OC Cells Research Articles

B4GALT5 a sialylation-related genes associated with patient prognosis and immune microenvironment in ovarian cancer and pan-cancer

BackgroundOvarian cancer (OC) is the predominant primary tumor in the human reproductive system. Abnormal sialylation has a significant impact on tumor development, metastasis, immune evasion, angiogenesis, and treatment resistance. B4GALT5, a gene associated with sialylation, plays a crucial role in ovarian cancer, and may potentially affect clinicopathological characteristics and prognosis.MethodsWe conducted a comprehensive search across TIMER, GEPIA2, GeneMANIA, and Metascape to obtain transcription profiling data of ovarian cancer from The Cancer Genome Atlas (TCGA). The expression of B4GALT5 was test by immunohistochemistry. To investigate the impact of B4GALT5 on growth and programmed cell death in OC cells, we performed transwell assays and western blots.ResultsThe presence of B4GALT5 was strongly associated with an unfavorable outcome in OC. B4GALT5 significantly promoted the proliferation of OC cells. Upon analyzing gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), it was discovered that B4GALT5 played a crucial role in the extracellular matrix, particularly in collagen-containing structures, and exhibited correlations with ECM-receptor interactions, transcriptional dysregulation in cancer, as well as the interleukin-1 receptor signaling pathway. Furthermore, there is a clear link between B4GALT5 and the tumor immune microenvironment in OC. Moreover, B4GALT5 exhibits favorable expression levels across various types of cancers, including CHOL, KIRC, STAD and UCES.ConclusionIn conclusion, it is widely believed that B4GALT5 plays a pivotal role in the growth and progression of OC, with its heightened expression serving as an indicator of unfavorable outcomes. Moreover, B4GALT5 actively participates in shaping the cancer immune microenvironment within OC. This investigation has the potential to contribute significantly to a deeper understanding of the substantial involvement of B4GALT5 in human malignancies, particularly OCs.

Exosomal LINC00958 maintains ovarian cancer cell stemness and induces M2 macrophage polarization via Hedgehog signaling pathway and GLI1 protein

Long non-coding RNA (lncRNA) LINC00958 has been reported to promote many gynecological cancers, but its detailed function in OC remains unclear. Cancer stem cells (CSCs) and tumor-associated macrophages (TAMs) have been reported to participate in the occurrence and metastasis of cancers. We want to explore the effects of exosomal LINC00958 on cell stemness and macrophage polarization in OC. LINC00958 expression was first verified in OC cells and its function on cell stemness was verified by subcellular fractionation analysis, sphere formation assay and so on. Exosomal LINC00958 was secreted from OC cells and the model of M2 macrophage polarization was established to further verify the impact of exosomal LINC00958 on the cell stemness and macrophage polarization of OC cells using several mechanism experiments including flow cytometry, RNA pulldown, luciferase reporter assays and so on. LINC00958 was up-regulated in OC cells and exosomal LINC00958 enhanced the stem cell-like properties of OC cells and M2 macrophage polarization. Furthermore, LINC00958 combined with glioma-associated oncogene homolog 1 (GLI1) to activate Hedgehog pathway, thereby promoting M2 polarization. Exosomal LINC00958 maintained OC cell stemness and induced M2 polarization via the Hedgehog signaling pathway.

Ultrasound-responsive Bi2MoO6-MXene heterojunction as ferroptosis inducers for stimulating immunogenic cell death against ovarian cancer

BackgroundOvarian cancer (OC) has the highest fatality rate among all gynecological malignancies, necessitating the exploration of novel, efficient, and low-toxicity therapeutic strategies. Ferroptosis is a type of programmed cell death induced by iron-dependent lipid peroxidation and can potentially activate antitumor immunity. Developing highly effective ferroptosis inducers may improve OC prognosis.ResultsIn this study, we developed an ultrasonically controllable two-dimensional (2D) piezoelectric nanoagonist (Bi2MoO6-MXene) to induce ferroptosis. A Schottky heterojunction between Bi2MoO6 (BMO) and MXene reduced the bandgap width by 0.44 eV, increased the carrier-separation efficiency, and decreased the recombination rate of electron–hole pairs under ultrasound stimulation. Therefore, the reactive oxygen species yield was enhanced. Under spatiotemporal ultrasound excitation, BMO-MXene effectively inhibited OC proliferation by more than 90%, induced lipid peroxidation, decreased mitochondrial-membrane potential, and inactivated the glutathione peroxidase and cystathionine transporter protein system, thereby causing ferroptosis in tumor cells. Ferroptosis in OC cells further activated immunogenic cell death, facilitating dendritic cell maturation and stimulating antitumor immunity.ConclusionWe have succeeded in developing a highly potent ferroptosis inducer (BMO-MXene), capable of inhibiting OC progression through the sonodynamic-ferroptosis-immunogenic cell death pathway.Graphical

Adipocyte pyroptosis occurs in omental tumor microenvironment and is associated with chemoresistance of ovarian cancer

