High-grade Serous Carcinoma Cells Research Articles

Concurrent RB1 Loss and BRCA Deficiency Predicts Enhanced Immunologic Response and Long-term Survival in Tubo-ovarian High-grade Serous Carcinoma.

The purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC). RB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss. RB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial-mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. Co-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

Podocalyxin promotes the formation of compact and chemoresistant cancer spheroids in high grade serous carcinoma

High grade serous carcinoma (HGSC) metastasises primarily intraperitoneally via cancer spheroids. Podocalyxin (PODXL), an anti-adhesive transmembrane protein, has been reported to promote cancer survival against chemotherapy, however its role in HGSC chemoresistance is unclear. This study investigated whether PODXL plays a role in promoting chemoresistance of HGSC spheroids. We first showed that PODXL was expressed variably in HGSC patient tissues (n = 17) as well as in ovarian cancer cell lines (n = 28) that are more likely categorised as HGSC. We next demonstrated that PODXL-knockout (KO) cells proliferated more slowly, formed less compact spheroids and were more fragile than control cells. Furthermore, when treated with carboplatin and examined for post-treatment recovery, PODXL-KO spheroids showed significantly poorer cell viability, lower number of live cells, and less Ki-67 staining than controls. A similar trend was also observed in ascites-derived primary HGSC cells (n = 6)—spheroids expressing lower PODXL formed looser spheroids, were more vulnerable to fragmentation and more sensitive to carboplatin than spheroids with higher PODXL. Our studies thus suggests that PODXL plays an important role in promoting the formation of compact/hardy HGSC spheroids which are more resilient to chemotherapy drugs; these characteristics may contribute to the chemoresistant nature of HGSC.

Abstract 1254: Integration of human omics analysis and new syngeneic tumor organoid models reveals that aberrant RAS/PI3K crosstalk triggers refractoriness in high-grade serous carcinoma

Abstract Refractory high-grade serous carcinoma (HGSC) is one of the most severe clinical problems in gynecology, but we have barely grasped the underlying mechanism of aggressiveness and chemo-resistance. Previous study has shown that homologous recombination genes, BRCA1/2-unmutated HGSC patients possess the potential to be in refractory condition, and their primary response to chemotherapy was heterogenous; thus, there can be subtype-specific determinant factors hatching refractoriness in BRCA1/2-unmutated HGSC patients. To explore the determinant factors, we performed gene set variation analysis and comprehensive log-rank tests on TCGA advanced serous ovarian cancer datasets. As a result, regulation of small GTPase signaling prominently correlated with poor prognosis uniquely in BRCA1/2-unmutated HGSC patients (p-value < 0.001), and eventually, pathway clustering analysis by variational Bayesian Gaussian mixture model discovered aberrant RAS/PI3K crosstalk as peculiar poor prognostic signature of BRCA1/2-unmutated HGSC patients. To elucidate the mechanism of poor progression triggered by RAS/PI3K, we utilized our syngeneic HGSC organoid models newly established from murine fallopian tube epithelium by making Myc overexpressing, and knocking out Trp53 and Rb1; as a supplement, the propriety of these genetic alterations to develop HGSC models was backed up by genetic profiles of high-grade serous ovarian cancer patients in AACR Project GENIE. After that, we knocked out Nf1 and Pten whose deletions were detected relatively with high frequency in HGSC to induce aberrant RAS/PI3K crosstalk in the primitive HGSC model. In-vivo evaluation and growth comparison indicated that aberrant RAS/PI3K crosstalk transformed Brca1/2-unmutated HGSC modeling cells into aggressive phenotypes such as poor survival (p-value < 0.01) and high growth rate (p-value < 0.001). Chemo-sensitivity assay also showed that the genetic manipulation made Brca1/2-unmutated HGSC modeling cells more tolerant to anti-tumor agents like carboplatin (p-value < 0.001), paclitaxel (p-value < 0.01), and olaparib (p-value < 0.01). To investigate biological processes that induce the aggressive and chemo-resistant phenotype, we performed gene set enrichment analysis among our HGSC models. The result showed that aberrant RAS/PI3K crosstalk induces cell cycle acceleration and downregulation of apoptosis in the HGSC cells, which backed up aggressive phenotype and chemo-resistance in our HGSC models. In conclusion, aberrant RAS/PI3K crosstalk transforms Brca1/2-unmutated HGSC modeling cells more aggressive and chemo-resistant, which was well recapitulated by our HGSC modeling organoids. The integration of our computational approach and experimental models will lead us to detect peculiar therapeutic targets against refractory HGSC. Citation Format: Tomohiro Tamura, Kenta Masuda, Shimpei Nagai, Keiyo Imaeda, Juntaro Yamasaki, Eiji Sugihara, Hiroyuki Nobusue, Yuji Otsuki, Rui Yamaguchi, Kazuhiro Sakurada, Wataru Yamagami, Hideyuki Saya, Osamu Nagano. Integration of human omics analysis and new syngeneic tumor organoid models reveals that aberrant RAS/PI3K crosstalk triggers refractoriness in high-grade serous carcinoma [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 1254.

