Clinical and Translational Medicine

Integrated single‐cell RNA sequencing and spatial transcriptomics analysis reveals the tumour microenvironment in patients with endometrial cancer responding to anti‐PD‐1 treatment

p53 inhibits OTUD5 transcription to promote GPX4 degradation and induce ferroptosis in gastric cancer

AbstractBackgroundGastric cancer is one of the most prevalent malignant tumors within the digestive system, and ferroptosis playing a crucial role in its progression. Glutathione peroxidase 4 (GPX4), a key negative regulator of ferroptosis, is highly expressed in gastric cancer and contributes to tumor growth. Targeting the regulation of GPX4 has emerged as a promising approach to induce ferroptosis and develop effective therapy for gastric cancer.MethodsTo confirm that OTUD5 is a deubiquitinase of GPX4 and regulates ferroptosis, we performed Western blotting, Co‐IP, immunofluorescence, quantitative real‐time PCR, Ub assay and flow cytometry experiments. To explore the physiological function of OUTD5, we knocked out the Otud5 gene in the mouse gastric cancer cell line (MFC) using CRISPR‐Cas9 and eatablished the subcutaneous tumour model. Immunohistochemistry (IHC) analysis was used to inveatigate the pathological correlation in human gastric cancer.ResultsWe report that ovarian tumor domain‐containing 5 (OTUD5) interacts with, deubiquitylates and stabilizes GPX4. OTUD5 depletion destabilizes GPX4, promotes lipid peroxidation and sensitizes gastric cancer cells to ferroptosis. Moreover, the p53 activator nutlin‐3a suppresses OTUD5 transcription, leading to GPX4 degradation and ferroptosis of gastric cancer cells. Notably, only wild‐type p53 has the capacity to inhibit OTUD5 transcription, while p53 mutations or deficiencies correlate with increased OTUD5 expression, promoting gastric cancer progression. Additionally, OTUD5 silencing and nutlin‐3a‐induced GPX4 degradation enhances the sensitivity of gastric cancer cells to ferroptosis in vivo. Subsequently, the p53/OTUD5/GPX4 axis is confirmed in clinical gastric cancer samples.ConclusionCollectively, these findings elucidate a mechanism whereby p53 inactivation upregulates OTUD5 transcription to deubiquitylate and stablize GPX4, resulting in ferroptosis inhibition and gastric cancer progression. This discovery highlights the potential therapeutic value of targeting OTUD5 to promote ferroptosis in p53‐inactivated gastric cancer.Key points OTUD5 mediates GPX4 deubiquitination to regulate its stability. Deletion of OTUD5 promotes ferroptosis and inhibits tumor growth. Wild type p53 inhibits OTUD5 transcription, thereby promoting GPX4 degradation and inhibiting the development of gastric cancer. OTUD5, GPX4 expression and p53 activity are highly correlated and correlates with clinical progression in STAD.

Single‐cell landscape of the tumour immune microenvironment in human gynaecologic malignancies

Abstract Background The immune microenvironment of the three most common gynaecological malignancies—tubo‐ovarian cancer, endometrial cancer and cervical cancer—has not been systematically studied, limiting clinical application. Methods This study analyses 272 389 CD45 + immune cells by integrating publicly available single‐cell RNA sequencing (scRNA‐seq) data from 111 tumour and non‐malignant tissue samples. We identified distinct subsets within immune cells: 11 for monocytes/macrophages, six for CD4 T cells, eight for CD8 T cells and five for B cells, detailing their distribution, prevalence and distinct functions. Results A pro‐angiogenic macrophage subset linked to NF‐κB signalling was associated with worse clinical outcomes and an interferon‐primed macrophage subset correlated with improved survival by recruiting T cells through CXCL9/10/11 secretion, as confirmed by multi‐colour immunohistochemistry. T cells exhibited dynamic roles in tubo‐ovarian cancer, with CD8 Tex cells contributing to immune dysfunction and poor prognosis, while CD8 Trm cells in early‐stage tumours supported immune surveillance. Additionally, we identified co‐stimulatory and co‐inhibitory receptor interactions and classified distinct B cell subsets with varying prognostic implications. Conclusions This comprehensive analysis of the tumour immune microenvironment in gynaecological malignancies provides new insights into immune cell composition and function offering potential for optimising immunotherapies and improving clinical outcomes in these cancers.

Extrachromosomal circular DNA expressing miRNA promotes ovarian cancer progression

AbstractBackgroundExtrachromosomal circular DNA (eccDNA) has emerged as a critical driver of oncogenesis, yet its functional roles in high‐grade serous ovarian cancer (HGSOC) remain poorly characterized. This highlights the need for comprehensive investigations into the abundance, biogenesis, and functional implications of eccDNA in HGSOC.MethodsTo characterize eccDNA in HGSOC, we performed comprehensive Circle‐seq analysis to assess eccDNA abundance and genomic annotation in HGSOC tissues compared to normal ovarian tissue. For mechanistic validation of eccDNA biogenesis pathways, targeted knockdown experiments of microhomology‐mediated end‐joining (MMEJ) dependent on LIG3 and POLQ were conducted. Functional characterization of HGSOC‐specific eccDNA‐harboring precursor microRNAs (eccMIRs) included in vitro assays using HGSOC cells and in vivo tumor growth experiments.ResultsCircle‐seq analysis revealed a 13‐fold increase in eccDNA abundance in HGSOC compared to normal ovarian tissue, with significant enrichment in promoter and coding regions. The MMEJ pathway was identified as the predominant pathway for eccDNA biogenesis in HGSOC, supported by characteristic microhomologies at junction sites and validation via LIG3 and POLQ knockdown experiments. Notably, HGSOC‐specific eccDNA frequently contained functional eccMIRs (eccMIR3661, eccMIR618, and eccMIR2277), which generate oncogenic miRNAs. These miRNAs promote tumor progression by downregulating tumor suppressor genes and activating key oncogenic pathways. Functional assays confirmed that these eccMIRs significantly enhanced HGSOC cell proliferation, migration, and invasion in vitro and promoted tumor growth in vivo.ConclusionsThese results underscore eccDNA as an oncogenic driver in HGSOC through non‐coding RNA‐mediated regulatory mechanisms, revealing novel therapeutic opportunities for targeting eccDNA biogenesis in this aggressive malignancy.Key points This study revealed a 13‐fold increase of eccDNA in HGSOC compared to normal tissues, with significant enrichment in promoter and coding regions. eccDNA‐derived miRNAs (eccMIRs) were shown to enhance cancer cell proliferation, invasion, and tumor growth through the expression of oncogenic miRNA sequences. The study highlights the importance of the MMEJ pathway in eccDNA generation and proposes that targeting eccDNA biogenesis in this aggressive malignancy presents a novel therapeutic opportunity.

