Molecular Cancer

BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance

PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms △11 and △11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood.We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials.Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene's reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations.

Advances and obstacles of T cell-based immunotherapy in gynecological malignancies

Lactate accumulation induces H4K12la to activate super-enhancer-driven RAD23A expression and promote niraparib resistance in ovarian cancer

Ovarian cancer is a gynecological malignancy with the highest recurrence and mortality rates. Although niraparib can effectively affect its progression, the challenge of drug resistance remains. Herein, niraparib-resistant ovarian cancer cell lines were constructed to identify the abnormally activated enhancers and associated target genes via RNA in situ conformation sequencing. Notably, the target gene RAD23A was markedly upregulated in niraparib-resistant cells, and inhibiting RAD23A restored their sensitivity. Additionally, abnormal activation of glycolysis in niraparib-resistant cells induced lactate accumulation, which promoted the lactylation of histone H4K12 lysine residues. Correlation analysis showed that key glycolysis enzymes such as pyruvate kinase M and lactate dehydrogenase A were significantly positively correlated with RAD23A expression in ovarian cancer. Additionally, H4K12la activated the super-enhancer (SE) of niraparib and RAD23A expression via MYC transcription factor, thereby enhancing the DNA damage repair ability and promoting the drug resistance of ovarian cancer cells. Overall, the findings of this study indicate that lactic acid accumulation leads to lactylation of histone H4K12la, thereby upregulating SE-mediated abnormal RAD23A expression and promoting niraparib resistance in ovarian cancer cells, suggesting RAD23A as a potential therapeutic target for niraparib-resistant ovarian cancer.

Understanding gold nanoparticles and their attributes in ovarian cancer therapy

Ovarian cancer is one the deadliest disease wherein the survival rate is very low. Despite of advances in medical sciences, researches are still at the stage of infancy where patients are succumbing to this malignancy. Multidrug resistance, toxicity, mode of treatment related issues like catheter related complication poises a number of challenges to scientists worldwide. Novel therapy is now thus being focussed to sensitive the cells more towards the treatment. Gold nanoparticles (Au NPs), known for their high biocompatibility, and strong optical and magnetic responses, have emerged as promising agents for both the diagnosis and treatment of ovarian cancer. Owing to physical characteristics, AuNPs may be used as adjuvants in bioimaging, radiotherapy and fluorescence imaging. As a result, these characteristics substantially support AuNPs in biological domains. In addition to their therapeutic potential, Au NPs exhibit strong surface plasmon resonance (SPR) properties, enhancing imaging techniques for early detection of ovarian tumors. Furthermore, chemical properties such as Magnetic Resonance and Imaging Properties, X-ray imaging property, Two-photon or multiphoton imaging, and Optical coherence tomography (OCT) imaging properties enhance the use of Au NPs in diagnosis. This paper highlights the properties, targeting potential and diagnosis and treatment of ovarian cancer by Au NPs has been discussed.

Plasma-derived exosomal miR-15a-5p as a promising diagnostic biomarker for early detection of endometrial carcinoma

AbstractEndometrial cancer (EC) is a major cause of death among gynecologic malignancies. To improve early detection of EC in patients, we carried out a large plasma-derived exosomal microRNA (miRNA) studies for diagnostic biomarker discovery in EC. Small RNA sequencing was performed to identify candidate exosomal miRNAs as diagnostic biomarkers in 56 plasma samples from healthy subjects and EC patients. These miRNA candidates were further validated in 202 independent plasma samples by droplet digital PCR (ddPCR), 32 pairs of endometrial tumors and adjacent normal tissues by quantitative real-time PCR (qRT-PCR), and matched plasma samples of 12 patients before and after surgery by ddPCR. miR-15a-5p, miR-106b-5p, and miR107 were significantly upregulated in exomes isolated from plasma samples of EC patients compared with healthy subjects. Particularly, miR-15a-5p alone yielded an AUC value of 0.813 to distinguish EC patients with stage I from healthy subjects. The integration of miR-15a-5p and serum tumor markers (CEA and CA125) achieved a higher AUC value of 0.899. There was also a close connection between miR-15a-5p and clinical manifestations in EC patients. Its exosomal expression was not only associated with the depth of muscular infiltration and aggressiveness of EC, but also correlated with levels of reproductive hormones such as TTE and DHEAS. Collectively, plasma-derived exosomal miR-15a-5p is a promising and effective diagnostic biomarker for the early detection of endometrial cancer.

SELP+ TEC:CD8+ T cell crosstalk associates with improved radiotherapy efficacy in cervical cancer

P-selectin (SELP) expression in tumor cells has been implicated in promoting tumor progression and treatment resistance across various cancers. However, our prior study identified SELP expression in a specific subpopulation of endothelial cells within cervical cancer (CC) and potentially linked to anti-cancer immune response. The precise mechanisms by which SELP influences anti-cancer immunity and its involvement in radiotherapy response in CC, however, remain elusive. To address these gaps, this study analyzed tumor tissue samples from 205 CC patients undergoing radiotherapy, scRNA-seq data from 42,159 cells of eight patients, and bulk RNA-sequencing data from 187 radiotherapy-treated patients. The results revealed that elevated SELP expression in tumor endothelial cells (TECs) was significantly correlated with improved survival outcomes in patients treated with radiotherapy. The SELP

CircZFR/YTHDF3 axis drives lymph node metastasis in cervical cancer via FASN translation

Lymph node metastasis is a key driver of poor outcomes in cervical cancer. However, the molecular mechanisms of circular RNAs (circRNAs) driving cervical cancer lymph node metastasis remain unclear. We identified circZFR, fatty acid synthase (FASN) and YTH N6-methyladenosine RNA binding protein F3 (YTHDF3) protein expression in the cervical cancer patients with long and short disease-free survival (DFS). Functional experiments were performed to investigate the function of circZFR, FASN and YTHDF3 on cell migration and invasion. MeRIP-qPCR, RNA pulldown, RNA Immunoprecipitation (RIP), and Co-Immunoprecipitation (Co-IP) assays were executed to investigate the mechanism of circZFR regulating FASN protein expression. Our study reveals that elevated FASN protein is closely linked to metastasis and reduced survival, and identified a regulatory mechanism involving circular RNAs. We identified circZFR as a crucial regulator, significantly enhancing FASN protein expression. CircZFR overexpression was significantly correlated with accelerated lymph node metastasis and shortened DFS. Mechanistically, circZFR binds to the m These findings establish circZFR as a pivotal driver of cervical cancer progression and highlight its inhibition as a promising therapeutic strategy.