Abstract B069: Bone marrow-derived adipocytes promote ovarian cancer progression and metastasis

Abstract B069: Bone marrow-derived adipocytes promote ovarian cancer progression and metastasis

Abstract A079: G-CSF released from ovulation recruits and activates neutrophils in the peritoneum to aid transformation, seeding, and invasion of high-grade serous carcinoma

Abstract The incidence and mortality burden of ovarian high-grade serous carcinoma (HGSC) is increasing rapidly in Taiwan and many urbanized countries. The etiology of HGSC, especially that responsible for the rapid and advanced seeding of the tumor in the peritoneum, which causes late diagnosis and poor prognosis, is elusive. Epidemiological and molecular studies have demonstrated incessant ovulation is the main culprit of ovarian cancer. Our previous studies also elucidated some transforming factors in the ovulatory follicular fluid (FF). We unveiled the oncogenic activities of ovulation on the malignant transformation of fallopian tube fimbria epithelial cells (FTEC), the primary origin of HGSC. We hypothesized that ovulation could also promote the intraperitoneal spreading of tube originated HGSC. First, in both human and mouse specimens, we observed a dramatic influx of neutrophils in the peritoneal fluid during ovulation after LH surge. Ovulatory FF from IVF women, when injected to the mouse peritoneum cavity, readily induced neutrophil influx and activated neutrophil extracellular trap (NET) formation (NETosis). Among the different neutrophil activators in FF, we found G-CSF is responsible for the ROS and PAD4 increases and histone citrullination in NETosis. This FF-GCSF-induced NETosis increases anchoring independent growth (AIG), cell adhesions, and matrigel invasion of STIC-mimicking FTEC and HGSC cells. Interestingly, while the AIG-promoting activity of the ovulation-activated NET showed cell autonomous, the adhesion and invasion activities were non-autonomous conferred by the conditioned medium of the activated neutrophils. Shown by a FF plus tumor cell i.p. coinjection mouse xenograft model, intraperitoneal seeding was increased by the coinjection and was inhibited by the inhibitor (GSK) of NETosis. Taken together, we disclosed an unprecedented intraperitoneal neutrophil influx/activation activity of ovulation. This activity, presumably acting as an antimicrobial to prevent ascending infection during fertilization, is utilized to aid the peritoneal seedings of tubal-origin HGSC. Citation Format: Hsuan Shun Huang, Tang-Yuan Chu. G-CSF released from ovulation recruits and activates neutrophils in the peritoneum to aid transformation, seeding, and invasion of high-grade serous carcinoma [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 A079.