BackgroundOvarian carcinoma (OC) is a fatal malignancy, with most patients experiencing recurrence and resistance to chemotherapy. In contrast to hematogenous metastasizing tumors, ovarian cancer cells disseminate within the peritoneal cavity, especially the omentum. Previously, we reported omental crown-like structure (CLS) number is associated with poor prognosis of advanced-stage OC. CLS that have pathologic features of a dead or dying adipocyte was surrounded by several macrophages is well known a histologic hallmark for inflammatory adipose tissue. In this study, we attempted to clarify the interaction between metastatic ovarian cancer cells and omental CLS, and to formulate a therapeutic strategy for advanced-stage ovarian cancer.MethodsA three-cell (including OC cells, adipocytes and macrophages) coculture model was established to mimic the omental tumor microenvironment (TME) of ovarian cancer. Caspase-1 activity, ATP and free fatty acids (FFA) levels were detected by commercial kits. An adipocyte organoid model was established to assess macrophages migration and infiltration. In vitro and in vivo experiments were performed for functional assays and therapeutic effect evaluations. Clinical OC tissue samples were collected for immunochemistry stain and statistics analysis.ResultsIn three-cell coculture model, OC cells-derived IL-6 and IL-8 could induce the occurrence of pyroptosis in omental adipocytes. The pyroptotic adipocytes release ATP to increase macrophage infiltration, release FFA into TME, uptake by OC cells to increase chemoresistance. From OC tumor samples study, we demonstrated patients with high gasdermin D (GSDMD) expression in omental adipocytes is highly correlated with chemoresistance and poor outcome in advanced-stage OC. In animal model, by pyroptosis inhibitor, DSF, effectively retarded tumor growth and prolonged mice survival.ConclusionsOmental adipocyte pyroptosis may contribute the chemoresistance in advanced stage OC. Omental adipocytes could release FFA and ATP through the GSDMD-mediate pyroptosis to induce chemoresistance and macrophages infiltration resulting the poor prognosis in advanced-stage OC. Inhibition of adipocyte pyroptosis may be a potential therapeutic modality in advanced-stage OC with omentum metastasis.

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

Ovarian 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.

Sodium citrate targeting Ca2+/CAMKK2 pathway exhibits anti-tumor activity through inducing apoptosis and ferroptosis in ovarian cancer

IntroductionOvarian cancer (OC) is known for its high mortality rate. Although sodium citrate has anti-tumor effects in various cancers, its effect and mechanism in OC remain unclear. ObjectivesTo analyze the inhibitory effect of sodium citrate on ovarian cancer cells and the underlying mechanism. MethodsCell apoptosis was examined by TUNEL staining, flow cytometry, and ferroptosis was examined intracellular Fe2+, MDA, LPO assays, respectively. Cell metabolism was examined by OCR and ECAR measurements. Immunoblotting and immunoprecipitation were used to elucidate the mechanism. ResultsThis study suggested that sodium citrate not only promoted ovarian cancer cell apoptosis but also triggeredferroptosis, manifested as elevated levels of Fe2+, LPO, MDA andlipid ROS production. On one hand, sodium citrate treatment led to a decrease of Ca2+ content in the cytosol by chelatingCa2+, which further inhibited the Ca2+/CAMKK2/AKT/mTOR signaling, thereby suppressing HIF1α-dependent glycolysis pathway and inducing cell apoptosis. On the other hand, the chelation of Ca2+ by sodium citrate resulted in inactivation of CAMKK2 and AMPK, leading to increase of NCOA4-mediated ferritinophagy, causing increased intracellular Fe2+ levels. More importantly, the inhibition of Ca2+/CAMKK2/AMPK signaling pathway reduced the activity of the MCU and Ca2+ concentration within the mitochondria, resulting in an increase in mitochondrial ROS. Additionally, metabolomic analysis indicated that sodium citrate treatment significantly increased de novo lipid synthesis. Altogether, these factors contributed to ferroptosis. As expected, Ca2+ supplementation successfully reversed the cell death and decreased tumor growth induced by sodium citrate. Inspiringly, it was found that coadministration of sodium citrate increased the sensitivity of OC cells to chemo-drugs. ConclusionThese results revealed that the sodium citrate exerted its anti-cancer activity by inhibiting Ca2+/CAMKK2-dependent cell apoptosis and ferroptosis. Sodium citrate will hopefully serve as a prospective compound for OC treatment and for improvingthe efficacy of chemo-drugs.

PITPNA-AS1 Inhibits Cell Proliferation and Migration in Ovarian Cancer by Regulating the MIR-223-3p/RHOB Axis.

Ovarian cancer is a fatal gynecologic malignancy. Long non-coding RNA (lncRNA) has been verified to serve as key regulator in ovarian cancer tumorigenesis. The aim of the study was to study the functions and mechanism of lncRNA PITPNA-AS1 in ovarian cancer cellular process. Clinical ovarian cancer samples were collected and stored at an academic medical center. Cellular fractionation assays and fluorescence in situ hybridization were conducted to locate PITPNA-AS1 in OC cells. TUNEL staining, colony-forming assays, and Transwell assays were performed for evaluating cell apoptosis as well as proliferative and migratory abilities. Western blot was conducted for quantifying protein levels of epithelialmesenchymal transition markers. The binding relation between genes was verified by RNA pulldown, RNA immunoprecipitation, and luciferase reporter assays. Gene expression levels in ovarian cancer tissues and cells were subjected to RT-qPCR. PITPNA-AS1 level was downregulated in ovarian cancer samples and cells. PITPNA-AS1 overexpression contributed to the accelerated ovarian cancer cell apoptosis and inhibited cell migration, proliferation, and epithelial-mesenchymal transition process. In addition, PITPNA-AS1 interacted with miR-223-3p to regulate RHOB. RHOB knockdown partially counteracted the repressive impact of PITPNA-AS1 on ovarian cancer cell activities. PITPNA-AS1 inhibited ovarian cancer cellular behaviors by targeting miR-223-3p and regulating RHOB.