Functional genomics pipeline identifies CRL4 inhibition for the treatment of ovarian cancer

AbstractBackgroundThe goal of precision oncology is to find effective therapeutics for every patient. Through the inclusion of emerging therapeutics in a high‐throughput drug screening platform, our functional genomics pipeline inverts the common paradigm to identify patient populations that are likely to benefit from novel therapeutic strategies.ApproachUtilizing drug screening data across a panel of 46 cancer cell lines from 11 tumor lineages, we identified an ovarian cancer‐specific sensitivity to the first‐in‐class CRL4 inhibitors KH‐4‐43 and 33‐11. CRL4 (i.e., Cullin‐4 RING E3 ubiquitin ligase) is known to be dysregulated in a variety of cancer contexts, making it an attractive therapeutic target. Unlike proteasome inhibitors that are associated with broad toxicity, CRL4 inhibition offers the potential for tumor‐specific effects.ResultsWe observed that CRL4 inhibition negatively regulates core gene signatures that are upregulated in ovarian tumors and significantly slowed tumor growth as compared to the standard of care, cisplatin, in OVCAR8 xenografts. Building on this, we performed combination drug screening in conjunction with proteomic and transcriptomic profiling to identify ways to improve the antitumor effects of CRL4 inhibition in ovarian cancer models. CRL4 inhibition consistently resulted in activation of the mitogen‐activated protein kinase (MAPK) signaling cascade at both the transcriptomic and protein levels, suggesting that survival signaling is induced in response to CRL4 inhibition. These observations were concordant with the results of the combination drug screens in seven ovarian cancer cell lines that showed CRL4 inhibition cooperates with MEK inhibition. Preclinical studies in OVCAR8 and A2780 xenografts confirmed the therapeutic potential of the combination of KH‐4‐43 and trametinib, which extended overall survival and slowed tumor progression relative to either single agent or the standard of care.ConclusionsTogether, these data demonstrate the prospective utility of functional modeling pipelines for therapeutic development and underscore the clinical potential of CRL4 inhibition in the ovarian cancer context.Highlights A precision medicine pipeline identifies ovarian cancer sensitivity to CRL4 inhibitors. CRL4 inhibition induces activation of MAPK signalling as identified by RNA sequencing, proteomics, and phosphoproteomics. Inhibitor combinations that target both CRL4 and this CRL4 inhibitor‐induced survival signalling enhance ovarian cancer sensitivity to treatment.

BIN2 inhibition suppress ovarian cancer progression meanwhile protect ovarian function through downregulating HDAC1 and RPS6 phosphorylation respectively

Ecological and evolutionary dynamics to design and improve ovarian cancer treatment

AbstractOvarian cancer ecosystems are exceedingly complex, consisting of a high heterogeneity of cancer cells. Development of drugs such as poly ADP‐ribose polymerase (PARP) inhibitors, targeted therapies and immunotherapies offer more options for sequential or combined treatments. Nevertheless, mortality in metastatic ovarian cancer patients remains high because cancer cells consistently develop resistance to single and combination therapies, urging a need for treatment designs that target the evolvability of cancer cells. The evolutionary dynamics that lead to resistance emerge from the complex tumour microenvironment, the heterogeneous populations, and the individual cancer cell's plasticity. We propose that successful management of ovarian cancer requires consideration of the ecological and evolutionary dynamics of the disease. Here, we review current options and challenges in ovarian cancer treatment and discuss principles of tumour evolution. We conclude by proposing evolutionarily designed strategies for ovarian cancer, with the goal of integrating such principles with longitudinal, quantitative data to improve the treatment design and management of drug resistance.Key points/Highlights Tumours are ecosystems in which cancer and non‐cancer cells interact and evolve in complex and dynamic ways. Conventional therapies for ovarian cancer inevitably lead to the development of resistance because they fail to consider tumours’ heterogeneity and cellular plasticity. Eco‐evolutionarily designed therapies should consider cancer cell plasticity and patient‐specific characteristics to improve clinical outcome and prevent relapse.

Single‐molecule epiallelic profiling of DNA derived from routinely collected Pap specimens for noninvasive detection of ovarian cancer

AbstractRecent advances in molecular analyses of ovarian cancer have revealed a wealth of promising tumour‐specific biomarkers, including protein, DNA mutations and methylation; however, reliably detecting such alterations at satisfactorily high sensitivity and specificity through low‐cost methods remains challenging, especially in early‐stage diseases. Here we present PapDREAM, a new approach that enables detection of rare, ovarian‐cancer‐specific aberrations of DNA methylation from routinely‐collected cervical Pap specimens. The PapDREAM approach employs a microfluidic platform that performs highly parallelized digital high‐resolution melt to analyze locus‐specific DNA methylation patterns on a molecule‐by‐molecule basis at or near single CpG‐site resolution at a fraction (< 1/10th) of the cost of next‐generation sequencing techniques. We demonstrate the feasibility of the platform by assessing intermolecular heterogeneity of DNA methylation in a panel of methylation biomarker loci using DNA derived from Pap specimens obtained from a cohort of 43 women, including 18 cases with ovarian cancer and 25 cancer‐free controls. PapDREAM leverages systematic multidimensional bioinformatic analyses of locus‐specific methylation heterogeneity to improve upon Pap‐specimen‐based detection of ovarian cancer, demonstrating a clinical sensitivity of 50% at 99% specificity in detecting ovarian cancer cases with an area under the receiver operator curve of 0.90. We then establish a logistic regression model that could be used to identify high‐risk patients for subsequent clinical follow‐up and monitoring. The results of this study support the utility of PapDREAM as a simple, low‐cost screening method with the potential to integrate with existing clinical workflows for early detection of ovarian cancer.Key points We present a microfluidic platform for detection and analysis of rare, heterogeneously methylated DNA within Pap specimens towards detection of ovarian cancer. The platform achieves high sensitivity (fractions <0.00005%) at a suitably low cost (∼$25) for routine screening applications. Furthermore, it provides molecule‐by‐molecule quantitative analysis to facilitate further study on the effect of heterogeneous methylation on cancer development.

CKAP4 and mutant p53 cooperatively abrogate cell cycle checkpoint to induce genotoxic resistance in ovarian cancer

Reciprocal crosstalk between Th17 and mesothelial cells promotes metastasis‐associated adhesion of ovarian cancer cells

AbstractBackgroundIL‐17A and TNF synergistically promote inflammation and tumorigenesis. Their interplay and impact on ovarian carcinoma (OC) progression are, however, poorly understood. We addressed this question focusing on mesothelial cells, whose interaction with tumor cells is known to play a pivotal role in transcoelomic metastasis formation.MethodsFlow‐cytometry and immunohistochemistry experiments were employed to identify cellular sources of IL‐17A and TNF. Changes in transcriptomes and secretomes were determined by bulk and single cell RNA sequencing as well as affinity proteomics. Functional consequences were investigated by microscopic analyses and tumor cell adhesion assays. Potential clinical implications were assessed by immunohistochemistry and survival analyses.ResultsWe identified Th17 cells as the main population of IL‐17A‐ and TNF producers in ascites and detected their accumulation in early omental metastases. Both IL‐17A and its receptor subunit IL‐17RC were associated with short survival of OC patients, pointing to a role in clinical progression. IL‐17A and TNF synergistically induced the reprogramming of mesothelial cells towards a pro‐inflammatory mesenchymal phenotype, concomitantly with a loss of tight junctions and an impairment of mesothelial monolayer integrity, thereby promoting cancer cell adhesion. IL‐17A and TNF synergistically induced the Th17‐promoting cytokines IL‐6 and IL‐1β as well as the Th17‐attracting chemokine CCL20 in mesothelial cells, indicating a reciprocal crosstalk that potentiates the tumor‐promoting role of Th17 cells in OC.ConclusionsOur findings reveal a novel function for Th17 cells in the OC microenvironment, which entails the IL‐17A/TNF‐mediated induction of mesothelial‐mesenchymal transition, disruption of mesothelial layer integrity and consequently promotion of OC cell adhesion. These effects are potentiated by a positive feedback loop between mesothelial and Th17 cells. Together with the observed clinical associations and accumulation of Th17 cells in omental micrometastases, our observations point to a potential role in early metastases formation and thus to new therapeutic options.