Abstract PR-010: Dual targeting of IDO1/TDO2 inhibits tumor progression and attenuates the immune suppressive tumor microenvironment

Abstract Introduction: High-grade serous carcinoma (HGSC) tumors of the ovary, fallopian tube, or peritoneum are predicted to be immunogenic since the presence of tumor-infiltrating lymphocytes conveys a better prognosis. However, the efficacy of immunotherapies has been limited due to the immune-suppressed tumor immune microenvironment (TIME), which is established through tumor metabolism and immune-suppressive metabolites that directly affect immune cell function by depleting nutrients and activating immune-suppressive transcriptional programs. The tryptophan (TRP) catabolism pathway is a critical regulator of immune-suppressed TIME; however, clinical trials targeting the pathway to date have demonstrated limited response. Note there are two structurally distinct rate-limiting TRP catabolizing enzymes, Indoleamine 2,3-Dioxygenase (IDO1) and Tryptophan 2,3-Dioxygenase (TDO2), that evolved separately to catabolize tryptophan into Kynurenine (KYN). Most TRP catabolism inhibitors (e.g., Epacadostat) have primarily targeted IDO1, which may explain the mild anti-tumor effects of Epacadostat. Thus, we hypothesize that targeting both enzymes involved in TRP catabolism will alleviate the immune-suppressed TIME. Methods: We assessed primary human ascites fluid (IRB #07-0395) with global untargeted metabolomics. Using a panel of HGSC cell lines, we assessed genetic (small hairpin-mediated knockdown and overexpression) and pharmacologic (Epacadostat [IDO1 inhibitor], AT-0174 [IDO1/TDO2 inhibitor], 66cl4 [TDO2 inhibitor], StemRegenin 1 [AhR inhibitor]) modulation of IDO1, TDO2, and aryl hydrocarbon receptor (AhR, signal transducer of KYN). Proliferation assays in 2D and 3D. We examined the response of IDO1/TDO2 inhibition in two syngeneic murine models (ID8 Tp53-/- and BR-Luc Tp53-/-, Brca1-/-, myr-AKT, MYC). Multispectral immunohistochemistry (mIHC) and flow cytometry defined the TIME. Results: Elevated KYN levels are correlated to an inflamed TIME. The depletion of TRP and increased KYN negatively affect the anti-tumor immune response. HGSC cell lines and clinical outcomes depend more on TDO2 than IDO1. In contrast to knocking down IDO1 and AhR, TDO2 knockdown inhibited HGSC cell growth, and overexpression of TDO2 induced cancer cell proliferation. Targeting both IDO1 and TDO2 significantly inhibited TRP catabolism. Notably, only the combined IDO1/TDO2 inhibition resulted in the downregulation of PD-L1. In a syngeneic HGSC murine model, a first-in-class orally available dual IDO1/TDO2 inhibitor (AT-0174) significantly inhibited tumor progression, reduced tumor-associated macrophages, and reduced expression of immune-suppressive proteins, including PD-L1, on immune and tumor cells. Conclusions: These studies demonstrate the importance of TDO2 and the potential of AT-0174 to overcome an immune-suppressed TIME. The findings represent a novel approach to targeting TRP catabolism that may improve tumor response rates to immune therapy. Citation Format: Lyndsey Crump, Jessica Floyd, Elizabeth Woodruff, Mike Bickerdike, Li-Wei Kuo, Miriam Post, Silviu Itescu, Jennifer Richer, Benjamin G. Bitler. Dual targeting of IDO1/TDO2 inhibits tumor progression and attenuates the immune suppressive tumor microenvironment [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-010.

Abstract A081: Chromobox 2 (CBX2) drives an immunosuppressive tumor microenvironment in high grade serous carcinoma (HGSC)

