Oncogene

Metabolic reprogramming of ovarian cancer involves ACSL1-mediated metastasis stimulation through upregulated protein myristoylation

As a result of the hostile microenvironment, metabolic alterations are required to enable the malignant growth of cancer cells. To understand metabolic reprogramming during metastasis, we conducted shotgun proteomic analysis of highly metastatic (HM) and non-metastatic (NM) ovarian cancer cells. The results suggest that the genes involved in fatty-acid (FA) metabolism are upregulated, with consequent increases of phospholipids with relatively short FA chains (myristic acid, MA) in HM cells. Among the upregulated proteins, ACSL1 expression could convert the lipid profile of NM cells to that similar of HM cells and make them highly aggressive. Importantly, we demonstrated that ACSL1 activates the AMP-activated protein kinase and Src pathways via protein myristoylation and finally enhances FA beta oxidation. Patient samples and tissue microarray data also suggested that omentum metastatic tumours have higher ACSL1 expression than primary tumours and a strong association with poor clinical outcome. Overall, our data reveal that ACSL1 enhances cancer metastasis by regulating FA metabolism and myristoylation.

ZEB2 facilitates peritoneal metastasis by regulating the invasiveness and tumorigenesis of cancer stem-like cells in high-grade serous ovarian cancers

AbstractPeritoneal metastasis is a common issue in the progression of high-grade serous ovarian cancers (HGSOCs), yet the underlying mechanism remains unconfirmed. We demonstrated that ZEB2, the transcription factor of epithelial–mesenchymal transition (EMT), was upregulated in ascites cells from HGSOC patients and in CD133+cancer stem-like cells (CSLCs) from epithelial ovarian cancer (EOC) cell lines. SiRNA-mediated knockdown of ZEB2 in EOC cells decreased the percentage of CSLCs and reduced the colony forming potential, cell invasion capacity and expression of pluripotent genes Oct4 and Nanog. Inhibition of ZEB2 also induced cellular apoptosis and impacted the tumorigenicity of ovarian CSLCs. The mesenchymal markers N-cadherin and vimentin were downregulated, while the epithelial marker E-cadherin was upregulated after ZEB2 knockdown. MiR-200a, a molecule that downregulates ZEB2, had the opposite effect of ZEB2 expression in EOC-CSLCs. A retrospective study of 98 HGSOC patients on the relationship of ascites volume, pelvic and abdominal metastasis, International Federation of Gynecology and Obstetrics (FIGO) stage and the malignant involvement of abdominal organs and lymph nodes was performed. Patients with high expression of ZEB2 in tumour tissues had a higher metastasis rate and a poorer prognosis than those with low expression. The parameters of ZEB2 expression and ascites volume were strongly linked with the prognostic outcome of HGSOC patients and had higher hazard ratios. These findings illustrated that ZEB2 facilitates the invasive metastasis of EOC-CSLCs and can predict peritoneal metastasis and a poor prognosis in HGSOC patients.

Loss of EIF4G2 mediates aggressiveness in distinct human endometrial cancer subpopulations with poor survival outcome in patients

Abstract The non-canonical translation initiation factor EIF4G2 plays essential roles in cellular stress responses via translation of selective mRNA cohorts. Currently there is limited and conflicting information regarding its involvement in cancer development and progression. Here we assessed its role in endometrial cancer (EC), in a cohort of 280 EC patients across different types, grades, and stages, and found that low EIF4G2 expression highly correlated with poor overall- and recurrence-free survival in Grade 2 EC patients, monitored over a period of up to 12 years. To establish a causative connection between low EIF4G2 expression and cancer progression, we stably knocked-down EIF4G2 in two human EC cell lines in parallel. EIF4G2 depletion resulted in increased resistance to conventional therapies and increased the prevalence of molecular markers for aggressive cell subsets, altering their transcriptional and proteomic landscapes. Prominent among the proteins with decreased abundance were Kinesin-1 motor proteins, KIF5B and KLC1, 2, 3. Multiplexed imaging of the EC patient tumor cohort showed a correlation between decreased expression of the kinesin proteins, and poor survival in patients with tumors of certain grades and stages. These findings reveal potential novel biomarkers for Grade 2 EC with ramifications for patient stratification and therapeutic interventions.

Exploring the therapeutic use and outcome of antibody-drug conjugates in ovarian cancer treatment

Abstract Ovarian cancer remains a leading cause of cancer-related deaths due to late-stage diagnosis and treatment resistance. While surgery and chemotherapy are standard treatments, challenges such as platinum resistance, tumor heterogeneity, and limited therapeutic options persist. Antibody-drug conjugates (ADCs) have emerged as a promising therapeutic strategy for treating ovarian cancer (OC), particularly in cases of platinum-resistant ovarian cancer (PROC). In OC, various ADCs targeting antigens such as folate receptor alpha (FRα), trophoblast cell surface antigen 2 (TROP-2), mesothelin (MSLN), sodium-dependent phosphate transport protein 2B (NaPi2b), and human epidermal growth factor receptor 2 (HER2) have shown encouraging preclinical results and significant clinical activity. However, challenges like antigen heterogeneity, off-target toxicity, and resistance mechanisms remain. This review highlights the current ADCs used in the clinic for the treatment of ovarian cancer, their challenges, and the future potential of ADC-based therapies in overcoming resistance and improving patient outcomes.

A safe haven for cancer cells: tumor plus stroma control by DYRK1B

Abstract The development of resistance remains one of the biggest challenges in clinical cancer patient care and it comprises all treatment modalities from chemotherapy to targeted or immune therapy. In solid malignancies, drug resistance is the result of adaptive processes occurring in cancer cells or the surrounding tumor microenvironment (TME). Future therapy attempts will therefore benefit from targeting both, tumor and stroma compartments and drug targets which affect both sides will be highly appreciated. In this review, we describe a seemingly paradoxical oncogenic mediator with this potential: The dual-specificity tyrosine-phosphorylation regulated kinase 1B (DYRK1B). DYRK1B promotes proliferative quiescence and yet is overexpressed or amplified in many hyperproliferative malignancies including ovarian cancer and pancreatic cancer. In particular the latter disease is a paradigmatic example for a therapy-recalcitrant and highly stroma-rich cancer entity. Here, recent evidence suggests that DYRK1B exerts its oncogenic features by installing a protective niche for cancer cells by directly affecting cancer cells but also the TME. Specifically, DYRK1B not only fosters cell-intrinsic processes like cell survival, chemoresistance, and disease recurrence, but it also upregulates TME and cancer cell-protective innate immune checkpoints and down-modulates anti-tumoral macrophage functionality. In this article, we outline the well-established cell-autonomous roles of DYRK1B and extend its importance to the TME and the control of the tumor immune stroma. In summary, DYRK1B appears as a single novel key player creating a safe haven for cancer cells by acting cell-intrinsically and—extrinsically, leading to the promotion of cancer cell survival, chemoresistance, and relapse. Thus, DYRK1B appears as an attractive drug target for future therapeutic approaches.

Schlafen 11 further sensitizes BRCA-deficient cells to PARP inhibitors through single-strand DNA gap accumulation behind replication forks

Abstract The preferential response to PARP inhibitors (PARPis) in BRCA-deficient and Schlafen 11 (SLFN11)-expressing ovarian cancers has been documented, yet the underlying molecular mechanisms remain unclear. As the accumulation of single-strand DNA (ssDNA) gaps behind replication forks is key for the lethality effect of PARPis, we investigated the combined effects of SLFN11 expression and BRCA deficiency on PARPi sensitivity and ssDNA gap formation in human cancer cells. PARPis increased chromatin-bound RPA2 and ssDNA gaps in SLFN11-expressing cells and even more in cells with BRCA1 or BRCA2 deficiency. SLFN11 was co-localized with chromatin-bound RPA2 under PARPis treatment, with enhanced recruitment in BRCA2-deficient cells. Notably, the chromatin-bound SLFN11 under PARPis did not block replication, contrary to its function under replication stress. SLFN11 recruitment was attenuated by the inactivation of MRE11. Hence, under PARPi treatment, MRE11 expression and BRCA deficiency lead to ssDNA gaps behind replication forks, where SLFN11 binds and increases their accumulation. As ovarian cancer patients who responded (progression-free survival >2 years) to olaparib maintenance therapy had a significantly higher SLFN11-positivity than short-responders (<6 months), our findings provide a mechanistic understanding of the favorable responses to PARPis in SLFN11-expressing and BRCA-deficient tumors. It highlight the clinical implications of SLFN11.

Shallow whole genome sequencing approach to detect Homologous Recombination Deficiency in the PAOLA-1/ENGOT-OV25 phase-III trial

AbstractThe bevacizumab (bev)/olaparib (ola) maintenance regimen was approved for BRCA1/2-mutated (BRCAmut) and Homologous Recombination Deficient (HRD) high-grade Advanced Ovarian Cancer (AOC) first line setting, based on a significantly improved progression-free survival (PFS) compared to bev alone in the PAOLA-1/ENGOT-ov25 trial (NCT02477644), where HRD was detected by MyChoice CDx PLUS test. The academic shallowHRDv2 test was developed based on shallow whole-genome sequencing as an alternative to MyChoice. Analytical and clinical validities of shallowHRDv2 as compared to MyChoice on 449 PAOLA-1 tumor samples are presented. The overall agreement between shallowHRDv2 and MyChoice was 94% (369/394). Less non-contributive tests were observed with shallowHRDv2 (15/449; 3%) than with MyChoice (51/449; 11%). Patients with HRD tumors according to shallowHRDv2 (including BRCAmut) showed a significantly prolonged PFS with bev+ola versus bev (median PFS: 65.7 versus 20.3 months, hazard ratio (HR): 0.36 [95% CI: 0.24–0.53]). This benefit was significant also for BRCA1/2 wild-type tumors (40.8 versus 19.5 months, HR: 0.45 [95% CI: 0.26–0.76]). ShallowHRDv2 is a performant, clinically validated, and cost-effective test for HRD detection.

Loss of LECT2 promotes ovarian cancer progression by inducing cancer invasiveness and facilitating an immunosuppressive environment

AbstractLeukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine that can bind to several receptors and mediate distinct molecular pathways in various cell settings. Changing levels of LECT2 have been implicated in multiple human disease states, including cancers. Here, we have demonstrated reduced serum levels of LECT2 in patients with epithelial ovarian cancer (EOC) and down-regulated circulating Lect2 as the disease progresses in a syngeneic mouse ID8 EOC model. Using the murine EOC model, we discovered that loss of Lect2 promotes EOC progression by modulating both tumor cells and the tumor microenvironment. Lect2 inhibited EOC cells’ invasive phenotype and suppressed EOC’s transcoelomic metastasis by targeting c-Met signaling. In addition, Lect2 downregulation induced the accumulation and activation of myeloid-derived suppressor cells (MDSCs). This fostered an immunosuppressive microenvironment in EOC by inhibiting T-cell activation and skewing macrophages toward an M2 phenotype. The therapeutic efficacy of programmed cell death-1 (PD-1)/PD-L1 pathway blockade for the ID8 model was significantly hindered. Overall, our data highlight multiple functions of Lect2 during EOC progression and reveal a rationale for synergistic immunotherapeutic strategies by targeting Lect2.

Loss of LATS1 and LATS2 promotes ovarian tumor formation by enhancing AKT activity and PD-L1 expression

High-grade serous ovarian cancer (HGSOC) is the deadliest and most common subtype of ovarian cancer. Unfortunately, most patients develop recurrence and, ultimately, resistance to standard platinum chemotherapy. Large tumor suppressors LATS1 and LATS2, the core Hippo signaling kinases, have been implicated in various cancer types, including ovarian cancer. The mechanism by which LATS1/2 suppresses ovarian cancer progression is currently elusive, but the expression of LATS1/2 is frequently reduced or lost in these cancers. In this study, we demonstrate that the inactivation of LATS1/2 is sufficient to transform normal mouse ovarian epithelium into tumorigenic cells associated with increased cell proliferation, invasion, and stemness and epithelial-mesenchymal transition (EMT) characteristics. The knockout of Lats1/2 in the epithelial cells also leads to higher expression levels of the immune checkpoint molecule PD-L1, suggesting a regulatory role of LATS1/2 in modulating immune responses and immune evasion. In addition to the loss of LATS1/2 activating the downstream transcriptional coactivators YAP and TAZ, PI3K-AKT activity was also increased, likely contributing to enhanced tumor proliferation and survival. The stimulatory effect of Lats1/2 knockout on cell proliferation can be partially reversed by treatment with the AKT inhibitor MK2206. Treatment with verteporfin, a potent inhibitor of YAP/TAZ, decreases ovarian tumor progression and reduces the activated AKT in the tumors. In summary, this study uncovers several biological mechanisms for the initiation of HGSOC and identifies LATS1/2 as potential prognostic indicators and therapeutic targets.

CBX7 binds the E-box to inhibit TWIST-1 function and inhibit tumorigenicity and metastatic potential

Deaths from ovarian cancer usually occur when patients succumb to overwhelmingly numerous and widespread micrometastasis. Whereas epithelial-mesenchymal transition is required for epithelial ovarian cancer cells to acquire metastatic potential, the cellular phenotype at secondary sites and the mechanisms required for the establishment of metastatic tumors are not fully determined. Using in vitro and in vivo models we show that secondary epithelial ovarian cancer cells (sEOC) do not fully reacquire the molecular signature of the primary epithelial ovarian cancer cells from which they are derived. Despite displaying an epithelial morphology, sEOC maintains a high expression of the mesenchymal effector, TWIST-1. TWIST-1 is however transcriptionally nonfunctional in these cells as it is precluded from binding its E-box by the PcG protein, CBX7. Deletion of CBX7 in sEOC was sufficient to reactivate TWIST-1-induced transcription, prompt mesenchymal transformation, and enhanced tumorigenicity in vivo. This regulation allows secondary tumors to achieve an epithelial morphology while conferring the advantage of prompt reversal to a mesenchymal phenotype upon perturbation of CBX7. We also describe a subclassification of ovarian tumors based on CBX7 and TWIST-1 expression, which predicts clinical outcomes and patient prognosis.

Initialization of epithelial cells by tumor cells in a metastatic microenvironment

Inhibition of PFKFB3 induces cell death and synergistically enhances chemosensitivity in endometrial cancer

AbstractThe advanced or recurrent endometrial cancer (EC) has a poor prognosis because of chemoresistance. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a glycolytic enzyme, is overexpressed in a variety of human cancers and plays important roles in promoting tumor cell growth. Here, we showed that high expression of PFKFB3 in EC cell lines is associated with chemoresistance. Pharmacological inhibition of PFKFB3 with PFK158 and or genetic downregulation of PFKFB3 dramatically suppressed cell proliferation and enhanced the sensitivity of EC cells to carboplatin (CBPt) and cisplatin (Cis). Moreover, PFKFB3 inhibition resulted in reduced glucose uptake, ATP production, and lactate release. Notably, we found that PFK158 with CBPt or Cis exerted strong synergistic antitumor activity in chemoresistant EC cell lines, HEC-1B and ARK-2 cells. We also found that the combination of PFK158 and CBPt/Cis induced apoptosis- and autophagy-mediated cell death through inhibition of the Akt/mTOR signaling pathway. Mechanistically, we found that PFK158 downregulated the CBPt/Cis-induced upregulation of RAD51 expression and enhanced CBPt/Cis-induced DNA damage as demonstrated by an increase in γ-H2AX levels in HEC-1B and ARK-2 cells, potentially revealing a means to enhance PFK158-induced chemosensitivity. More importantly, PFK158 treatment, either as monotherapy or in combination with CBPt, led to a marked reduction in tumor growth in two chemoresistant EC mouse xenograft models. These data suggest that PFKFB3 inhibition alone or in combination with standard chemotherapy may be used as a novel therapeutic strategy for improved therapeutic efficacy and outcomes of advanced and recurrent EC patients.

MCP-1/CCR-2 axis in adipocytes and cancer cell respectively facilitates ovarian cancer peritoneal metastasis

Ovarian cancer selective metastasizes to the omentum contributing to the poor prognosis associated with ovarian cancer. However, the mechanism underlining this propensity and therapeutic approaches to counter this process has not been fully elucidated. Here, we show that MCP-1 produced by omental adipocytes binding to its cognate receptor CCR-2 on ovarian cancer cells facilitates migration and omental metastasis by activating the PI3K/AKT/mTOR pathway and its downstream effectors HIF-1α and VEGF-A in cell lines, xenografts, and transgenic murine models. MCP-1 antibody significantly decreased tumor burden and increased survival of mice in vivo. Interestingly, metformin decreased omental metastasis at least partially by inhibiting MCP-1 secretion from adipocytes independent of direct effects on cancer cells. Together this suggests a novel target of MCP-1/CCR-2 axis that could benefit ovarian cancer patients.

