High-grade Serous Ovarian Cancer Tissue Research Articles

Synchronous profiling of mRNA N6-methyladenosine modifications and mRNA expression in high-grade serous ovarian cancer: a pilot study

This study aimed to synchronously determine epitranscriptome-wide RNA N6-methyladenosine (m6A) modifications and mRNA expression profile in high grade serous ovarian cancer (HGSOC). The methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to comprehensively examine the m6A modification profile and the RNA-sequencing (RNA-seq) was performed to analyze the mRNA expression profile in HGSOC and normal fallopian tube (FT) tissues. Go and KEGG analyses were carried out in the enrichment of those differentially methylated and expressed genes. MeRIP-seq data showed 53,794 m6A methylated peaks related to 19,938 genes in the HGSOC group and 51,818 m6A peaks representing 19,681 genes in the FT group. RNA-seq results revealed 2321 upregulated and 2486 downregulated genes in HGSOC. Conjoint analysis of MeRIP-seq and RNA-seq data identified differentially expressed genes in which 659 were hypermethylated (330 up- and 329 down-regulated) and 897 were hypomethylated (475 up- and 422 down-regulated). Functional enrichment analysis indicated that these differentially modulated genes are involved in pathways related to cancer development. Among methylation regulators, the m6A eraser (FTO) expression was significantly lower, but the m6A readers (IGF2BP2 and IGF2BP3) were higher in HGSOC, which was validated by the subsequent real-time PCR assay. Exploration through public databases further corroborated their possible clinical application of certain methylation regulators and differentially expressed genes. For the first time, our study screens the epitranscriptome-wide m6A modification and expression profiles of their modulated genes and signaling pathways in HGSOC. Our findings provide an alternative direction in exploring the molecular mechanisms of ovarian pathogenesis and potential biomarkers in the diagnosis and predicting the prognosis of the disease.

Screening and identification of serum exosomal protein ZNF587B in liquid biopsy for ovarian cancer diagnosis.

Addressing the critical challenge of early ovarian cancer (OC) detection, our study focuses on identifying novel biomarkers by analyzing preoperative peripheral blood exosomes from high-grade serous ovarian cancer (HGSC) patients and healthy controls. Utilizing high-performance liquid chromatography-mass spectrometry-based quantitative proteomics, we isolated and analyzed peripheral blood exosomes to identify differentially expressed proteins (DEPs). This comprehensive analysis, supported by gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) database assessments, revealed 28 proteins with decreased abundance and 33 with increased abundance in HGSC patients compared to controls. Notably, Zinc Finger Protein 587B (ZNF587B) exhibited a significant reduction in abundance, confirmed by decreased mRNA and protein levels in HGSC and normal ovarian tissues, consistent with omes exosomal protein expression levels. Immunohistochemical staining further confirmed reduced ZNF587B protein levels in HGSC tissues. The significant correlation between ZNF587B expression levels and tumor stage underscores its potential as a valuable biomarker for early liquid biopsy screening of OC. Our findings suggest ZNF587B plays a crucial role in early HGSC detection, highlighting the importance of further research to validate its clinical utility and improve ovarian cancer patient outcomes.

Reclassify High-Grade Serous Ovarian Cancer Patients Into Different Molecular Subtypes With Discrepancy Prognoses and Therapeutic Responses Based on Cancer-Associated Fibroblast-Enriched Prognostic Genes.

Cancer-associated fibroblasts (CAFs) play critical roles in the metastasis and therapeutic response of high-grade serous ovarian cancer (HGSC). Our study intended to select HGSC patients with unfavorable prognoses and therapeutic responses based on CAF-enriched prognostic genes. The bulk RNA and single-cell RNA sequencing (scRNA-seq) data of tumor tissues were collected from the TCGA and GEO databases. The infiltrated levels of immune and stromal cells were estimated by multiple immune deconvolution algorithms and verified through immunohistochemical analysis. The univariate Cox regression analyses were used to identify prognostic genes. Gene Set Enrichment Analysis (GSEA) was conducted to annotate enriched gene sets. The Genomics of Drug Sensitivity in Cancer (GDSC) database was used to explore potential alternative drugs. We found the infiltered levels of CAFs were remarkedly elevated in advanced and metastatic HGSC tissues and identified hundreds of genes specifically enriched in CAFs. Then we selected 6 CAF-enriched prognostic genes based on which HGSC patients were reclassified into 2 subclusters with discrepancy prognoses. Further analysis revealed that the HGSC patients in cluster-2 tended to undergo poor responses to traditional chemotherapy and immunotherapy. Subsequently, we selected 24 novel potential therapeutic drugs for cluster-2 HGSC patients. Moreover, we discovered a positive correlation of infiltrated levels between CAFs and monocytes/macrophages in HGSC tissues. Collectively, our study successfully reclassified HGSC patients into 2 different subgroups that have discrepancy prognoses and responses to current therapeutic methods.