Cell surface CD55 traffics to the nucleus leading to cisplatin resistance and stemness by inducing PRC2 and H3K27 trimethylation on chromatin in ovarian cancer

Abstract Background Platinum resistance is the primary cause of poor survival in ovarian cancer (OC) patients. Targeted therapies and biomarkers of chemoresistance are critical for the treatment of OC patients. Our previous studies identified cell surface CD55, a member of the complement regulatory proteins, drives chemoresistance and maintenance of cancer stem cells (CSCs). CSCs are implicated in tumor recurrence and metastasis in multiple cancers. Methods Protein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing. Results CD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2. Conclusions For the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry.

Identification of miPEP133 as a novel tumor-suppressor microprotein encoded by miR-34a pri-miRNA

Abstract Background Very few proteins encoded by the presumed non-coding RNA transcripts have been identified. Their cellular functions remain largely unknown. This study identifies the tumor-suppressor function of a novel microprotein encoded by the precursor of miR-34a. It consists of 133 amino acid residues, thereby named as miPEP133 (pri-microRNA encoded peptide 133). Methods We overexpressed miPEP133 in nasopharyngeal carcinoma (NPC), ovarian cancer and cervical cancer cell lines to determine its effects on cell growth, apoptosis, migration, or invasion. Its impact on tumor growth was evaluated in a xenograft NPC model. Its prognostic value was analyzed using NPC clinical samples. We also conducted western blot, immunoprecipitation, mass spectrometry, confocal microscopy and flow cytometry to determine the underlying mechanisms of miPEP133 function and regulation. Results miPEP133 was expressed in normal human colon, stomach, ovary, uterus and pharynx. It was downregulated in cancer cell lines and tumors. miPEP133 overexpression induced apoptosis in cancer cells and inhibited their migration and invasion. miPEP133 inhibited tumor growth in vivo. Low miPEP133 expression was an unfavorable prognostic marker associated with advanced metastatic NPC. Wild-type p53 but not mutant p53 induced miPEP133 expression. miPEP133 enhanced p53 transcriptional activation and miR-34a expression. miPEP133 localized in the mitochondria to interact with mitochondrial heat shock protein 70kD (HSPA9) and prevent HSPA9 from interacting with its binding partners, leading to the decrease of mitochondrial membrane potential and mitochondrial mass. Conclusion miPEP133 is a tumor suppressor localized in the mitochondria. It is a potential prognostic marker and therapeutic target for multiple types of cancers.

Downregulation of MYPT1 increases tumor resistance in ovarian cancer by targeting the Hippo pathway and increasing the stemness

Abstract Background Ovarian cancer is one of the most common and malignant cancers, partly due to its late diagnosis and high recurrence. Chemotherapy resistance has been linked to poor prognosis and is believed to be linked to the cancer stem cell (CSC) pool. Therefore, elucidating the molecular mechanisms mediating therapy resistance is essential to finding new targets for therapy-resistant tumors. Methods shRNA depletion of MYPT1 in ovarian cancer cell lines, miRNA overexpression, RT-qPCR analysis, patient tumor samples, cell line- and tumorsphere-derived xenografts, in vitro and in vivo treatments, analysis of data from ovarian tumors in public transcriptomic patient databases and in-house patient cohorts. Results We show that MYPT1 (PPP1R12A), encoding myosin phosphatase target subunit 1, is downregulated in ovarian tumors, leading to reduced survival and increased tumorigenesis, as well as resistance to platinum-based therapy. Similarly, overexpression of miR-30b targeting MYPT1 results in enhanced CSC-like properties in ovarian tumor cells and is connected to the activation of the Hippo pathway. Inhibition of the Hippo pathway transcriptional co-activator YAP suppresses the resistance to platinum-based therapy induced by either low MYPT1 expression or miR-30b overexpression, both in vitro and in vivo. Conclusions Our work provides a functional link between the resistance to chemotherapy in ovarian tumors and the increase in the CSC pool that results from the activation of the Hippo pathway target genes upon MYPT1 downregulation. Combination therapy with cisplatin and YAP inhibitors suppresses MYPT1-induced resistance, demonstrating the possibility of using this treatment in patients with low MYPT1 expression, who are likely to be resistant to platinum-based therapy.

circFBXO7/miR-96-5p/MTSS1 axis is an important regulator in the Wnt signaling pathway in ovarian cancer

Abstract Background CircRNAs are a novel class of evolutionarily conserved noncoding RNA molecules that form covalently closed continuous loop structures without 5′ caps and 3′ poly(A) tails. Accumulating evidence suggests that circRNAs play important regulatory roles in cancer and are promising biomarkers for cancer diagnosis and prognosis, as well as targets for cancer therapy. In this study, we identify and explore the role of a novel circRNA, circFBXO7, in ovarian cancer. Methods rRNA-depleted RNA-sequencing was performed to identify differentially expressed circRNAs between ovarian cancerous and normal tissues. qRT-PCR and single-molecule RNA in-situ hybridization was used to quantify circFBXO7 expression in tumor tissues. The association of circFBXO7 expression with patient prognosis was evaluated by Kaplan–Meier survival analysis. The biological function of circFBXO7 was also investigated using loss-of-function and gain-of-function assays in vivo and in vitro. Luciferase reporter and TOP/FOP-Flash reporter assays were then conducted together with RNA immunoprecipitation and western blot to assess the circFBXO7/miR-96-5p/MTSS1/Wnt/β-catenin axis. Results circFBXO7 was downregulated in ovarian cancer which was associated with poor prognosis. Biologically, circFBXO7 overexpression significantly suppressed ovarian cancer cell proliferation, migration, and invasion in vitro, and inhibited tumor growth and metastasis in vivo, whereas its knockdown exerted an opposite role. Mechanistically, circFBXO7 functioned as a competing endogenous RNA for miR-96-5p to regulate the expression of MTSS1. Consequently, downregulation of MTSS1 led to excessive accumulation of β-catenin and increased phosphorylation of GSK3β, leading to the translocation of β-catenin to the nucleus, thereby activating the Wnt/β-catenin signaling pathway and ultimately promoting ovarian cancer progression. Conclusions Our findings indicate that circFBXO7 acts as a bone fide tumor suppressor in ovarian cancer and that the circFBXO7/miR-96-5p/MTSS1 axis is an important regulator in the Wnt/β-catenin signaling pathway which may provide a promising target for ovarian cancer therapy.