MicroRNA-150-5p promotes cell motility by inhibiting c-Myb-mediated Slug suppression and is a prognostic biomarker for recurrent ovarian cancer

Treatment of ovarian cancer (OvCa) remains challenging owing to its high recurrence rates. Detachment of cancer cells into the peritoneal fluid plays a key role in OvCa relapse, but how this occurs remains incompletely understood. Here we examined global miRNA expression profiles of paired primary/recurrent OvCa specimens and identified a novel biomarker, microRNA-150-5p (miR-150-5p), that was significantly upregulated in 16 recurrent OvCa tissues compared with their matched primary specimens. Analyses of cohorts from two other groups confirmed that expression of miR-150-5p was associated with early relapse and poor survival of OvCa patients. Inhibition of miR-150-5p significantly inhibited the migration and invasion of OvCa cells and induced a mesenchymal-epithelial transition (MET) phenotype. We demonstrated that the proto-oncogene, MYB, is an miR-150-5p target in OvCa cells and that the miR-150-5p/c-Myb/Slug axis plays important roles in regulating epithelial-mesenchymal transition (EMT) in OvCa cells. Expression of MYB was significantly correlated with good clinical outcome in OvCa and was negatively correlated with Slug expression in late-stage clinical specimens. These results suggest that miR-150-5p upregulation mediates the progression of recurrent OvCa by targeting the c-Myb/Slug pathway. Inhibition of miR-150-5p may serve as a new therapeutic strategy for preventing recurrence of OvCa.

ULK1 promotes metastatic progression in experimental models of epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is a leading cause of gynecological cancer mortality, driven largely by late diagnosis and chemoresistance. While autophagy is critical for EOC spheroid survival during metastasis, the role of ULK1, a key regulator of autophagy, in EOC progression remains unclear. To investigate this, we utilized CRISPR/Cas9 technology to delete ULK1 in EOC cell lines OVCAR8, HEYA8, ES2 and the fallopian tube epithelial cell line FT190. ULK1 loss and autophagy disruption were confirmed in EOC spheroids, with reduced Beclin-1 phosphorylation, impaired LC3 processing, and p62 accumulation. ULK1 knockout decreased EOC spheroid cell viability via increased apoptosis, and impaired matrix-bound organoid growth, offering new insights into ULK1 activity in affecting EOC tumor growth and spread. These findings were supported by in vivo xenograft models, in which ULK1 loss significantly reduced tumor burden and metastatic potential. ULK1 requirement during metastasis was supported by diminished invasive capacity of ULK1 knockout spheroid cells in mesothelial clearance assays. To investigate ULK1 mechanisms contributing to EOC tumor progression and metastasis, we conducted proteomic analyses of OVCAR8 spheroids, which revealed ULK1 loss disrupted critical pathways, including MEK-MAPK, PI3K-AKT-mTOR, and apoptosis regulation. Although ULK1 knockout failed to synergize with standard-of-care chemotherapeutics, it significantly enhanced sensitivity to MEK and mTOR inhibition. Analysis of ovarian cancer datasets demonstrates that high ULK1 mRNA correlates with a poorer 10-year overall and progression-free survival; in fact, its expression is further elevated in metastases as compared with primary tumors and normal tissue. Treatment of metastatic patient-derived organoids with the clinical ULK1 inhibitor DCC-3116, MEK inhibitor trametinib, or mTORC1/2 inhibitor AZD-8055 reduced viability in a subset of these samples, reflecting inter-patient heterogeneity and need for biomarker-guided selection. Overall, this study highlights ULK1 as a critical regulator of multiple steps of EOC disease progression, underscoring its potential as a therapeutic target in advanced ovarian cancer.

SLC7A5-ERBB2 axis drives olaparib resistance via de novo lipid synthesis in ovarian cancer

In the treatment of ovarian cancer, PARP inhibitors such as olaparib have shown promise, yet resistance in advanced cases remains a significant challenge. Our study identified SLC7A5 as a key gene associated with olaparib resistance through differential gene expression and prognostic analysis. High SLC7A5 expression was found to correlate with poor prognosis. In vivo and in vitro experiments revealed that SLC7A5 enhances olaparib resistance by stabilizing ACLY and promoting de novo lipid synthesis via the ERBB2 axis, independent of leucine. Mechanistically, SLC7A5 upregulates ERBB2 transcription through ELK1, and ERBB2 competes with CUL3 to prevent ACLY degradation. These findings suggest that targeting SLC7A5 may reverse olaparib resistance, offering new strategies for combination therapies and improving clinical outcomes in ovarian cancer treatment.

Phosphorylation of FOXK2 at Thr13 and Ser30 by PDK2 sustains glycolysis through a positive feedback manner in ovarian cancer

AbstractOvarian cancer is one of the most common gynecological malignant tumors with insidious onset, strong invasiveness, and poor prognosis. Metabolic alteration, particularly aerobic glycolysis, which is tightly regulated by transcription factors, is associated with the malignant behavior of OC. We screened FOXK2 in this study as a key transcription factor that regulates glycolysis in OC. FOXK2 is overly expressed in OC, and poor prognosis is predicted by overexpression. FOXK2 promotes OC cell proliferation both in vitro and in vivo and cell migration in vitro. Further studies showed that PDK2 directly binds to the forkhead-associated (FHA) domain of FOXK2 to phosphorylate FOXK2 at Thr13 and Ser30, thereby enhancing the transcriptional activity of FOXK2. FOXK2 transcriptionally regulates the expression of PDK2, thus forming positive feedback to sustain glycolysis in OC cells.

m6A-modified circSTX6 as a key regulator of cervical cancer malignancy via SPI1 and IL6/JAK2/STAT3 pathways

In recent years, circRNAs have garnered increasing attention for their role in cervical cancer. However, the functions of many newly identified circRNAs remain unclear and require further exploration. In this study, we investigated the expression and oncogenic potential of the novel circRNA circSTX6 in cervical cancer. Our findings revealed that circSTX6 is highly expressed in cervical cancer (CC) and is significantly associated with poor patient prognosis, promoting cell survival, proliferation, invasion, and migration. Mechanistically, circSTX6 enhances the stability of the transcription factor SPI1 by binding to it, thereby upregulating IL6 transcription and activating the JAK2/STAT3 signaling pathway. Additionally, METTL3-mediated N6-methyladenosine (m6A) modification stabilizes circSTX6 through recognition by YTHDC1, forming a positive feedback regulatory loop among METTL3, circSTX6, and SPI1. These findings not only deepen our understanding of the biological mechanisms underlying CC but also highlight circSTX6 as a potential target for molecular therapies.

S1PR1 regulates ovarian cancer cell senescence through the PDK1-LATS1/2-YAP pathway

Abstract Cell senescence deters the activation of various oncogenes. Induction of senescence is, therefore, a potentially effective strategy to interfere with vital processes in tumor cells. Sphingosine-1-phosphate receptor 1 (S1PR1) has been implicated in various cancer types, including ovarian cancer. The mechanism by which S1PR1 regulates ovarian cancer cell senescence is currently elusive. In this study, we demonstrate that S1PR1 was highly expressed in human ovarian cancer tissues and cell lines. S1PR1 deletion inhibited the proliferation and migration of ovarian cancer cells. S1PR1 deletion promoted ovarian cancer cell senescence and sensitized ovarian cancer cells to cisplatin chemotherapy. Exposure of ovarian cancer cells to sphingosine-1-phosphate (S1P) increased the expression of 3-phosphatidylinositol-dependent protein kinase 1 (PDK1), decreased the expression of large tumor suppressor 1/2 (LATS1/2), and induced phosphorylation of Yes-associated protein (p-YAP). Opposite results were obtained in S1PR1 knockout cells following pharmacological inhibition. After silencing LATS1/2 in S1PR1-deficient ovarian cancer cells, senescence was suppressed and S1PR1 expression was increased concomitantly with YAP expression. Transcriptional regulation of S1PR1 by YAP was confirmed by chromatin immunoprecipitation. Accordingly, the S1PR1-PDK1-LATS1/2-YAP pathway regulates ovarian cancer cell senescence and does so through a YAP-mediated feedback loop. S1PR1 constitutes a druggable target for the induction of senescence in ovarian cancer cells. Pharmacological intervention in the S1PR1-PDK1-LATS1/2-YAP signaling axis may augment the efficacy of standard chemotherapy.

Dual-target CAR-T therapy for ovarian cancer: synergistic targeting of MSLN and B7H3 enhances anti-tumor efficacy and overcomes antigen heterogeneity

Ovarian cancer (OC) remains a lethal malignancy with limited treatment options owing to antigen heterogeneity and an immunosuppressive tumor microenvironment (TME). Here, we designed a unique chimeric Antigen Receptor T-Cell (CAR-T) construct (B4M3) that integrates an anti-MSLN scFv linked to the CD3ζ activation domain and an anti-B7H3 scFv linked to the 4-1BB co-stimulatory domain. In vitro, B4M3 CAR-T cells exhibited robust cytotoxicity against OC cell lines with enhanced degranulation (CD107a) and efficient tumor cell killing, even at low effector-to-target ratios. In vivo, B4M3 CAR-T cells significantly inhibited tumor growth and prolonged survival and demonstrated superior tumor infiltration and persistence in OC xenograft models. Imaging mass cytometry (IMC) revealed that B4M3 treatment reshaped the TME, increased cytotoxic T lymphocyte (CTL) infiltration, and reduced regulatory T cells (Tregs). Mechanistically, B4M3 therapy upregulated TGF-β, promoting Th17 differentiation and CTL recruitment, thereby enhancing anti-tumor immunity. Our findings demonstrate that B4M3 CAR-T cells effectively address antigen heterogeneity and enhance therapeutic efficacy in OC, thereby offering a promising strategy for solid tumor immunotherapy.

ARID1A: gene, protein, and function in endometrial cancer

ARID1A, a key structural subunit of the SWI/SNF chromatin remodeling complex, is the most frequently mutated SWI/SNF subunit in cancer with most mutations occurring in endometrial cancer. In a multitude of malignancies, loss of ARID1A protein correlates with poor patient prognosis, increased metastasis, and changes to key cancer pathways such as genomic instability. Despite this, little work has been done to deduce the molecular role of ARID1A in endometrial cancer progression and prognosis, and much of the present work is conflicting data. There is a growing body of work that shows a discordance between ARID1A mutation status and expression of ARID1A protein in endometrioid-type endometrial tumors. Several other malignancies have found that alternative mechanisms of ARID1A protein regulation can confer ARID1A protein loss. Therefore, relying solely on ARID1A sequencing may overlook a cohort of endometrial cancer patients with absence of ARID1A protein. With endometrial cancer being one of the sole malignancies increasing in both incidence and patient mortality since the mid-2000s, it is of upmost importance to assess the impacts and potential prognostic use of commonly mutated proteins such as ARID1A. This review will highlight the critical role of ARID1A in endometrial cancer pathogenesis, its potential therapeutic vulnerabilities, and emphasizes the need to move beyond ARID1A mutation as a sole diagnostic marker to elucidate its molecular and clinical implications in endometrial cancer.

LINC00654 promotes ovarian cancer progression by facilitating nuclear export of HuR and stabilizing oncogenic mRNAs

Ovarian cancer (OC) remains a significant challenge in oncology due to its late diagnosis and poor prognosis. Emerging evidence suggests that long non-coding RNAs (lncRNAs) play critical roles in cancer biology. Herein, we reported that LINC00654 was highly expressed in OC tissues and correlated with poor patient prognosis. In addition, LINC00654 silencing restrained OC cell proliferation and migration in vitro and in vivo. Mechanically, LINC00654 was identified to directly interact with Human antigen R (HuR), a known RNA-binding protein, through RNA pull-down, RNA immunoprecipitation (RIP), and cross‑linking immunoprecipitation (CLIP). Further analysis revealed that LINC00654 could induce the translocation of HuR from the nucleus to the cytosol, where it regulated the stability of its target oncogenes, such as VASH2. The stabilization of VASH2 subsequently activated the TGF-β pathway, which is known to play a critical role in cancer progression. Taken together, these findings establish a specific mechanism by which LINC00654 interacts with HuR, facilitates its nuclear export, and stabilizes VASH2, thereby activating the TGF-β pathway and promoting OC progression. This insight into LINC00654's role in OC provides potential therapeutic targets for intervention.

m6A-driven NAT10 translation facilitates fatty acid metabolic rewiring to suppress ferroptosis and promote ovarian tumorigenesis through enhancing ACOT7 mRNA acetylation

RNA epigenetic modifications have been implicated in cancer progression. However, the interplay between distinct RNA modifications and its role in cancer metabolism remain largely unexplored. Our study demonstrates that N-acetyltransferase 10 (NAT10) is notably upregulated in ovarian cancer (OC), correlating with poor patient prognosis. IGF2BP1 enhances the translation of NAT10 mRNA in an m

Aberrant granulosa cell-fate related to inactivated p53/Rb signaling contributes to granulosa cell tumors and to FOXL2 downregulation in the mouse ovary

Ovarian granulosa cell tumors (GCTs) are indolent tumors of the ovary affecting women at all ages and potentially displaying late recurrence. Even if there is still little information regarding the mechanisms involved in GCT development and progression, FOXL2 would be a major tumor suppressor gene in granulosa cells. We analyzed the mechanisms underlying GCT initiation and progression by using mice with targeted expression of SV40 large T-antigen in granulosa cells (AT mouse), which develop GCTs. Consistent with patients, AT mice with developing GCTs displayed increased levels in circulating anti-Müllerian hormone (AMH), estradiol and androgens, as well as decreased FOXL2 protein abundance. Very few mice developed metastases (1 out of 30). In situ analyses revealed that GCT initiation resulted from both increased granulosa cell survival and proliferation in large antral follicles. Tumorigenesis was associated with the combined inactivation of p53 and Rb pathways, as shown by the impaired expression of respective downstream targets regulating cell apoptosis and proliferation, i.e., Bax, Bak, Gadd45a, Ccna2, Ccne1, E2f1, and Orc1. Importantly, the expression of FOXL2 was still present in newly developed GCTs and its downregulation only started during GCT growth. Collectively, our experiments provide evidence that disrupted p53/Rb signaling can drive tumor initiation and growth. This model challenges the current paradigm that impaired FOXL2 signaling is a major switch of granulosa cell tumorigenesis, albeit possibly contributing to tumor growth.

Context-dependent activation of SIRT3 is necessary for anchorage-independent survival and metastasis of ovarian cancer cells

Tumor cells must alter their antioxidant capacity for maximal metastatic potential. Yet the antioxidant adaptations required for ovarian cancer transcoelomic metastasis, which is the passive dissemination of cells in the peritoneal cavity, remain largely unexplored. Somewhat contradicting the need for oxidant scavenging are previous observations that expression of SIRT3, a nutrient stress sensor and regulator of mitochondrial antioxidant defenses, is often suppressed in many primary tumors. We have discovered that this mitochondrial deacetylase is specifically upregulated in a context-dependent manner in cancer cells. SIRT3 activity and expression transiently increased following ovarian cancer cell detachment and in tumor cells derived from malignant ascites of high-grade serous adenocarcinoma patients. Mechanistically, SIRT3 prevents mitochondrial superoxide surges in detached cells by regulating the manganese superoxide dismutase (SOD2). This mitochondrial stress response is under dual regulation by SIRT3. SIRT3 rapidly increases SOD2 activity as an early adaptation to cellular detachment, which is followed by SIRT3-dependent increases in SOD2 mRNA during sustained anchorage-independence. In addition, SIRT3 inhibits glycolytic capacity in anchorage-independent cells thereby contributing to metabolic changes in response to detachment. While manipulation of SIRT3 expression has few deleterious effects on cancer cells in attached conditions, SIRT3 upregulation and SIRT3-mediated oxidant scavenging are required for anoikis resistance in vitro following matrix detachment, and both SIRT3 and SOD2 are necessary for colonization of the peritoneal cavity in vivo. Our results highlight the novel context-specific, pro-metastatic role of SIRT3 in ovarian cancer.

UCHL3 promotes ovarian cancer progression by stabilizing TRAF2 to activate the NF-κB pathway

The inflammatory response plays an important role in carcinogenesis. However, the functional role and mechanism of the UCHL3-associated inflammatory response in ovarian cancer remain to be characterized. Here, we report that increased expression of UCHL3 facilitates tumourigenesis by targeting TRAF2 protein, thereby enhancing the inflammatory response. The expression of UCHL3 is elevated in ovarian cancer patients and is associated with an unfavourable prognosis. Genetic ablation of UCHL3 was found to markedly block ovarian cancer cell proliferation, viability and migration both in vitro and in vivo. Mechanistically, luciferase pathway screening results show that NF-κB signalling is clearly activated compared with other pathways. UCHL3 was found to activate NF-κB signalling by deubiquitinating and stabilizing TRAF2, leading to tumourigenesis. Our results indicate that highly expressed UCHL3 enhances inflammation by stabilizing TRAF2, which in turn facilitates tumourigenesis in ovarian cancer, and that UCHL3 is a potential target for ovarian cancer patients with increased inflammation.