MiR-451a suppresses the proliferation and migration of high-grade serous ovarian cancer by targeting RAB5A through the Ras/Raf/MEK/ERK pathway.

Ovarian cancer is one of the most common cancers in women. Profiles changes of microRNAs (miRNAs) are closely linked to malignant tumors. In the present study, we investigated expression of miR-451a in high-grade serous ovarian cancer (HGSOC). We also investigated the potential pathological roles and the likely mechanism of miR-451a in the development of HGSOC using animal models and cell lines. Using bioinformatics techniques and a real-time PCR, we analyzed differently expressed miRNAs in HGSOC compared to normal tissue. MTT (i.e. 3-[4, 5-dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide), EDU (i.e. 5-ethynyl-2'-deoxyuridine) and transwell assays were performed to investigate the effect of miR-451a on the proliferation and migration of HGSOC SKOV-3 cells. A dual luciferase reporter assay was performed to verify the targeting relationship of miR-451 and RAB5A (one of the Rab GTPase proteins that regulates endocytosis and vesicle transport). Also, we analyzed levels of the RAB5A mRNA and protein by real-time PCR, western blotting and immunohistochemistry assays in HGSOC cells and tissues. Finally, we performed in vivo experiments using HGSOC mice. miR-451a was substantially upregulated in HGSOC and associated with favorable clinical characteristics. miR-451a knockdown significantly increased growth and metastasis of HGSOC cell line SKOV-3 through Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling. In addition, RAB5A, an early endosome marker, was shown to be a direct target of miR-451a. Moreover, RAB5A is correlated with unfavorable clinical features and shows independent prognostic significance in HGSOC. We found that the miR-451a/RAB5A axis is associated with tumorigenesis and progression through the Ras/Raf/MEK/ERK pathway, providing prognostic indicators and therapeutic targets for patients with HGSOC.

CD133, CD47, and PD-L1 Expression in Ovarian High-grade Serous Carcinoma and Its Association with Metastatic Disease: A Cross-sectional Study.

Ovarian cancer is a primary cause of cancer-related death in women. At the time of diagnosis, the majority of ovarian malignancies had metastasized. It is believed that cancer stem cells (CSCs) and immune evasion play a crucial role in the metastatic process. The objective of this study was to describe the expression profiles of cluster of differentiation (CD)133, CD47, and programmed death ligand 1 (PD-L1) in high-grade serous ovarian cancer (HGSC) as commonly utilized markers for CSCs and immune evasion. Using an immunohistochemical procedure, 51 HGSC tissue samples were stained with anti-CD133, anti-CD47, and anti-PDL1 antibodies. The samples contained 31 HGSC with metastases and 20 HGSC absent metastases. The expression of CD133, CD47, and PD-L1 was compared between groups. Strong expression of CD133 and CD47 was seen in 52% and 66% of tissue samples, respectively. Twenty of the thirty-one patients with metastases had a significant level of CD133 expression, with a p-value of 0.039. CD47 expression was increased in 26 of 31 samples with metastatic disease. A 62.7 percent of samples were negative for PD-L1 expression, significantly inversely correlated with HGSC metastatic disease (p=0.023). Although there was no significant association between CD133, CD47, or PD-L1 expression and age, Tumor Infiltrating Lymphocytes demonstrated a significantly varied relationship. Our findings suggested that expression of CD133, CD47, and PD-L1 may have dynamically increased as the primary lesion progressed to the metastatic lesion, implying that these proteins may be involved in the progression of high-grade serous ovarian cancer from the primary to the metastatic stage.