Potential clinical utility of liquid biopsies in ovarian cancer

AbstractBackgroundOvarian cancer (OC) is the most lethal gynecologic malignancy worldwide. One of the main challenges in the management of OC is the late clinical presentation of disease that results in poor survival. Conventional tissue biopsy methods and serological biomarkers such as CA-125 have limited clinical applications. Liquid biopsy is a novel sampling method that analyzes distinctive tumour components released into the peripheral circulation, including circulating tumour DNA (ctDNA), circulating tumour cells (CTCs), cell-free RNA (cfRNA), tumour-educated platelets (TEPs) and exosomes. Increasing evidence suggests that liquid biopsy could enhance the clinical management of OC by improving early diagnosis, predicting prognosis, detecting recurrence, and monitoring response to treatment. Capturing the unique tumour genetic landscape can also guide treatment decisions and the selection of appropriate targeted therapies. Key advantages of liquid biopsy include its non-invasive nature and feasibility, which allow for serial sampling and longitudinal monitoring of dynamic tumour changes over time. In this review, we outline the evidence for the clinical utility of each liquid biopsy component and review the advantages and current limitations of applying liquid biopsy in managing ovarian cancer. We also highlight future directions considering the current challenges and explore areas where more studies are warranted to elucidate its emerging clinical potential.

N6-methyladenosine-modified circPLPP4 sustains cisplatin resistance in ovarian cancer cells via PIK3R1 upregulation

Abstract Background Cisplatin (CDDP) is the first-line chemotherapeutic strategy to treat patients with ovarian cancer (OC). The development of CDDP resistance remains an unsurmountable obstacle in OC treatment and frequently induces tumor recurrence. Circular RNAs (circRNAs) are noncoding RNAs with important functions in cancer progression. Whether circRNAs function in CDDP resistance of OC is unclear. Methods Platinum-resistant circRNAs were screened via circRNA deep sequencing and examined using in situ hybridization (ISH) in OC. The role of circPLPP4 in CDDP resistance was assessed by clone formation and Annexin V assays in vitro, and by OC patient-derived xenografts and intraperitoneal tumor models in vivo . The mechanism underlying circPLPP4-mediated activation of miR-136/PIK3R1 signaling was examined by luciferase reporter assay, RNA pull-down, RIP, MeRIP and ISH. Results circPLPP4 was remarkably upregulated in platinum resistant OC. circPLPP4 overexpression significantly enhanced, whereas circPLPP4 silencing reduced, OC cell chemoresistance. Mechanistically, circPLPP4 acts as a microRNA sponge to sequester miR-136, thus competitively upregulating PIK3R1 expression and conferring CDDP resistance. The increased circPLPP4 level in CDDP-resistant cells was caused by increased RNA stability, mediated by increased N6-methyladenosine (m 6 A) modification of circPLPP4 . In vivo delivery of an antisense oligonucleotide targeting circPLPP4 significantly enhanced CDDP efficacy in a tumor model. Conclusions Our study reveals a plausible mechanism by which the m 6 A -induced circPLPP4/ miR-136/ PIK3R1 axis mediated CDDP resistance in OC, suggesting that circPLPP4 may serve as a promising therapeutic target against CDDP resistant OC. A circPLPP4-targeted drug in combination with CDDP might represent a rational regimen in OC.

Minimally invasive approaches for the early detection of endometrial cancer

AbstractEndometrial cancer (EC) is one of the most common gynecologic cancers and its incidence is rising globally. Although advanced EC has a poor prognosis; diagnosing EC at an earlier stage could improve long-term patient outcomes. However, there is no consensus on the early detection strategies for EC and the current diagnostic practices such as transvaginal ultrasound, hysteroscopy and endometrial biopsy are invasive, costly and low in specificity. Thus, accurate and less invasive screening tests that detect EC in women with early stages of the disease are needed. Current research has revolutionized novel EC early detection methodologies in many aspects. This review aims to comprehensively characterizes minimally invasive screening techniques that can be applied to EC in the future, and fully demonstrate their potential in the early detection of EC.

Integrated multi-omics identifies a CD54+ iCAF-ITGAL+ macrophage niche driving immunosuppression via CXCL8-PDL1 axis in cervical cancer

Cervical cancer (CC) remains a formidable clinical challenge, particularly in advanced stages where immune checkpoint blockade yields suboptimal responses. Despite the established role of the tumor microenvironment (TME) in fostering immunosuppression, the precise mechanisms of stroma-immune crosstalk in CC remain elusive. Leveraging single-cell RNA sequencing of 77,221 cells from CC and normal cervical tissues, we uncovered a tumor-enriched subpopulation of inflammatory cancer-associated fibroblasts (iCAFs) marked by elevated CD54 expression (CD54

Proteogenomic characterization of molecular and cellular targets for treatment-resistant subtypes in locally advanced cervical cancers

We report proteogenomic analysis of locally advanced cervical cancer (LACC). Exome-seq data revealed predominant alterations of keratinization-TP53 regulation and O-glycosylation-TP53 regulation axes in squamous and adeno-LACC, respectively, compared to in early-stage cervical cancer. Integrated clustering of mRNA, protein, and phosphorylation data identified six subtypes (Sub1-6) of LACC among which Sub3, 5, and 6 showed the treatment-resistant nature with poor local recurrence-free survival. Elevated immune and extracellular matrix (ECM) activation mediated by activated stroma (PDGFD and CXCL1

NSUN6-mediated 5-methylcytosine modification of NDRG1 mRNA promotes radioresistance in cervical cancer