Mutational profiling of mitochondrial DNA reveals an epithelial ovarian cancer‐specific evolutionary pattern contributing to high oxidative metabolism

AbstractBackgroundEpithelial ovarian cancer (EOC) heavily relies on oxidative phosphorylation (OXPHOS) and exhibits distinct mitochondrial metabolic reprogramming. Up to now, the evolutionary pattern of somatic mitochondrial DNA (mtDNA) mutations in EOC tissues and their potential roles in metabolic remodelling have not been systematically elucidated.MethodsBased on a large somatic mtDNA mutation dataset from private and public EOC cohorts (239 and 118 patients, respectively), we most comprehensively characterised the EOC‐specific evolutionary pattern of mtDNA mutations and investigated its biological implication.ResultsMutational profiling revealed that the mitochondrial genome of EOC tissues was highly unstable compared with non‐cancerous ovary tissues. Furthermore, our data indicated the delayed heteroplasmy accumulation of mtDNA control region (mtCTR) mutations and near‐complete absence of mtCTR non‐hypervariable segment (non‐HVS) mutations in EOC tissues, which is consistent with stringent negative selection against mtCTR mutation. Additionally, we observed a bidirectional and region‐specific evolutionary pattern of mtDNA coding region mutations, manifested as significant negative selection against mutations in complex V (ATP6/ATP8) and tRNA loop regions, and potential positive selection on mutations in complex III (MT‐CYB). Meanwhile, EOC tissues showed higher mitochondrial biogenesis compared with non‐cancerous ovary tissues. Further analysis revealed the significant association between mtDNA mutations and both mitochondrial biogenesis and overall survival of EOC patients.ConclusionsOur study presents a comprehensive delineation of EOC‐specific evolutionary patterns of mtDNA mutations that aligned well with the specific mitochondrial metabolic remodelling, conferring novel insights into the functional roles of mtDNA mutations in EOC tumourigenesis and progression.

17‐β‐estradiol reduces surface PD‐L1 expression in estrogen receptor‐positive breast cancer but not type 1 endometrial cancer cells

FGFR2 mutations promote endometrial cancer progression through dual engagement of EGFR and Notch signalling pathways

AbstractBackgroundMutations in the receptor tyrosine kinase gene fibroblast growth factor receptor 2 (FGFR2) occur at a high frequency in endometrial cancer (EC) and have been linked to advanced and recurrent disease. However, little is known about how these mutations drive carcinogenesis.MethodsDifferential transcriptomic analysis and two‐step quantitative real‐time PCR (qRT‐PCR) assays were applied to identify genes differentially expressed in two cohorts of EC patients carrying mutations in the FGFR2 gene as well as in EC cells harbouring mutations in the FGFR2. Candidate genes and target signalling pathways were investigated by qRT‐PCR assays, immunohistochemistry and bioinformatics analysis. The functional roles of differently regulated genes were analysed using in vitro and in vivo experiments, including 3D‐orthotypic co‐culture systems, cell proliferation and migration protocols, as well as colony and focus formation assays together with murine xenograft tumour models. The molecular mechanisms were examined using CRISPR/Cas9‐based loss‐of‐function and pharmacological approaches as well as luciferase reporter techniques, cell‐based ectodomain shedding assays and bioinformatics analysis.ResultsWe show that common FGFR2 mutations significantly enhance the sensitivity to FGF7‐mediated activation of a disintegrin and metalloprotease (ADAM)17 and subsequent transactivation of the epidermal growth factor receptor (EGFR). We further show that FGFR2 mutants trigger the activation of ADAM10‐mediated Notch signalling in an ADAM17‐dependent manner, highlighting for the first time an intimate cooperation between EGFR and Notch pathways in EC. Differential transcriptomic analysis in EC cells in a cohort of patients carrying mutations in the FGFR2 gene identified a strong association between FGFR2 mutations and increased expression of members of the Notch pathway and ErbB receptor family. Notably, FGFR2 mutants are not constitutively active but require FGF7 stimulation to reprogram Notch and EGFR pathway components, resulting in ADAM17‐dependent oncogenic growth.ConclusionsThese findings highlight a pivotal role of ADAM17 in the pathogenesis of EC and provide a compelling rationale for targeting ADAM17 protease activity in FGFR2‐driven cancers.

IGF2BP2 binding to CPSF6 facilitates m6A‐mediated alternative polyadenylation of PUM2 and promotes malignant progression in ovarian cancer

AbstractBackgroundN6‐methyladenosine (m6A) and alternative polyadenylation (APA) are common posttranscriptional regulatory mechanisms in eukaryotes. However, the m6A‐dependent mechanism of APA regulation in ovarian cancer (OC) is still unclear.MethodsThe correlation between m6A and APA was analyzed by using RNA methylation sequencing of OC cells and single‐cell sequencing of clinical samples from public databases. To explore the core regulatory factors that served as a bridge between m6A and APA, we employed RNA pull‐down with biotin‐labelled m6A, immunoprecipitation, mass spectrometry, western blot, protein purification and GST pull‐down assays. Furthermore, the important target genes were screened by PAS‐seq, eCLIP‐seq, RIP‐seq and meRIP‐seq, and verified by RT‐qPCR, 3′RACE, RNA stability, and dual luciferase reporter assays. Multiple phenotypic experiments were conducted to evaluate the function of the IGF2BP2‐PUM2 axis in vitro and in vivo.ResultsThis study found that the m6A was correlated with the APA and affected the 3′end processing in OC. The APA regulator CPSF6 tended to bind the m6A‐modified transcripts in OC cells. Mechanistically, we demonstrated that the m6A reader IGF2BP2 KH1‐4 domains could directly bind to the CPSF6‐RS domain to regulate the 3′end processing of OC. Furthermore, sequencing revealed that the m6A was highly enriched in the 3′UTR near the proximal polyadenylation signal (PAS), which promotes the use of proximal PAS and leads to 3′UTR shortening. PUM2 was carried m6A and recognized by IGF2BP2, and CPSF6 was recruited at the proximal polyadenylation signal (pPAS) to generate the short‐3′UTR transcript. The short PUM2 transcript was more stable than the long transcript, which promoted the malignant progression of OC.ConclusionsWe revealed a novel mechanism in which the m6A could regulate the APA processing of pre‐mRNAs by crosstalk of IGF2BP2 and CPSF6. This study provides a potential strategy for the effective treatment of OC.Highlights The interaction between m6A and APA is mediated by the m6A regulator IGF2BP2 and the APA factor CPSF6. The transcripts harboring m6A modification tend to use the proximal polyadenylation signal (PAS) in ovarian cancer (OC). PUM2 promotes the malignant progression of OC through its m6A methylation and APA processing.

HIF‐1α‐induced long noncoding RNA LINC02776 promotes drug resistance of ovarian cancer by increasing polyADP‐ribosylation

AbstractBackgroundChemoresistance remains a major hurdle in ovarian cancer (OC) treatment, as many patients eventually develop resistance to platinum‐based chemotherapy and/or PARP inhibitors (PARPi).MethodsWe performed transcriptome‐wide analysis by RNA sequencing (RNA‐seq) data of platinum‐resistant and ‐sensitive OC tissues. We demonstrated the role of LINC02776 in platinum resistance in OC cells, mice models and patient‐derived organoid (PDO) models.ResultsWe identify the long noncoding RNA LINC02776 as a critical factor of platinum resistance. Elevated expression of LINC02776 is observed in platinum‐resistant OC and serves as an independent prognostic factor for OC patients. Functionally, silencing LINC02776 reduces proliferation and DNA damage repair in OC cells, thereby enhancing sensitivity to platinum and PARPi in both xenograft mouse models and patient‐derived organoid (PDO) models with acquired chemoresistance. Mechanistically, LINC02776 binds to the catalytic domain of poly (ADP‐ribose) polymerase 1 (PARP1), promoting PARP1‐dependent polyADP‐ribosylation (PARylation) and facilitating homologous recombination (HR) restoration. Additionally, high HIF‐1α expression in platinum‐resistant tissues further stimulates LINC02776 transcription.ConclusionsOur findings suggest that targeting LINC02776 represents a promising therapeutic strategy for OC patients who have developed resistance to platinum or PARPi.Key points LINC02776 promotes OC cell proliferation by regulating DNA damage and apoptosis signaling pathways. LINC02776 binds PARP1 to promote DNA damage‐triggered PARylation in OC cells. LINC02776 mediates cisplatin and olaparib resistance in OC cells by enhancing PARP1‐mediated PARylation activity and regulating the PARP1‐mediated HR pathway. The high expression of LINC02776 is induced by HIF‐1α in platinum‐resistant OC cells and tissues.