Abstract Introduction: Epigenetic reader and component of Polycomb Repressor Complex 1 (PRC1), Chromobox 2 (CBX2) is expressed in greater than 75% of high-grade serous carcinoma (HGSC) tumors. Increased CBX2 expression conveys a poorer disease-free and overall survival. CBX2 knockdown leads to increased chemotherapy sensitivity and attenuates stem-like properties, while CBX2 overexpression is sufficient to promote chemoresistance. In an HGSC immune competent murine model, knockdown of CBX2 decreased tumor progression. We sought to explore the impact of modulation of CBX2 on the tumor immune microenvironment (TIME), understanding that TIME plays a critical role in disease progression and development of therapy resistance. Methods: We performed an exploration of existing data sets, including The Cancer Genome Atlas (TCGA), TIMERv2, and Carcinoma EcoTyper. We used HGSC cell lines (OVCAR4 and PEO1) with knockdown of CBX2 and RNA-sequencing. KEGG pathway analysis performed. A syngeneic ID8 mouse model was completed with shCBX2 compared to shControl. Nanostring Immuno-Oncology Panel and Multispectral Immunohistochemistry (mIHC) were completed to evaluate the TIME. Cell lines with genetic CBX2 modulation were utilized to create spheroids to recapitulate HGSC growth and confocal microscopy was employed to evaluate macrophage recruitment. Monocyte recruitment and macrophage polarization were evaluated using flow cytometry. Results: We found that existing data sets demonstrate a shift in TIME dependent on up or downregulation of CBX2. For instance, high CBX2 is associated with decreased CD8+ T cells (14.69% vs. 4.89%, p=0.0032) and increased undifferentiated macrophages (14.77 vs. 29.93%, p=0.006). Also, CBX2 is a defining feature of the Cell State Type 6 Carcinoma EcoType, which is associated with poor prognosis. RNA sequencing identified an association between CBX2 and CCL2, a cytokine known to recruit immunosuppressive macrophages. Pathway analysis of differentially expressed genes demonstrates an enrichment of several immune signatures. CBX2 knockdown in the ID8 syngeneic mouse model led to decreased tumor progression compared to control (11.50 mg. vs 51.63 mg, p=0.0001). NanoString analysis of the specimens suggest a shift in immune cell composition, with an increase in macrophages. mIHC defined the spatial relationships in this composition and confirmed increase in macrophage infiltration (28.64% vs. 34.60%, p=0.0231). Confocal microscopy and co-culture experiments to explore monocyte recruitment, found that both knockdown and overexpression of CBX2 lead to increased recruitment and infiltration of macrophages. Flow cytometry of macrophages cultured with CBX2 overexpressing cells showed an increase in M2-like (CD206+) macrophages and decreased phagocytosis activity. Conclusion: Increased CBX2 expression leads to increased recruitment and polarization of M2-like macrophages leading to an immunosuppressive TIME. Targeting of CBX2 may serve to modulate the TIME to enhance the efficacy of immune therapies. Citation Format: Lindsay W. Brubaker, Ritsuko Iwanaga, Tomomi M Yamamoto, Elizabeth R. Woodruff, Nicole Marjon, Siddhartha Mitra, Benjamin G. Bitler. Chromobox 2 (CBX2) drives an immunosuppressive tumor microenvironment in high grade serous carcinoma (HGSC) [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 A081.

Abstract C055: Withaferin A offers novel targeted immunotherapy against high-grade serous ovarian carcinoma

Abstract Background: Tumor-induced immunosuppression and cell proliferation are the hallmarks of progression of malignant tumors including high-grade serous ovarian carcinoma (HGSOC), a fatal malignancy of women. Natural killer (NK) cell plays critical roles in killing tumor cells in the tumor vicinity as it does not require antigen presentation. Our earlier studies showed the frequency of tumor infiltrating NK cells is significantly low as compared with that of tumor stroma suggesting that tumor-induced factors may be involved in inhibition of NK cell infiltration, and the tumor continue to metastasize. Cytokines including IL-10/IL-15 are used to stimulate NK cells and tumor continuously secretes IL-10 or IL-15 and it is possible that excessive exposure to these cytokines induces cytokine inducible SH2-containing protein (CISH), a marker of NK cell exhaustion. We have shown earlier that root powder of Ashwagandha (ASH), an anti-stress natural herb improved NK cell population and reduced tumor progression. The goal of this study was to determine whether Withaferin A (WFA), an active ingredient of ASH, targets the induction of CISH or cyclin D1 (CCND1), a stimulator cell growth and prevented tumor progression. Materials & methods: Study-1: An exploratory study was conducted with clinical specimens which included normal ovaries (n=5) and ovarian high grade serous carcinoma (HGSC) at both early and late stages (n=5 each) and expression levels of CISH and CCND1 were examined. Study-2: A prospective study using the laying hen model of OVCA supplemented with or without 2% dietary ASH root powder for 90 days (n=7 each;normal, early and late-stage). Normal and tumor ovarian tissues were collected at the end of the study and used for analysis. Both the clinical and hen specimens were used for immunohistochemistry, western blotting and gene expression assays to determine expression levels of CISH and CCND1. In vitro studies were also conducted with WFA on the OVCAR3, an HGSC cell line, where multiple doses were tested, and expression of markers previously mentioned were examined. Data were collected and analyzed by ANOVA and unpaired t-test and considered significant when P<0.05. Results: Frequency of CISH-expressing cells in both clinical and hen specimens in normal, early and late-stages were localized. Compared to normal, increased expression of both CISH and CCND1 was seen in early-stages and increased further through late stages. Similarly, strong expression of both CISH and CCND1 was seen in proteomic and genomic studies. ASH supplementation is hens compared to untreated showed a significant decrease (P<0.001) in CISH expression as well as in CCND1 gene expression studies. In vitro studies WFA treatment significantly decreased expression of both markers (P<0.05). Conclusion: The results of this study suggest that both CISH and CCND1 are involved in progression of OVCA and dietary supplementation with ASH or treatment with WFA may be an effective bi-targeted immunotherapy against HGSOC. Support: Ovarian Cancer Spore, Roswell Park Citation Format: Jasmin C. Acosta, Animesh Barua. Withaferin A offers novel targeted immunotherapy against high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C055.