Abstract Background Radioresistance is the leading cause of death in advanced cervical cancer (CC). Dysregulation of RNA modification has recently emerged as a regulatory mechanism in radiation and drug resistance. We aimed to explore the biological function and clinical significance of 5-methylcytosine (m5C) in cervical cancer radiosensitivity. Methods The abundance of RNA modification in radiotherapy-resistant and sensitive CC specimens was quantified by liquid chromatography-tandem mass spectrometry. The essential RNA modification-related genes involved in CC radiosensitivity were screened via RNA sequencing. The effect of NSUN6 on radiosensitivity was verified in CC cell lines, cell-derived xenograft (CDX), and 3D bioprinted patient-derived organoid (PDO). The mechanisms of NSUN6 in regulating CC radiosensitivity were investigated by integrative m5C sequencing, mRNA sequencing, and RNA immunoprecipitation. Results We found a higher abundance of m5C modification in resistant CC samples, and NSUN6 was the essential m5C-regulating gene concerning radiosensitivity. NSUN6 overexpression was clinically correlated with radioresistance and poor prognosis in cervical cancer. Functionally, higher NSUN6 expression was associated with radioresistance in the 3D PDO model of cervical cancer. Moreover, silencing NSUN6 increased CC radiosensitivity in vivo and in vitro. Mechanistically, NDRG1 was one of the downstream target genes of NSUN6 identified by integrated m5C-seq, mRNA-seq, and functional validation. NSUN6 promoted the m5C modification of NDRG1 mRNA, and the m5C reader ALYREF bound explicitly to the m5C-labeled NDRG1 mRNA and enhanced NDRG1 mRNA stability. NDRG1 overexpression promoted homologous recombination-mediated DNA repair, which in turn led to radioresistance in cervical cancer. Conclusions Aberrant m5C hypermethylation and NSUN6 overexpression drive resistance to radiotherapy in cervical cancer. Elevated NSUN6 expression promotes radioresistance in cervical cancer by activating the NSUN6/ALYREF-m5C-NDRG1 pathway. The low expression of NSUN6 in cervical cancer indicates sensitivity to radiotherapy and a better prognosis.

EZH2 mediated metabolic rewiring promotes tumor growth independently of histone methyltransferase activity in ovarian cancer

Abstract Background Enhancer of zeste homolog 2 (EZH2), the key catalytic subunit of polycomb repressive complex 2 (PRC2), is overexpressed and plays an oncogenic role in various cancers through catalysis-dependent or catalysis-independent pathways. However, the related mechanisms contributing to ovarian cancer (OC) are not well understood. Methods The levels of EZH2 and H3K27me3 were evaluated in 105 OC patients by immunohistochemistry (IHC) staining, and these patients were stratified based on these levels. Canonical and noncanonical binding sites of EZH2 were defined by chromatin immunoprecipitation sequencing (ChIP-Seq). The EZH2 solo targets were obtained by integrative analysis of ChIP-Seq and RNA sequencing data. In vitro and in vivo experiments were performed to determine the role of EZH2 in OC growth. Results We showed that a subgroup of OC patients with high EZH2 expression but low H3K27me3 exhibited the worst prognosis, with limited therapeutic options. We demonstrated that induction of EZH2 degradation but not catalytic inhibition profoundly blocked OC cell proliferation and tumorigenicity in vitro and in vivo. Integrative analysis of genome-wide chromatin and transcriptome profiles revealed extensive EZH2 occupancy not only at genomic loci marked by H3K27me3 but also at promoters independent of PRC2, indicating a noncanonical role of EZH2 in OC. Mechanistically, EZH2 transcriptionally upregulated IDH2 to potentiate metabolic rewiring by enhancing tricarboxylic acid cycle (TCA cycle) activity, which contributed to the growth of OC. Conclusions These data reveal a novel oncogenic role of EZH2 in OC and identify potential therapeutic strategies for OC by targeting the noncatalytic activity of EZH2.

ESM1 enhances fatty acid synthesis and vascular mimicry in ovarian cancer by utilizing the PKM2-dependent warburg effect within the hypoxic tumor microenvironment

Abstract Background The hypoxic tumor microenvironment is a key factor that promotes metabolic reprogramming and vascular mimicry (VM) in ovarian cancer (OC) patients. ESM1, a secreted protein, plays an important role in promoting proliferation and angiogenesis in OC. However, the role of ESM1 in metabolic reprogramming and VM in the hypoxic microenvironment in OC patients has not been determined. Methods Liquid chromatography coupled with tandem MS was used to analyze CAOV3 and OV90 cells. Interactions between ESM1, PKM2, UBA2, and SUMO1 were detected by GST pull-down, Co-IP, and molecular docking. The effects of the ESM1-PKM2 axis on cell glucose metabolism were analyzed based on an ECAR experiment. The biological effects of the signaling axis on OC cells were detected by tubule formation, transwell assay, RT‒PCR, Western blot, immunofluorescence, and in vivo xenograft tumor experiments. Results Our findings demonstrated that hypoxia induces the upregulation of ESM1 expression through the transcription of HIF-1α. ESM1 serves as a crucial mediator of the interaction between PKM2 and UBA2, facilitating the SUMOylation of PKM2 and the subsequent formation of PKM2 dimers. This process promotes the Warburg effect and facilitates the nuclear translocation of PKM2, ultimately leading to the phosphorylation of STAT3. These molecular events contribute to the promotion of ovarian cancer glycolysis and vasculogenic mimicry. Furthermore, our study revealed that Shikonin effectively inhibits the molecular interaction between ESM1 and PKM2, consequently preventing the formation of PKM2 dimers and thereby inhibiting ovarian cancer glycolysis, fatty acid synthesis and vasculogenic mimicry. Conclusion Our findings demonstrated that hypoxia increases ESM1 expression through the transcriptional regulation of HIF-1α to induce dimerization via PKM2 SUMOylation, which promotes the OC Warburg effect and VM.