A cuproptosis-related signature predicts prognosis and indicates cross-talk with immunocyte in ovarian cancer

PurposeCuproptosis, programmed cell death by intracellular copper-mediated lipoylated protein aggregation, is involved in various tumorigenesis and drug resistance abilities by mediating the tumor microenvironment. Previous studies have demonstrated that serum copper levels are higher in OC patients than in normal subjects. However, the exact relationship between cuproptosis and ovarian cancer progression remains to be further elucidated.MethodsThe Cancer Genome Atlas (TCGA) and gene expression omnibus (GEO) datasets were utilized to establish a cuproptosis-related prognostic signature in ovarian cancer. Subsequently, the bulk RNA-seq analysis and single-cell RNA-seq analysis were used to identify the relationship between signature with immune cell infiltration, chemotherapy, and cuproptosis-related scoring (CuRS) system. Finally, the potential biological functional roles of target genes in cuproptosis were validated in vitro.ResultsBy using LASSO-Cox regression analysis to establish the cuproptosis-related prognostic model, our works demonstrated the accuracy and efficiency of our model in the TCGA (583 OC patients) and GEO (260 OC patients) OC cohorts, and the high-scoring groups showed worse survival outcomes. Notably, there were substantial differences between the high and low-risk groups in extensive respects, such as the activating transcription factors, cell pseudotime features, cell intercommunication patterns, immunocytes infiltration, chemotherapy response, and potential drug resistance. KIF26B was selected to construct a prognostic model from the identified 33 prognosis-related genes, and high expression of KIF26B predicted poorer prognosis in ovarian cancer. Ultimately, further in vitro experiments demonstrated that KIF26B participated in the proliferation and cisplatin resistance of OC cells. Knockdown of KIF26B increased the sensitivity of OC cells to elesclomol, a cuproptosis agonists.ConclusionThis study constructed a new cuproptosis-related gene signature that has a good prognostic capacity in assessing the outcome of OC patients. This study enhances our understanding of cuproptosis associated with ovarian cancer aggressiveness, cross-talk with immunocytes, and serves as a novel chemotherapy strategy.

Circ-TFRC downregulation suppresses ovarian cancer progression via miR-615-3p/IGF2 axis regulation

BackgroundOvarian cancer (OC) is a malignancy among female globally. Circular RNAs (circRNAs) are a family of circular endogenous RNAs generated from selective splicing, which take part in many traits. Former investigation suggested that circ-TFRC was abnormally expressed in breast cancer (BC). Further, the role of circ-TFRC to the progress of OC remains unclear. So, the aim of this study was to reveal the regulatory mechanism of circ-TFRC.MethodsOur team made the luciferase reporter assay to validate circ-TFRC downstream target. Transwell migration assay, 5-ethynyl-20-deoxyuridine, and cell counting kit-8 were applied to investigate both proliferation and migration. In vivo tumorigenesis and metastasis assays were performed to investigate the circ-TFRC role in OC.ResultsThe outputs elucidated that circ-TFRC expression incremented in OC cells and tissues. circ-TFRC downregulation inhibited OC cell proliferation as well as migration in in vivo and in vitro experiments. The luciferase results validated that miR-615-3p and IGF2 were circ-TFRC downstream targets. IGF2 overexpression or miR-615-3p inhibition reversed OC cell migration after circ-TFRC silencing. Also, IGF2 overexpression reversed OC cell migration and proliferation post miR-615-3p upregulation.ConclusionResults demonstrate that circ-TFRC downregulation inhibits OC progression and metastasis via IGF2 expression regulation and miR-615-3psponging.

PAX8-AS1/microRNA-25–3p/LATS2 regulates malignant progression of ovarian cancer via Hippo signaling