Abstract A054: Interleukin-16 is a novel target to prevent age-associated epigenetic changes leading to malignant transformation associated with ovarian high-grade serous carcinoma (HGSOC)

Abstract Low-grade chronic inflammation and persistent oxidative stress are hallmarks of malignant transformation. High-grade serous ovarian carcinoma (HGSOC) is a fatal malignancy of women, and the median age of its incidence is 60-65 years. Our earlier studies showed enhanced expression of interleukin 16 (IL-16, an inflammatory cytokine), its receptor CD9, and glucose-regulated protein (GRP78, a marker of cellular stress and inhibitor of DNA damage repair mechanism) in postmenopausal ovaries and patients with HGSOC as compared with premenopausal subjects. It is possible that these age-associated increases in IL-16, CD9, and GRP78 expression may contribute to HGSOC-related epigenetic modifications of gH2A histone family member X (gH2AX, a marker for double-stranded DNA breaks), and histone deacetylase 1 (HDAC1), involved in DNA repair. The goal of this study was to examine if age-associated such changes lead to HGSOC-associated malignant transformation and if so, to determine mechanisms involved in such transformation. Materials and Methods: Exploratory study: Archived tissue specimens were selected for immunohistochemical, immunoblotting, genomic studies, and immunoassays based on the review of their hematoxylin and eosin staining and final pathological reports. Specimens were grouped as normal premenopausal women (30-50 years old), normal postmenopausal women (55-85 years old), and ovarian high-grade serous carcinoma (HGSC). In vitro study: human ovarian surface epithelium (HOSE) cells were treated with or without recombinant human IL-16 (rhIL-16) and recombinant human GRP78 (rhGRP78), and small interfering RNA-GRP78 (siGRP78) for 24 hours or 48 hours. Nuclear fractionations from treated- or untreated HOSE cells or OVCAR3 (HGSOC cell line) cells were extracted. Results: Compared to premenopausal women, a subset of postmenopausal women exhibited significantly elevated expression (P<0.01) of inflammatory markers IL-16, and its receptor CD9, as well as a marker of oxidative stress (GRP78). Additionally, markers of epigenetic changes, including gH2AX and HDAC1, presented similar patterns of increase in their expression. These observations were also consistent in HGSOC patients. Enhanced expression of GRP78, gH2AX, HDAC1, and CD9 in rhIL-16 treated HOSE cells like OVCAR3 cells and their reduction in siGRP78 treated HOSE cells suggests that age-associated increase in IL-16 in postmenopausal women may be a risk factor for malignant transformation and GRP78 may be a mechanism for IL-16-induced HGSOC development. Thus, IL-16 and GRP78 represent novel targets that may be used to develop novel targeted therapies to prevent HGSOC. Expression of gH2AX and HDAC1 increased in rhGRP78-treated HOSE cells. Conclusion: The results of this study suggest that age-associated increases in IL-16 (a marker of chronic inflammation) and GRP78 (a marker of oxidative stress) may contribute to the predisposition for HGSOC-associated malignant transformation. IL-16 and GRP78 offer potential targets for the prevention of HGSOC in women. Support: The Portes Foundation Citation Format: Jessica Ramirez, Animesh Barua. Interleukin-16 is a novel target to prevent age-associated epigenetic changes leading to malignant transformation associated with ovarian high-grade serous carcinoma (HGSOC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A054.

TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

A crucial requirement for metastasis formation in ovarian high-grade serous carcinoma (HGSC) is the disruption of the protective peritoneal mesothelium. Using co-culture systems of primary human cells, we discovered that tumor-associated NK cells induce TRAIL-dependent apoptosis in mesothelial cells via death receptors DR4 and DR5 upon encounter with activated Tcells. Upregulation of TRAIL expression in NK cells concomitant with enhanced cytotoxicity toward mesothelial cells was driven predominantly by T-cell-derived TNFα, as shown by affinity proteomics-based analysis of the Tcell secretome in conjunction with functional studies. Consistent with these findings, we detected apoptotic mesothelial cells in the peritoneal fluid of HGSC patients. In contrast to mesothelial cells, HGSC cells express negligible levels of both DR4 and DR5 and are TRAIL resistant, indicating cell-type-selective killing by NK cells. Our data point to a cooperative action of T and NK in breaching the mesothelial barrier in HGSC patients.

Insulin-like growth factor (IGF) and hepatocyte growth factor (HGF) in follicular fluid cooperatively promote the oncogenesis of high-grade serous carcinoma from fallopian tube epithelial cells: Dissection of the molecular effects.

Incessant ovulation is believed to be a potential cause of epithelial ovarian cancer (EOC). Our previous investigations have shown that insulin-like growth factor(IGF2) and hepatocyte growth factor(HGF) in the ovulatory follicular fluid (FF) contributed to the malignant transformation initiated by p53 mutations. Here we examined the individual and synergistic impacts of IGF2 and HGF on enhancing the malignant properties of high-grade serous carcinoma (HGSC), the most aggressive type of EOC, and its precursor lesion, serous tubal intraepithelial carcinoma (STIC). In a mouse xenograft co-injection model, we observed that FF co-injection induced tumorigenesis of STIC-mimicking cells, FE25. Co-injection with IGF2 or HGF partially recapitulated the tumorigenic effects of FF, but co-injection with both resulted in a higher tumorigenic rate than FF. We analyzed the different transformation phenotypes influenced by these FF growth signals through receptor inhibition. The IGF signal was necessary for clonogenicity, while the HGF signal played a crucial role in the migration and invasion of STIC and HGSC cells. Both signals were necessary for the malignant phenotype of anchoring-independent growth but had little impact on cell proliferation. The downstream signals responsible for these HGF activities were identified as the tyrosine-protein kinase Met (cMET)/mitogen-activated protein kinase and cMET/AKT pathways. Together with the previous finding that the FF-IGF2 could mediate clonogenicity and stemness activities via the IGF-1R/AKT/mammalian target of rapamycin and IGF-1R/AKT/NANOG pathways, respectively, this study demonstrated the cooperation of the FF-sourced IGF and HGF growth signals in the malignant transformation and progression of HGSC through both common and distinct signaling pathways. These findings help develop targeted prevention of HGSC.

Ovarian high-grade serous carcinoma cells with low SMARCA4 expression and high SMARCA2 expression contribute to platinum resistance.