HNRNPH1-stabilized LINC00662 promotes ovarian cancer progression by activating the GRP78/p38 pathway

AbstractNumerous studies suggest an important role for copy number alterations (CNAs) in cancer progression. However, CNAs of long intergenic noncoding RNAs (lincRNAs) in ovarian cancer (OC) and their potential functions have not been fully investigated. Here, based on analysis of The Cancer Genome Atlas (TCGA) database, we identified in this study an oncogenic lincRNA termed LINC00662 that exhibited a significant correlation between its CNA and its increased expression. LINC00662 overexpression is highly associated with malignant features in OC patients and is a prognostic indicator. LINC00662 significantly promotes OC cell proliferation and metastasis in vitro and in vivo. Mechanistically, LINC00662 is stabilized by heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1). Moreover, LINC00662 exerts oncogenic effects by interacting with glucose-regulated protein 78 (GRP78) and preventing its ubiquitination in OC cells, leading to activation of the oncogenic p38 MAPK signaling pathway. Taken together, our results define an oncogenic role for LINC00662 in OC progression mediated via GRP78/p38 signaling, with potential implications regarding therapeutic targets for OC.

COL11A1 activates cancer-associated fibroblasts by modulating TGF-β3 through the NF-κB/IGFBP2 axis in ovarian cancer cells

Ovarian cancer has a unique tumor microenvironment (TME) that enables cancer-associated fibroblasts (CAFs) to interact with cellular and matrix constituents and influence tumor development and migration into the peritoneal cavity. Collagen type XI alpha 1 (COL11A1) is overexpressed in CAFs; therefore this study examines its role during CAF activation in epithelial ovarian cancer (EOC). Coculturing human ovarian fibroblasts (HOFs) with high COL11A1-expressing EOC cells or exposure to the conditioned medium of these cells prompted the expression of COL11A1 and CAF phenotypes. Conversely, coculturing HOFs with low COL11A1-expressing EOC cells or COL11A1-knockdown abrogated COL11A1 overexpression and secretion, in addition to CAF activation. Increased p-SP1 expression attributed to COL11A1-mediated extracellular signal-regulated kinase activation (ERK) induced p65 translocation into the nucleus and augmented its binding to the insulin-like growth factor binding protein 2 (IGFBP2) promoter, ultimately inducing TGF-β3 activation. The CAF-cancer cell crosstalk triggered interleukin-6 release, which in turn promoted EOC cell proliferation and invasiveness. These in vitro results were confirmed by in vivo findings in a mouse model, showing that COL11A1 overexpression in EOC cells promoted tumor formation and CAF activation, which was inhibited by TGF-β3 antibody. Human tumors with high TGF-β3 levels showed elevated expression of COL11A1 and IGFBP2, which was associated with poor survival. Our findings suggest the possibility that anti-TGF-β3 treatment strategy may be effective in targeting CAFs in COL11A1-positive ovarian tumors.

Exploiting synthetic lethality to target BRCA1/2-deficient tumors: where we stand

The principle of synthetic lethality, which refers to the loss of viability resulting from the disruption of two genes, which, individually, do not cause lethality, has become an attractive target approach due to the development and clinical success of Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). In this review, we present the most recent findings on the use of PARPi in the clinic, which are currently approved for second-line therapy for advanced ovarian and breast cancer associated with mutations of BRCA1 or BRCA2 (BRCA1/2) genes. PARPi efficacy, however, appears to be limited by acquired and inherent resistance, highlighting the need for alternative and synergistic targets to eliminate these tumors. Here, we explore other identified synthetic lethal interactors of BRCA1/2, including DNA polymerase theta (POLQ), Fanconi anemia complementation group D2 (FANDC2), radiation sensitive 52 (RAD52), Flap structure-specific endonuclease 1 (FEN1), and apurinic/apyrimidinic endodeoxyribonuclease 2 (APE2), as well as other protein and nonprotein targets, for BRCA1/2-mutated cancers and their implications for future therapies. A wealth of information now exists for phenotypic and functional characterization of these novel synthetic lethal interactors of BRCA1/2, and leveraging these findings can pave the way for the development of new targeted therapies for patients suffering from these cancers.

Tumour-derived exosomal piR-25783 promotes omental metastasis of ovarian carcinoma by inducing the fibroblast to myofibroblast transition

Ovarian carcinoma inherently possesses a distinct metastatic organotropism for the adipose-rich omentum, contributing to disease progression. Although the premetastatic microenvironment (PMM) has been known to often play a prometastatic role during the process, incomplete mechanistic insight into PMM formation has prevented its therapeutic targeting. Omental fibroblasts can be activated by tumour cells to differentiate into myofibroblasts, termed the fibroblast-to-myofibroblast transition (FMT), which, in turn, enhances cancer aggressiveness. Here, we report crosstalk between cancer cells and omental fibroblasts through exosomal piR-25783, which fuels tumour metastasis. Tumour cell-secreted exosomal piR-25783 activates the TGF-β/SMAD2/SMAD3 pathway in fibroblasts and promotes the FMT in the omentum along with the secretion of various cytokines and elevation of proliferative, migratory, and invasive properties, contributing to the formation of PMMs. Furthermore, piR-25783-induced myofibroblasts promote tumour implantation and growth in the omentum. In addition, the overexpression of piR-25783 in ovarian carcinoma is associated with unfavourable clinicopathological characteristics and shorter survival. In this study, we provide molecular, functional, and translational evidence suggesting that exosomal piR-25783 plays an important role in the formation of PMMs and the development of metastatic diseases in vitro and in vivo and may serve as a potential therapeutic target for ovarian carcinoma with metastasis.

Prognostic immunologic signatures in epithelial ovarian cancer

Epithelial Ovarian Cancer (EOC) is a deadly gynecologic malignancy in which patients frequently develop recurrent disease following initial platinum-taxane chemotherapy. Analogous to many other cancer subtypes, EOC clinical trials have centered upon immunotherapeutic approaches, most notably programmed cell death 1 (PD-1) inhibitors. While response rates to these immunotherapies in EOC patients have been low, evidence suggests that ovarian tumors are immunogenic and that immune-related genomic profiles can serve as prognostic markers. This review will discuss recent advances in the development of immune-based prognostic signatures in EOC that predict patient clinical outcomes, as well as emphasize specific research areas that need to be addressed to drive this field forward.

Distinct roles of treatment schemes and BRCA2 on the restoration of homologous recombination DNA repair and PARP inhibitor resistance in ovarian cancer

Poly (ADP-ribose) polymerase inhibitors (PARPis) represent a major advance in ovarian cancer, now as a treatment and as a maintenance therapy in the upfront and recurrent settings. However, patients often develop resistance to PARPis, underlining the importance of dissecting resistance mechanisms. Here, we report different dosing/timing schemes of PARPi treatment in BRCA2-mutant PEO1 cells, resulting in the simultaneous development of distinct resistance mechanisms. PARPi-resistant variants PEO1/OlaJR, established by higher initial doses and short-term PARPi treatment, develops PARPi resistance by rapidly restoring functional BRCA2 and promoting drug efflux activity. In contrast, PEO1/OlaR, developed by lower initial doses with long-term PARPi exposure, shows no regained BRCA2 function but a mesenchymal-like phenotype with greater invasion ability, and exhibits activated ATR/CHK1 and suppressed EZH2/MUS81 signaling cascades to regain HR repair and fork stabilization, respectively. Our study suggests that PARPi resistance mechanisms can be governed by treatment strategies and have a molecular basis on BRCA2 functionality. Further, we define different mechanisms that may serve as useful biomarkers to assess subsequent treatment strategies in PARPi-resistant ovarian cancer.

JAK2/ULK1 axis promotes cervical cancer progression by autophagy induction and SRPK1 phosphorylation

Cervical cancer is the most common gynecologic cancer. Autophagy is involved in the progression of CCa. ULK1 is a crucial kinase in autophagy initiation. However, few studies have investigated the role of ULK1 phosphorylation at tyrosine residues in the progression of CCa, and the underlying mechanism remains elusive. In this study, we demonstrated that JAK2 is a novel upstream kinase that phosphorylates ULK1 at the tyrosine site. JAK2 interacts with and phosphorylates ULK1 at Tyr1007. The phosphorylation of ULK1 at Y1007 increases its activity and stability, activates autophagy, and promotes the progression of CCa. We further showed that the phosphorylation of ULK1 at Y1007 is a predictive marker of CCa patient outcome. Furthermore, we identified SRPK1 as a potential downstream substrate of ULK1 to promote the progression of CCa. Our research sheds light on the molecular mechanism of CCa progression, through JAK2/ULK1 axis, and emphasizes the phosphorylation of ULK1 at Y1007 as a predictor of CCa.

SOX17 and PAX8 constitute an actionable lineage-survival transcriptional complex in ovarian cancer

Müllerian tissue-specific oncogenes, prototyped by PAX8, underlie ovarian tumorigenesis and represent unique molecular vulnerabilities. Further delineating such lineage-dependency factors and associated therapeutic implications would provide valuable insights into ovarian cancer biology and treatment. In this study, we identified SOX17 as a new lineage-survival master transcription factor, which shared co-expression pattern with PAX8 in epithelial ovarian carcinoma. Genetic disruption of SOX17 or PAX8 analogously inhibited neoplastic cell viability and downregulated a spectrum of lineage-related transcripts. Mechanistically, we showed that SOX17 physically interacted with PAX8 in cultured cell lines and clinical tumor specimens. The two nuclear proteins bound to overlapping genomic regions and regulated a common set of downstream genes, including those involved in cell cycle and tissue morphogenesis. In addition, we revealed that small-molecule inhibitors of transcriptional cyclin-dependent kinases (CDKs) effectively reduced SOX17 and PAX8 expression. ZSQ1722, a novel orally bioavailable CDK12/13 covalent antagonist, exerted potent anti-tumor activity in xenograft models. These findings shed light on an actionable lineage-survival transcriptional complex in ovarian cancer, and facilitated drug discovery by generating a serial of candidate compounds to pharmacologically target this difficult-to-treat malignancy.

ROS-regulated phosphorylation of ITPKB by CAMK2G drives cisplatin resistance in ovarian cancer

Platinum resistance accounts for much of the high mortality and morbidity associated with ovarian cancer. Identification of targets with significant clinical translational potential remains an unmet challenge. Through a high-throughput synthetical lethal screening for clinically relevant targets using 290 kinase inhibitors, we identify calcium/calmodulin-dependent protein kinase II gamma (CAMK2G) as a critical vulnerability in cisplatin-resistant ovarian cancer cells. Pharmacologic inhibition of CAMK2G significantly sensitizes ovarian cancer cells to cisplatin treatment in vitro and in vivo. Mechanistically, CAMK2G directly senses ROS, both basal and cisplatin-induced, to control the phosphorylation of ITPKB at serine 174, which directly regulates ITPKB activity to modulate cisplatin-induced ROS stress. Thereby, CAMK2G facilitates the adaptive redox homeostasis upon cisplatin treatment and drives cisplatin resistance. Clinically, upregulation of CAMK2G activity and ITPKB pS174 correlates with cisplatin resistance in human ovarian cancers. This study reveals a key kinase network consisting of CAMK2G and ITPKB for ROS sense and scavenging in ovarian cancer cells to maintain redox homeostasis, offering a potential strategy for cisplatin resistance treatment.

Single-cell RNA-seq recognized the initiator of epithelial ovarian cancer recurrence

Epithelial ovarian cancers (EOCs) are sensitive to chemotherapy but will ultimately relapse and develop drug resistance. The origin of EOC recurrence has been elusive due to intra-tumor heterogeneity. Here we performed single-cell RNA sequencing (scRNA-seq) in 13,369 cells from primary, untreated peritoneal metastasis, and relapse tumors. We used time-resolved analysis to chart the developmental sequence of cells from the metastatic tumors, then traced the earliest replanting cells back to the primary tumors. We discovered seven distinct subpopulations in primary tumors where the CYR61

CD147 supports paclitaxel resistance via interacting with RanBP1

AbstractThough the great success of paclitaxel, the variable response of patients to the drug limits its clinical utility and the precise mechanisms underlying the variable response to paclitaxel remain largely unknown. This study aims to verify the role and the underlying mechanisms of CD147 in paclitaxel resistance. Immunostaining was used to analyze human non-small-cell lung cancer (NSCLC) and ovarian cancer tissues. RNA-sequencing was used to identify downstream effectors. Annexin V-FITC/propidium iodide and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to detect apoptosis. Co-immunoprecipitation (Co-IP), fluorescence resonance energy transfer (FRET) and surface plasmon resonance (SPR) were performed to determine protein interactions. Fluorescence recovery after photobleaching (FRAP) was performed to measure the speed of microtubule turnover. Xenograft tumor model was established to evaluate sensitivity of cancer cells to paclitaxel in vivo. In vitro and in vivo assays showed that silencing CD147 sensitized the cancer cells to paclitaxel treatment. CD147 protected cancer cells from paclitaxel-induced caspase-3 mediated apoptosis regardless of p53 status. Truncation analysis showed that the intracellular domain of CD147 (CD147ICD) was indispensable for CD147-regulated sensitivity to paclitaxel. Via screening the interacting proteins of CD147ICD, Ran binding protein 1 (RanBP1) was identified to interact with CD147ICD via its C-terminal tail. Furthermore, we showed that RanBP1 mediated CD147-regulated microtubule stability and dynamics as well as response to paclitaxel treatment. These results demonstrated that CD147 regulated paclitaxel response by interacting with the C-terminal tail of RanBP1 and targeting CD147 may be a promising strategy for preventing paclitaxel resistant.

PARP1-stabilised FOXQ1 promotes ovarian cancer progression by activating the LAMB3/WNT/β-catenin signalling pathway

Metastasis is an important factor that causes ovarian cancer (OC) to become the most lethal malignancy of the female reproductive system, but its molecular mechanism is not fully understood. In this study, through bioinformatics analysis, as well as analysis of tissue samples and clinicopathological characteristics and prognosis of patients in our centre, it was found that Forkhead box Q1 (FOXQ1) was correlated with metastasis and prognosis of OC. Through cell function experiments and animal experiments, the results show that FOXQ1 can promote the progression of ovarian cancer in vivo and in vitro. Through RNA-seq, chromatin immunoprecipitation sequencing (ChIP-seq), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), Western blotting (WB), quantitative real-time polymerase chain reaction (qRT‒PCR), immunohistochemistry (IHC), luciferase assay, and ChIP-PCR, it was demonstrated that FOXQ1 can mediate the WNT/β-catenin pathway by targeting the LAMB promoter region. Through coimmunoprecipitation (Co-IP), mass spectrometry (MS), ubiquitination experiments, and immunofluorescence (IF), the results showed that PARP1 could stabilise FOXQ1 expression via the E3 ubiquitin ligase Hsc70-interacting protein (CHIP). Finally, the whole mechanism pathway was verified by animal drug combination experiments and clinical specimen prognosis analysis. In summary, our results suggest that PARP1 can promote ovarian cancer progression through the LAMB3/WNT/β-catenin pathway by stabilising FOXQ1 expression.

BRD4 inhibition sensitizes cervical cancer to radiotherapy by attenuating DNA repair

Cisplatin-based chemoradiotherapy is the recommended treatment for local advanced cervical cancer, but radioresistance remains one of the most important and unresolved clinical problems. Investigations have revealed aberrant epigenetic modifications as one of the chief culprits for the development of radioresistance. Here, we attempt to identify a radiosensitizer from an epigenetic drug synergy screen and explore the underlying mechanism. We integrated epigenetic inhibitors and radiotherapy in cervical cancer cell lines to identify potential radiosensitizers. We further verified the sensitization effect of the drug and the function of its target gene both in vitro and in vivo. Finally, we validated the clinical significance of its target gene in clinical cervical cancer specimens. We identified JQ1, a BRD4 inhibitor, as a potent radiosensitizer. Functional assays demonstrated that repressing BRD4 activity led to significant radiosensitization and potentiation of DNA damage in cervical cancer cell lines. By using RNA-seq to determine JQ1-mediated changes in transcription, we identified RAD51AP1 as a major BRD4 target gene involved in radiosensitivity. A dual-luciferase reporter assay and ChIP-qPCR showed that BRD4 binds to the promoter region of RAD51AP1 and promotes its transcription, whereas this activity was attenuated by BRD4 inhibition. The in vivo experiments also suggested a synergy between BRD4 inhibition and radiotherapy. High BRD4 expression was found to be related to a worse prognosis and radiation resistance. BRD4 inhibition sensitizes cervical cancer to radiotherapy by inhibiting RAD51AP1 transcription. The combination of JQ1 with radiotherapy merits further evaluation as a therapeutic strategy for improving local control in cervical cancer.

Downregulation of NUDT21 contributes to cervical cancer progression through alternative polyadenylation

Nudix Hydrolase 21 (NUDT21), an alternative polyadenylation (APA)-regulatory protein, exhibits tumor-suppressive effects. However, its role in cervical cancer (CxCa) remains unknown. In the present study, we found that NUDT21 expression was reduced in CxCa tissues and cells, and NUDT21 levels were highly associated with the clinical prognosis of patients with CxCa. Knockdown of NUDT21 promoted CxCa cell proliferation, migration, and invasion in vitro, as well as tumorigenesis and lung metastasis in vivo. Overexpression of NUDT21 produces the opposite effects. Moreover, we performed polyadenylation site sequencing (PAS-Seq) and identified 457 transcripts with lengthened 3' untranslated regions (3' UTRs) upon NUDT21 overexpression. In particular, NUDT21 modulated the expression of several genes involved in fatty acid metabolism and the Wnt and NF-κB signaling pathways in CxCa development. Taken together, our study demonstrated that the APA regulatory effect of NUDT21 is an important mechanism for CxCa suppression.