BackgroundOvarian cancer (OC) is a frequent malignancy of the female reproductive system. Recently, the aberrant expression of numerous lncRNAs has been confirmed as a key factor for cancer development. The regulatory role of PAX8-AS1 in some cancers has been investigated, but its role in OC progression remains unclear. This study focuses on the role and molecular mechanism of PAX8-AS1 in the malignant progression of OC. MethodsBioinformatics means were adopted to analyze the expression of PAX8-AS1, microRNA-25–3p, and LATS2 in OC tissues and the binding sites between the three. qRT-PCR was employed to determine the expression of these genes in OC cells. CCK-8, colony formation, scratch healing, and Transwell assays were used to see cell viability, proliferation, migration, and invasion, respectively. Fluorescence in situ Hybridization was performed to probe the subcellular localization of PAX8-AS1. Western blot was applied to evaluate the expression and phosphorylation levels of YAP and TAZ, and an immunofluorescence assay was used to detect the translocation of them. Dual luciferase assay was applied to validate the binding relationship between PAX8-AS1 and microRNA-25–3p, as well as between microRNA-25–3p and LATS2. ResultsPAX8-AS1 and LATS2 were lowly expressed. MicroRNA-25–3p was highly expressed in OC. PAX8-AS1 was expressed in cytoplasm and regulated LATS2 expression by sponging microRNA-25–3p. Overexpressing PAX8-AS1 can suppress the malignant behaviors of OC cells, whereas treatment with microRNA-mimic can reverse these results. In addition, the phosphorylation levels of YAP and TAZ increased upon oe-LATS2 treatment, and oe-LATS2 could promote YAP and TAZ translocate from the nucleus to cytoplasm. Rescue experiments demonstrated that sh-PAX8-AS1 fostered malignant progression of OC, which was reversed by simultaneous oe-LATS2. ConclusionIn summary, PAX8-AS1/microRNA-25–3p/LATS2 regulated the malignant progression of OC through Hippo signaling, which suggested that PAX8-AS1/microRNA-25–3p/LATS2 axis may be a novel target for OC treatment.

Mesothelin-based CAR-T cells exhibit potent antitumor activity against ovarian cancer

BackgroundOvarian cancer (OC) is characterized by its rapid growth and spread which, accompanied by a low 5-year survival rate, necessitates the development of improved treatments. In ovarian cancer, the selective overexpression of Mucin-16 (MUC16, CA125) in tumor cells highlights its potential as a promising target for developing anti-tumor therapies. However, the potential effectiveness of CAR-T cell therapy that targets MUC16 in ovarian cancer cells is unknown.MethodsThe expression of MUC16 in viable OC cells was detected using immunofluorescence and flow cytometry techniques. A MSLN-CAR construct, comprising the MUC16-binding polypeptide region of mesothelin (MSLN), a CD8 hinge spacer and transmembrane domain, 4-1BB, and CD3ζ endo-domains; was synthesized and introduced into T cells using lentiviral particles. The cytotoxicity of the resultant CAR-T cells was evaluated in vitro using luciferase assays. Cytokine release by CAR-T cells was measured using enzyme-linked immunosorbent assays. The anti-tumor efficacy of the CAR-T cells was subsequently assessed in mice through both systemic and local administration protocols.ResultsMSLN-CAR T cells exhibited potent cytotoxicity towards OVCAR3 cells and their stem-like cells that express high levels of MUC16. Also, MSLN-CAR T cells were inefficient at killing SKOV3 cells that express low levels of MUC16, but were potently cytotoxic to such cells overexpressing MUC16. Moreover, MSLN-CAR T cells delivered via tail vein or peritoneal injection could shrink OVCAR3 xenograft tumors in vivo, with sustained remission observed following peritoneal delivery of MSLN-CAR T cells.ConclusionsCollectively, these results suggested that MSLN-CAR T cells could potently eliminate MUC16- positive ovarian cancer tumor cells both in vitro and in vivo, thereby providing a promising therapeutic intervention for MUC16-positive patients.

MARCH5 promotes aerobic glycolysis to facilitate ovarian cancer progression via ubiquitinating MPC1

MARCH5 is a ring-finger E3 ubiquitin ligase located in the outer membrane of mitochondria. A previous study has reported that MARCH5 was up-regulated and contributed to the migration and invasion of OC cells by serving as a competing endogenous RNA. However, as a mitochondrial localized E3 ubiquitin ligase, the function of MARCH5 in mitochondrial-associated metabolism reprogramming in human cancers remains largely unexplored, including OC. We first assessed the glycolysis effect of MARCH5 in OC both in vitro and in vivo. Then we analyzed the effect of MARCH5 knockdown or overexpression on respiratory activity by evaluating oxygen consumption rate, activities of OXPHOS complexes and production of ATP in OC cells with MARCH5. Co-immunoprecipitation, western-blot, and in vitro and vivo experiments were performed to investigate the molecular mechanisms underlying MARCH5-enhanced aerobic glycolysis s in OC. In this study, we demonstrate that the abnormal upregulation of MARCH5 is accompanied by significantly increased aerobic glycolysis in OC. Mechanistically, MARCH5 promotes aerobic glycolysis via ubiquitinating and degrading mitochondrial pyruvate carrier 1 (MPC1), which mediates the transport of cytosolic pyruvate into mitochondria by localizing on mitochondria outer membrane. In line with this, MPC1 expression is significantly decreased and its downregulation is closely correlated with unfavorable survival. Furthermore, in vitro and in vivo assays revealed that MARCH5 upregulation-enhanced aerobic glycolysis played a critical role in the proliferation and metastasis of OC cells. Taken together, we identify a MARCH5-regulated aerobic glycolysis mechanism by degradation of MPC1, and provide a rationale for therapeutic targeting of aerobic glycolysis via MARCH5-MPC1 axis inhibition.