Genome‐wide profiling of N6‐methyladenosine‐modified pseudogene‐derived long noncoding RNAs reveals the tumour‐promoting and innate immune‐restraining function of RPS15AP12 in ovarian cancer

AbstractBackgroundPseudogene‐derived lncRNAs are widely dysregulated in cancer. Technological advancements have facilitated the functional characterization of increasing pseudogenes in cancer progression. However, the association between pseudogenes and RNA N6‐methyladenosine (m6A) modification in cancer, as well as the underlying mechanisms, remains largely unexplored.MethodsWe analyzed the expression of 12 146 pseudogenes and comprehensively examined the m6A modification of RNAs derived from them and their paralogs. Through integrative analysis of multi‐omics data, we explored the associations between pseudogene dysregulation and m6A, identifying critical pseudogenes involved in HGSOC progression. Tumour promotion role of RPS15AP12 and its cognate parent gene was characterized by cell proliferation, transwell assays, and scratch assays in ovarian cells and xenograft nude mice. RNA decay assays were used to reveal the participation of m6A in decreasement of RPS15AP12 lncRNA stability. Luciferase reporter assays were performed to verify that RPS15AP12 enhances RPS15A expression by competitively binding to miR‐96‐3p. Western blot and phosphorylation assays were performed to investigate the impairment of RPS15AP12 towards the sensors of MAVS (RIG‐I and MDA5), and downstream p‐TBK1 and p‐IRF3. Finally, ELISA assays were performed to explore the regulatory role of RPS15AP12 in IFN‐β expression.ResultsM6A is distributed across over a thousand pseudogenes, and hypomethylation leads to their upregulation in HGSOC. We identified a processed pseudogene, RPS15AP12, upregulated by FTO‐mediated m6A demethylation. RPS15AP12 enhances the growth ability and metastatic capabilities of ovarian cancer (OC) cells via functioning as a competitive endogenous RNA (ceRNA) for its host gene, RPS15A, through the sequestration of miR‐96‐3p. Importantly, the deletion of RPS15AP12 diminishes the expression of RPS15A, leading to the upregulation of anti‐tumour immune responses by activating RIG‐I and MDA5 and downstream p‐TBK1 and p‐IRF3 as well as IFN‐β levels.ConclusionOur findings expand the understanding of m6A‐modulated pseudogenes in tumour growth and anti‐tumour innate immunity in OC.Key Points Genome‐wide profiling reveals the redistribution of m6A modification on pseudogene‐derived lncRNAs and m6A redistribution‐relevant dysregulation of pseudogenes in HGSOC. RPS15AP12, as a representative processed pseudogene, is up‐regulated by FTO‐mediated demethylation and acts as a miRNA sponge to promote RPS15A expression via competitively binding to miR‐96‐3p. RPS15AP12/RPS15A axis inhibits MAVS sensors (RIG‐I and MDA5) and downstream IFN‐β levels in ovarian cancer.

Targeting KIF23 inhibits cell proliferation and primary chemoresistance in cervical cancer by inactivating the MYH9/MCM2/PCNA pathway

Abstract Background Kinesin family member 23 (KIF23) is recognised as an important tumour promoter involved in the pathogenesis of various cancers. However, its role and underlying molecular mechanisms in regulating cervical cancer (CC) growth and primary chemoresistance remain to be fully elucidated. Methods The expression and prognostic significance of KIF23 were initially assessed through bioinformatic analyses and subsequently validated in clinical specimens. To evaluate the effects of KIF23 on cell proliferation and cisplatin (DDP) sensitivity in CC cells, in vitro and in vivo experiments were conducted using CRISPR/Cas9 knockout, overexpression and mouse xenograft models. Co‐immunoprecipitation, protein half‐life assays and ubiquitination assays were employed to elucidate the interactions and regulatory mechanisms involving KIF23, myosin heavy chain 9 (MYH9), minichromosome maintenance protein 2 (MCM2) and proliferating cell nuclear antigen (PCNA), thereby revealing the molecular basis of KIF23‐mediated CC progression and primary chemoresistance. Results KIF23 is highly expressed in CC tissues and is significantly correlated with poor prognosis and DDP resistance in patients. The knockout of KIF23 inhibited cell proliferation, induced G1‐phase arrest and enhanced chemosensitivity to DDP. Mechanistically, the C‐terminal domain of KIF23 was found to directly bind to the myosin tail domain of MYH9. This interaction stabilises MYH9 by recruiting deubiquitinase 7 (ubiquitin‐specific protease 7 [USP7]), which removes K48‐linked ubiquitin chains. The consequent upregulation of MYH9 promoted the recruitment of ubiquitin‐specific protease 15 (USP15) to deubiquitinate MCM2, thereby preventing its degradation. Lysine 469 (K469) of MCM2 was identified as the key site for MYH9‐induced deubiquitination. Furthermore, elevated MCM2 levels enhanced its binding to PCNA, thereby promoting CC cell proliferation. Conclusions These findings demonstrated that elevated KIF23 levels act as an unfavorable prognostic factor for CC by promoting cell proliferation and primary chemoresistance via the activation of the MYH9/MCM2/PCNA axis. Thus, KIF23 may represent a promising therapeutic target for improving clinical outcomes in CC. Highlights Cisplatin treatment induces KIF23 expression in a concentration‐ and time‐dependent manner. KIF23 recruits USP7, which removes the K48‐linked ubiquitin chain of MYH9, thereby stabilising MYH9 and facilitating its nuclear transport. MYH9 recruits USP15, thereby stabilising MCM2, which, in turn, regulates the G1/S phase transition by binding to PCNA. Targeting the KIF23/MYH9/MCM2/PCNA axis sensitises cervical cancer cells to cisplatin.

The WNK1–ERK5 route plays a pathophysiological role in ovarian cancer and limits therapeutic efficacy of trametinib

AbstractBackgroundThe dismal prognosis of advanced ovarian cancer calls for the development of novel therapies to improve disease outcome. In this regard, we set out to discover new molecular entities and to assess the preclinical effectiveness of their targeting.MethodsCell lines, mice and human ovarian cancer samples were used. Proteome profiling of human phosphokinases, in silico genomic analyses, genetic (shRNA and CRISPR/Cas9) and pharmacological strategies as well as an ex vivo human preclinical model were performed.ResultsWe identified WNK1 as a highly phosphorylated protein in ovarian cancer and found that its activation or high expression had a negative impact on patients’ survival. Genomic analyses showed amplification of WNK1 in human ovarian tumours. Mechanistically, we demonstrate that WNK1 exerted its action through the MEK5–ERK5 signalling module in ovarian cancer. Loss of function, genetic or pharmacological experiments, demonstrated anti‐proliferative and anti‐tumoural effects of the targeting of the WNK1–MEK5–ERK5 route. Additional studies showed that this pathway modulated the anti‐tumoural properties of the MEK1/2 inhibitor trametinib. Thus, treatment with trametinib activated the WNK1–MEK5–ERK5 route, raising the possibility that this effect may limit the therapeutic benefit of ERK1/2 targeting in ovarian cancer. Moreover, in different experimental settings, including an ex vivo patient‐derived model consisting of ovarian cancer cells cultured with autologous patient sera, we show that inhibition of WNK1 or MEK5 increased the anti‐proliferative and anti‐tumour efficacy of trametinib.ConclusionsThe present study uncovers the participation of WNK1–MEK5–ERK5 axis in ovarian cancer pathophysiology, opening the possibility of acting on this pathway with therapeutic purposes. Another important finding of the present study was the activation of that signalling axis by trametinib, bypassing the anti‐tumoural efficacy of this drug. That fact should be considered in the context of the use of trametinib in ovarian cancer.