Platinum resistance is a major obstacle to the treatment of ovarian cancer and is correlated with poor clinical outcomes. Intratumor heterogeneity plays a key role in chemoresistance. Recent studies have emphasized the contributions of genetic and epigenetic factors to the development of intratumor heterogeneity. Although the clinical significance of multi-subunit chromatin remodeler, switch/sucrose nonfermenting (SWI/SNF) complexes in cancers has been reported, the impacts of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4/subfamily A, member 2 (SMARCA4/A2) expression patterns in human cancer tissues have not been fully elucidated. Here, we show that low expression of SMARCA4 and high expression of SMARCA2 are associated with platinum resistance in ovarian high-grade serous carcinoma (HGSC) cells. We used fluorescence multiplex immunohistochemistry (fmIHC) to study resected specimens; we examined heterogeneity in human HGSC tissues at the single-cell level, which revealed that the proportion of cells with the SMARCA4low /SMARCA2high phenotype was positively correlated with clinical platinum-resistant recurrence. We used stable transfection of SMARCA2 and siRNA knockdown of SMARCA4 to generate HGSC cells with the SMARCA4low /SMARCA2high phenotype; these cells had the greatest resistance to carboplatin. Bioinformatics analyses revealed that the underlying mechanism involved in substantial alterations to chromatin accessibility and resultant fibroblast growth factor (FGF) signaling activation, MAPK pathway activation, BCL2 overexpression, and reduced carboplatin-induced apoptosis; these were confirmed by in vitro functional experiments. Furthermore, in vivo experiments in an animal model demonstrated that combination therapy with carboplatin and a fibroblast growth factor receptor (FGFR) inhibitor promoted cell death in HGSC xenografts. Taken together, these observations reveal a specific subpopulation of HGSC cells that is associated with clinical chemoresistance, which may lead to the establishment of a histopathological prediction system for carboplatin response. Our findings may facilitate the development of novel therapeutic strategies for platinum-resistant HGSC cells. © 2023 The Pathological Society of Great Britain and Ireland.

5P Physiologically relevant treatment models to investigate epigenetic mechanisms driving platinum resistance in ovarian high grade serous carcinoma

The prognosis for patients with platinum-resistant ovarian High Grade Serous Carcinoma (HGSC) remains poor. BriTROC-1 data indicate that genomic alterations alone cannot explain acquired platinum resistance. We investigate epigenetic changes that may drive platinum resistance in HGSC. Using in an in vitro two-dimensional model, we recreated multiple cycles of chemotherapy. We utilised both established cell lines and primary cultures derived from HGSC ascites. Following cell characterisation (p53, PAX8 IHC), carboplatin sensitivity was assessed (sulforhodamine B assay). Cells were then pulsed with four cycles of carboplatin (50μM for 6 hours) with a week of recovery between each cycle. Methylation (Illumina 850k array), transcriptomic (RNAseq) and chromatin accessibility (ATACseq) assays were performed. Cells were also imaged using Stochastic Optical Reconstruction Microscopy. Firstly, we focused on RNAseq analysis to evaluate genes (and related biological processes) that are consistently enriched following carboplatin cycles and the unique genes that are conversely enriched at cycle level. We attempted to identify uniformly up-/down-regulated pathways across the different cultures. These data were compared with the same results obtained from the OvCar4, an established HGSC cell line and IVR01, an in-vivo resistant OvCar4 derivative. The ATACseq and methylation analyses are still ongoing, aiming to identify possible concordant changes in the chromatin accessibility and the methylation patterns that will allow us to clarify the changes observed in transcriptomic. The preliminary data obtained from the RNAseq analysis show that the highest number of enriched genes seems to occur after the first Carboplatin cycle, with a lower number of progressively enriched genes across all other ones. No constant biological processes were identified for the moment, across the different primary cell cultures. Further comparisons will be certainly needed. Understanding the epigenetic landscape of HGSC using physiologically relevant models will allow us to identify possible therapeutic targets that could eventually prevent platinum resistance.

Abstract A015: Age-associated molecular changes may predispose the ovary to malignant transformation leading to ovarian cancer (OVCA)