Abstract 2838: ZNFX1 is a master regulator and tumor suppressor in epigenetically induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer

Abstract Immunomodulatory agents are an important recent advance in cancer therapy, but utility is often limited by tumor immune evasion mechanisms. Strategic therapeutic activation of intracellular antiviral immune responses offers an opportunity to reverse immune evasion mechanisms and improve treatment outcomes. Anti-cancer agents such as, DNA methyltransferase inhibitors (DNMTis) induce re-expression of endogenous retroviruses (ERVs), leading to cytosolic double-stranded RNA (dsRNA) accumulation that activates interferon/inflammasome signaling. Moreover, poly (ADP ribose) polymerase inhibitors (PARPi) increase cytosolic dsDNA, activating stimulator of interferon (IFN) genes (STING). We reported that DNMTis in combination with PARPis induce STING-dependent signaling in a process termed pathogen mimicry response (PMR). Mitochondria (mt) are an important gateway for antiviral inflammasome signaling, but are not fully understood in cancer. Our studies of the little-known gene, NFX1-type zinc finger-containing 1 (ZNFX1), show that it acts as a master nucleic acid (dsRNA/DNA) sensor for mt gateway function. Bioinformatics analysis in primary ovarian tumors from TCGA and clinical trial RNAseq datasets, shows that increased ZNFX1 expression tracks with tumor stage and grade but inversely correlates with a mt dysfunction signature. In studies of high-grade and endometrial serous carcinoma (OC) cell lines (N=3), transfection of dsRNA/DNA mimics or DNMTi azacytidine (AZA) and PARPi (talazoparib [TAL]) treatments induce increased ZNFX1 expression and binding to mt antiviral protein (MAVs) localized on the mt outer membrane. Functional studies of mt dysfunction in OC cells, show that dsRNA/DNA as well as above viral mimicry drugs increase mt reactive oxygen species (ROS) as measured by mitosox flow cytometry. AZA and TAL treatments also increase fragmented mtDNA and oxidative mtDNA base damage, as measured by long range PCR and 8-oxoguanine (8-oxoG) ELISA assays. These drug treatments also increase release of mtDNA into the cytosol, resulting in STING-dependent inflammasome signaling and cytokine release. Notably, ZNFX1 knockout (KO) attenuates these dynamics, including in bioinformatics of RNA seq data, thus defining this gene as essential for IFN/inflammasome signaling induced by mtDNA damage. Further pathway analysis of ZNFX1 KO vs wild-type (WT) RNAseq data suggest increased proliferation and epithelial to mesenchymal transition (EMT) that was validated by in vitro and in vivo assays. Not only is ZNFX1 a master regular of the above mt dynamics, but it is a tumor suppressor in OC. Abrogating ZNFX1 expression in OC cells triggers increased cell proliferation, invasive capacity and, in-vivo, increased tumor growth. This work then suggests novel approaches to improve immune therapy responses in OC through manipulating ZNFX1. Citation Format: Lora Stojanovic, Rachel Abbotts, Kaushlendra Tripathi, Collin M. Coon, Sheng Liu, Jun Wan, Michael Topper, Stephen Baylin, Kenneth Nephew, Feyruz Rassool. ZNFX1 is a master regulator and tumor suppressor in epigenetically induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2838.

Abstract 4650: RalBP1, a novel approach for ovarian cancer therapy

Abstract Ovarian cancer (OC) is the most devastating gynecologic cancer. The ever-climbing incidence rate of OC makes it the most lethal female reproductive disorder. Triggering apoptosis is one of the potential strategies to overcome chemoresistance. Recent evidence suggests that the failure of drug-induced apoptosis may be an underlying cause of drug-resistance. Molecular mechanisms of failed apoptosis in chemoresistant OC cells include p53 mutations, abnormal expression of the Bcl2 family proteins, and upregulation of other inhibitors of apoptosis that block caspases and stabilize the mitochondrial permeability pore. RalBP1 (Ral-binding protein) is a required stress-defense protein for OC, and its specific depletion or inhibition will cause apoptosis in cancer cells expressing RalBP1. RalBP1 is a multifunctional 76 kDa protein encoded as a splice variant in the human RALBP1 gene (18p11.22). RalBP1 is an ATP dependent efflux transporter for glutathione (GSH) conjugates (GS-E) of products of oxidative stress, like 4HNE and GSH-conjugates of chemotherapy drugs. Our recent studies indicate that upon depletion/inhibition of RalBP1, malignant cells are significantly more sensitive to apoptosis than non-malignant cells, suggesting that the stress-protective, anti-apoptotic activity of RalBP1 is necessary for malignant cells. This differential sensitivity to RalBP1 translates into tumor regression, a lack of toxicity, and the prolonged survival of mice bearing subcutaneous OC cells in which RalBP1 is inhibited by anti-RalBP1 IgG or depleted using RalBP1 antisense oligonucleotides. These studies demonstrate that inhibition or depletion of RalBP1 exerts anti-neoplastic effects in OC and indicate that this GS-E and xenobiotic transporter occupies an important position in the hierarchy of stress defense mechanisms necessary for cancer cell survival. Overall, the present research focuses on hypothesis-driven studies to define novel relationships between RalBP1 and signaling pathways, and to determine the suitability of RalBP1 as an anticancer target for OC. Citation Format: Sharad S. Singhal, Sravani Ramisetty, Atish Mohanty, Prakash Kulkarni, David Horne, Sanjay Awasthi, Ravi Salgia. RalBP1, a novel approach for ovarian cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4650.