Proteomic landscaping of high‐grade serous ovarian carcinoma identifies stearoyl‐CoA desaturase 5 as a potential predictive biomarker for poly(ADP‐ribose) polymerase inhibitor response

Spatially resolved proteomics surveys the chemo‐refractory proteins related to high‐grade serous ovarian cancer

AbstractHigh‐grade serous ovarian carcinoma (HGSC) is a lethal malignancy characterized by high incidence, mortality, and chemoresistance. However, its molecular drivers are unknown. In this study, spatially resolved proteomics was applied to 1144 formalin‐fixed paraffin‐embedded tissue spots obtained by laser capture microdissection from 10 patients with HGSC and divergent carboplatin‐paclitaxel (CP) responses. Specific sampling revealed stroma‐driven tumour heterogeneity, identifying 642 tumour‐specific and 180 stroma‐specific proteins, with 505 CP‐responsive therapeutic targets. Most of these protein signatures represented previously unreported associations with chemoresistance in HGSCs. Two clinically significant spatial proteomic maps were generated by introducing tumour (TS) and chemical (CS) scores. TS analysis revealed conserved tissue architecture across CP response groups, whereas CS mapping revealed pretreatment metabolic reprogramming (rather than proliferation) as the defining feature of chemo‐resistant tumours, challenging current resistance paradigms. Immunohistochemical validation of HGSC tissue microarrays confirmed the spatial proteomic localization of TFRC and PDLIM3, which are linked to tumour progression, while establishing their novel role as chemotherapy resistance biomarkers through this study, with broader predictive potential observed across additional targets in the discovery cohort. This study developed a spatially resolved proteomic framework to enhance the diagnostic and therapeutic strategies for HGSC.Key points HGSC intra‐tumour heterogeneity is predominantly driven by stroma, as revealed by spatial proteomic compartmentalization (tumour/stroma). Spatial proteomics expands the therapeutic target database, enabling prediction of platinum‐based chemotherapy response. Chemo‐resistant patients exhibit pre‐treatment metabolic activation rather than proliferative signatures. TFRC (iron transport) and PDLIM3 (cytoskeletal remodelling) are spatially validated as chemo‐response biomarkers.

Parkin regulates IGF2BP3 through ubiquitination in the tumourigenesis of cervical cancer

AbstractBackgroundInsulin‐like growth Factor 2 mRNA‐binding protein 3 (IGF2BP3) is a highly conserved RNA‐binding protein and plays a critical role in regulating posttranscriptional modifications.MethodsImmunoprecipitation was used to examine the interaction of Parkin and IGF2BP3. Mass spectrometry was performed to identify the ubiquitination sites of IGF2BP3. RNA‐immunoprecipitation was conducted to examine the target genes of IGF2BP3. Xenograft mouse model was constructed to determine the tumorigenesis of IGF2BP3.ResultsIGF2BP3 expression is negatively correlated with Parkin expression in human cervical cancer cells and tissues. Parkin directly interacts with IGF2BP3, and overexpression of Parkin causes the proteasomal degradation of IGF2BP3, while knockdown of PARK2 increases the protein levels of IGF2BP3. Mechanistically, in vivo and in vitro ubiquitination assays demonstrated that Parkin is able to ubiquitinate IGF2BP3. Moreover, the ubiquitination site of IGF2BP3 was identified at K213 in the first KH domain of IGF2BP3. IGF2BP3 mutation results in the loss of its oncogenic function as an m6A reader, resulting in the inactivation of the phosphoinositide 3‐kinase (PI3K) and mitogen‐activated protein kinase (MAPK) signalling pathways. In addition, IGF2BP3 mutation results in the attenuation of Parkin‐mediated mitophagy, indicating its inverse role in regulating Parkin. Consequently, the tumourigenesis of cervical cancer is also inhibited by IGF2BP3 mutation.ConclusionIGF2BP3 is ubiquitinated and regulated by the E3 ubiquitin ligase Parkin in human cervical cancer and ubiquitination modification plays an important role in modulating IGF2BP3 function. Thus, understanding the role of IGF2BP3 in tumourigenesis could provide new insights into cervical cancer therapy.

Basal cell adhesion molecule promotes metastasis‐associated processes in ovarian cancer

AbstractBackgroundBasal cell adhesion molecule (BCAM) is a laminin α5 (LAMA5) binding membrane‐bound protein with a putative role in cancer. Besides full‐length BCAM1, an isoform lacking most of the cytoplasmic domain (BCAM2), and a soluble form (sBCAM) of unknown function are known. In ovarian carcinoma (OC), all BCAM forms are abundant and associated with poor survival, yet BCAM's contribution to peritoneal metastatic spread remains enigmatic.MethodsBiochemical, omics‐based and real‐time cell assays were employed to identify the source of sBCAM and metastasis‐related functions of different BCAM forms. OC cells, explanted omentum and a mouse model of peritoneal colonisation were used in loss‐ and gain‐of‐function experiments.ResultsWe identified ADAM10 as a major BCAM sheddase produced by OC cells and identified proteolytic cleavage sites proximal to the transmembrane domain. Recombinant soluble BCAM inhibited single‐cell adhesion and migration identically to membrane‐bound isoforms, confirming its biological activity in OC. Intriguingly, this seemingly anti‐tumorigenic potential of BCAM contrasts with a novel pro‐metastatic function discovered in the present study. Thus, all queried BCAM forms decreased the compactness of tumour cell spheroids by inhibiting LAMA5 – integrin β1 interactions, promoted spheroid dispersion in a three‐dimensional collagen matrix, induced clearance of mesothelial cells at spheroid attachment sites in vitro and enhanced invasion of spheroids into omental tissue both ex vivo and in vivo.ConclusionsMembrane‐bound BCAM as well as sBCAM shed by ADAM10 act as decoys rather than signalling receptors to modulate metastasis‐related functions. While BCAM appears to have tumour‐suppressive effects on single cells, it promotes the dispersion of OC cell spheroids by regulating LAMA5‐integrin‐β1‐dependent compaction and thereby facilitating invasion of metastatic target sites. As peritoneal dissemination is majorly mediated by spheroids, these findings offer an explanation for the association of BCAM with a poor clinical outcome of OC, suggesting novel therapeutic options.

Ensemble biomarkers for guiding anti‐angiogenesis therapy for ovarian cancer using deep learning

Single‐cell transcriptomes reveal heterogeneity of high‐grade serous ovarian carcinoma

AbstractBackgroundHigh‐grade serous ovarian carcinoma (HGSOC) is the most common and aggressive histotype of epithelial ovarian cancer. The heterogeneity and molecular basis of this disease remain incompletely understood.MethodsTo address this question, we have performed a single‐cell transcriptomics analysis of matched primary and metastatic HGSOC samples.ResultsA total of 13 571 cells are categorized into six distinct cell types, including epithelial cells, fibroblast cells, T cells, B cells, macrophages, and endothelial cells. A subset of aggressive epithelial cells with hyperproliferative and drug‐resistant potentials is identified. Several new markers that are highly expressed in epithelial cells are characterized, and their roles in ovarian cancer cell growth and migration are further confirmed. Dysregulation of multiple signaling pathways, including the translational machinery, is associated with ovarian cancer metastasis through the trajectory analysis. Moreover, single‐cell regulatory network inference and clustering (SCENIC) analysis reveals the gene regulatory networks and suggests the JUN signaling pathway as a potential therapeutic target for treatment of ovarian cancer, which is validated using the JUN/AP‐1 inhibitor T‐5224. Finally, our study depicts the epithelial‐fibroblast cell communication atlas and identifies several important receptor‐ligand complexes in ovarian cancer development.ConclusionsThis study uncovers new molecular features and the potential therapeutic target of HGSOC, which would advance the understanding and treatment of the disease.