Abstract C092: Genetic profiling of high grade serous ovarian cancer among women of African ancestry

Abstract (A) AN INTRODUCTORY SENTENCE INDICATING THE PURPOSES OF THE STUDY: Ovarian cancer (OC) is a heterogeneous disease with high grade serous ovarian cancer (HGSOC) as the commonest, the most aggressive tumor, which is often diagnosed at advanced stages, and with poor overall survival. Unfortunately, like many other cancer diagnoses, women of West African ancestry (Black women) with ovarian cancer experience worse outcomes than White women. In the US, Black women have higher morbidity and mortality rates and higher un-staged or unclassified tumors compared with White women, resulting in undertreatment with subsequent compromise in progression-free survival. Genomic instability is one of the most common underlying features of ovarian cancer and defective DNA damage repair has been implicated. The goal is to uncover canonical genes associated with the aggressive behavior of HGSOC in a cohort of black women that could explain the biology of this tumor. (B) A BRIEF DESCRIPTION OF PERTINENT EXPERIMENTAL PROCEDURES: Formalin fixed paraffin embedded (FFPE) HGSOC tissue blocks from Nigeria in West Africa (WAB), Bahama in Caribbean (CBB), and Miami in USA (USB) were identified and selected after rigorous review and confirmation with immunohistochemical (IHC) markers when needed. Integration of digital histopathology and IHC markers (estrogen receptor, progesterone receptor, Ki67 and P53), whole exome sequence, RNA sequence of the FFPE samples was used to identify the proportion of genomic instability and defective DNA damage repair genes. We use spatial genomic library on tissue microarray (TMA) slide prepared on GeoMx digital spatial profiler (DSP) Nanostring platform to study the biology of the tumor and the tumor microenvironment (TME) and proteogenomics.(C) A SUMMARY OF THE NEW, UNPUBLISHED DATA: The unsupervised principal component analysis of the tumor showed a distinct population of WAB and USB while the CBB showed admixture population. BACH2, ARG1, NAFT2 and FRA10A1 are commonly differentially expressed genes among the cohort. There is low expression of routine IHC markers among the WAB compared to the other groups. Conclusion: There is variation on gene expression by nativity and we hope to explore this variation for therapeutic purpose among black women with ovarian cancer. Citation Format: Ayodele Omotoso, Uchenna Ezenkwa, Raleigh Butler, Melissa Castillo, Alex Sanchez-Covarrubias, Matthew Schlumbrecht, Andre Pinto, Bala Audu, Sophia George. Genetic profiling of high grade serous ovarian cancer among women of African ancestry [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C092.

In situ profiling reveals metabolic alterations in the tumor microenvironment of ovarian cancer after chemotherapy

In this study, we investigated the metabolic alterations associated with clinical response to chemotherapy in patients with ovarian cancer. Pre- and post-neoadjuvant chemotherapy (NACT) tissues from patients with high-grade serous ovarian cancer (HGSC) who had poor response (PR) or excellent response (ER) to NACT were examined. Desorption electrospray ionization mass spectrometry (DESI-MS) was performed on sections of HGSC tissues collected according to a rigorous laparoscopic triage algorithm. Quantitative MS-based proteomics and phosphoproteomics were performed on a subgroup of pre-NACT samples. Highly abundant metabolites in the pre-NACT PR tumors were related to pyrimidine metabolism in the epithelial regions and oxygen-dependent proline hydroxylation of hypoxia-inducible factor alpha in the stromal regions. Metabolites more abundant in the epithelial regions of post-NACT PR tumors were involved in the metabolism of nucleotides, and metabolites more abundant in the stromal regions of post-NACT PR tumors were related to aspartate and asparagine metabolism, phenylalanine and tyrosine metabolism, nucleotide biosynthesis, and the urea cycle. A predictive model built on ions with differential abundances allowed the classification of patients’ tumor responses as ER or PR with 75% accuracy (10-fold cross-validation ridge regression model). These findings offer new insights related to differential responses to chemotherapy and could lead to novel actionable targets.

Function of m5C RNA methyltransferase NOP2 in high-grade serous ovarian cancer

ABSTRACT RNA methyltransferase nucleolar protein p120 (NOP2), commonly referred to as NOP2/Sun RNA methyltransferase family member 1 (NSUN1), is involved in cell proliferation and is highly expressed in various cancers. However, its role in high-grade serous ovarian cancer (HGSOC) remains unclear. Our study investigated the expression of NOP2 in HGSOC tissues and normal fimbria tissues, and found that NOP2 was significantly upregulated in HGSOC tissues. Our experiments showed that NOP2 overexpression promoted cell proliferation in vivo and in vitro and increased the migration and invasion ability of HGSOC cells in vitro. Furthermore, we identified Rap guanine nucleotide exchange factor 4 (RAPGEF4) as a potential downstream target of NOP2 in HGSOC. Finally, our findings suggest that the regulation of NOP2 and RAPGEF4 may depend on m5C methylation levels.