METTL3 potentiates progression of cervical cancer by suppressing ER stress via regulating m6A modification of TXNDC5 mRNA

N6-methyladenosine (m6A) is the most abundant chemical modification on mRNA and plays significant roles in many bioprocesses. However, the functions of m6A on cervical cancer (CC) tumorigenesis remain unclear. Here we found methyltransferase-like 3 (METTL3), a core member of the m6A methyltransferase family, was greatly upregulated as an independent prognostic factor in CC. Mechanistically, the transcription factor ETS1 recruited P300 and WDR5 which separately mediated H3K27ac and H3K4me3 histone modification in the promoter of METTL3 and induced METTL3 transcription activation. Furthermore, we identified TXNDC5 as a target of METTL3-mediated m6A modification through MeRIP-seq, and revealed that METTL3-mediated TXNDC5 expression relied on the m6A reader-dependent manner. Functionally, we verified that METTL3 promoted proliferation and metastasis of CC cells by regulating of TXNDC5 expression through in vitro and in vivo experiments. In addition, our study verified the effect of METTL3/TXNDC5 axis on ER stress. Taken together, METTL3 facilitates the malignant progression of CC, suggesting that METTL3 might be a potential prognostic biomarker and therapeutic target for CC.

Downregulation of LNMAS orchestrates partial EMT and immune escape from macrophage phagocytosis to promote lymph node metastasis of cervical cancer

AbstractEpithelial-mesenchymal transition (EMT) is an essential step to drive the metastatic cascade to lymph nodes (LNs) in cervical cancer cells. However, few of them metastasize successfully partially due to increased susceptibility to immunosurveillance conferred by EMT. The precise mechanisms of cancer cells orchestrate EMT and immune evasion remain largely unexplored. In this study, we identified a lncRNA termed lymph node metastasis associated suppressor (LNMAS), which was downregulated in LN-positive cervical cancer patients and correlated with LN metastasis and prognosis. Functionally, LNMAS suppressed cervical cancer cells metastasis in vitro and in vivo. Mechanistically, LNMAS exerts its metastasis suppressive activity by competitively interacting with HMGB1 and abrogating the chromatin accessibility of TWIST1 and STC1, inhibiting TWIST1-mediated partial EMT and STC1-dependent immune escape from macrophage phagocytosis. We further demonstrated that the CpG sites in the promoter region of LNMAS was hypermethylated and contributed to the downregulation of LNMAS. Taken together, our results reveal the essential role of LNMAS in the LN metastasis of cervical cancer and provide mechanistic insights into the regulation of LNMAS in EMT and immune evasion.

High serum LDL promotes EMT and stemness through LDLR/FOXQ1/NF-κB1 pathway in epithelial ovarian cancer

Epithelial ovarian cancer (EOC), the deadliest gynecological malignancy, is increasingly linked to dysregulated lipid metabolism. Nevertheless, the involvement of circulating low-density lipoprotein (LDL) in ovarian cancer progression remains controversial. Analyses of single-cell RNA sequencing and clinical data demonstrated a positive correlation between elevated LDL levels and EOC progression. Mechanistically, LDL internalized via LDL receptor (LDLR) enhanced epithelial-mesenchymal transition (EMT) and stemness in ovarian cancer cells, driven by the upregulation of the key transcription factor FOXQ1. Intriguingly, our investigations unveiled a novel transcriptional complex comprising FOXQ1/β-Catenin/ADNP. Both β-Catenin and ADNP interacted with FOXQ1 at the Forkhead domain, where FOXQ1 bound to the NF-κB1 gene promoter to enhance transcriptional activation. Notably, β-Catenin and ADNP were identified for the first time as competitive repressors within this regulatory axis. These findings were further corroborated in vivo using an ovarian cancer xenograft metastasis model, as well as in human pathological specimens, highlighting LDL-driven metastasis via FOXQ1 upregulation. Collectively, LDL promotes ovarian cancer metastasis through LDLR/FOXQ1/NF-κB1 axis. Furthermore, we discover a novel transcriptional complex, where FOXQ1 acts as the central regulator while β-Catenin/ADNP serve as co-repressors. These insights suggest that modulating serum LDL levels or targeting FOXQ1 may offer promising strategies to curb ovarian cancer progression. LDL promotes ovarian cancer metastasis through LDLR/FOXQ1/NF-κB1 axis. High serum LDL uptake mediated by LDLR enhances EMT and stemness of ovarian cancer cells via upregulating FOXQ1 expression. Both β-Catenin and ADNP interact with FOXQ1 in the Forkhead domain (FH), also where FOXQ1 binds to NF-κB1 gene promoter to active its transcription, suggesting that β-Catenin and ADNP may act as competitive repressors in this novel transcriptional regulatory complex. Thus, controlling serum LDL levels and targeting FOXQ1 may be effective interventions for preventing metastasis in women with ovarian cancer.

LEF1 confers resistance to DNA-damaging chemotherapies through upregulation of PARP1 and NUMA1 in ovarian cancer

Resistance to platinum-based drugs and PARP inhibitors (PARPi) is the leading cause of treatment failure in epithelial ovarian cancer (EOC). This study aimed to identify resistance mechanisms shared by both. Using bioinformatic analyses, EOC tissues, primary tumor cells and organoids, and chemoresistant cell lines, we identified lymphoid enhancer-binding factor 1 (LEF1) as a candidate, whose expression was increased in both platinum-resistant and PARPi-resistant tumors. Moreover, LEF1 deficiency increased EOC cell sensitivity to cisplatin and PARPi in vitro and in vivo. Mechanistically, LEF1 knockdown promoted double-strand breaks and significantly impaired both homologous recombination and nonhomologous end joining by directly downregulating the transcription of PARP1 and NUMA1. In addition, the LEF1 inhibitor niclosamide increased ovarian cancer sensitivity to Cisplatin and PARPi in patient-derived organoids and Niraparib-resistant cell lines. These findings indicate that LEF1 is a potential therapeutic target for overcoming resistance to chemotherapy based on platinum and PARPi in EOC.

Activated MAFB in ovarian cancer promotes cytoskeletal remodeling and immune microenvironment suppression by interfering with m6A modifications through WTAP competition

The tumor microenvironment (TME) coordinates cancer progression through complex transcriptional networks, but the molecular mechanisms controlling immune evasion in ovarian cancer remain elusive. Here, by integrating immune dysfunction characteristics across multiple clinical cohorts and single-cell transcriptomics, we identified MAFB as a major regulator of ovarian cancer progression. MAFB expression exhibited stage-dependent elevation and was associated with immune checkpoint characteristics. Mechanistically, MAFB competitively binds to the core component WTAP of the m6A methyltransferase complex, thereby antagonizing the degradation of target gene mRNAs (WNT5A, CD55). This atypical regulatory axis leads to persistent expression of the target genes, further coordinating tumor cell invasiveness and immune landscape remodeling through cytoskeletal protein reorganization, M2 macrophage polarization, and regulatory T cell infiltration. Correlative analyses in patient cohorts and therapeutic effects in preclinical models support the clinical relevance of this pathway. Our findings uncover a novel mechanism by which MAFB promotes ovarian cancer progression through cytoskeletal remodeling and immune suppression, connecting transcriptional regulation with epitranscriptomic modifications, and identify the MAFB-WTAP-CD55 axis as a potential therapeutic target in ovarian cancer.

SNRPB-mediated regulation of DDX39A splicing promotes ovarian cancer progression by regulating α6 integrin subunit expression

Dysfunction or aberrant expression of DEAD-box RNA helicases might play a role in the initiation and progression of human cancers. Nevertheless, the key regulator and underlying molecular mechanism have yet to be fully elucidated in ovarian cancer. This study identified DDX39A as one of the prominently upregulated genes in ovarian cancer through a systematic analysis of RNA helicase expression profiles using the CPTAC and TCGA ovarian cancer datasets. High expression of DDX39A was confirmed in paraffin-embedded ovarian cancer samples. Specifically, elevated DDX39A expression was found to be associated with poor overall survival in ovarian cancer patients. Antisense oligonucleotide-mediated DDX39A silencing led to a decrease in the proliferation capacity of a CDX model and a PDX model. Furthermore, DDX39A expression is regulated by the splicing factor SNRPB. SNRPB depletion or DDX39A knockdown induced the retention of DDX39A introns 6 and 8 to generate the noncoding transcript DDX39A-209, which yielded premature termination codons and resulted in nonsense-mediated RNA decay and decreased expression of the DDX39A protein. DDX39A silencing reduced the proliferative and metastatic capacities of SNRPB-overexpressing cells, indicating that DDX39A mediates the oncogenic function of SNRPB in ovarian cancer cells. In addition, RNA-Seq data analysis revealed that DDX39A promotes the proliferation and metastasis of ovarian cancer cells through the regulation of exon skipping of ITGA6 to produce the oncogenic ITGA6A transcript. These findings suggest that the SNRPB/DDX39A/ITGA6 axis plays critically important role in the progression of ovarian cancer, which increases our understanding of the role of DEAD-box RNA helicases and provides a viable therapeutic target for ovarian cancer.

SPTLC2 drives an EGFR-FAK-HBEGF signaling axis to promote ovarian cancer progression

The epidermal growth factor receptor (EGFR) signaling pathway is frequently associated with ovarian cancer (OC) progression. However, inhibition of EGFR signaling in OC patients achieved limited therapeutic effects, highlighting the need to define the mechanism of EGFR deregulation in OC development. Herein we showed that serine palmitoyltransferase long chain base subunit 2 (SPTLC2) acts as a positive regulator in the EGFR signaling pathway in OC. Phenotypically, depletion of SPTLC2 suppressed clonogenic growth and migration of OC cells in vitro and in ovo, as well as metastasis in OC xenograft models, whereas overexpression of SPTLC2 yielded opposite effects. Mechanistically, SPTLC2 drives an EGFR-FAK-HBEGF signaling axis via binding with EGFR. Notably, the serine palmitoyltransferase activity of SPTLC2 is critical for regulation of the EGFR-FAK-HBEGF signaling axis and activity in OC progression. Clinically, high SPTLC2 expression is associated with high-grade serous ovarian cancer and metastasis. Collectively, our findings establish an oncogenic role of SPTLC2 in OC growth and progression though upregulation of EGFR signaling and suggest that SPTLC2 represents a potential therapeutic target in EGFR-driven ovarian cancer patients.

CypA/TAF15/STAT5A/miR-514a-3p feedback loop drives ovarian cancer metastasis

Cyclophilin A (CypA) is a peptidyl-prolyl isomerase that participates in multiple cancer events, but the molecular mechanisms of abnormal expression and regulation of CypA in ovarian cancer (OC) have never been considered. This study identifies CypA as a key driver of epithelial-mesenchymal transition (EMT) in ovarian cancer and explores the mechanisms that underly this process. We show that CypA is upregulated in tissues and serum of ovarian cancer patients and that CypA overexpression correlates with poor prognosis. CypA facilitates tumor growth and metastasis in vivo in subcutaneous tumor xenograft and abdominal metastatic models, and in vitro studies suggest a mechanism, showing that CypA accelerates ovarian cancer cell epithelial-mesenchymal transition by activating a PI3K/AKT signaling pathway. Mechanistic studies showed that STAT5A binds pri-miR-514a-3p and inhibits its activity, whereas miR-514a-3p directly binds to the 3'-UTR of CypA to suppress its expression, resulting in STAT5A promoting the expression of CypA, forming the STAT5A/miR-514a-3p/CypA axis. Furthermore, immunoprecipitates and mass spectrometry analysis identifies a CypA interaction with TAF15 that stabilizes TAF15 by suppressing its proteasome degradation and promotes its entry into the nucleus. While STAT5A is positively regulated by TAF15. Our findings identify a novel feedback loop for CypA that drives EMT and ovarian tumor growth and metastasis via a TAF15/STAT5A/miR-514a-3p pathway in ovarian cancer and facilitates the release of CypA into the extracellular, which provides a promising therapeutic target for OC treatment and a diagnostic biomarker.

SGK1 suppresses ferroptosis in ovarian cancer via NRF2-dependent and -independent pathways

USP33 facilitates the ovarian cancer progression via deubiquitinating and stabilizing CBX2

Post-translational modifications of proteins play a pivotal role in both the initiation and progression of ovarian cancer. Despite the recognition of USP33 as a significant factor in various cancers, its specific function and underlying mechanisms in ovarian cancer remain elusive. Proteomics and ubiquitinomics approaches were coupled to screen novel substrate proteins directly regulated by USP33. Our findings unveil that USP33 was observed to eliminate K27- and K48-linked ubiquitin chains from CBX2 at the K277 position. Notably, acetylation of CBX2 at K199, catalyzed by lysine acetyltransferase GCN5, was found to enhance its interaction with USP33, subsequently promoting further deubiquitination and stabilization. Functionally, our experiments demonstrate that USP33 significantly enhances ovarian cancer proliferation and metastasis in a CBX2-dependent manner. Furthermore, analysis revealed a direct positive correlation between the expression levels of USP33 and CBX2 proteins in human specimens, with elevated levels being associated with reduced survival rates in ovarian cancer patients. These findings elucidate the mechanism by which USP33 augments ovarian cancer progression through the stabilization of CBX2, underscoring the USP33-CBX2 axis as a promising therapeutic target in ovarian cancer management.

Transcription factor ZIC2 regulates the tumorigenic phenotypes associated with both bulk and cancer stem cells in epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy in North America. Current therapeutic regimens are ineffective against advanced EOC. A better understanding of the molecular mechanisms that regulate the biology of EOC will be a critical step toward developing more efficacious therapies against EOC. Herein, we demonstrate that elevated expression of transcription factor ZIC2 was associated with lower survival of EOC patients. Knockout of endogenous ZIC2 in EOC cells attenuated the tumorigenic phenotypes associated with both bulk and cancer stem cells in vitro and in vivo, indicating a pro-tumorigenic role of ZIC2 in EOC. On the other hand, however, overexpression of ZIC2 in EOC cells that do not express endogenous ZIC2 promoted cell migration and sphere formation, but inhibited cell growth and colony formation in vitro and tumor growth in vivo, indicating that the role for ZIC2 in EOC is context dependent. Our transcriptomic analysis showed that ZIC2-regulated genes were involved in multiple biological processes and signaling pathways associated with tumor progression. In conclusion, our findings reveal a context-dependent role for ZIC2 in regulating tumorigenic phenotypes in EOC, providing evidence that ZIC2 can be a potential therapeutic target for EOCs that express a high level of ZIC2.

The splicing factor WBP11 mediates MCM7 intron retention to promote the malignant progression of ovarian cancer

Accumulating studies suggest that splicing factors play important roles in many diseases including human cancers. Our study revealed that WBP11, a core splicing factor, is highly expressed in ovarian cancer (OC) tissues and associated with a poor prognosis. WBP11 inhibition significantly impaired the proliferation and mobility of ovarian cancer cells in vitro and in vivo. Furthermore, FOXM1 transcriptionally activated WBP11 expression by directly binding to its promoter in OC cells. Importantly, RNA-seq and alternative splicing event analysis revealed that WBP11 silencing decreased the expression of MCM7 by regulating intron 4 retention. MCM7 inhibition attenuated the increase in malignant behaviors of WBP11-overexpressing OC cells. Overall, WBP11 was identified as an oncogenic splicing factor that contributes to malignant progression by repressing intron 4 retention of MCM7 in OC cells. Thus, WBP11 is an oncogenic splicing factor with potential therapeutic and prognostic implications in OC.

Ascites exosomal lncRNA PLADE enhances platinum sensitivity by inducing R-loops in ovarian cancer

RUNX1-IT1 acts as a scaffold of STAT1 and NuRD complex to promote ROS-mediated NF-κB activation and ovarian cancer progression

Dysregulated expression of long-stranded non-coding RNAs is strongly associated with carcinogenesis. However, the precise mechanisms underlying their involvement in ovarian cancer pathogenesis remain poorly defined. Here, we found that lncRNA RUNX1-IT1 plays a crucial role in the progression of ovarian cancer. Patients with high RUNX1-IT1 expression had shorter survival and poorer outcomes. Notably, knockdown of RUNX1-IT1 suppressed the proliferation, migration and invasion of ovarian cancer cells in vitro, and reduced the formation of peritoneum metastasis in vivo. Mechanistically, RUNX1-IT1 bound to HDAC1, the core component of the NuRD complex, and STAT1, acting as a molecular scaffold of the STAT1 and NuRD complex to regulate intracellular reactive oxygen homeostasis by altering the histone modification status of downstream targets including GPX1. Consequently, RUNX1-IT1 activated NF-κB signaling and altered the biology of ovarian cancer cells. In conclusion, our findings demonstrate that RUNX1-IT1 promotes ovarian malignancy and suggest that targeting RUNX1-IT1 represents a promising therapeutic strategy for ovarian cancer treatment.