Abstract 6657: Oncolytic Vaccinia Virus Carrying OPCML Tumor Suppressor is active in Epithelial Ovarian Cancer

Abstract Objective: Epithelial Ovarian cancer (EOC) has the highest mortality rate among gynecologic malignancies, necessitating novel therapeutic approaches. Viral oncolytic immunotherapy with recombinant oncolytic vaccinia virus (OVV) offers promise for advanced-stage cancer treatment. OPCML is a GPI-anchored 3 Ig-domain tumor suppressor inactivated by somatic methylation in 87% of EOC. OVV with OPCML (OVV-OPCML) gene insertion may have potential as a therapeutic agent. This study explores the oncolytic, signaling-inhibitory, anti-angiogenic and synergistic immunotherapeutic properties of OVV-OPCML in EOC treatment, evaluating its therapeutic potential. Methods: To create OVV-OPCML, the virulent vaccinia virus Copenhagen strain F3L site was modified with OPCML cDNA, confirmed by qPCR and western blot. Oncolytic potential was assessed through in vitro and in vivo EOC cell infection with OVV-OPCML. Real-time cell analysis (RTCA) assessed cell proliferation. Xenograft tumor models in nude mice were used to evaluate in vivo oncolytic activity, while OVV-OPCML combined with anti-PD-1 treatment was tested using a syngeneic orthotopic EOC mouse model (ID8) in C57BL/6 immunocompetent mice. Serum IFN-γ and TNF-α were quantified via ELISA. Receptor Tyrosine Kinases, angiogenesis and phosphorylation together with immune markers were examined by western blotting and by multiplex immunohistochemistry (mIHC) analysis of tumor sections. Results: OVV-OPCML selectively infected OC cells, leading to viral replication and tumor cell death, while sparing normal ovarian cells in-vitro. OPCML protein expression increased post-infection. OVV-OPCML reduced ERBB2, EGFR, and FGFR1 expression in SKVO3 OV cells. Intratumoral or intraperitoneal (IP) OVV-OPCML administration in human ovarian cancer nude mouse models reduced tumor growth and improved survival. IP in-vivo OVV-OPCML in combination with anti-PD-1 treatment in vivo, in the IP ID8 syngeneic model demonstrated synergy with survival improvement. H&E staining showed tumor cell damage without significant harm to normal tissues. Protein expression of ERBB2, EGFR, FGFR1, and their phosphorylated forms decreased. CD31, VEGFR2, p-VEGFR2 (Tyr1175), and p-VEGFR2 (Tyr1214) downregulation suggested tumor angiogenesis disruption. OVV-OPCML treatment elevated serum TNF-α and INF-γ levels and increased CD8+ T cells and PD-L1 expression in OVV-OPCML treated syngeneic tumors. Conclusions: OPCML gene enhances OVV selectivity against OC cells through direct oncolysis and OPCML protein production, inhibiting specific RTKs, suppressing angiogenesis, and activating antitumor immune responses synergistically with anti-PD-1. OVV-OPCML therapy shows promise as a targeted strategy for ovarian cancer treatment. Citation Format: Yong Tang, Yingzhao Liu, Mingjing Deng, Fei Huang, Jiahu Wang, Paul Blake, Hani Gabra, Qi Wang. Oncolytic Vaccinia Virus Carrying OPCML Tumor Suppressor is active in Epithelial Ovarian Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6657.

Abstract PR-006: A novel bispecific NK cell engager targeting FSHR and Siglec-7 displays potent anti-tumor immunity against ovarian cancer