C5aR1 blockade reshapes immunosuppressive tumor microenvironment and synergizes with immune checkpoint blockade therapy in high-grade serous ovarian cancer

ABSTRACT High-grade serous ovarian cancer (HGSC), with a modest response to immune checkpoint blockade (ICB) targeting PD-1/PD-L1 monotherapy, is densely infiltrated by M2-polarized tumor-associated macrophages (TAMs) and regulatory T (Treg) cells. The complement C5a/C5aR1 axis contributes to the programming of the immunosuppressive phenotype of TAMs in solid tumors and represents a promising immunomodulatory target for treating HGSCs. Here, we aimed to identify the relevance of C5aR1 in prognosis, immune microenvironment, and immunotherapy response in HGSCs. The expression and relationship of C5aR1 with tumor-infiltrating immune cells were assessed by immunohistochemistry and flow cytometry in the training cohort (n = 120) and fresh HGSC tissues (n = 36). Transcriptomic analyses of the xenografts delineated the mechanisms driving the immunomodulatory activity of PMX53, an orally bioavailable C5aR1 inhibitor. Therapeutic relevance was confirmed in ex vivo tumor cultures and The Cancer Genome Atlas (TCGA) datasets. C5aR1 expression independently predicted dismal prognosis and was linked to the immunoevasive subtype of HGSC, characterized by increased infiltration of pro-tumor cells (Treg cells, M2-polarized macrophages, and neutrophils) and impaired CD8+T functions. PMX53 antagonized subcutaneous tumor growth, modulated immunosuppressive mechanisms and synergized with aPD-1 in several tumor types. Single-cell RNA-seq analysis revealed predominant C5aR1 expression in TAMs, with an immunosuppressive-related expression signature in C5aR1+TAMs. Furthermore, the combination of C5aR1 and PD-L1 was associated with specific molecular characteristics and matched clinical response annotations. Therefore, the abundance of C5aR1 could predict an inferior prognosis in HGSCs, and incorporating PD-L1 may serve as a novel predictive biomarker to guide therapeutic options.

FAK inhibitor PF-562271 inhibits the migration and proliferation of high-grade serous ovarian cancer cells through FAK and FAK mediated cell cycle arrest.

Focal adhesion kinase (FAK) is a promising therapeutic target for various cancers and its inhibitor development is in full swing. PF-562271 is a classic FAK inhibitor that has shown promising preclinical data and has been found to exhibit an anti-migration effect on some cancer cells. However, its anticancer effect on high-grade serous ovarian cancer (HGSOC) has not been reported. In this study, we evaluated the anti-migration and anti-proliferation effects of PF-562271 against HGSOC SKOV3 and A2780 cells, as well as the underlying mechanism. The results demonstrated that FAK was overexpressed in clinical HGSOC tissues and was positively correlated with the pathological progression of HGSOC. Moreover, HGSOC patients with high FAK expression levels exhibited low survival rates. PF-562271 treatment significantly inhibited the cell adhesion and migration of SKOV3 and A2780 cells by inhibiting p-FAK expression and decreasing the FA surface area. Additionally, PF-562271 treatment inhibited colony formation and induced cell senescence through G1 phase cell cycle arrest mediated DNA replication inhibition. Taken together, the findings demonstrated that FAK inhibitor PF-562271 significantly inhibits HGSOC cell adhesion, migration, and proliferation process through FAK and/or FAK mediated cell cycle arrest, and suggested that PF-562271 could serve as a potential oncotherapeutic agent for HGSOC targeting treatment.

Oncogene-mediated nuclear accumulation of lactate promotes epigenetic alterations to induce cancer cell proliferation.

Homeobox gene families are associated with embryonic development and organogenesis. Pieces of evidence suggest that these Homeobox genes are also crucial in facilitating oncogenesis when mutated or overexpressed. Paired homeodomain transcription factor-2 (PITX2), one of the members of this family, is involved in oncogenic regulation apart from its different development regulatory functions. PITX2 has been earlier shown to induce ovarian cancer cell proliferation through the activation of different signaling cascades. Increased cancer cell proliferation requires a constant supply of nutrients for both adenosine triphosphate and biomass synthesis, which is facilitated by altered cancer cell metabolism that includes enhanced glucose uptake and increased glycolytic rate. This present study highlights the involvement of PITX2 in enhancing the cellular glycolysis pathway in ovarian cancer cells through protein kinase B-phosphorylation (phospho-AKT). PITX2 expression correlates positively with that of the glycolytic rate-determining enzyme, lactate dehydrogenase-A (LDHA), in both high-grade serous ovarian cancer tissues and common ovarian cancer cell lines. Interestingly, transient localization of enzymatically active LDHA in the nucleus was observed in PITX2-overexpressed ovarian cancer cells. This nuclear LDHA produces higher concentrations of the glycolytic end product, lactate, which accumulates in the nuclear compartment resulting in decreased histone deacetylase (HDAC1/2) expression and increased histone acetylation at H3/H4. However, the mechanistic details of lactate-HDAC interaction are still elusive in the earlier reports. Our in silico studies elaborated on the interaction dynamics of lactate in the HDAC catalytic core through ligand-binding studies and molecular dynamics simulation approaches. Blocking lactate production by silencing LDHA reduced cancer cell proliferation. Thus, PITX2-induced epigenetic changes can lead to high cellular proliferation and increase the size of tumors in syngeneic mice as well. Taken together, this is the first report of its kind to show that the developmental regulatory homeobox gene PITX2 could enhance oncogenesis through enhanced glycolysis of tumor cells followed by epigenetic modifications.