Core fucosylation of NCEH1 by FUT8 promotes progression of high-grade serous ovarian cancer by driving tumor-associated macrophage M2 polarization

High-grade serous ovarian cancer (HGSC) is the most aggressive subtype of ovarian epithelial cancer (OEC), with characters of late-stage diagnosis, high recurrence rate, and poor survival outcomes. Fucosyltransferase 8 (FUT8) is responsible for α1,6-core fucosylation biosynthesis, and aberrant FUT8/α1,6-core fucosylation level is involved in tumor progression. However, the roles and mechanisms of protein FUT8 and α1,6-core fucosylation in HGSC tumorigenesis and progression remain elusive. Here, our study confirms that elevated levels of FUT8/α1,6-core fucose in the tissues and serum of HGSC patients, and the elevation is associated with poor patient prognosis. By applying glycoproteomic assay, we globally screen and identify NCEH1 as the specific scaffold protein of α1,6-core fucosylation. Alpha 1,6-core fucose modification stabilizes NCEH1 by preventing its degradation through proteasomal pathway. Importantly, combined with non-targeted metabolomics analysis, α1,6-core fucosylated NCEH1 facilitates LPA secretion, driving M2-like polarization of tumor-associated macrophages in the tumor microenvironment, thus leading to oncogenesis and peritoneal metastasis of HGSC in vitro and in vivo. These findings broaden the understanding of FUT8/α1,6-core fucosylation/NCEH1 in HGSC progression and metastasis, and offer glycosylated diagnostic indicators and targets for therapeutic strategies in HGSC.

TFAP2C protects against ferroptosis in ovarian cancer through the KEAP1-NRF2 axis by recruiting HDAC1/2

Ferroptosis, a distinct form of programmed cell death characterized by the iron-dependent aberrant buildup of lipid peroxides, has emerged as a promising approach in cancer therapy. The KEAP1-NRF2 axis serves as a critical regulator of ferroptosis, exerting its suppressive effects by preserving cellular redox homeostasis and orchestrating the transcriptional activation of downstream antioxidant genes. NRF2 hyperactivation is frequently observed across multiple cancer types and is associated with tumor progression and therapeutic resistance. Here, we identified the transcription factor TFAP2C as a novel regulator of the KEAP1-NRF2 signaling pathway in ovarian cancer (OC). TFAP2C knockdown inactivated KEAP1-NRF2 signaling, consequently reducing cell viability while inducing the accumulation of reactive oxygen species (ROS) and ferrous iron (Fe²⁺). Additionally, a decrease in the mitochondrial membrane potential (MMP) was observed upon TFAP2C knockdown. These alterations collectively triggered ferroptosis, thereby inhibiting the progression of OC to some extent. Moreover, NRF2 knockdown partially attenuated the pro-proliferative and ferroptosis-resistant phenotypes driven by TFAP2C overexpression in OC. ChIP and dual-luciferase reporter gene assays confirmed that TFAP2C transcriptionally repressed KEAP1 expression, thereby weakening the ubiquitination degradation of NRF2 by KEAP1. The upregulation of TFAP2C expression stabilized the NRF2 protein, activated the NRF2-dependent transcriptional program, and strengthened cellular antioxidant defenses, ultimately conferring resistance to ferroptosis. Mechanistically, TFAP2C bound to the promoter region of KEAP1 and recruited histone deacetylases 1/2 (HDAC1/2), resulting in the deacetylation of H3K27 and subsequent transcriptional repression of KEAP1. In summary, our mechanistic investigations revealed TFAP2C as a novel oncogenic driver in OC and a key regulator of ferroptosis via its epigenetic modulation of the KEAP1-NRF2 axis. These findings highlight TFAP2C as a potential therapeutic target for ferroptosis-inducing therapies in OC patients with high TFAP2C expression.

Tumor cell-intrinsic PD-1 in malignant ascites drives ovarian cancer progression via MAPK/ERK signaling

Programmed cell death protein 1 (PD-1), an immune checkpoint primarily expressed on T cells, plays a critical role in mediating tumor immune evasion. However, the role of PD-1 in non-immune cells remains poorly understood. Here, we report tumor cell-intrinsic PD-1 expression in malignant ascites from ovarian cancer patients. Using murine ovarian cancer models, we demonstrate that PD-1 directly promotes ovarian cancer progression. Moreover, malignant ascites markedly upregulates PD-1 expression in ID8 ovarian cancer cells, acting as a pathological amplifier that exacerbates PD-1-mediated oncogenic signaling cascades, including enhanced proliferation and metastasis both in vitro and in vivo. Mechanistically, soluble PD-L1 (sPD-L1) in ascites interacts with tumor cell-intrinsic PD-1, activating the MAPK/ERK signaling pathway through enhanced phosphorylation of ERK1/2. In contrast, PD-1 inhibition, achieved by genetic knockout or antibody blockade, reverses these tumor-promoting effects. Furthermore, pharmacological inhibition of phosphorylated ERK1/2 counteracts the tumor progression mediated by the PD-1 and prolongs survival in murine ovarian cancer models. Our study uncovers a previously unrecognized tumor-intrinsic PD-1-ERK signaling axis in ovarian cancer, that accelerates tumorigenesis and provides new insights and perspectives for PD-1/PD-L1 immune checkpoint therapy in ovarian cancer.

SPOP/NOLC1/B4GALT1 signaling axis enhances paclitaxel resistance in endometrial cancer by inducing O-dysglycosylation

The effective treatment of paclitaxel-resistant patients remains a major challenge. We found that nucleolar and coiled body phosphoprotein 1 (NOLC1) was highly expressed in the paclitaxel-resistant endometrial cancer (ECa) cells and pathological tissue of ECa patients, which could promote the occurrence and progression of ECa cells. Mechanistically, we confirmed that the E3 ubiquitin ligase substrate-binding adaptor SPOP mediates the ubiquitination and degradation of NOLC1, thereby maintaining normal protein levels. However, ECa-associated SPOP mutants abrogated the binding and ubiquitination of NOLC1, resulting in the accumulation of NOLC1, and ultimately promoting the proliferation, migration, and invasion of ECa cells. In addition, we demonstrated that NOLC1 could act as a transcriptional factor to activate the transcriptional expression of B4GALT1, ultimately leading to abnormal glycosylation metabolism. Moreover, knockdown of B4GALT1 can partly counteract the cancer-promoting effect caused by the overexpression of NOLC1 in vitro and in vivo. Based on these findings, an O-glycosylation inhibitor combined with paclitaxel could effectively improve the sensitivity of paclitaxel-resistant cells. In summary, we found that SPOP can negatively regulate the NOLC1-B4GALT1 signaling axis in ECa, whereas ECa-associated SPOP mutants lead to abnormal activation of this signaling axis, leading to glycosylation metabolism disorders. In addition, paclitaxel combined with B4GALT1-KD or glycosylation inhibitors can significantly inhibit the growth of paclitaxel-resistant endometrial cancer cells.

PPP2R1A mutations cause ATR inhibitor sensitivity in ovarian clear cell carcinoma

Identification of ARID1A/ATR synthetic lethality led to ATR inhibitor phase II trials in ovarian clear cell carcinoma (OCCC), a cancer of unmet need. Using multiple CRISPR-Cas9 mutagenesis and interference screens, we show that inactivation of protein phosphatase 2A (PP2A) subunits, including PPP2R1A, enhance ATRi sensitivity in ARID1A mutant OCCC. Analysis of a new OCCC cohort indicates that 52% possess oncogenic PPP2R1A p.R183 mutations and of these, one half possessed both ARID1A as well as PPP2R1A mutations. Using CRISPR-prime editing to generate new isogenic models of PPP2R1A mutant OCCC, we found that PPP2R1A p.R183W and p.R183P mutations cause ATRi-induced S phase stress, premature mitotic entry, genomic instability and ATRi sensitivity in OCCC tumour cells. p.R183 mutation also enhanced both in vitro and in vivo ATRi sensitivity in preclinical models of ARID1A mutant OCCC. These results argue for the assessment of PPP2R1A mutations as a biomarker of ATRi sensitivity.

Loss-of-function mutations of SOX17 lead to YAP/TEAD activation-dependent malignant transformation in endometrial cancer

Aberrant hyperactivation of the Hippo pathway effector YAP/TEAD complex causes tissue overgrowth and tumorigenesis in various cancers, including endometrial cancer (EC). The transcription factor SOX17 (SRY [sex-determining region Y]-box 17) is frequently mutated in EC; however, SOX17 mutations are rare in other cancer types. The molecular mechanisms underlying SOX17 mutation-induced EC tumorigenesis remain poorly understood. Here, we demonstrate that SOX17 serves as a tumor suppressor to restrict the proliferation, migration, invasion, and anchorage-independent growth of EC cells, partly by suppressing the transcriptional outputs of the Hippo-YAP/TEAD pathway. SOX17 binds to TEAD transcription factors through its HMG domain and attenuates the DNA-binding ability of TEAD. SOX17 loss by inactivating mutations leads to the malignant transformation of EC cells, which can be reversed by small-molecule inhibitors of YAP/TEAD or cabozantinib, an FDA-approved drug targeting the YAP/TEAD transcriptional target AXL. Our findings reveal novel molecular mechanisms underlying Hippo-YAP/TEAD pathway-driven EC tumorigenesis, and suggest potential therapeutic strategies targeting the Hippo-YAP/TEAD pathway in SOX17-mutated EC.

Plasma exosomes from endometrial cancer patients contain LGALS3BP to promote endometrial cancer progression

Endometrial cancer (EC) is a common gynaecological cancer worldwide. Exosomes, secreted by living cells and detected in various body fluids, can exchange information between organs and compartments to affect cellular functions, such as proliferation, apoptosis, migration and angiogenesis. We hypothesise that plasma exosomal contents are altered during cancer progression and promote cancer growth and angiogenesis by delivering biomolecules to cancer and vascular endothelial cells. In this study, circulating exosomes derived from EC patients and age-matched healthy people were acquired by commercial kits. Cell counting kit-8, Transwell and Matrigel tube formation assays showed that circulating exosomes from EC patients promote EC cell growth and human umbilical vein endothelial cell (HUVEC) angiogenesis. Next, proteomic analysis and ELISA revealed that plasma exosomal lectin galactoside-binding soluble 3 binding protein (LGALS3BP) increased during EC progression. Moreover, to explore the function of exosomal LGALS3BP, we acquired exosomes containing high levels of LGALS3BP by overexpressing LGALS3BP in human embryonic kidney 293 cells, and we demonstrated that highly contained exosomal LGALS3BP contributed to EC cell proliferation and migration and HUVEC functions via the activation of the PI3K/AKT/VEGFA signalling pathway both in vitro and in vivo. Finally, high LGALS3BP expression was observed in human EC tissue, which indicated a poor prognosis. In addition, immunohistochemical analysis of human EC tissues revealed that LGALS3BP expression was correlated with VEGFA expression and blood vessel density. Hence, we proposed that plasma exosomes containing LGALS3BP contributed to EC growth and angiogenesis during EC progression, which also provided a novel perspective on EC diagnosis and prognosis.

A positive feedback loop of OTUD1 and c-Jun driven by leptin expedites stemness maintenance in ovarian cancer

Cancer stem cells (CSCs) are closely associated with drug resistance and recurrence in ovarian cancer patients. Although leptin is a high-risk factor for ovarian cancer and promotes stemness maintenance, a therapeutic strategy that counteracts the downstream signaling pathway of leptin remains elusive. Herein, the deubiquitinase OTUD1 was identified as a critical regulator of leptin in maintaining OCSCs properties. Mechanistically, leptin treatment significantly increased the chromatin enrichment of the transcription factor c-Jun, including the OTUD1 gene enhancer, which was sufficient to increase the OTUD1 protein level and subsequently cause OTUD1 aggresome formation, ASK1 recruitment and JNK/c-Jun pathway activation. The resultant positive feedback loop of c-Jun and OTUD1 was required for OCSCs stemness maintenance. Notably, the disruption of the positive feedback loop by targeting c-Jun or ASK1/JNK with T-5224, selonsertib, or ibrutinib markedly inhibited the leptin-induced stemness maintenance of OCSCs and tumorigenicity. Our findings reveal a crucial mechanism for leptin-mediated stemness maintenance and indicate that targeting c-Jun or the identified positive feedback loop has translational potential for ovarian cancer patients.

LOXL2 reduces susceptibility to PARP inhibitors by promoting super-enhancer-regulated DNA damage repair in high-grade serous ovarian cancer

Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionized the treatment of homologous recombination-deficient (HRD) tumors, yet their efficacy in homologous recombination-proficient (HRP) tumors is still limited. Here, we pinpoint lysyl oxidase-like 2 (LOXL2) as a key epigenetic regulator driving PARPi resistance. Our study demonstrate that elevated LOXL2 expression correlates with poor prognosis and disease recurrence in high-grade serous ovarian cancer (HGSOC) patients. Functional studies reveal that LOXL2 depletion or pharmacological inhibition synergizes with PARPi to suppress HRP models of both ovarian and breast cancer. Mechanistically, LOXL2 directly interacts with and transcriptionally activates BRD4, a core component of the super-enhancer complex, thereby amplifying the expression of DNA damage repair (DDR) genes such as MDC1, KAT5, and USP7. Strikingly, LOXL2 inhibition induces a "BRCAness" phenotype in HRP tumors, rendering them more susceptible to PARPi by impairing DDR capacity. Combining BET inhibitors with PARPi abrogates LOXL2-mediated resistance, underscoring BRD4 dependency in this process. Our findings establish LOXL2 as a druggable epigenetic target to overcome PARPi resistance in HRP models of multiple tumor types, presenting a therapeutic strategy independent of HR status and holding significant clinical potential for expanding PARPi benefits to a broader patient population.

Rictor orchestrates β-catenin/FOXO balance by maintaining redox homeostasis during development of ovarian cancer

Rictor/mTORC2 has been demonstrated to have important roles in cancer development and progression in a number of solid and hematologic malignancies. However, little is known about the role of Rictor/mTORC2 in ovarian cancer pathophysiology. Herein, using conditional Rictor knockout mice, we were able to demonstrate that Rictor deletion disrupted glutathione metabolism through AKT/Nrf2 signaling pathway and induced intracellular oxidative stress during the malignant transformation of Kras/Pten-mutant ovarian surface epithelial cells. Elevated reactive oxygen species and activated FOXO3a in Rictor-deleted cells strikingly shifts the functional interaction of β-catenin from TCF to FOXO3a, which strongly inhibits classical Wnt/β-catenin signaling. Our findings emphasize a pivotal role for Rictor in orchestrating crosstalk between the PI3K/AKT and Wnt/β-catenin signaling in the development of ovarian cancer. Illustration of Rictor/mTORC2 in promoting tumor onset by regulating glutathione metabolism and mediating oncogenic signaling.

GSK3β and UCHL3 govern RIPK4 homeostasis via deubiquitination to enhance tumor metastasis in ovarian cancer

Receptor-interacting protein kinase 4 (RIPK4) is increasingly recognized as a pivotal player in ovarian cancer, promoting tumorigenesis and disease progression. Despite its significance, the posttranslational modifications dictating RIPK4 stability in ovarian cancer remain largely uncharted. In this study, we first established that RIPK4 levels are markedly higher in metastatic than in primary ovarian cancer tissues through single-cell sequencing. Subsequently, we identified UCHL3 as a key deubiquitinase that regulates RIPK4. We elucidate the mechanism that UCHL3 interacts with and deubiquitinates RIPK4 at the K469 site, removing the K48-linked ubiquitin chain and thus enhancing RIPK4 stabilization. Intriguingly, inhibition of UCHL3 activity using TCID leads to increased RIPK4 ubiquitination and degradation. Furthermore, we discovered that GSK3β-mediated phosphorylation of RIPK4 at Ser420 enhances its interaction with UCHL3, facilitating further deubiquitination and stabilization. Functionally, RIPK4 was found to drive the proliferation and metastasis of ovarian cancer in a UCHL3-dependent manner both in vitro and in vivo. Importantly, positive correlations between RIPK4 and UCHL3 protein expression levels were observed, with both serving as indicators of poor prognosis in ovarian cancer patients. Overall, this study uncovers a novel pathway wherein GSK3β-induced phosphorylation of RIPK4 strengthens its interaction with UCHL3, leading to increased deubiquitination and stabilization of RIPK4, thereby promoting ovarian cancer metastasis. These findings offer new insights into the molecular underpinnings of ovarian cancer and highlight potential therapeutic targets for enhancing antitumor efficacy.

PG545 sensitizes ovarian cancer cells to PARP inhibitors through modulation of RAD51-DEK interaction

AbstractPG545 (Pixatimod) is a highly sulfated small molecule known for its ability to inhibit heparanase and disrupt signaling mediated by heparan-binding-growth factors (HB-GF). Previous studies indicated that PG545 inhibits growth factor-mediated signaling in ovarian cancer (OC) to enhance response to chemotherapy. Here we investigated the previously unidentified mechanisms by which PG545 induces DNA damage in OC cells and found that PG545 induces DNA single- and double-strand breaks, reduces RAD51 expression in an autophagy-dependent manner and inhibits homologous recombination repair (HRR). These changes accompanied the ability of PG545 to inhibit endocytosis of the heparan-sulfate proteoglycan interacting DNA repair protein, DEK, leading to DEK sequestration in the tumor microenvironment (TME) and loss of nuclear DEK needed for HRR. As a result, PG545 synergized with poly (ADP-ribose) polymerase inhibitors (PARPis) in OC cell lines in vitro and in 55% of primary cultures of patient-derived ascites samples ex vivo. Moreover, PG545/PARPi synergy was observed in OC cells exhibiting either de novo or acquired resistance to PARPi monotherapy. PG545 in combination with rucaparib also generated increased DNA damage, increased antitumor effects and increased survival of mice bearing HRR proficient OVCAR5 xenografts compared to monotherapy treatment in vivo. Synergistic antitumor activity of the PG545/rucaparib combination was likewise observed in an immunocompetent syngeneic ID8F3 OC model. Collectively, these results suggest that targeting DEK-HSPG interactions in the TME through the use of PG545 may be a novel method of inhibiting DNA repair and sensitizing cells to PARPis.