OLFM4 promotes the progression of intestinal metaplasia through activation of the MYH9/GSK3β/β-catenin pathway

Abstract Background Intestinal metaplasia (IM) is classified into complete intestinal metaplasia (CIM) and incomplete intestinal metaplasia (IIM). Patients diagnosed with IIM face an elevated susceptibility to the development of gastric cancer, underscoring the critical need for early screening measures. In addition to the complexities associated with diagnosis, the exact mechanisms driving the progression of gastric cancer in IIM patients remain poorly understood. OLFM4 is overexpressed in several types of tumors, including colorectal, gastric, pancreatic, and ovarian cancers, and its expression has been associated with tumor progression. Methods In this study, we used pathological sections from two clinical centers, biopsies of IM tissues, precancerous lesions of gastric cancer (PLGC) cell models, animal models, and organoids to explore the role of OLFM4 in IIM. Results Our results show that OLFM4 expression is highly increased in IIM, with superior diagnostic accuracy of IIM when compared to CDX2 and MUC2. OLFM4, along with MYH9, was overexpressed in IM organoids and PLGC animal models. Furthermore, OLFM4, in combination with Myosin heavy chain 9 (MYH9), accelerated the ubiquitination of GSK3β and resulted in increased β-catenin levels through the Wnt signaling pathway, promoting the proliferation and invasion abilities of PLGC cells. Conclusions OLFM4 represents a novel biomarker for IIM and could be utilized as an important auxiliary means to delimit the key population for early gastric cancer screening. Finally, our study identifies cell signaling pathways involved in the progression of IM.

Tumor-secreted exosomal miR-141 activates tumor-stroma interactions and controls premetastatic niche formation in ovarian cancer metastasis

AbstractBackgroundMetastatic colonization is one of the critical steps in tumor metastasis. A pre-metastatic niche is required for metastatic colonization and is determined by tumor-stroma interactions, yet the mechanistic underpinnings remain incompletely understood.MethodsPCR-based miRNome profiling, qPCR, immunofluorescent analyses evaluated the expression of exosomal miR-141 and cell-to-cell communication. LC-MS/MS proteomic profiling and Dual-Luciferase analyses identified YAP1 as the direct target of miR-141. Human cytokine profiling, ChIP, luciferase reporter assays, and subcellular fractionation analyses confirmed YAP1 in modulating GROα production. A series of in vitro tumorigenic assays, an ex vivo model and Yap1 stromal conditional knockout (cKO) mouse model demonstrated the roles of miR-141/YAP1/GROα/CXCR1/2 signaling cascade. RNAi, CRISPR/Cas9 and CRISPRi systems were used for gene silencing. Blood sera, OvCa tumor tissue samples, and tissue array were included for clinical correlations.ResultsHsa-miR-141-3p (miR-141), an exosomal miRNA, is highly secreted by ovarian cancer cells and reprograms stromal fibroblasts into proinflammatory cancer-associated fibroblasts (CAFs), facilitating metastatic colonization. A mechanistic study showed that miR-141 targeted YAP1, a critical effector of the Hippo pathway, reducing the nuclear YAP1/TAZ ratio and enhancing GROα production from stromal fibroblasts. Stromal-specific knockout (cKO) of Yap1 in murine models shaped the GROα-enriched microenvironment, facilitating in vivo tumor colonization, but this effect was reversed after Cxcr1/2 depletion in OvCa cells. The YAP1/GROα correlation was demonstrated in clinical samples, highlighting the clinical relevance of this research and providing a potential therapeutic intervention for impeding premetastatic niche formation and metastatic progression of ovarian cancers.ConclusionsThis study uncovers miR-141 as an OvCa-derived exosomal microRNA mediating the tumor-stroma interactions and the formation of tumor-promoting stromal niche through activating YAP1/GROα/CXCRs signaling cascade, providing new insight into therapy for OvCa patients with peritoneal metastases.

CircMUC16 promotes autophagy of epithelial ovarian cancer via interaction with ATG13 and miR-199a

Abstract Background Circular RNA (circRNA) has been proven to play a significant role in multiple types of cancer. However, the expression and role of circRNAs in epithelial ovarian cancer (EOC) remains elusive. Methods CircRNA and mRNA expression profiles of EOC were screened with sequencing analysis. Gene silencing and over-expression were used to study circRNA function. Cell proliferation and Matrigel invasion assays were used to detect cell proliferation and invasion, respectively. The expression of circRNAs, mRNAs and miRNAs was detected using qPCR. The location of circRNAs was detected using FISH. The expression of proteins was detected using western blot and immunohistochemistry. Results CircMUC16 had increased expression in EOC tissues as compared to healthy ovarian tissues. The expression of circMUC16 was linked to the progression in stage and grade of EOC. Hence, silencing circMUC16 suppressed autophagy flux of SKOV3 cells. In contrast, ectopic expression of circMUC16 promoted autophagy flux of A2780 cells. CircMUC16-mediated autophagy exacerbated EOC invasion and metastasis. Mechanistically, circMUC16 could directly bind to miR-199a-5p and relieve suppression of target Beclin1 and RUNX1. In turn, RUNX1 elevated the expression of circMUC16 via promotion of its transcription. CircMUC16 could directly bind to ATG13 and promote its expression. Conclusion This study demonstrated that circMUC16 regulated Beclin1 and RUNX1 by sponging miR-199a-5p. The data suggested that circMUC16 could be a potential target for EOC diagnosis and therapy.

PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

Abstract Background Poly (ADP-ribose) polymerase inhibitor (PARPi) resistance poses a significant challenge in ovarian carcinoma (OC). While the role of DOT1L in cancer and chemoresistance is acknowledged, its specific role in PARPi resistance remains unclear. This study aims to elucidate the molecular mechanism of DOT1L in PARPi resistance in OC patients. Methods This study analyzed the expression of DOT1L in PARPi-resistant cell lines compared to sensitive ones and correlated it with clinical outcomes in OC patients. Comprehensive in vitro and in vivo functional experiments were conducted using cellular and mouse models. Molecular investigations, including RNA sequencing, chromatin immunoprecipitation (ChIP) and Cleavage Under Targets and Tagmentation (CUT&Tag) assays, were employed to unravel the molecular mechanisms of DOT1L-mediated PARPi resistance. Results Our investigation revealed a robust correlation between DOT1L expression and clinical PARPi resistance in non-BRCA mutated OC cells. Upregulated DOT1L expression in PARPi-resistant tissues was associated with diminished survival in OC patients. Mechanistically, we identified that PARP1 directly binds to the DOT1L gene promoter, promoting transcription independently of its enzyme activity. PARP1 trapping induced by PARPi treatment amplified this binding, enhancing DOT1L transcription and contributing to drug resistance. Sequencing analysis revealed that DOT1L plays a crucial role in the transcriptional regulation of PLCG2 and ABCB1 via H3K79me2. This established the PARP1-DOT1L-PLCG2/ABCB1 axis as a key contributor to PARPi resistance. Furthermore, we discovered that combining a DOT1L inhibitor with PARPi demonstrated a synergistic effect in both cell line-derived xenograft mouse models (CDXs) and patient-derived organoids (PDOs). Conclusions Our results demonstrate that DOT1L is an independent prognostic marker for OC patients. The PARP1-DOT1L/H3K79me2-PLCG2/ABCB1 axis is identified as a pivotal contributor to PARPi resistance. Targeted inhibition of DOT1L emerges as a promising therapeutic strategy for enhancing PARPi treatment outcomes in OC patients.