FGF19 promotes cell autophagy and cisplatin chemoresistance by activating MAPK signaling in ovarian cancer.

Chemotherapy is one of the primary treatments for ovarian cancer patients. Autophagy has been linked to chemotherapy resistance in tumor cells. Recent studies have suggested that fibroblast growth factor 19 (FGF19) may be involved in the onset and progression of malignancies. However, the relationship between FGF19 and autophagy in ovarian cancer is still unknown. Next-generation sequencing (NGS) was conducted to analyze gene mutation profiles of 62 cases of high grade serous ovarian cancer (HGSOC). Fluorescence in situ hybridization (FISH) was performed to validate the amplification of FGF19 in HGSOC tissues. Quantitative PCR (qPCR) and immunohistochemistry (IHC) were used to analyze the difference of FGF19 in mRNA and protein expression. Meanwhile, bioinformatics techniques were used to analyze the expression profiles of FGF19 and the correlation with prognosis. Besides, immunofluorescence, transmission electron microscopy and Cell Counting Kit 8 (CCK-8) were used to investigate the potential mechanisms. In this study, we found that FGF19 promotes cisplatin resistance in ovarian cancer cells by inducing autophagy. NGS analysis of 62 HGSOC cases identified a significantly amplified gene, FGF19. In addition, the expression level of FGF19 in ovarian cancer samples was higher than that in normal samples. FISH results showed a positive correlation between amplification and expression of FGF19. Knockdown of FGF19 inhibited the cell autophagy through decrease in the expression of LC3 and Beclin 1, and increase in the expression of SQSTM1/p62. Furthermore, we observed that p38 MAPK phosphorylation was down-regulated after FGF19 knockdown. IFN-γ, a potential p38 MAPK activator, counteracted the inhibition of cell autophagy and the anti-proliferation effect of cisplatin induced by FGF19 knockdown in ovarian cancer cells. FGF19 increases autophagy and chemoresistance in ovarian cancer by activating the p38 MAPK pathway. These results could point to FGF19 being a potential therapeutic target for ovarian cancer.

3P NaPi2b expression in high grade serous ovarian cancer: Results from combined data sets

NaPi2b is a sodium-dependent phosphate transporter broadly expressed in high-grade serous ovarian cancer (HGSOC), with limited expression in normal tissues. Uptifitamab rilsodotin (UpRi) is a first-in-class NaPi2b-targeting antibody drug conjugate (ADC) with a novel scaffold-linker-payload that enables high drug-to-antibody ratio and controlled bystander effect. NaPi2b is a new target currently being studied in ovarian cancer; understanding its expression and prevalence across HGSOCs is key in developing a biomarker-driven therapy. Multiple datasets were used to address the question of NaPi2b expression and prevalence. In all datasets, NaPi2b expression was assessed by IHC with NaPi2b positivity defined as a tumor proportion score ≥75. Sample sets included commercially procured HGSOC representing synchronous sampling of primary and metastatic lesions, matched metachronous samples (“archival” and “fresh”) from the Ph1b UpRi trial and HGSOC tissues sampled at multiple time points throughout the course of their disease. Our results suggest high prevalence of NaPi2b positive expression in HGSOC. In the UpRi Ph1b clinical trial, 64% (50/78; 95% CI 52.4 - 74.7) of patients were determined to be NaPi2b positive, as determined by the GLP (good laboratory practices) assay. When NaPi2b expression was examined from synchronous sampling of primary and metastatic lesions (N=18 pairs), as well as matched metachronous samples (N=56 pairs), there was high concordance rate ranging from 72-76% regardless of whether expression was evaluated over time or between anatomic locations. NaPi2b is a stable biomarker that is highly expressed in the majority of HGSOC patients. Expression remains stable over time - regardless of biopsy sites or treatment history. These data are consistent with data previously published from an investigational companion diagnostic assay study from 398 unique HGSOC tissue samples suggesting a prevalence of 59% (Patel et al., USCAP 2022). These findings support early NaPi2b testing and the continued development of NaPi2b-directed agents for HGSOC. Further analyses from ongoing trials and data sets will provide additional information on NaPi2b expression.