Abstract Ovarian cancer (OC) is the deadliest gynecologic malignancy, with the highest mortality among cancers of the female reproductive system. Because of the close interaction between the OC cells and tumor microenvironment (TME), development of approaches which not only target tumor cells but also can maintain their anti-tumor function in this TME is of high importance. Uniquely, Follicle stimulating hormone receptor (FSHR) represents such an important target with specificity for OC and ovarian granulosa cells. We have recently developed monoclonal antibodies (mAbs) that specifically recognize human FSHR on the surface of multiple OC. We characterized them, selected potent anti-FSHR mAb: D2AP11 for detailed investigation and immune engineering in the form of a bispecific T cell engager (D2AP11-TCE) (Bordoloi et al., JCI Insight, 2022). Since most OCs are extremely aggressive and have restricted treatment choices, engaging additional immune effector components may further help. We have developed mAbs against PAN NK marker; Siglec 7 in humanized mice. We identified several Siglec-7 mAbs through high throughput screening and down-selected the pertinent clones. By using a highly potent anti-Siglec-7 clone DB7.2, we have developed a novel NK cell engager (NKCE): DB7.2xD2AP11 NKCE, by linking two antibody binding fragments (scFVs) arranged to bind FSHR, the OC antigen and hman Siglec-7, the NK marker. The NKCE was efficiently expressed in vitro and showed potent binding to both FSHR overexpressing K562 cells as well as Siglec-7 overexpressed HEK293T cells. This novel NKCE resulted in robust and specific killing of multiple OC targets in vitro in the presence of human PBMCs or NK cells, from multiple donors. The OC targets evaluated for killing by the NKCE include high grade serous adenocarcinoma, ovarian clear cell adenocarcinoma and ovarian cystadenocarcinoma lines which carry different mutations; BRCA1&2, AKT, TP53, PIK3CA, BRAF etc. and exhibit resistance against a wide array of cancer drug targets; PARP, HSP90, HDAC, MTORC, DNA alkylating agents, EGFR, PI3K, and WEE1, among others. DB7.2xD2AP11 NKCE displayed killing with EC50-142.87 pM and 236.6 pM against target OVISE-FSHR and OVCAR3 cells respectively, indicating its potency. Further, DNA encoded DB7.2xD2AP11 NKCE significantly attenuated the tumor burden in OVCAR3-FSHR and OVISE challenged humanized mice model. Additionally, this novel NKCE significantly enhanced the median survival of OVCAR3-FSHR challenged mice, suggesting in vivo potency. These data support further research on this novel NKCE as crucial tools for treating OC as well as targeted therapies for other malignancies. Of note, it is the first study of the development of Siglec-7 or any Siglec family member as a bispecific immune engager for targeted immunotherapy. Citation Format: Devivasha Bordoloi, Abhijeet J. Kulkarni, Opeyemi S. Adeniji, Pratik S. Bhojnagarwala, Shushu Zhao, Candice Ionescu, Mohamed bdel-Mohsen, David B. Weiner. A novel bispecific NK cell engager targeting FSHR and Siglec-7 displays potent anti-tumor immunity against ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-006.

Abstract B068: FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models

Abstract The unfavorable prognosis of high-grade serous ovarian cancer, HGSOC, may be driven by a small subset of ovarian cancer stem cells (OCSC), which are endowed with tumor-initiating ability and are intrinsically resistant to cytotoxic treatments. These characteristics are sustained by signals coming from the tumor microenvironment (TME). As a common site of HGSOC presentation is the omentum, we partially recapitulated the architecture of human omentum in vitro using 3D co-cultures of primary human omental fibroblasts and mesothelial cells. Patient-derived HGSOC cells were then integrated in these organotypic models, either as bulk cancer cells or as OCSC from the same patients. After co-culture in this model, malignant cells were re-isolated by FACS sorting and subjected to whole-transcriptome RNA sequencing, comparing them to control cells that have not been in contact with the in vitro TME. Our data revealed transcriptional reprogramming induced by the in vitro TME in HGSOC cells. We found a number of pathways differentially regulated in the bulk population of OC cells vs OCSC, shedding light on the impact of the TME on OC stemness. Moreover, we identified a panel of transcription factors (TFs) specifically altered by the in vitro TME in OCSC. These TF-regulated gene networks represent OCSC vulnerabilities and, hence, potential targets in relapsing tumors. Among these, we focused on FOXM1, a TF with pleiotropic functions in cancer cells, whose activity is altered in 84% of ovarian cancer patients. The FOXM1 pathway was strongly induced by the in vitro TME specifically in OCSC. Mechanistically, the contact with the in vitro TME triggered a FAK/YAP axis which, in turn, accounted for the induction of FOXM1 and its downstream target genes. Moreover, the pharmacological inhibition of FOXM1 significantly impaired OCSC fitness, supporting, on one hand, the key role of TME in orchestrating OC stemness and suggesting, on the other, a new approach for the eradication of OCSC from the omental niche. Overall, our patient-derived organotypic platform offers the opportunity to unravel the impact of the TME on OCSC at the molecular level in experimental models with clinical relevance and can be exploited to design innovative HGSOC-eradicating treatments. Citation Format: Chiara Battistini, Hilary A. Kenny, Valentina Nieddu, Valentina Melocchi, Alessandra Decio, Beatrice Malacrida, Alessia Gatto, Fabrizio Bianchi, Raffaella Giavazzi, Nicoletta Colombo, Frances R. Balkwill, Ernst Lengyel, Ugo Cavallaro. FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B068.

Combined inhibition of HER2 and VEGFR synergistically improves therapeutic efficacy via PI3K-AKT pathway in advanced ovarian cancer