Loss of exosomal miR‐26a‐5p contributes to endometrial cancer lymphangiogenesis and lymphatic metastasis

Bevacizumab in ovarian cancer: Clinical data and predictive and prognostic biomarkers

Abstract Angiogenesis, driven by the vascular endothelial growth factor (VEGF)/VEGFR signalling axis under hypoxic conditions, is one of the hallmarks of ovarian cancer (OC), contributing to tumour progression, metastatic dissemination and immune evasion. Hypoxia‐induced angiogenic signalling sustains tumour growth and shapes an immunosuppressive tumour microenvironment, while homologous recombination deficiency (HRD) has been associated with increased tumour hypoxia and pro‐angiogenic signalling. Conversely, VEGF pathway inhibition may exacerbate DNA damage and modulate immune cell trafficking, providing a strong biological rationale for synergy between anti‐angiogenic agents, PARP inhibitors (PARPi), and immune checkpoint inhibitors. Bevacizumab, a humanised monoclonal antibody targeting VEGF‐A, represents a pivotal therapeutic agent in OC management by inhibiting tumour angiogenesis and inducing transient vascular normalisation. Its clinical efficacy has been demonstrated as maintenance therapy in the first‐line setting, alone or in combination with PARPi for HRD‐positive disease, and in the recurrent setting both in platinum‐sensitive and platinum‐resistant disease. Despite these benefits, variability in patient response highlights the unmet need for validated predictive biomarkers. Circulating, tissue‐based and molecular biomarkers have been investigated, including angiogenic factors (Tie2/Ang1 axis, interleukin‐6 [IL‐6] and chitinase‐3‐like protein [YKL‐40]), VEGF‐A isoforms, microvessel density, EGFR/ADAM17 signalling, angiomiRs and transcriptional subtypes with mesenchymal and proliferative phenotypes showing greater sensitivity to anti‐angiogenic strategies. Although HRD status holds prognostic relevance and selected microRNAs show emerging potential, no biomarker has yet been validated to predict benefit from bevacizumab in clinical practice. Translational analyses from the MITO16A/MaNGO OV‐2 program, highlight challenges such as assay standardisation, multiplicity correction and external validation, while identifying tumour immune infiltration patterns, TP53 mutation classes and composite HRD assessments as areas of further investigation. In conclusion, bevacizumab remains an integral component of OC treatment. Future progress will depend on biomarker‐driven, prospectively designed clinical trials and the integration of multi‐omic data and machine learning approaches to enable precision application of anti‐angiogenic strategies, maximising clinical benefit while minimising toxicity.

Identification of biomarkers complementary to homologous recombination deficiency for improving the clinical outcome of ovarian serous cystadenocarcinoma

AbstractOvarian cancer patients with homologous recombination deficiency (HRD) tumors would benefit from PARP inhibitor (PARPi) therapy. However, patients with HRD tumors account for less than 50% of the whole cohort, so new biomarkers still need to be developed. Based on the data from the SNP array and somatic mutation profiles in the ovarian cancer genome, we found that high frequency of actionable mutations existed in patients with non‐HRD tumors. Through transcriptome analysis, we identified that a downstream target of the cGAS‐STING pathway, CXCL11, was upregulated in HRD tumors and could be used as a predictor of survival outcome. Further comprehensive analysis of the tumor immune microenvironment (TIME) revealed that CXCL11 expression signature was closely correlated with cytotoxic cells, neoantigen load and immune checkpoint blockade (ICB). Clinical trial data confirmed that the expression of CXCL11 could be used as a biomarker for anti‐PD‐1/PD‐L1 therapy. Finally, in vivo and in vitro experiments showed that cancer cells with PARPi treatment increased the expression of CXCL11. Collectively, our study not only provides biomarkers of ovarian cancer complementary to the HRD score but also introduces a potential new perspective for identifying prognostic biomarkers of immunotherapy.

Phase II clinical trial of nirogacestat in patients with relapsed ovarian granulosa cell tumours

Abstract Background Adult ovarian granulosa cell tumours (GCT) are the most common subtype of ovarian sex cord‐stromal tumours. Forkhead transcription factor FOXL2 is required for development and function of normal granulosa cells, including proliferation and ovarian hormone synthesis. A single somatic missense mutation in FOXL2 , c.402C > G (p.Cys134Trp), has previously been identified in the majority of GCT and is a pathognomonic marker for this tumour type. NOTCH activation contributes to GCT survival in preclinical models, and NOTCH2 and NOTCH3 are critical for embryonic development of the ovary and function of the ovarian follicle. Nirogacestat is a potent, selective, noncompetitive inhibitor of gamma secretase, which inhibits NOTCH pathway signalling. Treatment of GCT with nirogacestat was predicted to inhibit granulosa cell survival. Methods A Phase II clinical trial was conducted to assess antitumour activity of nirogacestat in adult patients with relapsed/refractory ovarian GCT (NCT05348356). This study enrolled 53 patients; all were evaluable for efficacy and safety. Endpoints included objective response rate by Response Evaluation Criteria in Solid Tumors v1.1 and 6‐month progression‐free survival (PFS6). Fresh or archival tumour samples were analysed for mutational profiling. Results Patients received a median of 5 prior lines of therapy (range, 1–13) and a median of 3.7 months of treatment (range, 0–20 months). A decrease in tumour burden was seen in 16 (30%) patients; however, there were no confirmed objective responses. Thirty‐one (58%) patients had stable disease; 18 (34%) had progressive disease. Eleven (21%) patients achieved PFS6. No correlations with disease stability were found with baseline clinical characteristics. All 3 patients who had an activating NOTCH1 mutation achieved PFS6. Conclusions In patients with heavily pretreated GCT, nirogacestat treatment resulted in durable disease stabilisation of at least 7 weeks for 58% of patients, with 21% achieving PFS6, including the 3 patients whose tumours had an activating NOTCH1 mutation. Key points This Phase II clinical trial of a rare tumour achieved its enrolment target in under 1 year and completed primary analysis within 2 years. 87% (46 of 53 patients who received nirogacestat) had fresh or archival biopsies that were analysed by next‐generation sequencing for mutational profiling. Of the 3 patients with activating NOTCH1 mutations, all achieved 6‐month progression‐free survival (PFS6); 8 other patients also achieved PFS6 but did not share a common mutation.