Clinical significance of SPOCK2 expression signature for high-grade serous ovarian cancer patients.

Background: SPOCK2 is a member of the SPOCK family, a 424-amino acid protein that binds to glycosaminoglycans to form proteoglycans. The purpose of this study was to explore expression profile of SPOCK2, and evaluate prognostic potential and its correlation with immune infiltration in high-grade serous ovarian cancer (HGSOC). Methods: Expression of SPOCK2 mRNA and protein between normal and tumor tissues were analyzed using the Cancer Genome Atlas database (TCGA), Gene Expression Omnibus (GEO), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and the Human Protein Atlas (HPA) databases. Receiver operating characteristic (ROC) curve was used to evaluate diagnostic performance of SPOCK2. Kaplan-Meier method and Cox regression analysis were conducted to assess the effect of SPOCK2 on survival. Nomogram was used to predict the impact of SPOCK2 on prognosis. LinkedOmics were used to find correlated genes and perform functional enrichment analyses. The relationships between SPOCK2 and tumor infiltrating lymphocytes (TILs) were determined by tumor-immune system interaction database (TISIDB) and GSVA package (V1.34.0). Results: SPOCK2 was highly expressed in HGSOC tissue compared to normal tissue at both mRNA (p < 0.001) and protein (p = 0.03) levels. The area under the curve (AUC) is 0.894 (CI: 0.865–0.923). Kaplan-Meier analysis showed that HGSOC patients with high-level SPOCK2 mRNA expression had a worse overall survival (OS) than those with a low expression (HR = 1.45, p = 0.005). Univariate logistic regression analysis found that age, primary therapy outcome, tumor status, tumor residual, and SPOCK2 expression level were significantly associated with OS (p < 0.05). The nomogram model indicated an effective predictive performance of SPOCK2. Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) term analyses showed that SPOCK2 were mainly involved in regulating extracellular matrix. Immune infiltration analysis showed that SPOCK2 may correlate with abundance of TILs. Conclusion: SPOCK2 has potentials to estimate diagnosis and prognosis for HGSOC and is involved in regulating extracellular matrix and immune cell infiltration.

CCNE1 is a potential target of Metformin for tumor suppression of ovarian high-grade serous carcinoma

ABSTRACT High-grade serous ovarian cancer (HGSOC) is the most common and malignant type of ovarian cancer, accounting for 70%–80% of mortality. However, the treatment of HGSOC has improved little in the past few decades. Metformin is the first-line medication for the treatment of type 2 diabetes and has now gained more attention in cancer treatment. In this study, we sought to identify potential hub genes that metformin could target in the treatment of HGSOC. We downloaded GSE69428 and GSE69429 in the Gene Expression Omnibus database and performed the bioinformatics analysis. Subsequently, we analyzed the effect of Metformin in HGSOC through biological experiments. Molecular simulation docking was used to predict the interaction of Metformin and CCNE1. We chose CCNE1 for the study based on bioinformatics analysis, literature studies, and preliminary data. We evaluated that CCNE1 is overexpressed in HGSOC tissues and found that HGSOC cells with high CCNE1 expression increase sensitivity to Metformin treatment in the analysis of cell proliferation and anchorage-independent growth. Metformin could inhibit the expression of CCNE1, which is associated with the anti-proliferative effect of tumor cells. Moreover, Metformin could ameliorate the tumor growth in syngeneic orthotopic transplantation mouse models and xenograft tumorigenesis models. Furthermore, molecular simulation docking showed that Metformin may bind to CCNE1 protein, suggesting that CCNE1 could be a potential target for Metformin. Our data revealed that Metformin has antitumor effects on ovarian cancer and CCNE1 could be a potential target for Metformin.

Single-Cell RNA Sequencing Reveals the Tissue Architecture in Human High-Grade Serous Ovarian Cancer.