Amyloid-like p53 as prognostic biomarker in serous ovarian cancer—a study of the OVCAD consortium

TP53 is the most commonly mutated gene in cancer and has been shown to form amyloid-like aggregates, similar to key proteins in neurodegenerative diseases. Nonetheless, the clinical implications of p53 aggregation remain unclear. Here, we investigated the presence and clinical relevance of p53 aggregates in serous ovarian cancer (OC). Using the p53-Seprion-ELISA, p53 aggregates were detected in 46 out of 81 patients, with a detection rate of 84.3% in patients with missense mutations. High p53 aggregation was associated with prolonged progression-free survival. We found associations of overall survival with p53 aggregates, but they did not reach statistical significance. Interestingly, p53 aggregation was significantly associated with elevated levels of p53 autoantibodies and increased apoptosis, suggesting that high levels of p53 aggregates may trigger an immune response and/or exert a cytotoxic effect. To conclude, for the first time, we demonstrated that p53 aggregates are an independent prognostic marker in serous OC. P53-targeted therapies based on the amount of these aggregates may improve the patient's prognosis.

The splicing factor SNRPB promotes ovarian cancer progression through regulating aberrant exon skipping of POLA1 and BRCA2

Splicing factors play a crucial role in the initiation and development of various human cancers. SNRPB, a core spliceosome component, regulates pre-mRNA alternative splicing. However, its function and underlying mechanism in ovarian cancer remain unclear. This study identified SNRPB as a critical driver of ovarian cancer through TCGA and CPTAC database analysis. SNRPB was highly upregulated in fresh frozen ovarian cancer tissues compared with normal fallopian tubes. Immunohistochemistry revealed that SNRPB expression was increased in formalin-fixed, paraffin-embedded ovarian cancer sections and was positively correlated with a poor prognosis for ovarian cancer. Functionally, SNRPB knockdown suppressed ovarian cancer cell proliferation and invasion, and overexpression exerted opposite effects. SNRPB expression increased after cisplatin treatment, and silencing SNRPB sensitized ovarian cancer cells to cisplatin. KEGG pathway analysis revealed that the differentially expressed genes (DEGs) were mainly enriched in DNA replication and homologous recombination, and almost all DEGs related to DNA replication and homologous recombination were downregulated after SNRPB knockdown according to RNA-seq. Exon 3 skipping of the DEGs DNA polymerase alpha 1 (POLA1) and BRCA2 was induced by SNRPB silencing. Exon 3 skipping of POLA1 yielded premature termination codons and led to nonsense-mediated RNA decay (NMD); exon 3 skipping of BRCA2 led to loss of the PALB2 binding domain, which is necessary for homologous recombination, and increased ovarian cancer cell cisplatin sensitivity. POLA1 or BRCA2 knockdown partially impaired the increased malignancy of SNRPB-overexpressing ovarian cancer cells. Moreover, miR-654-5p was found to reduce SNRPB mRNA expression by directly binding to the SNRPB 3'-UTR. Overall, SNRPB was identified as an important oncogenic driver that promotes ovarian cancer progression by repressing exon 3 skipping of POLA1 and BRCA2. Thus, SNRPB is a potential treatment target and prognostic marker for ovarian cancer.

MAP4K4 promotes ovarian cancer metastasis through diminishing ADAM10-dependent N-cadherin cleavage

AbstractPeritoneal metastasis is a key feature of advanced ovarian cancer, but the critical protein required for ovarian cancer metastasis and progression is yet to be defined. Thus, an unbiased high throughput and in-depth study is warranted to unmask the mechanism. Transcriptomic sequencing of paired primary ovarian tumors and metastases unveiled that MAP4K4, a serine/threonine kinase belongs to the Ste20 family of kinases, was highly expressed in metastatic sites. Increased MAP4K4 expression in metastasis was further validated in other independent patients, with higher MAP4K4 expression associated with poorer survival, higher level of CA125 and more advanced FIGO stage. Down regulation of MAP4K4 inhibited cancer cell adhesion, migration, and invasion. Notably, MAP4K4 was found to stabilize N-cadherin. Further results showed that MAP4K4 mediated phosphorylation of ADAM10 at Ser436 results in suppression of N-cadherin cleavage by ADAM10, leading to N-cadherin stabilization. Pharmacologic inhibition of MAP4K4 abrogated peritoneal metastases. Overall, our data reveal MAP4K4 as a significant promoter in ovarian cancer metastasis. Targeting MAP4K4 may be a potential therapeutic approach for ovarian cancer patients.

Exploitation of ATP-sensitive potassium ion (KATP) channels by HPV promotes cervical cancer cell proliferation by contributing to MAPK/AP-1 signalling

Abstract Persistent infection with high-risk human papillomaviruses (HPVs) is the causal factor in multiple human malignancies, including >99% of cervical cancers and a growing proportion of oropharyngeal cancers. Prolonged expression of the viral oncoproteins E6 and E7 is necessary for transformation to occur. Although some of the mechanisms by which these oncoproteins contribute to carcinogenesis are well-characterised, a comprehensive understanding of the signalling pathways manipulated by HPV is lacking. Here, we present the first evidence to our knowledge that the targeting of a host ion channel by HPV can contribute to cervical carcinogenesis. Through the use of pharmacological activators and inhibitors of ATP-sensitive potassium ion (K ATP ) channels, we demonstrate that these channels are active in HPV-positive cells and that this activity is required for HPV oncoprotein expression. Further, expression of SUR1, which forms the regulatory subunit of the multimeric channel complex, was found to be upregulated in both HPV+ cervical cancer cells and in samples from patients with cervical disease, in a manner dependent on the E7 oncoprotein. Importantly, knockdown of SUR1 expression or K ATP channel inhibition significantly impeded cell proliferation via induction of a G1 cell cycle phase arrest. This was confirmed both in vitro and in in vivo tumourigenicity assays. Mechanistically, we propose that the pro-proliferative effect of K ATP channels is mediated via the activation of a MAPK/AP-1 signalling axis. A complete characterisation of the role of K ATP channels in HPV-associated cancer is now warranted in order to determine whether the licensed and clinically available inhibitors of these channels could constitute a potential novel therapy in the treatment of HPV-driven cervical cancer.

Resistance to the CHK1 inhibitor prexasertib involves functionally distinct CHK1 activities in BRCA wild-type ovarian cancer

AbstractHigh grade serous ovarian cancer (HGSOC) is a fatal gynecologic malignancy in the U.S. with limited treatment options. New therapeutic strategies include targeting of the cell cycle checkpoints, e.g., ATR and CHK1. We recently reported a promising clinical activity of the CHK1 inhibitor (CHK1i) prexasertib monotherapy inBRCAwild-type (BRCAwt) HGSOC patients. In this study, biopsies of treated patients and cell line models were used to investigate possible mechanisms of resistance to CHK1i. We report that BRCAwt HGSOC develops resistance to prexasertib monotherapy via a prolonged G2 delay induced by lower CDK1/CyclinB1 activity, thus preventing cells from mitotic catastrophe and cell death. On the other hand, we noted CHK1’s regulation on RAD51-mediated homologous recombination (HR) repair was not altered in CHK1i-resistant cells. Therefore, CHK1i sensitizes CHK1i-resistant cells to DNA damaging agents such as gemcitabine or hydroxyurea by inhibition of HR. In summary, our results demonstrate new mechanistic insights of functionally distinct CHK1 activities and highlight a potential combination treatment approach to overcome CHK1i resistance in BRCAwt HGSOC.

Splicing factor proline- and glutamine-rich (SFPQ) protein regulates platinum response in ovarian cancer-modulating SRSF2 activity

AbstractIn epithelial ovarian cancer (EOC), response to platinum (PT)-based chemotherapy dictates subsequent treatments and predicts patients’ prognosis. Alternative splicing is often deregulated in human cancers and can be altered by chemotherapy. Whether and how changes in alternative splicing regulation could impact on the response of EOC to PT-based chemotherapy is still not clarified. We identified the splicing factor proline and glutamine rich (SFPQ) as a critical mediator of response to PT in an unbiased functional genomic screening in EOC cells and, using a large cohort of primary and recurrent EOC samples, we observed that it is frequently overexpressed in recurrent PT-treated samples and that its overexpression correlates with PT resistance. At mechanistic level, we show that, under PT treatment, SFPQ, in complex with p54nrb, binds and regulates the activity of the splicing factor SRSF2. SFPQ/p54nrb complex decreases SRSF2 binding to caspase-9 RNA, favoring the expression of its alternative spliced antiapoptotic form. As a consequence, SFPQ/p54nrb protects cells from PT-induced death, eventually contributing to chemoresistance. Overall, our work unveils a previously unreported SFPQ/p54nrb/SRSF2 pathway that in EOC cells plays a central role in regulating alternative splicing and PT-induced apoptosis and that could result in the design of new possible ways of intervention to overcome PT resistance.

XCL1 expression correlates with CD8-positive T cells infiltration and PD-L1 expression in squamous cell carcinoma arising from mature cystic teratoma of the ovary

AbstractMolecular characteristics of carcinoma arising from mature cystic teratoma of the ovary (MCT) remain unclear due to its rarity. We analyzed RNA-sequencing data of 2322 pan-cancer [1378 squamous cell carcinomas (SCC), 6 adenosquamous carcinomas (ASC), and 938 adenocarcinomas (AC)] including six carcinomas arising from MCT (four SCCs, one ASC, and one AC). Hierarchical clustering and principal component analysis showed that gene expression profiles of carcinomas arising from MCT were different between each histological type and that gene expression profiles of SCCs arising MCT (MCT-SCCs) was apparently similar to those of lung SCCs. By epidermis-associated pathways activity based on gene set enrichment analysis, 1030 SCCs were divided into two groups: epidermis-signature high (head and neck, esophagus, and skin) and low (cervix, lung, and MCT). In addition to pan-SCC transcriptome analysis, cytokeratin profiling based on immunohistochemistry in the independent samples of 21 MCT-SCCs clarified that MCT-SCC dominantly expressed CK18, suggesting the origin of MCT-SCC was columnar epithelium. Subsequently, we investigated differentially expressed genes in MCT-SCCs compared with different SCCs and identified XCL1 was specifically overexpressed in MCT-SCCs. Through immunohistochemistry analysis, we identified XCL1 expression on tumor cells in 13/24 (54%) of MCT-SCCs but not in MCTs. XCL1 expression was also significantly associated with the number of tumor-infiltrating CD8-positive T cells and PD-L1 expression on tumor cells. XCL1 produced by tumor cells may induce PD1/PD-L1 interaction and dysfunction of CD8-positive T cells in tumor microenvironment. XCL1 expression may be a novel biomarker for malignant transformation of MCT into SCC and a biomarker candidate for therapeutic response to an anti-PD1/PD-L1 therapy.

DDR2-regulated arginase activity in ovarian cancer-associated fibroblasts promotes collagen production and tumor progression

AbstractOvarian cancer has poor survival outcomes particularly for advanced stage, metastatic disease. Metastasis is promoted by interactions of stromal cells, such as cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME), with tumor cells. CAFs play a key role in tumor progression by remodeling the TME and extracellular matrix (ECM) to result in a more permissive environment for tumor progression. It has been shown that fibroblasts, in particular myofibroblasts, utilize metabolism to support ECM remodeling. However, the intricate mechanisms by which CAFs support collagen production and tumor progression are poorly understood. In this study, we show that the fibrillar collagen receptor, Discoidin Domain Receptor 2 (DDR2), promotes collagen production in human and mouse omental CAFs through arginase activity. CAFs with high DDR2 or arginase promote tumor colonization in the omentum. In addition, DDR2-depleted CAFs had decreased ornithine levels leading to decreased collagen production and polyamine levels compared to WT control CAFs. Tumor cell invasion was decreased in the presence CAF conditioned media (CM) depleted of DDR2 or arginase-1, and this invasion defect was rescued in the presence of CM from DDR2-depleted CAFs that constitutively overexpressed arginase-1. Similarly, the addition of exogenous polyamines to CM from DDR2-depleted CAFs led to increased tumor cell invasion. We detected SNAI1 protein at the promoter region of the arginase-1 gene, and DDR2-depleted CAFs had decreased levels of SNAI1 protein at the arginase-1 promoter region. Furthermore, high stromal arginase-1 expression correlated with poor survival in ovarian cancer patients. These findings highlight how DDR2 regulates collagen production by CAFs in the tumor microenvironment by controlling the transcription of arginase-1, and CAFs are a major source of arginase activity and L-arginine metabolites in ovarian cancer models.

ΔNp63α exerts antitumor functions in cervical squamous cell carcinoma

The molecular basis underlying the aggressive nature and excessive proliferation of cervical squamous cancer cell remains unclear. ΔNp63α is the predominant isotype of p63 expressed in the epithelia and regulates epithelial cell differentiation. The pro-/anti-tumor role of ΔNp63α in different kinds of solid tumors remains controversial and the precise molecular mechanisms are still elusive. In this study, we uncovered the molecular functions of ΔNp63α in cervical squamous cell carcinoma to clarify its roles as a tumor suppressor. We demonstrated that ΔNp63α suppressed cell migration, invasiveness, and tumor growth in SiHa and ME-180 cells with both in vivo and in vitro assays. Mechanistic investigation via RNA-sequencing and chromatin immunoprecipitation-sequencing revealed that ΔNp63α exerted its antitumor capacity via regulating the expression of a cohort of cell junction genes. Further, we showed that ZNF385B and CLDN1 were two direct ΔNp63α targets with significant relevance to cervical squamous cell carcinoma examined in cell cultures, tumor xenografts, and clinic tumors. We also demonstrated that ΔNp63α downregulated NFATC1 to reduce cisplatin resistance. These findings shed new lights on functions of ΔNp63α in tumors and providing novel insights in targeted therapy of cervical cancers.

Obesity-induced extracellular vesicles proteins drive the endometrial cancer pathogenesis: therapeutic potential of HO-3867 and Metformin

Abstract Endometrial cancer (EC) is the leading gynecologic malignancy in the United States with obesity implicated in 57% of cases. This research investigates the molecular complexities of extracellular vesicles (EV) secretion as carriers of oncogenic protein and their involvement in obesity-mediated EC. An understanding of these mechanisms is pivotal for unraveling pathways relevant to obesity-associated EC, thereby guiding the development of innovative prevention and treatment strategies. Our exploration revealed a significant increase in EV secretion carrying oncogenic proteins (TMEM205, STAT5, and FAS) in adipose and uterine tissues/serum samples from obese EC patients compared to control (without cancer). We identified alterations in EV-regulating proteins (Rab7, Rab11, and Rab27a) in obesity-mediated EC patients, adipose/uterine tissues, and serum samples. Through a 24-week analysis of the effects of a 45% kcal high-fat diet (HFD) on mice, we observed increased body weight, increased adipose tissue, enlarged uterine horns, and increased inflammation in the HFD group. This correlated with elevated levels of EV secretion and increased expression of oncogenic proteins TMEM205, FAS, and STAT5 and downregulation of the tumor suppressor gene PIAS3 in adipose and uterine tissues. Furthermore, our study confirmed that adipocyte derived EV increased EC cell proliferation, migration and xenograft tumor growth. Additionally, we identified that the small molecule inhibitors (HO-3867) or Metformin inhibited EV secretion in vitro and in vivo, demonstrating significant inhibition of high glucose or adipocyte-mediated EC cell proliferation and a reduction in body weight and adipose tissue accumulation when administered to HFD mice. Moreover, HO-3867 or Metformin treatment inhibited HFD induced hyperplasia (precursor of EC) by altering the expression of EV-regulated proteins and decreasing oncogenic protein expression levels. This study provides critical insights into the mechanisms underpinning obesity-mediated EV secretion with oncogenic protein expression, shedding light on their role in EC pathogenesis. Additionally, it offers pre-clinical evidence supporting the initiation of novel studies for EV-targeted therapies aimed at preventing obesity-mediated EC.