Single‐cell transcriptomics identify TNFRSF1B as a novel T‐cell exhaustion marker for ovarian cancer

AbstractBackground:Ovarian cancer (OC) patients routinely show poor immunotherapeutic response due to the complex tumour microenvironment (TME). It is urgent to explore new immunotherapeutic markers.Methods:Through the single‐cell RNA sequencing (scRNA‐seq) analyses on high‐grade serous OC (HGSOC), moderate severity borderline tumour and matched normal ovary, we identified a novel exhausted T cells subpopulation that related to poor prognosis in OC. Histological staining, multiple immunofluorescences, and flow cytometry were applied to validate some results from scRNA‐seq. Furthermore, a tumour‐bearing mice model was constructed to investigate the effects of TNFRSF1B treatment on tumour growth in vivo.Results:Highly immunosuppressive TME in HGSOC is displayed compared to moderate severity borderline tumour and matched normal ovary. Subsequently, a novel exhausted subpopulation of CD8+TNFRSF1B+ T cells is identified, which is associated with poor survival. In vitro experiments demonstrate that TNFRSF1B is specifically upregulated on activated CD8+ T cells and suppressed interferon‐γ secretion. The expression of TNFRSF1B on CD8+T cells is closely related to OC clinical malignancy and is a marker of poor prognosis through 140 OC patients’ verification. In addition, the blockade of TNFRSF1B inhibits tumour growth via profoundly remodeling the immune microenvironment in the OC mouse model.Conclusions:Our transcriptomic results analyzed by scRNA‐seq delineate a high‐resolution snapshot of the entire tumour ecosystem of OC TME. The major applications of our findings were an exhausted subpopulation of CD8+TNFRSF1B+ T cells for predicting OC patient prognosis and the potential therapeutic value of TNFRSF1B. These findings demonstrated the clinical value of TNFRSF1B as a potential immunotherapy target and extended our understanding of factors contributing to immunotherapy failure in OC.

Synergistic inhibition of progesterone receptor‐A/B signalling by simvastatin and mifepristone in human uterine leiomyomas

VAMP7‐mediated autophagy regulates cervical cancer progression associated with persistent HPV16 infection

Abstract Backgroud Persistent infection with high‐risk human papillomavirus type 16 (HPV16) is a principal etiological factor in cervical cancer. Nevertheless, the molecular events linking HPV16‐associated lesion progression to malignant transformation remain insufficiently characterized, particularly those involving vesicular trafficking and autophagy regulation. Methods Proteomic analysis was conducted across five stages of HPV16‐associated cervical lesion progression to identify differentially expressed proteins. The expression of vesicle‐associated membrane protein 7 (VAMP7) was validated in cervical tissue specimens and cellular models. Gain‐ and loss‐of‐function approaches were employed to assess the effects of VAMP7 on cellular proliferation, migration, invasion, and apoptosis. Autophagic activity was evaluated by LC3 lipidation, autophagosome accumulation, and analysis of SNARE complexrelated proteins. The in vivo effects of VAMP7 were examined using xenograft tumor models. Results VAMP7 demonstrated dynamic expression changes during cervical lesion progression, characterized by decreased expression in HPV16‐positive non‐malignant tissues and a gradual increase with disease severity, reaching the highest levels in advanced cervical cancer. Functionally, VAMP7 enhanced proliferation, migration, and invasion while inhibiting apoptosis in cervical cancer cells, whereas distinct effects were observed in non‐tumor cervical epithelial cells. Mechanistically, VAMP7 regulated autophagic flux through modulation of SNARE‐mediated vesicle fusion, resulting in altered autophagosome accumulation and autophagy‐related signaling. In xenograft models, VAMP7 overexpression significantly promoted tumor growth and increased the expression of autophagy‐associated markers. Conclusion These data indicate that dysregulation of VAMP7‐mediated autophagy contributes to cervical carcinogenesis in an HPV16‐associated context. VAMP7 may represent a potential therapeutic target for the treatment of cervical cancer. Key points VAMP7 displays dynamic expression changes during HPV16‐associated cervical lesion progression. VAMP7 promotes malignant phenotypes of cervical cancer cells by regulating autophagic flux via SNARE‐mediated vesicle fusion. Dysregulated VAMP7‐mediated autophagy contributes to cervical carcinogenesis in an HPV16‐associated context.

Tailored chemotherapy: Innovative deep‐learning model customizing chemotherapy for high‐grade serous ovarian carcinoma

Human tumour vessel heterogeneity in ovarian cancer and its association with response to neoadjuvant chemotherapy

Potential of peptide‐engineered exosomes with overexpressed miR‐92b‐3p in anti‐angiogenic therapy of ovarian cancer

AbstractIntroductionExosomal microRNA (miRNA) as a mediator of intercellular communication plays an essential part in tumor‐relevant angiogenesis. Therapy against angiogenesis has been demonstrated to have a remarkable antitumor efficacy in various malignancies, but not as expected in ovarian cancer.MethodsExosomes were isolated by ultracentrifugation. Exosomal miRNA sequencing and gene function experiments were used to identify the differential expressed miRNAs in exosomes and their mRNA targets. SKOV3 cell line that stably overexpressed miR‐92b‐3p was constructed by lentivirus. In vitro, angiogenesis was analyzed by tube formation assay and migration assay. The angiogenic and antitumor effects in vivo were assessed in zebrafish and nude mouse models. Combination index was calculated to assess the synergetic inhibition of angiogenesis between miR‐92b‐3p and Apatinib. Peptides were conjugated with exosomal membranes to obtain engineered exosomes.ResultsOvarian cancer cell‐derived exosomes facilitated the angiogenesis and migration capability of vascular endothelial cells in vitro and in vivo. The expression of miR‐92b‐3p was much lower in ovarian cancer cell‐derived exosomes than that in immortalized ovarian epithelial cell‐derived exosomes. The exosomal miR‐92b‐3p modulated tumor‐associated angiogenesis via targeting SOX4. Besides, Peptide‐engineered exosomes with overexpressed miR‐92b‐3p showed the stronger abilities of anti‐angiogenesis and antitumor than parental exosomes, whether alone or combined with Apatinib.ConclusionsOur findings demonstrate the effect and mechanism of exosomal miR‐92b‐3p from ovarian cancer cells on tumor‐associated angiogenesis and the potential of artificially generated exosomes with overexpressed miR‐92b‐3p to be used as anti‐angiogenic agent, which may provide a new approach for anti‐angiogenic therapy of ovarian cancer.

Identification of potent SENP1 inhibitors that inactivate SENP1/JAK2/STAT signaling pathway and overcome platinum drug resistance in ovarian cancer

Discovery and characterization of potent And‐1 inhibitors for cancer treatment

AbstractAcidic nucleoplasmic DNA‐binding protein 1 (And‐1), an important factor for deoxyribonucleic acid (DNA) replication and repair, is overexpressed in many types of cancer but not in normal tissues. Although multiple independent studies have elucidated And‐1 as a promising target gene for cancer therapy, an And‐1 inhibitor has yet to be identified. Using an And‐1 luciferase reporter assay to screen the Library of Pharmacologically Active Compounds (LOPAC) in a high throughput screening (HTS) platform, and then further screen the compound analog collection, we identified two potent And‐1 inhibitors, bazedoxifene acetate (BZA) and an uncharacterized compound [(E)‐5‐(3,4‐dichlorostyryl)benzo[c][1,2]oxaborol‐1(3H)‐ol] (CH3), which specifically inhibit And‐1 by promoting its degradation. Specifically, through direct interaction with And‐1 WD40 domain, CH3 interrupts the polymerization of And‐1. Depolymerization of And‐1 promotes its interaction with E3 ligase Cullin 4B (CUL4B), resulting in its ubiquitination and subsequent degradation. Furthermore, CH3 suppresses the growth of a broad range of cancers. Moreover, And‐1 inhibitors re‐sensitize platinum‐resistant ovarian cancer cells to platinum drugs in vitro and in vivo. Since BZA is an FDA approved drug, we expect a clinical trial of BZA‐mediated cancer therapy in the near future. Taken together, our findings suggest that targeting And‐1 by its inhibitors is a potential broad‐spectrum anti‐cancer chemotherapy regimen.