BackgroundOvarian cancer (OC) is a prevalent malignancy in the female reproductive system, and developing effective targeted therapies for this disease remains challenging. The aim of this study was to use clinically-relevant OC models to evaluate the therapeutic effectiveness of RC48, an antibody-drug conjugate (ADC) targeting HER2, either alone or in combination with the VEGFR inhibitor Cediranib Maleate (CM), for the treatment of advanced OC.MethodsOC tumor specimens and cell lines were analyzed to determine HER2 and VEGFR expression by Western blot, immunocytochemistry and immunofluorescence. Moreover, the OC cell lines, cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models were treated with RC48 and/or CM and then subjected to cell proliferation, viability, apoptosis, and tumor growth analyses to evaluate the feasibility of combination therapy for OC both in vitro and in vivo. Additionally, RNA-Seq was performed to investigate the critical mechanism underlying the combination therapy of RC48 and CM.ResultsOur results demonstrated that RC48 alone effectively targeted and inhibited the growth of HER2-positive OC tumors in both cell lines and PDX models. Furthermore, the combination of RC48 and CM synergistically induced tumor regression in human OC cell lines, as well as CDX and PDX models. Mechanistically, we observed that the combination treatment inhibited the growth of OC cells involved inducing apoptosis and suppressing cell motility. RNA-seq analysis provided further mechanistic insights and revealed that co-administration of RC48 and CM downregulated multiple cancer-related pathways, including the AKT/mTOR pathway, cell cycle, and cell proliferation. Notably, our data further confirmed that the PI3K-AKT pathway played a key role in the inhibition of proliferation triggered by combinational treatment of RC48 and CM in OC cells.ConclusionsThese findings provide a preclinical framework supporting the potential of dual targeting HER2 and VEGFR as a promising therapeutic strategy to improve outcomes in patients with OC.

Upregulation of CALD1 predicted a poor prognosis for platinum-treated ovarian cancer and revealed it as a potential therapeutic resistance target

BackgroundOvarian cancer (OC) has the worst prognosis among gynecological malignancies, most of which are found to be in advanced stage. Cell reduction surgery based on platinum-based chemotherapy is the current standard of treatment for OC, but patients are prone to relapse and develop drug resistance. The objective of this study was to identify a specific molecular target responsible for platinum chemotherapy resistance in OC.ResultsWe screened the protein-coding gene Caldesmon (CALD1), expressed in cisplatin-resistant OC cells in vitro. The prognostic value of CALD1 was evaluated using survival curve analysis in OC patients treated with platinum therapy. The diagnostic value of CALD1 was verified by drawing a Receiver Operating Characteristic (ROC) curve using clinical samples from OC patients. This study analyzed data from various databases including Gene Expression Omnibus (GEO), Human Protein Atlas (HPA), The Cancer Cell Line Encyclopedia (CCLE), The Cancer Genome Atlas (TCGA), GEPIA 2, UALCAN, Kaplan–Meier (KM) plotter, LinkedOmics database, and String. Different expression genes (DEGs) between cisplatin-sensitive and cisplatin-resistant cells were acquired respectively from 5 different datasets of GEO. CALD1 was selected as a common gene from 5 groups DEGs. Online data analysis of HPA and CCLE showed that CALD1 was highly expressed in both normal ovarian tissue and OC. In TCGA database, high expression of CALD1 was associated with disease stage and venous invasion in OC. Patients with high CALD1 expression levels had a worse prognosis under platinum drug intervention, according to Kaplan–Meier (KM) plotter analysis. Analysis of clinical sample data from GEO showed that CALD1 had superior diagnostic value in distinguishing patients with platinum "resistant" and platinum "sensitive" (AUC = 0.816), as well as patients with worse progression-free survival (AUC = 0.741), and those with primary and omental metastases (AUC = 0.811) in ovarian tumor. At last, CYR61 was identified as a potential predictive molecule that may play an important role alongside CALD1 in the development of platinum resistance in OC.ConclusionsCALD1, as a member of cytoskeletal protein, was associated with poor prognosis of platinum resistance in OC, and could be used as a target protein for mechanism study of platinum resistance in OC.

SETDB1 promotes progression through upregulation of SF3B4 expression and regulates the immunity in ovarian cancer

BackgroundOvarian cancer (OC) is the most lethal gynecologic malignant tumour. The mechanism promoting OC initiation and progression remains unclear. SET domain bifurcated histone lysine methyltransferase 1(SETDB1) acts as an oncogene in a variety of tumours. This study aims to explore the role of SETDB1 in OC.MethodsGEO, TCGA, CSIOVDB and CPTAC databases jointly analysed SETDB1 mRNA and protein expression. Effect of SETDB1 expression on the clinical prognosis of OC patients was analysed through online Kaplan‒Meier plotter and CSIOVDB database. Then, the effect of SETDB1 in OC cells progression and mobility was examined using MTT, EdU, colony formation and transwell assay. Additionally, Cistrome DB database was used to visualize the binding of SETDB1 protein and splicing factor 3b subunit 4 (SF3B4) promoter, and dual-luciferase reporter gene assay was performed to confirm the interaction. Finally, bioinformatics analysis was employed to reveal the relationship between SETDB1 and the microenvironment of OC.ResultsIn the present study, we found that SETDB1 was obviously upregulated in OC and its overexpression predicted poor prognosis of OC patients. Then, we verified that SETDB1 promoted the progression and motility of OC cells in vitro. Knockdown of SETDB1 had the opposite effect. Further research showed that SETDB1 acted as a transcription factor to activate SF3B4 expression. SF3B4 knockdown impaired the effect of SETDB1 to promote the proliferative capacity and motility of OC cells. Finally, the results of bioinformatics analysis confirmed that SETDB1 regulated the immune microenvironment of ovarian cancer.ConclusionSETDB1 promoted ovarian cancer progression by upregulating the expression of SF3B4 and inhibiting the tumour immunity. SETDB1 may be a promising prognostic and therapeutic marker for OC.