer cells in matrigel Boyden chamber assays. Conclusions: Our findings support a role of leptin as one of the factors driving the detrimental effect of obesity in high-grade serous ovarian cancer. Leptin induces a pro-inflammatory reinforcement loop between cancer cell and macrophage that favors ovarian cancer progression by turning the macrophage tumor-suppressive phenotype into a pro-tumorigenic one and prompting an inflammatory microenvironment. (Research funded by Fondecyt 1120292). Citation Format: Mauricio A. Cuello, Lorena Abarzua-Catalan, Sumie Kato, Isidora Solar, Claudia Arancibia. Leptin induces a pro-inflammatory macrophage-cancer cell reinforcement loop that favors high-grade serous ovarian cancer progression among overweight/obese women. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B61.

Differential Wnt11 Expression Related to Wnt5a in High- and Low-grade Serous Ovarian Cancer: Implications for Migration, Adhesion and Survival

Wnt is a powerful signaling pathway that plays a crucial role in cell fate determination, survival, proliferation and motility during development, in adult tissues and cancer. The aims of the present study were three fold: i) to assess Wnt11 immunoexpression and its possible relationship with Wnt5a in high- and low-grade human serous ovarian cancer (HGSC and LGSC) specimens; ii) to assess Wnt11 expression levels in Wnt5a overexpressing SKOV-3 cells; iii) to reveal the role of Wnt11 in viability, adhesion, migration and invasion of SKOV-3 cells using recombinant human Wnt11 (rhWnt11). Immunohistochemistry revealed a significant difference in Wnt11 expression between HGSC and LGSC groups (p=0.001). Moreover, a positive correlation was observed between Wnt5a and Wnt11 expression in the HGSC (r=0.713, p=0.001), but not the LGSC group. The expression of Wnt11 was decreased by 35% in Wnt5a overexpressing cells (SKOV-3/Wnt5a) compared to mock controls. Similarly Wnt11 expression levels were decreased by 47% in the presence of exogenous Wnt5a compared to untreated cells. In the presence of rhWnt11, 31% increased cell viability (p<0.001) and 21% increased cell adhesion to matrigel (p<0.01) were observed compared to control. Cell migration was increased by 1.6-fold with rhWnt11 as revealed by transwell migration assay (p<0.001). However, 45% decreased cell invasion was observed in the presence of rhWnt11 compared to control (p<0.01). Our results may suggest that differential Wnt11 immunoexpression in HGSC compared to LGSC could play important roles in serous ovarian cancer progression and may be modulated by Wnt5a expression levels.

Androgen receptor protein levels are significantly reduced in serous ovarian carcinomas compared with benign or borderline disease but are not altered by cancer stage or metastatic progression.

The androgen receptor (AR) is expressed in a majority of ovarian carcinomas, but its role in disease development remains unclear. In this study, AR and a novel AR molecular chaperone called small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) were investigated to assess their potential role in ovarian carcinogenesis. First, an AR and SGTA-positive ovarian cancer cell line was identified to examine whether SGTA influenced AR subcellular localization. Next, relative protein levels of AR and SGTA were measured in two sets of clinical samples: (1) 46 serous ovarian carcinomas (stages I-IV), 9 serous borderline tumors, and 11 benign ovarian tumors; and (2) 24 patient-matched stage III primary and metastatic serous ovarian tumors. Ablation of SGTA protein in OVCAR3 cells significantly increased AR nuclear localization under basal (p ≤ 0.001) and androgen-stimulated (p ≤ 0.001) conditions. In the first clinical set, AR levels were significantly lower in early- (I/II) and late-stage (III/IV) cancers compared with benign (p ≤ 0.001) but not borderline ovarian tumors. SGTA alone did not discriminate between groups but the AR/SGTA ratio was significantly lower in carcinomas and borderline tumors compared with benign tumors (p ≤ 0.001 and 0.015, respectively). In the second clinical set, matched primary and metastatic serous ovarian cancers did not significantly differ for any parameter measured. Collectively, our results suggest that SGTA can influence AR signaling in ovarian cancer cells and that AR signaling capacity may be reduced with the development but not metastatic progression of serous ovarian cancer.

Discovery of serum biomarkers implicated in the onset and progression of serous ovarian cancer in a rat model using iTRAQ technique

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy, and early tumor detection is the most promising approach for improving the EOC survival rate. The goal of this study was to identify the biomarkers underlying ovarian carcinogenesis. To mimic the onset and progression of human ovarian cancer, we established a rat model of ovarian neoplasm by implanting 7,12-dimethylbenz(a)anthracene (DMBA)-coated silk cloth strips onto the ovaries. Sera collected from rats bearing serous ovarian carcinoma (SOC) at baseline, 12 and 24 weeks after DMBA treatment and from controls were analyzed using iTRAQ combined with two-dimensional liquid chromatography and tandem mass spectrometry. The data were analyzed with ProteinPilot software for peptide matching, protein identification, and protein quantitation. Ingenuity pathway analysis software was used to identify the canonical pathways and biological interaction networks of differentially expressed proteins. The cumulative ovarian tumor incidence rate reached 75% at 32 weeks after DMBA treatment. Out of all tumors, 94% were EOC, and 51% of the EOC cases were SOC. A total of 225 unique, non-redundant proteins were identified with 95% confidence. Twenty-seven differentially expressed proteins were significantly up- or down-regulated during the early or advanced carcinogenesis of SOC. Fifteen proteins were previously reported to be involved in ovarian cancer, and 12 proteins, including MMRN1, SERPINC1, TLN1, AHSG, PLG, APOA2, HPX, APOC1, APOC2, FERMT3, FETUB and HBB, were identified for the first time in our study. The discovery of these differentially expressed proteins provides valuable clues for understanding the molecular mechanism underlying the dynamic carcinogenic process of ovarian cancer. These proteins could be used as diagnostic biomarkers for early detection, disease monitoring and therapeutic targets.

Gene expression profiling of advanced‐stage serous ovarian cancers distinguishes novel subclasses and implicates ZEB2 in tumor progression and prognosis

To elucidate the mechanisms of rapid progression of serous ovarian cancer, gene expression profiles from 43 ovarian cancer tissues comprising eight early stage and 35 advanced stage tissues were carried out using oligonucleotide microarrays of 18,716 genes. By non-negative matrix factorization analysis using 178 genes, which were extracted as stage-specific genes, 35 advanced stage cases were classified into two subclasses with superior (n = 17) and poor (n = 18) outcome evaluated by progression-free survival (log rank test, P = 0.03). Of the 178 stage-specific genes, 112 genes were identified as showing different expression between the two subclasses. Of the 48 genes selected for biological function by gene ontology analysis or Ingenuity Pathway Analysis, five genes (ZEB2, CDH1, LTBP2, COL16A1, and ACTA2) were extracted as candidates for prognostic factors associated with progression-free survival. The relationship between high ZEB2 or low CDH1 expression and shorter progression-free survival was validated by real-time RT-PCR experiments of 37 independent advanced stage cancer samples. ZEB2 expression was negatively correlated with CDH1 expression in advanced stage samples, whereas ZEB2 knockdown in ovarian adenocarcinoma SKOV3 cells resulted in an increase in CDH1 expression. Multivariate analysis showed that high ZEB2 expression was independently associated with poor prognosis. Furthermore, the prognostic effect of E-cadherin encoded by CDH1 was verified using immunohistochemical analysis of an independent advanced stage cancer samples set (n = 74). These findings suggest that the expression of epithelial-mesenchymal transition-related genes such as ZEB2 and CDH1 may play important roles in the invasion process of advanced stage serous ovarian cancer.