The multicellular signalling network of ovarian cancer metastases

AbstractBackgroundTranscoelomic spread is the major route of metastasis of ovarian high‐grade serous carcinoma (HGSC) with the omentum as the major metastatic site. Its unique tumour microenvironment with its large populations of adipocytes, mesothelial cells and immune cells establishes an intercellular signaling network that is instrumental for metastatic growth yet poorly understood.MethodsBased on transcriptomic analysis of tumour cells, tumour‐associated immune and stroma cells we defined intercellular signaling pathways for 284 cytokines and growth factors and their cognate receptors after bioinformatic adjustment for contaminating cell types. The significance of individual components of this network was validated by analysing clinical correlations and potentially pro‐metastatic functions, including tumour cell migration, pro‐inflammatory signal transduction and TAM expansion.ResultsThe data show an unexpected prominent role of host cells, and in particular of omental adipocytes, mesothelial cells and fibroblasts (CAF), in sustaining this signaling network. These cells, rather than tumour cells, are the major source of most cytokines and growth factors in the omental microenvironment (n = 176 vs. n = 13). Many of these factors target tumour cells, are linked to metastasis and are associated with a short survival. Likewise, tumour stroma cells play a major role in extracellular‐matrix‐triggered signaling. We have verified the functional significance of our observations for three exemplary instances. We show that the omental microenvironment (i) stimulates tumour cell migration and adhesion via WNT4 which is highly expressed by CAF; (ii) induces pro‐tumourigenic TAM proliferation in conjunction with high CSF1 expression by omental stroma cells and (iii) triggers pro‐inflammatory signaling, at least in part via a HSP70–NF‐κB pathway.ConclusionsThe intercellular signaling network of omental metastases is majorly dependent on factors secreted by immune and stroma cells to provide an environment that supports ovarian HGSC progression. Clinically relevant pathways within this network represent novel options for therapeutic intervention.

Assessment of the immune landscapes of advanced ovarian cancer in an optimized in vivo model

AbstractBackgroundOvarian cancer (OC) is typically diagnosed late, associated with high rates of metastasis and the onset of ascites during late stage disease. Understanding the tumor microenvironment and how it impacts the efficacy of current treatments, including immunotherapies, needs effective in vivo models that are fully characterized. In particular, understanding the role of immune cells within the tumor and ascitic fluid could provide important insights into why OC fails to respond to immunotherapies. In this work, we comprehensively described the immune cell infiltrates in tumor nodules and the ascitic fluid within an optimized preclinical model of advanced ovarian cancer.MethodsGreen Fluorescent Protein (GFP)‐ID8 OC cells were injected intraperitoneally into C57BL/6 mice and the development of advanced stage OC monitored. Nine weeks after tumor injection, mice were sacrificed and tumor nodules analyzed to identify specific immune infiltrates by immunohistochemistry. Ascites, developed in tumor bearing mice over a 10‐week period, was characterized by mass cytometry (CyTOF) to qualitatively and quantitatively assess the distribution of the immune cell subsets, and their relationship to ascites from ovarian cancer patients.ResultsTumor nodules in the peritoneal cavity proved to be enriched in T cells, antigen presenting cells and macrophages, demonstrating an active immune environment and cell‐mediated immunity. Assessment of the immune landscape in the ascites showed the predominance of CD8+, CD4+, B–, and memory T cells, among others, and the coexistance of different immune cell types within the same tumor microenvironment.ConclusionsWe performed, for the first time, a multiparametric analysis of the ascitic fluid and specifically identify immune cell populations in the peritoneal cavity of mice with advanced OC. Data obtained highlights the impact of CytOF as a diagnostic tool for this malignancy, with the opportunity to concomitantly identify novel targets, and define personalized therapeutic options.

COLGALT2 is overexpressed in ovarian cancer and interacts with PLOD3

LRRK2 inhibition potentiates PARP inhibitor cytotoxicity through inhibiting homologous recombination‐mediated DNA double strand break repair

AbstractPARP inhibitors induce DNA lesions, the repair of which are highly dependent on homologous recombination (HR), and preferentially kill HR‐ deficient cancers. However, cancer cells have developed several mechanisms to transform HR and confer drug resistance to PARP inhibition. Therefore, there is a great clinical interest in exploring new therapies that induce HR deficiency (HRD), thereby sensitizing cancer cells to PARP inhibitors. Here, we found that GSK2578215A, a high‐selective and effective leucine‐rich repeat kinase 2 (LRRK2) inhibitor, or LRRK2 depletion suppresses HR preventing the recruitment of RAD51 to DNA damage sites through disruption of the interaction of RAD51 and BRCA2. Moreover, LRRK2 inhibition or depletion increases the susceptibility of ovarian cancer cells to Olaparib in vitro and in vivo. In clinical specimens, LRRK2 high expression is high related with advanced clinical characteristics and poor survival of ovarian cancer patients. All these findings indicate ovarian cancers expressing high levels of LRRK2 are more resistant to treatment potentially through promoting HR. Furthermore, combination treatment with an LRRK2 and PARP inhibitor may be a novel strategy to improve the effectiveness of LRRK2 expression ovarian cancers.

MTHFR inhibits TRC8‐mediated HMOX1 ubiquitination and regulates ferroptosis in ovarian cancer

Integrative analysis identifies three molecular subsets in ovarian cancer

PI3K/mTOR inhibition induces tumour microenvironment remodelling and sensitises pS6high uterine leiomyosarcoma to PD‐1 blockade

AbstractBackgroundUterine leiomyosarcomas (uLMS) are aggressive tumours with poor prognosis and limited treatment options. Although immune checkpoint blockade (ICB) has proven effective in some ‘challenging‐to‐treat’ cancers, clinical trials showed that uLMS do not respond to ICB. Emerging evidence suggests that aberrant PI3K/mTOR signalling can drive resistance to ICB. We therefore explored the relevance of the PI3K/mTOR pathway for ICB treatment in uLMS and explored pharmacological inhibition of this pathway to sensitise these tumours to ICB.MethodsWe performed an integrated multiomics analysis based on TCGA data to explore the correlation between PI3K/mTOR dysregulation and immune infiltration in 101 LMS. We assessed response to PI3K/mTOR inhibitors in immunodeficient and humanized uLMS patient‐derived xenografts (PDXs) by evaluating tumour microenvironment modulation using multiplex immunofluorescence. We explored response to single‐agent and a combination of PI3K/mTOR inhibitors with PD‐1 blockade in humanized uLMS PDXs. We mapped intratumoural dynamics using single‐cell RNA/TCR sequencing of serially collected biopsies.ResultsPI3K/mTOR over‐activation (pS6high) associated with lymphocyte depletion and wound healing immune landscapes in (u)LMS, suggesting it contributes to immune evasion. In contrast, PI3K/mTOR inhibition induced profound tumour microenvironment remodelling in an ICB‐resistant humanized uLMS PDX model, fostering adaptive anti‐tumour immune responses. Indeed, PI3K/mTOR inhibition induced macrophage repolarisation towards an anti‐tumourigenic phenotype and increased antigen presentation on dendritic and tumour cells, but also promoted infiltration of PD‐1+ T cells displaying an exhausted phenotype. When combined with anti‐PD‐1, PI3K/mTOR inhibition led to partial or complete tumour responses, whereas no response to single‐agent anti‐PD‐1 was observed. Combination therapy reinvigorated exhausted T cells and induced clonal hyper‐expansion of a cytotoxic CD8+ T‐cell population supported by a CD4+ Th1 niche.ConclusionsOur findings indicate that aberrant PI3K/mTOR pathway activation contributes to immune escape in uLMS and provides a rationale for combining PI3K/mTOR inhibition with ICB for the treatment of this patient population.

Somatic mutations and CRISPR/Cas9 library screening integrated analysis identifies cervical cancer drug‐resistant pathways

Hypermethylated PCDHGB7 as a universal cancer only marker and its application in early cervical cancer screening

Development of a deep learning‐based nomogram for predicting lymph node metastasis in cervical cancer: A multicenter study

RAB2A promotes cervical cancer progression as revealed by comprehensive analysis of HPV integration and proteome in longitudinal cervical samples