The heterogeneity of high-grade serous ovarian cancer (HGSOC) is not well studied, which severely hinders clinical treatment of HGSOC. Thus, it is necessary to characterize the heterogeneity of HGSOC within its tumor microenvironment (TME). The tumors of 7 treatment-naïve patients with HGSOC at early or late stages and five age-matched nonmalignant ovarian samples were analyzed by deep single-cell RNA sequencing (scRNA-seq). A total of 59,324 single cells obtained from HGSOC and nonmalignant ovarian tissues were sequenced by scRNA-seq. Among those cells, tumor cells were characterized by a set of epithelial-to-mesenchymal transition (EMT)-associated gene signatures, in which a combination of NOTCH1, SNAI2, TGFBR1, and WNT11 was further selected as a genetic panel to predict the poor outcomes of patients with HGSOC. Matrix cancer-associated fibroblasts (mCAF) expressing α-SMA, vimentin, COL3A, COL10A, and MMP11 were the dominant CAFs in HGSOC tumors and could induce EMT properties of ovarian cancer cells in the coculture system. Specific immune cell subsets such as C7-APOBEC3A M1 macrophages, CD8+ TRM, and TEX cells were preferentially enriched in early-stage tumors. In addition, an immune coinhibitory receptor TIGIT was highly expressed on CD8+ TEX cells and TIGIT blockade could significantly reduce ovarian cancer tumor growth in mouse models. Our transcriptomic results analyzed by scRNA-seq delineate an ecosystemic landscape of HGSOC at early or late stages with a focus on its heterogeneity with TME. The major applications of our findings are a four-EMT gene model for prediction of HGSOC patient outcomes, mCAFs' capability of enhancing ovarian cancer cell invasion and potential therapeutic value of anti-TIGIT treatment.

Model Cell Lines and Tissues of Different HGSOC Subtypes Differ in Local Estrogen Biosynthesis.

Simple SummaryOvarian cancer (OC) comprises a heterogeneous group of hormone-dependent diseases with very high mortality. Estrogens have been shown to promote the progression of OC; however, their exact role in OC subtypes remains unknown. Here, we investigated the local estrogen biosynthesis in OC. We performed targeted transcriptomics and estrogen metabolism analyses in high-grade serous OC (HGSOC) cell lines that differed in chemoresistance status and compared these data with publicly available transcriptome and proteome data for HGSOC tissues. In HGSOC cells, estrogen metabolism decreased with increasing chemoresistance. In highly chemoresistant cells and platinum-resistant HGSOC tissues, HSD17B14 expression was increased. Proteome data showed differential levels of HSD17B10, SULT1E1, CYP1B1, and NQO1 between the four HGSOC subtypes. Our results confirm that estrogen biosynthesis differs between different HGSOC cell models and possibly between different HGSOC subtypes. Such differentially expressed enzymes have potential as targets in the search of new treatment options.Ovarian cancer (OC) is highly lethal and heterogeneous. Several hormones are involved in OC etiology including estrogens; however, their role in OC is not completely understood. Here, we performed targeted transcriptomics and estrogen metabolism analyses in high-grade serous OC (HGSOC), OVSAHO, Kuramochi, COV632, and immortalized normal ovarian epithelial HIO-80 cells. We compared these data with public transcriptome and proteome data for the HGSOC tissues. In all model systems, high steroid sulfatase expression and weak/undetected aromatase (CYP19A1) expression indicated the formation of estrogens from the precursor estrone-sulfate (E1-S). In OC cells, the metabolism of E1-S to estradiol was the highest in OVSAHO, followed by Kuramochi and COV362 cells, and decreased with increasing chemoresistance. In addition, higher HSD17B14 and CYP1A2 expressions were observed in highly chemoresistant COV362 cells and platinum-resistant tissues compared to those in HIO-80 cells and platinum-sensitive tissues. The HGSOC cell models differed in HSD17B10, CYP1B1, and NQO1 expression. Proteomic data also showed different levels of HSD17B10, CYP1B1, NQO1, and SULT1E1 between the four HGSOC subtypes. These results suggest that different HGSOC subtypes form different levels of estrogens and their metabolites and that the estrogen-biosynthesis-associated targets should be further studied for the development of personalized treatment.

Ovarian cancer immunogenicity is governed by a narrow subset of progenitor tissue-resident memory T-cells