FBW7 suppresses ovarian cancer development by targeting the N6-methyladenosine binding protein YTHDF2

Abstract Background The tumor suppressor FBW7 is the substrate recognition component of the SCF E3-ubiquitin ligase complex that mediates proteolytic degradation of various oncogenic proteins. However, the role of FBW7 in ovarian cancer progression remains inadequately understood. Methods IP-MASS, co-IP, immunohistochemistry, and western blotting were used to identify the potential substrate of FBW7 in ovarian cancer. The biological effects of FBW7 were investigated using in vitro and in vivo models. LC/MS was used to detect the m6A levels in ovarian cancer tissues. MeRIP-Seq and RNA-Seq were used to assess the downstream targets of YTHDF2. Results We unveil that FBW7 is markedly down-regulated in ovarian cancer tissues and its high expression is associated with favorable prognosis and elevated m6A modification levels. Consistently, ectopic FBW7 inhibits ovarian cancer cell survival and proliferation in vitro and in vivo, while ablation of FBW7 empowers propagation of ovarian cancer cells. In addition, the m6A reader protein, YTHDF2, is identified as a novel substrate for FBW7. FBW7 counteracts the tumor-promoting effect of YTHDF2 by inducing proteasomal degradation of the latter in ovarian cancer. Furthermore, YTHDF2 globally regulates the turnover of m6A-modified mRNAs, including the pro-apoptotic gene BMF. Conclusions Our study has demonstrated that FBW7 suppresses tumor growth and progression via antagonizing YTHDF2-mediated BMF mRNA decay in ovarian cancer.

HnRPD/AUF1 facilitates human ovarian cancer progression through activating FLI1 and maintaining cisplatin resistance

Exploiting synthetic lethality in epithelial ovarian cancer: multi-dimensional approaches beyond DNA damage repair

Folate-engineered chitosan nanoparticles: next-generation anticancer nanocarriers

A comprehensive overview of ovarian cancer stem cells: correlation with high recurrence rate, underlying mechanisms, and therapeutic opportunities

Ovarian cancer is one of the most lethal gynecological malignancies, with a recurrence rate of 70-80%, particularly in patients diagnosed at advanced stages (stage III or IV), where the five-year survival rate falls below 30%. A key driver of this recurrence is the presence of cancer stem cells (CSCs), which exhibit resistance to chemotherapy and possess the capacity for self-renewal, plasticity, and tumor regeneration. The tumor microenvironment (TME) plays a crucial role in maintaining ovarian cancer stem cells (OCSCs) by providing nutrient and oxygen gradients, extracellular matrix (ECM) interactions, immune cell modulation, and support from cancer-associated fibroblasts (CAFs). CAFs secrete growth factors, cytokines, and ECM components that create a pro-tumorigenic niche, promoting CSC maintenance, invasion, and chemoresistance. Additionally, dysregulation of critical signaling pathways, including WNT, NOTCH, PI3K/AKT/mTOR, TGF-β, JAK/STAT, Hedgehog, NF-κB, and Hippo, supports CSC stemness, plasticity, maintenance, and adaptability, thereby increasing their survival and progression. Numerous inhibitors targeting these pathways have shown promise in preclinical studies. This review discusses the molecular mechanisms underlying CSC-mediated recurrence in ovarian cancer and highlights emerging therapeutic strategies. Particular emphasis is placed on the potential of combination therapies involving routine platinum or taxane based regimens with OCSC inhibitors to overcome chemoresistance, reduce recurrence rates, and improve survival outcomes for patients with advanced-stage ovarian cancer.

Proapoptotic activity of JNK-sensitive BH3-only proteins underpins ovarian cancer response to replication checkpoint inhibitors

Recent studies indicate that replication checkpoint modulators (RCMs) such as inhibitors of CHK1, ATR, and WEE1 have promising monotherapy activity in solid tumors, including platinum-resistant high grade serous ovarian cancer (HGSOC). However, clinical response rates are generally below 30%. While RCM-induced DNA damage has been extensively examined in preclinical and clinical studies, the link between replication checkpoint interruption and tumor shrinkage remains incompletely understood. Here we utilized HGSOC cell lines and patient-derived xenografts (PDXs) to study events leading from RCM treatment to ovarian cancer cell death. These studies show that RCMs increase CDC25A levels and CDK2 signaling in vitro, leading to dysregulated cell cycle progression and increased replication stress in HGSOC cell lines independent of homologous recombination status. These events lead to sequential activation of JNK and multiple BH3-only proteins, including BCL2L11/BIM, BBC3/PUMA and the BMF, all of which are required to fully initiate RCM-induced apoptosis. Activation of the same signaling pathway occurs in HGSOC PDXs that are resistant to poly(ADP-ribose) polymerase inhibitors but respond to RCMs ex vivo with a decrease in cell number in 3-dimensional culture and in vivo with xenograft shrinkage or a significantly diminished growth rate. These findings identify key cell death-initiating events that link replication checkpoint inhibition to antitumor response in ovarian cancer.