Circulating extracellular vesicles protein expression for early prediction of platinum-resistance in high-grade serous ovarian cancer

Abstract Platinum resistance in high-grade serous ovarian carcinoma (HGSOC) portends a poor prognosis. Although initial platinum-based chemotherapy response rates are high, 15-20% of patients demonstrate primary resistance to platinum therapy and almost all patients will develop platinum resistance in the recurrent setting. No predictive or diagnostic biomarkers have been utilized specific to platinum resistance. This study aimed to identify candidate biomarkers for platinum resistance in HGSOC using an extracellular vesicle (EV) based approach. We found differentially expressed and distinct EV proteins, namely TMEM205 and CFH, in patients with platinum-resistant (PR) HGSOC compared to those of platinum-sensitive (PS) patients, utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). Expression of these EV proteins were validated in patient-derived PR cell lines as well as in clinically relevant mouse models of HGSOC post-platinum therapy. We corroborated these findings using serum samples from patients with PS and PR-HGSOC. Both EV CFH and EV TMEM205 exhibited excellent diagnostic capability for PR as noted by receiver operating characteristic curves with area under the curve values of 0.95 and 0.84, respectively. The high diagnostic performance of TMEM205 and CFH within EVs compared to the relatively poor performance of conventional serum proteins such as Ca125 suggests their robust potential as non-invasive biomarkers for detecting platinum resistance in HGSOC. Furthermore, the ROC curve for the combined biomarker demonstrated excellent diagnostic performance, with an AUC of 0.973, a true positive rate (TPR) of 0.938, and a false positive rate (FPR) of 0.062. Incorporating this multi-protein biomarker panel alongside established biomarkers further enhances diagnostic accuracy. Serum EV CFH and TMEM205 are promising biomarkers for early detection of platinum resistance in HGSOC and may highlight underlying chemoresistance mechanisms, offering potential future therapeutic targets.

Metabolic imaging distinguishes ovarian cancer subtypes and detects their early and variable responses to treatment

Abstract High grade serous ovarian cancer displays two metabolic subtypes; a high OXPHOS subtype that shows increased expression of genes encoding electron transport chain components, increased oxygen consumption, and increased chemosensitivity, and a low OXPHOS subtype that exhibits glycolytic metabolism and is more drug resistant. We show here in patient-derived organoids and in the xenografts obtained by their subcutaneous implantation that the low OXPHOS subtype shows higher lactate dehydrogenase activity and monocarboxylate transporter 4 expression than the high OXPHOS subtype and increased lactate labeling in 13C magnetic resonance spectroscopy (MRS) measurements of hyperpolarized [1-13C]pyruvate metabolism. There was no difference between the subtypes in PET measurements of 2-deoxy-2-[fluorine-18]fluoro-D-glucose ([18F]FDG) uptake. Both metabolic imaging techniques could detect the early response to Carboplatin treatment in drug-sensitive high OXPHOS xenografts and no response in drug-resistant in low OXPHOS xenografts. 13C magnetic resonance spectroscopic imaging of hyperpolarized [1-13C]pyruvate metabolism has the potential to be used clinically to distinguish low OXPHOS and high OXPHOS tumor deposits in HGSOC patients and to detect their differential responses to treatment.

METAP2 inhibits K48-linked ubiquitination of YTHDF2 to promote ovarian cancer progression

TP53 somatic evolution in cervical liquid-based cytology and blood from individuals with and without ovarian cancer and BRCA1 or BRCA2 germline mutations

Somatic TP53 mutations are prevalent in normal tissue but little is known about their association with cancer risk. Cervical liquid-based cytology (LBC), commonly known as Pap test, provides an accessible gynecological sample to test the value of TP53 somatic mutations as a biomarker for high-grade serous ovarian cancer (HGSC), a cancer type mostly driven by TP53 mutations. We used ultra-deep duplex sequencing to analyze TP53 mutations in LBC and blood samples from 70 individuals (30 with and 40 without HGSC) undergoing gynecologic surgery, 30 carrying BRCA1 or BRCA2 germline pathogenic variants (BRCApv). Only 30% of the tumor mutations were found in LBC samples. However, TP53 pathogenic mutations were identified in nearly all LBC and blood samples, with only 5.4% of mutations in LBC (20/368) also found in the corresponding blood sample. TP53 mutations were more abundant in LBC than in blood and increased with age in both sample types. BRCApv carriers with HGSC had more TP53 clonal expansions in LBC than BRCApv carriers without cancer. Our results show that, while not useful for direct cancer detection, LBC samples capture TP53 mutation burden in the gynecological tract, presenting potential value for cancer risk assessment in individuals at higher hereditary risk for ovarian cancer.

A CRISPR-based base-editing screen for the functional assessment of BRCA1 variants

Genetic mutations in BRCA1, which is crucial for the process of DNA repair and maintenance of genomic integrity, are known to increase markedly the risk of breast and ovarian cancers. Clinical genetic testing has been used to identify new BRCA1 variants; however, functional assessment and determination of their pathogenicity still poses challenges for clinical management. Here, we describe that CRISPR-mediated cytosine base editor, known as BE3, can be used for the functional analysis of BRCA1 variants. We performed CRISPR-mediated base-editing screening using 745 gRNAs targeting all exons in BRCA1 to identify loss-of-function variants and identified variants whose function has heretofore remained unknown, such as c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A. Our results show that CRISPR-mediated base editor is a powerful tool for the reclassification of variants of uncertain significance (VUSs) in BRCA1.

FBP1 regulates proliferation, metastasis, and chemoresistance by participating in C-MYC/STAT3 signaling axis in ovarian cancer

AbstractFructose-1,6-bisphosphatase (FBP1) is a rate-limiting enzyme in gluconeogenesis and an important tumor suppressor in human malignancies. Here, we aimed to investigate the expression profile of FBP1 in ovarian cancer, the molecular mechanisms that regulate FBP1 expression and to examine how the FBP1 regulatory axis contributes to tumorigenesis and progression in ovarian cancer. We showed that FBP1 expression was significantly decreased in ovarian cancer tissues compared with normal ovarian tissues, and low-FBP1 expression predicted poor prognosis in patients with ovarian cancer. The enhanced expression of FBP1 in ovarian cancer cell lines suppressed proliferation and 2-D/3-D invasion, reduced aerobic glycolysis, and sensitized cancer cells to cisplatin-induced apoptosis. Moreover, DNA methylation and C-MYC binding at the promoter inhibited FBP1 expression. Furthermore, through physical interactions with signal transducer and activator of transcription 3 (STAT3), FBP1 suppressed nuclear translocation of STAT3 and exerted its non-metabolic enzymatic activity to induce the dysfunction of STAT3. Thus, our study suggests that FBP1 may be a valuable prognostic predictor for ovarian cancer. C-MYC-dependent downregulation of FBP1 acted as a tumor suppressor via modulating STAT3, and the C-MYC/FBP1/STAT3 axis could be a therapeutic target.

NR1D1 regulation by Ran GTPase via miR4472 identifies an essential vulnerability linked to aneuploidy in ovarian cancer

AbstractWhile aneuploidy is a main enabling characteristic of cancers, it also creates specific vulnerabilities. Here we demonstrate that Ran inhibition targets epithelial ovarian cancer (EOC) survival through its characteristic aneuploidy. We show that induction of aneuploidy in rare diploid EOC cell lines or normal cells renders them highly dependent on Ran. We also establish an inverse correlation between Ran and the tumor suppressor NR1D1 and reveal the critical role of Ran/NR1D1 axis in aneuploidy-associated endogenous DNA damage repair. Mechanistically, we show that Ran, through the maturation of miR4472, destabilizes the mRNA of NR1D1 impacting several DNA repair pathways. We showed that NR1D1 interacts with both PARP1 and BRCA1 leading to the inhibition of DNA repair. Concordantly, loss of Ran was associated with NR1D1 induction, accumulation of DNA damages, and lethality of aneuploid EOC cells. Our findings suggest a synthetic lethal strategy targeting aneuploid cells based on their dependency to Ran.

A novel homeostatic loop of sorcin drives paclitaxel-resistance and malignant progression via Smad4/ZEB1/miR-142-5p in human ovarian cancer

AbstractThe primary chemotherapy of ovarian cancer (OC) often acquires chemoresistance. Sorcin (SRI), a soluble resistance-related calcium-binding protein, has been reported to be an oncogenic protein in cancer. However, the molecular mechanisms of SRI regulation and the role and aberrant expression of SRI in chemoresistant OC remain unclear. Here, we identified SRI as a key driver of paclitaxel (PTX)-resistance and explored its regulatory mechanism. Using transcriptome profiles, qRT-PCR, proteomics, Western blot, immunohistochemistry, and bioinformatics analyses, we found that SRI was overexpressed in PTX-resistant OC cells and the overexpression of SRI was related to the poor prognosis of patients. SRI was a key molecule required for growth, migration, and PTX-resistance in vitro and in vivo and was involved in epithelial–mesenchymal transition (EMT) and stemness. Mechanistic studies showed that miR-142-5p directly bound to the 3ʹ-UTR of SRI to suppress its expression, whereas a transcription factor zinc-finger E-box binding homeobox 1 (ZEB1) inhibited the transcription of miR-142-5p by directly binding to the E-box fragment in the miR-142 promoter region. Furthermore, ZEB1 was negatively regulated by SRI which physically interacted with Smad4 to block its translocation from the cytosol to the nucleus. Taken together, our findings unveil a novel homeostatic loop of SRI that drives the PTX-resistance and malignant progression via Smad4/ZEB1/miR-142-5p in human OC. Targeting this SRI/Smad4/ZEB1/miR-142-5p loop may reverse the PTX-resistance.

C/EBPβ promotes poly(ADP-ribose) polymerase inhibitor resistance by enhancing homologous recombination repair in high-grade serous ovarian cancer

AbstractPARP inhibitors (PARPi) are efficacious in treating high-grade serous ovarian cancer (HG-SOC) with homologous recombination (HR) deficiency. However, they exhibit suboptimal efficiency in HR-proficient cancers. Here, we found that the expression of CCAAT/enhancer-binding protein β (C/EBPβ), a transcription factor, was inversely correlated with PARPi sensitivity in vitro and in vivo, both in HR-proficient condition. High C/EBPβ expression enhanced PARPi tolerance; PARPi treatment in turn induced C/EBPβ expression. C/EBPβ directly targeted and upregulated multiple HR genes (BRCA1, BRIP1, BRIT1, and RAD51), thereby inducing restoration of HR capacity and mediating acquired PARPi resistance. C/EBPβ is a key regulator of the HR pathway and an indicator of PARPi responsiveness. Targeting C/EBPβ could induce HR deficiency and rescue PARPi sensitivity accordingly. Our findings indicate that HR-proficient patients may benefit from PARPi via targeting C/EBPβ, and C/EBPβ expression levels enable predicting and tracking PARPi responsiveness during treatment.

Targeting autocrine amphiregulin robustly and reproducibly inhibits ovarian cancer in a syngeneic model: roles for wildtype p53

AbstractOvarian cancer (OvCA) remains one of the most devastating malignancies, but treatment options are still limited. We report that amphiregulin (AREG) can serve as an effective and safe pharmacological target in a syngeneic murine model. AREG is highly abundant in abdominal fluids of patients with advanced OvCa. In immunocompetent animals, depletion or overexpression of AREG respectively prolonged or shortened animal survival. A new antibody we generated in AREG-knockout mice recognized murine AREG and reproducibly prolonged animal survival in the syngeneic model. The underlying mechanism likely involves binding of wildtype p53 to AREG’s promoter and autocrine activation of the epidermal growth factor receptor (EGFR), a step blocked by the antibody. Accordingly, depletion of p53 downregulated AREG secretion and conferred tolerance, whereas blocking an adaptive process involving CXCL1, which transactivates EGFR, might increase therapeutic efficacy. Consistent with these observations, analysis of OvCa patients revealed that high AREG correlates with poor prognosis of patients expressing wildtype TP53. In conclusion, clinical tests of the novel antibody are warranted; high AREG, normal TP53, and reduced CXCL1 activity might identify patients with OvCa who may derive therapeutic benefit.

The hidden role of paxillin: localization to nucleus promotes tumor angiogenesis

Paxillin (PXN), a key component of the focal adhesion complex, has been associated with cancer progression, but the underlying mechanisms are poorly understood. The purpose of this study was to elucidate mechanisms by which PXN affects cancer growth and progression, which we addressed using cancer patient data, cell lines, and orthotopic mouse models. We demonstrated a previously unrecognized mechanism whereby nuclear PXN enhances angiogenesis by transcriptionally regulating SRC expression. SRC, in turn, increases PLAT expression through NF-ĸB activation; PLAT promotes angiogenesis via LRP1 in endothelial cells. PXN silencing in ovarian cancer mouse models reduced angiogenesis, tumor growth, and metastasis. These findings provide a new understanding of the role of PXN in regulating tumor angiogenesis and growth.

Molecular disruption of DNA polymerase β for platinum sensitisation and synthetic lethality in epithelial ovarian cancers

AbstractTargeting PARP1 [Poly(ADP-Ribose) Polymerase 1] for synthetic lethality is a new strategy for BRCA germ-line mutated or platinum sensitive ovarian cancers. However, not all patients respond due to intrinsic or acquired resistance to PARP1 inhibitor. Development of alternative synthetic lethality approaches is a high priority. DNA polymerase β (Polβ), a critical player in base excision repair (BER), interacts with PARP1 during DNA repair. Here we show that polβ deficiency is a predictor of platinum sensitivity in human ovarian tumours. Polβ depletion not only increased platinum sensitivity but also reduced invasion, migration and impaired EMT (epithelial to mesenchymal transition) of ovarian cancer cells. Polβ small molecular inhibitors (Pamoic acid and NSC666719) were selectively toxic to BRCA2 deficient cells and associated with double-strand breaks (DSB) accumulation, cell cycle arrest and increased apoptosis. Interestingly, PARG [Poly(ADP-Ribose) Glycohydrolase] inhibitor (PDD00017273) [but not PARP1 inhibitor (Olaparib)] was synthetically lethal in polβ deficient cells. Selective toxicity to PDD00017273 was associated with poly (ADP-ribose) accumulation, reduced nicotinamide adenine dinucleotide (NAD+) level, DSB accumulation, cell cycle arrest and increased apoptosis. In human tumours, polβ-PARG co-expression adversely impacted survival in patients. Our data provide evidence that polβ targeting is a novel strategy and warrants further pharmaceutical development in epithelial ovarian cancers.

Suppression of DDX39B sensitizes ovarian cancer cells to DNA-damaging chemotherapeutic agents via destabilizing BRCA1 mRNA

Multiple RNA processing events including transcription, mRNA splicing, and export are delicately coordinated by the TREX complex. As one of the essential subunits, DDX39B couples the splicing and export machineries by recruiting ALYREF onto mRNA. In this study, we further explore the functions of DDX39B in handling damaged DNA, and unexpectedly find that DDX39B facilitates DNA repair by homologous recombination through upregulating BRCA1. Specifically, DDX39B binds to and stabilizes BRCA1 mRNA. DDX39B ensures ssDNA formation and RAD51 accumulation at DSB sites by maintaining BRCA1 levels. Without DDX39B being present, ovarian cancer cells exhibit hypersensitivity to DNA-damaging chemotherapeutic agents like platinum or PARPi. Moreover, DDX39B-deficient mice show embryonic lethality or developmental retardation, highly reminiscent of those lacking BRCA1. High DDX39B expression is correlated with worse survival in ovarian cancer patients. Thus, DDX39B suppression represents a rational approach for enhancing the efficacy of chemotherapy in BRCA1-proficient ovarian cancers.

Serum- and glucocorticoid- inducible kinase 2, SGK2, is a novel autophagy regulator and modulates platinum drugs response in cancer cells

AbstractFor many tumor types chemotherapy still represents the therapy of choice and many standard treatments are based on the use of platinum (PT) drugs. However, de novo or acquired resistance to platinum is frequent and leads to disease progression. In Epithelial Ovarian Cancer (EOC) patients, PT-resistant recurrences are very common and improving the response to treatment still represents an unmet clinical need. To identify new modulators of PT-sensitivity, we performed a loss-of-function screening targeting 680 genes potentially involved in the response of EOC cells to platinum. We found that SGK2 (Serum-and Glucocorticoid-inducible kinase 2) plays a key role in PT-response. We show here that EOC cells relay on the induction of autophagy to escape PT-induced death and that SGK2 inhibition increases PT sensitivity inducing a block in the autophagy cascade due to the impairment of lysosomal acidification. Mechanistically we demonstrate that SGK2 controls autophagy in a kinase-dependent manner by binding and inhibiting the V-ATPase proton pump. Accordingly, SGK2 phosphorylates the subunit V1H (ATP6V1H) of V-ATPase and silencing or chemical inhibition of SGK2, affects the normal autophagic flux and sensitizes EOC cells to platinum. Hence, we identified a new pathway that links autophagy to the survival of cancer cells under platinum treatment in which the druggable kinase SGK2 plays a central role. Our data suggest that blocking autophagy via SGK2 inhibition could represent a novel therapeutic strategy to improve patients’ response to platinum.