Abstract Despite repeated associations between T-cell infiltration and patient outcome, human ovarian cancer remains poorly responsive to immunotherapy. We report that hallmarks of tumor recognition in ovarian cancer-infiltrating T-cells are primarily restricted to tissue-resident memory (TRM) cells. In mouse models we found that TRM T-cells were better than the re-circulating counterpart at controlling tumor growth. Single-cell RNA/TCR/ATAC sequencing of 83,454 CD3+CD8+CD103+CD69+ TRM cells and 24,175 CD3+CD8+CD103− re-circulating TILs showed that progenitor (TCF1low) tissue-resident memory T-cells (TRMstem cells) arise from transitional recirculating T-cells, which depends on antigen affinity/persistence, resulting in oligoclonal, trogocytic, effector lymphocytes. This effector population develops into proliferative lymphocytes that eventually become exhausted TRMs. Immunohistochemistry of 122 high-grade serous ovarian cancer tissues showed that only TRMstem cells, but not re-circulating TCF1+ T-cells, predict ovarian cancer outcome. Therefore, ovarian cancer is indeed an immunogenic disease that depends on ~13% of CD8+ tumor-infiltrating T-cells (~3% of CD8+ clonotypes), which are primed against high-affinity antigens and maintain waves of effector TRM cells. Support for Shared Resources was provided by Cancer Center Support Grant (CCSG) CA076292 to H. Lee Moffitt Cancer Center and by CCSG CA010815 to The Wistar Institute. This study was supported by grants from NIH (R01CA157664, R01CA124515, R01CA178687, R01CA211913 and U01CA232758 to JRCG; R01CA184185 and RO1CA262121 to PCR.)

Molecular Mechanism of Gleditsiae Spina for the Treatment of High-Grade Serous Ovarian Cancer Based on Network Pharmacology and Pharmacological Experiments.

Background Gleditsiae Spina, widely used in traditional Chinese medicine, has a good curative effect on malignant tumors such as ovarian cancer, but the mechanism is not clear. So, we aimed to analyze the pharmacological mechanism of Gleditsiae Spina in the treatment of high-grade serous ovarian cancer (HGSC) based on network pharmacology and biological experiments. Methods The main active ingredients of Gleditsiae Spina were identified by high performance liquid chromatography (HPLC) and mass spectrometry (MS), and the active ingredients were performed by ADME screening. The component targets of Gleditsiae Spina were screened using the PharmMapper platform, and differentially expressed genes in normal and HGSC tissues were identified through the GEO database. Thereafter, the network of “active ingredient-targets” was constructed by cytoscape 3.7.2 software. The protein-protein interaction network was established by the BioGenet database to mine the potential protein function. Biological processes and pathways were analyzed through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. The binding ability of the core components of the Gleditsiae Spina and the core target of HGSC was verified by molecular docking and molecular dynamics simulation, and the therapeutic effect of Gleditsiae Spina was proved in vitro through cytotoxicity experiments. The effect of Gleditsiae Spina on the core pathway is obtained by western blotting. Results Gleditsiae Spina had cytotoxicity on HGSC based on network pharmacology and biological experiments. Luteolin, genistein, D-(+)-tryptophan, ursolic acid, and berberine are the identified core active ingredients of Gleditsiae Spina for regulating HGSC, with HPSE, PI3KCA, AKT1, and CTNNB1as the ideal targets. The prediction results were verified by molecular docking, molecular dynamic simulation, cell viability, and western blot analysis. Conclusion Gleditsiae Spina mainly downregulates the expression of heparanase and β-catenin to affect the composition of tumor cytoplasmic matrix and can regulate the PI3K-AKT pathway, integrating multiple targets and multiple pathways to play a therapeutic role. It also provides a theoretical basis for the prevention of ovarian cancer and its treatment using traditional Chinese medicine in the future.

ETV5-mediated upregulation of lncRNA CTBP1-DT as a ceRNA facilitates HGSOC progression by regulating miR-188-5p/MAP3K3 axis

High-grade serous ovarian cancer (HGSOC) is a common and lethal cancer of the female reproductive system. Long non-coding RNAs (lncRNAs) are aberrantly expressed in various cancers and play crucial roles in tumour progression. However, their function and molecular mechanism in HGSOC remain largely unknown. Based on public databases and bioinformatics analyses, the overexpression of lncRNA CTBP1-DT in HGSOC tissues was detected and validated in a cohort of HGSOC tissues. High expression of lncRNA CTBP1-DT was associated with poor prognosis and was an independent risk factor for survival. Overexpression of lncRNA CTBP1-DT promoted malignant biological behaviour of HGSOC cells, whereas its depletion induced growth arrest of HGSOC cells by vitro and in vivo assays. Mechanistically, lncRNA CTBP1-DT could competitively bind to miR-188-5p to protect MAP3K3 from degradation. Moreover, our results revealed that ETV5 could specifically interact with the promoter of lncRNA CTBP1-DT and activate its transcription. Collectively, these results reveal a novel ETV5/lncRNA CTBP1-DT/miR-188-5p/MAP3K3 pathway for HGSOC progression and suggest that lncRNA CTBP1-DT might be a potential biomarker and therapeutic target for HGSOC.