Unraveling the extracellular vesicle network: insights into ovarian cancer metastasis and chemoresistance

Ovarian cancer (OC) is one of the most prevalent and lethal gynecological malignancies, with high mortality primarily due to its aggressive nature, frequent metastasis, and resistance to standard therapies. Recent research has highlighted the critical role of extracellular vesicles (EVs) in these processes. EVs, secreted by living organisms and carrying versatile and bioactive cargoes, play a vital role in intercellular communication. Functionally, the transfer of cargoes orchestrates multiple processes that actively affect not only the primary tumor but also local and distant pre-metastatic niche. Furthermore, their unique biological properties position EVs as novel therapeutic targets and promising drug delivery systems, with potential profound implications for cancer patients.This review summarizes recent progress in EV biology, delving into the intricate mechanisms by which EVs contribute to OC metastasis and drug resistance. It also explores the latest advances and therapeutic potential of EVs in the clinical context of OC. Despite the progress made, EV research in OC remains in its nascent stages. Consequently, this review presents existing research limitations and suggests avenues for future investigation. Altogether, the review aims to elucidate the critical roles of EVs in OC and spotlight their promising potential in this field.

Induction of circulating ABCB1 transcripts under platinum-based chemotherapy indicates poor prognosis and a bone micrometastatic phenotype in ovarian cancer patients

The drug efflux transporter P-glycoprotein, encoded by the ABCB1 gene, promotes acquired chemoresistance. We explored the presence and clinical relevance of circulating cell-free ABCB1 transcripts (cfABCB1

Efficacy and safety of chiauranib in a combination therapy in platinum-resistant or refractory ovarian cancer: a multicenter, open-label, phase Ib and II study

Platinum-resistant or refractory ovarian cancer is a highly lethal gynecologic disease with limited treatment options. Chiauranib is a novel small-molecule selective inhibitor, which could effectively target multiple pathways including Aurora B and CSF-1R to inhibit cell cycle process and improve anti-tumor immune function, as long as VEGF pathway for tumor extinction. A phase II study was sequentially conducted after a phase Ib monotherapy study to evaluate the efficacy of chiauranib combined with chemotherapy. Chinese patients with recurrent ovarian cancer were enrolled. Eligible patients received chiauranib combined with a maximum of six cycles of chemotherapy: etoposide (CE group) or weekly-paclitaxel (CP group). Patients, who exhibited a complete or partial response, or stable disease following combo treatment, progressed to maintenance phase to receive chiauranib monotherapy. Primary endpoint was progression-free survival (PFS) according to RECIST v1.1. From November 2017 to March 2019, 25 patients were enrolled in a phase 1b study and a median PFS of 3.7 months (95% CI 1.8-NE) was achieved by chiauranib monotherapy. From July 2019 to December 2020, a total of 47 patients were enrolled in the phase II study. One CP patient did not receive the study drugs, and three patients withdrew before the first tumor assessment. Thus, 43 patients (CE group: 22 patients; CP group: 21 patients) were included in the evaluation. The median PFS was 5·4 months (95% CI 2·8-5·6) and 5·6 months (95% CI 3·4-7·0), respectively. This was the first study to evaluate chiauranib, a novel multi-targeted kinase inhibitor in patients with ovarian cancer. The administration of chiauranib along with etoposide or weekly-paclitaxel significantly enhanced the efficacy with manageable adverse events. This warrants further clinical studies on this novel treatment. A phase III study is promising and ongoing. ClinicaTrials.gov identifier: NCT03901118 (phase II) and NCT03166891 (phase Ib).

Multi-omics profiling reveal cells with novel oncogenic cluster, TRAP1low/CAMSAP3low, emerge more aggressive behavior and poor-prognosis in early-stage endometrial cancer

AbstractThe clinical heterogeneity of early-stage endometrial cancer (EC) is worthy of further study to identify high-quality prognostic markers and their potential role in aggressive tumor behavior. Mutation of TP53 was considered as an important primary triage in modified molecular typing for EC, it still cannot precisely predict the prognosis of EC. After proteomic analysis of cancer and para-cancerous tissues from 24 early-stage endometrioid EC patients with different survival outcomes, 13 differentially expressed proteins were screen out while 2 proteins enriched in p53 signaling pathway were further identified by single-cell transcriptome (scRNA-seq). Interestingly, tumor necrosis factor type-1 receptor-associated protein (TRAP1) and calmodulin-regulated spectrin-associated protein family member 3 (CAMSAP3) were found to be significantly downregulated in the specific cell cluster. Expectedly, the signature genes of TRAP1low/CAMSAP3low cluster included classical oncogenes. Moreover, close cellular interactions were observed between myeloid cells and the TRAP1low/CAMSAP3low cluster after systematically elucidating their relationship with tumor microenvironment (TME). The expression of TRAP1 and CAMSAP3 was verified by immunohistochemistry. Thus, a novel prediction model combining TRAP1, CAMSAP3 and TP53 was construct by multi-omics. Compared with the area under the curve, it demonstrated a significantly improvemrnt in the diagnostic efficacy in EC patients from TCGA bank. In conclusion, this work improved the current knowledge regarding the prognosis of early-stage EC through proteomics and scRNA-seq. These findings may lead to improvements in precise risk stratification of early-stage EC patients.

The role of distinct BRD4 isoforms and their contribution to high-grade serous ovarian carcinoma pathogenesis

AbstractHigh-grade serous ovarian carcinoma (HGSOC) is the most aggressive type of ovarian cancer, often diagnosed at advanced stages. Molecularly, HGSOC shows high degree of genomic instability associated with large number of genetic alterations. BRD4 is the 4th most amplified gene in HGSOC, which correlates with poor patients’ prognosis. BRD4 is constitutively expressed and generates two proteins, BRD4 long (BRD4-L) and BRD4 short (BRD4-S). Both isoforms contain bromodomains that bind to lysine-acetylated histones. Amongst other functions, BRD4 participates in chromatin organization, acetylation of histones, transcriptional control and DNA damage repair. In cancer patients with amplified BRD4, the increased activity of BRD4 is associated with higher expression of oncogenes, such as MYC, NOTCH3 and NRG1. BRD4-driven oncogenes promote increased tumor cells proliferation, genetic instability, epithelial-mesenchymal transition, metastasis and chemoresistance. Ablation of BRD4 activity can be successfully achieved with bromodomain inhibitors (BETi) and degraders, and it has been applied in pre-clinical and clinical settings. Inhibition of BRD4 function has an effective anti-cancer effect, reducing tumor growth whether ablated by single agents or in combination with other drugs. When combined with standard chemotherapy, BETi are capable of sensitizing highly resistant ovarian cancer cell lines to platinum drugs. Despite the evidence that BRD4 amplification in ovarian cancer contributes to poor patient prognosis, little is known about the specific mechanisms by which BRD4 drives tumor progression. In addition, newly emerging data revealed that BRD4 isoforms exhibit contradicting functions in cancer. Therefore, it is paramount to expand studies elucidating distinct roles of BRD4-L and BRD4-S in HGSOC, which has important implications on development of therapeutic approaches targeting BRD4.