Expression of the chrXq27.3 miRNA cluster in recurrent ovarian clear cell carcinoma and its impact on cisplatin resistance

AbstractOvarian clear cell carcinoma (OCCC) is a histological subtype of epithelial ovarian cancer and exhibits dismal prognosis due to chemoresistance. Moreover, only few effective therapeutic options exist for patients with recurrent OCCC, and an understanding of its molecular characteristics is essential for the development of novel therapeutic approaches. In the present study, we investigated unique MicroRNAs (miRNA) profiles in recurrent/metastatic OCCC and the role of miRNAs in cisplatin resistance. Comprehensive miRNA sequencing revealed that expression of several miRNAs, including miR-508-3p, miR-509-3p, miR-509-3-5p, and miR-514a-3p was remarkably less in recurrent cancer tissues when compared with that in paired primary cancer tissues. These miRNAs are located in the chrXq27.3 region on the genome. Moreover, its expression was negative in omental metastases in two patients with advanced OCCC. In vitro analyses revealed that overexpression of miR-509-3p and miR-509-3-5p reversed cisplatin resistance and yes-associated protein 1 (YAP1) was partially responsible for the resistance. Immunohistochemistry revealed that YAP1 expression was inversely correlated with the chrXq27.3 miRNA cluster expression. In conclusion, these findings suggest that alteration of the chrXq27.3 miRNA cluster could play a critical role in chemoresistance and miRNAs in the cluster and their target genes can be potential therapeutic targets.

Single-cell EMT-related transcriptional analysis revealed intra-cluster heterogeneity of tumor cell clusters in epithelial ovarian cancer ascites

Malignant ascites of epithelial ovarian cancer is a metastatic tumor microenvironment in which large amounts of disseminated single cells (DSCs) and disseminated tumor cell clusters (DTCCs) are commonly observed. The tumor cell clusters are known to be more aggressive than individual tumor cells in cancer metastasis; however, little is known about the mechanism. Applying single-cell epithelial-to-mesenchymal transition (EMT)-related transcriptional analysis in 120 DSCs and 195 intra-cluster cells from 27 DTCCs, we demonstrated that DTCCs were heterogeneous cellular units comprised of epithelial tumor cells, leukocytes, and cancer-associated fibroblasts (CAFs). Through the analysis of intra-DTCC heterogeneity, we identified that CAFs induced EMT of tumor cells via TGFβ signaling within the DTCC microenvironment. The activation of EMT program, in particular the upregulation of ZEB2, enabled the acquisition of additional chemoresistance and metastasis abilities of the intra-DTCC tumor cells, which resulted in the aggressiveness of DTCCs.

MicroRNA-200 family governs ovarian inclusion cyst formation and mode of ovarian cancer spread

Epidemiologic and histopathologic findings and the laying hen model support the long-standing incessant ovulation hypothesis and cortical inclusion cyst involvement in sporadic ovarian cancer development. MicroRNA-200 (miR-200) family is highly expressed in ovarian cancer. Herewith, we show that ovarian surface epithelial (OSE) cells with ectopic miR-200 expression formed stabilized cysts in three-dimensional (3D) organotypic culture with E-cadherin fragment expression and steroid hormone pathway activation, whereas ovarian cancer 3D cultures with miR-200 knockdown showed elevated TGF-β expression, mitotic spindle disorientation, increased lumenization, disruption of ROCK-mediated myosin II phosphorylation, and SRC signaling, which led to histotype-dependent loss of collective movement in tumor spread. Gene expression profiling revealed that epithelial-mesenchymal transition and hypoxia were the top enriched gene sets regulated by miR-200 in both OSE and ovarian cancer cells. The molecular changes uncovered by the in vitro studies were verified in both human and laying hen ovarian cysts and tumor specimens. As miR-200 is also essential for ovulation, our results of estrogen pathway activation in miR-200-expressing OSE cells add another intriguing link between incessant ovulation and ovarian carcinogenesis.

ERBB3-induced furin promotes the progression and metastasis of ovarian cancer via the IGF1R/STAT3 signaling axis

High-grade serous carcinoma, accounts for up to 70% of all ovarian cases. Furin, a proprotein convertase, is highly expressed in high-grade serous carcinoma of ovarian cancer patients, and its expression is even higher in tumor omentum than in normal omentum, the preferred site of ovarian cancer metastasis. The proteolytic actions of this cellular endoprotease help the maturation of several important precursors of protein substrates and its levels increase the risk of several cancer. We show that furin activates the IGF1R/STAT3 signaling axis in ovarian cancer cells. Conversely, furin knockdown downregulated IGF1R-β and p-STAT3 (Tyr705) expression. Further, silencing furin reduced tumor cell migration and invasion in vitro and tumor growth and metastasis in vivo. Collectively, our findings show that furin can be an effective therapeutic target for ovarian cancer prevention or treatment.

Inhibiting Importin 4-mediated nuclear import of CEBPD enhances chemosensitivity by repression of PRKDC-driven DNA damage repair in cervical cancer

AbstractCervical cancer (CC) remains highest in the mortality of female reproductive system cancers, while cisplatin (CDDP) resistance is the one of main reasons for the lethality. Preceding evidence has supported that karyopherins are associated with chemoresistance. In this study, we simultaneously compared CDDP-incomplete responders with CDDP-complete responders of CC patients and CDDP‐insensitive CC cell lines with CDDP‐sensitive group. We finally identified that DNA-PKcs (PRKDC) was related to CDDP sensitivity after overlapping in CC sample tissues and CC cell lines. Further functional assay revealed that targeting PRKDC by shRNA and NU7026 (specific PRKDC inhibitor) could enhance CDDP sensitivity in vitro and in vivo, which was mediated by impairing DNA damage repair pathway in CC. Mechanistically, we found that PRKDC was transcriptionally upregulated by CCAAT/enhancer-binding protein delta (CEBPD), while intriguingly, CDDP treatment strengthened the transcriptional activity of CEBPD to PRKDC. We further disclosed that Importin 4 (IPO4) augmented the nuclear translocation of CEBPD through nuclear localization signals (NLS) to activate PRKDC-mediated DNA damage repair in response to CDDP. Moreover, we demonstrated that IPO4 and CEBPD knockdown improved CDDP-induced cytotoxicity in vitro and in vivo. Together, we shed the novel insight into the role of IPO4 in chemosensitivity and provide a clinical translational potential to enhance CC chemosensitivity since the IPO4-CEBPD-PRKDC axis is actionable via NU7026 (PRKDC inhibitor) or targeting IPO4 in combination with CDDP.

PLAA suppresses ovarian cancer metastasis via METTL3-mediated m6A modification of TRPC3 mRNA

AbstractWide metastasis contributes to a high death rate in ovarian cancer, and understanding of the molecular mechanism helps to find effective targets for metastatic ovarian cancer therapy. It has been found that phospholipase A2-activating protein (PLAA) is inactivated in some cancers, but its role in cancer metastasis remains unknown. Here, we found that PLAA was significantly downregulated in ovarian cancer highly metastatic cell lines and patients, and the low expression of PLAA was associated with poorer prognosis and high-risk clinicopathological features of patients. PLAA inhibited the migration and invasion of ovarian cancer cells and metastasis of transplanted tumor in the orthotopic xenograft mouse model. Meanwhile, PLAA inhibited metastasis of ovarian cancer by inhibiting transient receptor potential channel canonical 3 (TRPC3)-mediated the intracellular Ca2+ level. Mechanistically, PLAA inhibited methyltransferase-like 3 (METTL3) expression through the ubiquitin-mediated degradation, and METTL3 stabilized TRPC3 mRNA expression via N6-methyladenosine (m6A) modification. Our study verified the function and mechanism of the PLAA-METTL3-TRPC3 axis involved in ovarian cancer metastasis, with a view to providing a potential therapeutic approach for ovarian cancer.

The tumor suppressor Parkin exerts anticancer effects through regulating mitochondrial GAPDH activity

Cancer cells preferentially utilize glycolysis for energy production, and GAPDH is a critical enzyme in glycolysis. Parkin is a tumor suppressor and a key protein involved in mitophagy regulation. However, the tumor suppression mechanism of Parkin has still not been elucidated. In this study, we identified mitochondrial GAPDH as a new substrate of the E3 ubiquitin ligase Parkin, which mediated GAPDH ubiquitination in human cervical cancer. The translocation of GAPDH into mitochondria was driven by the PINK1 kinase, and either PINK1 or GAPDH mutation prevented the accumulation of GAPDH in mitochondria. Parkin caused the ubiquitination of GAPDH at multiple sites (K186, K215, and K219) located within the enzyme-catalyzed binding domain of the GAPDH protein. GAPDH ubiquitination was required for mitophagy, and stimulation of mitophagy suppressed cervical cancer cell growth, indicating that mitophagy serves as a type of cell death. Mechanistically, PHB2 served as a key mediator in GAPDH ubiquitination-induced mitophagy through stabilizing PINK1 protein and GAPDH mutation resulted in the reduced distribution of PHB2 in mitophagic vacuole. In addition, ubiquitination of GAPDH decreased its phosphorylation level and enzyme activity and inhibited the glycolytic pathway in cervical cancer cells. The results of in vivo experiments also showed that the GAPDH mutation increased glycolysis in cervical cancer cells and accelerated tumorigenesis. Thus, we concluded that Parkin may exert its anticancer function by ubiquitinating GAPDH in mitochondria. Taken together, our study further clarified the molecular mechanism of tumor suppression by Parkin through the regulation of energy metabolism, which provides an experimental basis for the development of new drugs for the treatment of human cervical cancer.

WAPL induces cervical intraepithelial neoplasia modulated with estrogen signaling without HPV E6/E7

AbstractSince cervical cancer still afflicts women around the world, it is necessary to understand the underlying mechanism of cervical cancer development. Infection with HPV is essential for the development of cervical intraepithelial neoplasia (CIN). In addition, estrogen receptor signaling is implicated in the development of cervical cancer. Previously, we have isolated human wings apart-like (WAPL), which is expected to cause chromosomal instability in the process of HPV-infected precancerous lesions to cervical cancer. However, the role of WAPL in the development of CIN is still unknown. In this study, in order to elucidate the role of WAPL in the early lesion, we established WAPL overexpressing mice (WAPL Tg mice) and HPV E6/E7 knock-in (KI) mice. WAPL Tg mice developed CIN lesion without HPV E6/E7. Interestingly, in WAPL Tg mice estrogen receptor 1 (ESR1) showed reduction as compared with the wild type, but cell growth factors MYC and Cyclin D1 controlled by ESR1 expressed at high levels. These results suggested that WAPL facilitates sensitivity of ESR1 mediated by some kind of molecule, and as a result, affects the expression of MYC and Cyclin D1 in cervical cancer cells. To detect such molecules, we performed microarray analysis of the uterine cervix in WAPL Tg mice, and focused MACROD1, a co-activator of ESR1. MACROD1 expression was increased in WAPL Tg mice compared with the wild type. In addition, knockdown of WAPL induced the downregulation of MACROD1, MYC, and Cyclin D1 but not ESR1 expression. Furthermore, ESR1 sensitivity assay showed lower activity in WAPL or MACROD1 downregulated cells than control cells. These data suggested that WAPL increases ESR1 sensitivity by activating MACROD1, and induces the expression of MYC and Cyclin D1. Therefore, we concluded that WAPL not only induces chromosomal instability in cervical cancer tumorigenesis, but also plays a key role in activating estrogen receptor signaling in early tumorigenesis.

PRDM4 inhibits cell proliferation and tumorigenesis by inactivating the PI3K/AKT signaling pathway through targeting of PTEN in cervical carcinoma

AbstractPR domain zinc finger protein 4 (PRDM4) is a transcription factor that plays key roles in stem cell self-renewal and tumorigenesis. However, its biological role and exact mechanism in cervical cancer remain unknown. Here, both immunohistochemistry (IHC) and Western blot assays demonstrated that the expression of PRDM4 in cervical cancer tissues was much lower than that in the normal cervix. A xenograft assay showed that PRDM4 overexpression in the cervical cancer cell lines SiHa and HeLa dramatically inhibited cell proliferation and tumorigenic potential in vivo. Conversely, the silencing of PRDM4 promoted cervical cancer cell proliferation and tumorigenic potential. Mechanistically, PRDM4 induced cell cycle arrest at the transition from G0/G1 phase to S phase by upregulating p27 and p21 expression and downregulating Cyclin D1 and CDK4 expression. Furthermore, the PI3K/AKT signaling pathway was inactivated in PRDM4-overexpressing cells, which decreased the levels of p-AKT and upregulated the expression of PTEN, an inhibitor of the PI3K/AKT signaling pathway, at both the transcriptional and translational levels. Dual-luciferase reporter assays and qChIP assays confirmed that PRDM4 transactivated the expression of PTEN by binding to two specific regions in thePTENpromoter. Furthermore, PTEN silencing or a PTEN inhibitor rescued the cell defects induced by PRDM4 overexpression. Therefore, our data suggest that PRDM4 inhibits cell proliferation and tumorigenesis by downregulating the activity of the PI3K/AKT signaling pathway by directly transactivating PTEN expression in cervical cancer.

LGR6 activates the Wnt/β-catenin signaling pathway and forms a β-catenin/TCF7L2/LGR6 feedback loop in LGR6high cervical cancer stem cells

AbstractThe leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6) is considered to be a stem cell marker in many normal tissues and promotes tissue development, regeneration, and repair. LGR6 is also related to the initiation and progression of some malignant tumors. However, the role of LGR6 in cervical cancer has not been reported. Here, immunohistochemistry and western blotting showed that LGR6 was significantly upregulated in cervical cancer, compared with the normal cervix. By analyzing The Cancer Genome Atlas database, LGR6 was found to be correlated with a poor prognosis of cervical cancer. Then, a small population of LGR6high cells isolated by using the fluorescence-activated cell sorting exhibited enhanced properties of cancer stem cells including self-renewal, differentiation, and tumorigenicity. Moreover, RNA sequencing revealed that LGR6 was correlated with the Wnt signaling pathway and TOP/FOP, reverse transcription-PCR, and western blotting further proved that LGR6 could activate the Wnt/β-catenin signaling pathway. Interestingly, LGR6 upregulated the expression of TCF7L2 by activating the Wnt/β-catenin pathway. Then, TCF7L2 combining with β-catenin in the nucleus enhanced LGR6 transcription by binding the promoter of LGR6, which further activated the Wnt signaling to form a positive feedback loop. Thus, our study demonstrated that LGR6 activated a novel β-catenin/TCF7L2/LGR6-positive feedback loop in LGR6high cervical cancer stem cells (CSCs), which provided a new therapeutic strategy for targeting cervical CSCs to improve the prognosis of cervical cancer patients.

The deubiquitinase (DUB) USP13 promotes Mcl-1 stabilisation in cervical cancer

Abstract Protein ubiquitination is a critical regulator of cellular homeostasis. Aberrations in the addition or removal of ubiquitin can result in the development of cancer and key components of the ubiquitination machinery serve as oncogenes or tumour suppressors. An emerging target in the development of cancer therapeutics are the deubiquitinase (DUB) enzymes that remove ubiquitin from protein substrates. Whether this class of enzyme plays a role in cervical cancer has not been fully explored. By interrogating the cervical cancer data from the TCGA consortium, we noted that the DUB USP13 is amplified in ~15% of cervical cancer cases. We confirmed that USP13 expression was increased in cervical cancer cell lines, cytology samples from patients with cervical disease and in cervical cancer tissue. Depletion of USP13 inhibited cervical cancer cell proliferation. Mechanistically, USP13 bound to, deubiquitinated and stabilised Mcl-1, a pivotal member of the anti-apoptotic BCL-2 family. Furthermore, reduced Mcl-1 expression partially contributed to the observed proliferative defect in USP13 depleted cells. Importantly, the expression of USP13 and Mcl-1 proteins correlated in cervical cancer tissue. Finally, we demonstrated that depletion of USP13 expression or inhibition of USP13 enzymatic activity increased the sensitivity of cervical cancer cells to the BH3 mimetic inhibitor ABT-263. Together, our data demonstrates that USP13 is a potential oncogene in cervical cancer that functions to stabilise the pro-survival protein Mcl-1, offering a potential therapeutic target for these cancers.

Cyclin E1 overexpression sensitizes ovarian cancer cells to WEE1 and PLK1 inhibition

Abstract Cyclin E1 (CCNE1) amplification is associated with poor prognosis of ovarian carcinomas across histological subtypes. Inhibitors targeting PLK1 or WEE1 are emerging as promising therapeutic agents for cancer treatment that disrupt the critical G2/M checkpoint, leading to cancer cell death. However, biomarkers that predict the response to these inhibitors are not well defined. Here, we evaluated the efficacy of the PLK1 inhibitor, volasertib, and the WEE1 inhibitor, adavosertib, along with the biomarker potential of cyclin E1 in ovarian cancer cells. Both inhibitors suppressed the proliferation of cyclin E1-overexpressing cells to a greater extent than that of cells exhibiting low cyclin E1 expression. TP53 silencing did not increase the sensitivity to these inhibitors. In cyclin E1-overexpressing cells, PLK1 inhibition reduced the proportion of cells in the G1 phase and increased those in the G2/M and sub-G1 phases. WEE1 inhibition reduced G1 phase cells without a clear peak in the S-G2/M phase and increased the sub-G1 phase cells. Both inhibitors suppressed the growth of cyclin E1-overexpressing tumors in vivo. Taken together, cyclin E1 overexpression, regardless of TP53 status, may serve as a predictive biomarker for the efficacy of these inhibitors, offering potential personalized treatment strategies for ovarian cancer.