The CD47/TSP-1 axis: a promising avenue for ovarian cancer treatment and biomarker research

Abstract Background Ovarian cancer (OC) remains one of the most challenging and deadly malignancies facing women today. While PARP inhibitors (PARPis) have transformed the treatment landscape for women with advanced OC, many patients will relapse and the PARPi-resistant setting is an area of unmet medical need. Traditional immunotherapies targeting PD-1/PD-L1 have failed to show any benefit in OC. The CD47/TSP-1 axis may be relevant in OC. We aimed to describe changes in CD47 expression with platinum therapy and their relationship with immune features and prognosis. Methods Tumor and blood samples collected from OC patients in the CHIVA trial were assessed for CD47 and TSP-1 before and after neoadjuvant chemotherapy (NACT) and multiplex analysis was used to investigate immune markers. Considering the therapeutic relevance of targeting the CD47/TSP-1 axis, we used the CD47-derived TAX2 peptide to selectively antagonize it in a preclinical model of aggressive ovarian carcinoma. Results Significant reductions in CD47 expression were observed post NACT. Tumor patients having the highest CD47 expression profile at baseline showed the greatest CD4+ and CD8+ T-cell influx post NACT and displayed a better prognosis. In addition, TSP-1 plasma levels decreased significantly under NACT, and high TSP-1 was associated with a worse prognosis. We demonstrated that TAX2 exhibited a selective and favorable biodistribution profile in mice, localizing at the tumor sites. Using a relevant peritoneal carcinomatosis model displaying PARPi resistance, we demonstrated that post-olaparib (post-PARPi) administration of TAX2 significantly reduced tumor burden and prolonged survival. Remarkably, TAX2 used sequentially was also able to increase animal survival even under treatment conditions allowing olaparib efficacy. Conclusions Our study thus (1) proposes a CD47-based stratification of patients who may be most likely to benefit from postoperative immunotherapy, and (2) suggests that TAX2 is a potential alternative therapy for patients relapsing on PARP inhibitors.

Drug resistance in ovarian cancer: from mechanism to clinical trial

AbstractOvarian cancer is the leading cause of gynecological cancer-related death. Drug resistance is the bottleneck in ovarian cancer treatment. The increasing use of novel drugs in clinical practice poses challenges for the treatment of drug-resistant ovarian cancer. Continuing to classify drug resistance according to drug type without understanding the underlying mechanisms is unsuitable for current clinical practice. We reviewed the literature regarding various drug resistance mechanisms in ovarian cancer and found that the main resistance mechanisms are as follows: abnormalities in transmembrane transport, alterations in DNA damage repair, dysregulation of cancer-associated signaling pathways, and epigenetic modifications. DNA methylation, histone modifications and noncoding RNA activity, three key classes of epigenetic modifications, constitute pivotal mechanisms of drug resistance. One drug can have multiple resistance mechanisms. Moreover, common chemotherapies and targeted drugs may have cross (overlapping) resistance mechanisms. MicroRNAs (miRNAs) can interfere with and thus regulate the abovementioned pathways. A subclass of miRNAs, “epi-miRNAs”, can modulate epigenetic regulators to impact therapeutic responses. Thus, we also reviewed the regulatory influence of miRNAs on resistance mechanisms. Moreover, we summarized recent phase I/II clinical trials of novel drugs for ovarian cancer based on the abovementioned resistance mechanisms. A multitude of new therapies are under evaluation, and the preliminary results are encouraging. This review provides new insight into the classification of drug resistance mechanisms in ovarian cancer and may facilitate in the successful treatment of resistant ovarian cancer.

Homologous recombination deficiency (HRD) testing on cell-free tumor DNA from peritoneal fluid

Abstract Background Knowing the homologous recombination deficiency (HRD) status in advanced epithelial ovarian cancer (EOC) is vital for patient management. HRD is determined by BRCA1/BRCA2 pathogenic variants or genomic instability. However, tumor DNA analysis is inconclusive in 15–19% of cases. Peritoneal fluid, available in > 95% of advanced EOC cases, could serve as an alternative source of cell-free tumor DNA (cftDNA) for HRD testing. Limited data show the feasibility of cancer panel gene testing on ascites cfDNA but no study, to date, has investigated HRD testing. Methods We collected ascites/peritoneal washings from 53 EOC patients (19 from retrospective cohort and 34 from prospective cohort) and performed a Cancer Gene Panel (CGP) using NGS for TP53/HR genes and shallow Whole Genome Sequencing (sWGS) for genomic instability on cfDNA. Results cfDNA was detectable in 49 out of 53 patients (92.5%), including those with limited peritoneal fluid. Median cfDNA was 3700 ng/ml, with a turnaround time of 21 days. TP53 pathogenic variants were detected in 86% (42/49) of patients, all with HGSOC. BRCA1 and BRCA2 pathogenic variants were found in 14% (7/49) and 10% (5/49) of cases, respectively. Peritoneal cftDNA showed high sensitivity (97%), specificity (83%), and concordance (95%) with tumor-based TP53 variant detection. NGS CGP on cftDNA identified BRCA2 pathogenic variants in one case where tumor-based testing failed. sWGS on cftDNA provided informative results even when tumor-based genomic instability testing failed. Conclusion Profiling cftDNA from peritoneal fluid is feasible, providing a significant amount of tumor DNA. This fast and reliable approach enables HRD testing, including BRCA1/2 mutations and genomic instability assessment. HRD testing on cfDNA from peritoneal fluid should be offered to all primary laparoscopy patients.