Abstract Background: Ovarian cancer (OVCA) is a fatal malignancy of women which in most cases is a disease of postmenopausal women. Thus, age-associated changes in women may predispose them to malignancy. Persistent high levels of follicle stimulating hormone (FSH) is a feature of aging and we have shown that aging was associated with increased expression of inflammatory cytokine interleukin 16 (IL-16) and glucose regulatory protein (GRP78), a marker of cellular stress. It is possible that age-associated increased expression of IL-16 and GRP78 might be involved in production of mutagenic 8-OXO-2dG or its cognate enzyme OGG1 and malondialdehyde (MDA) as well as markers of DNA repair mechanisms. The goal of this study was to determine the mechanism of age-associated malignant transformation in the ovary. Materials and Methods: Exploratory study: Archived tissue specimens were selected for immunohistochemical, immunoblotting, genomic studies and immunoassays based on the review of their hematoxylin and eosin staining and final Pathological reports. Specimens were grouped as normal premenopausal women (30-50 years old), normal postmenopausal women (55-85 years old) and ovarian high grade serous carcinoma (HGSC). In vitro study: human ovarian surface epithelium (HOSE) cells were treated with or without FSH for 24 hours, and their cytoplasmic and nuclear fractionations together with that of OVCAR3 (HGSC cell line) cells were extracted. Expression of markers of inflammation (IL-16), oxidative stress (SOD2, GRP78), DNA adducts (8-OXO-2dG/OGG1, MDA) and epigenetics (HDAC1, gH2AX) and were examined. Significant differences in expression of different markers among different age groups and ovarian HGSC, and treated or control OVCAR3 cells were determined by ANOVA and t-tests. Results: Compared with premenopausal women, expression of markers of inflammation and oxidative stress including IL-16, GRP78, SOD2, 8-OXO-2dG/OGG1, MDA as well as markers of epigenetic changes including HDAC1 and gH2AX was significantly higher (<0.0001) in a subset of postmenopausal women. Similar patterns of expression were also observed in patients with ovarian HGSC. Nuclear expression of mutagenic DNA adducts (OGG1, MDA) and inhibitor of DNA damage repair mechanism (GRP78) enhanced during aging. Thus, age-associated inflammation and oxidative stress are predispositions to malignant transformation. This assumption is based on the observation from the treatment of HOSE cells with FSH which showed increased expression of the above markers as also observed in OVCAR3 cells. Conclusion: Expression of IL-16, GRP78, SOD2, 8-OXO-2dG/OGG1, MDA and HDAC1 increased in ovary during aging. These results suggest that aging is associated with persistent inflammation and oxidative stress in ovarian tissues, which predisposes these tissues to malignant transformation leading to OVCA development. Support: NIH/NCI: CA210370 Citation Format: Jessica Ramirez, Elizabeth Paris, Sanjib Basu, Animesh Barua. Age-associated molecular changes may predispose the ovary to malignant transformation leading to ovarian cancer (OVCA) [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A015.

Exosome Secretion and Epithelial-Mesenchymal Transition in Ovarian Cancer Are Regulated by Phospholipase D.

Phospholipase D (PLD) isoenzymes participate in a variety of cellular functions that are mostly attributed to phosphatidic acid (PA) synthesis. Dysregulation of PLD regulates tumor progression and metastasis, yet little is known about the underlying mechanism. We previously reported on the expression and clinical role of the PLD isoenzymes PLD1 and PLD2 in tubo-ovarian high-grade serous carcinoma (HGSC). In the present study, we investigated the biological function of PLD1 and PLD2 using the OVCAR-3 and OVCAR-8 HGSC cell lines. KO cell lines for both PLDs were generated using CRISPR/CAS9 technology and assayed for exosome secretion, spheroid formation, migration, invasion and expression of molecules involved in epithelial-mesenchymal transition (EMT) and intracellular signaling. Significant differences between PLD1 and PLD2 KO cells and controls were observed for all the above parameters, supporting an important role for PLD in regulating migration, invasion, metastasis and EMT.

Ovulation Enhances Intraperitoneal and Ovarian Seedings of High-Grade Serous Carcinoma Cells Originating from the Fallopian Tube: Confirmation in a Bursa-Free Mouse Xenograft Model.

Background: Recently, new paradigms for the etiology and origin of ovarian high-grade serous carcinoma (HGSC) have emerged. The carcinogens released during ovulation transform fallopian tube epithelial cells, exfoliating and metastasizing to the peritoneal organs, including the ovaries. Solid in vivo evidence of the paradigms in a mouse model is urgently needed but is hampered by the differing tubo-ovarian structures. In mice, there is a bursa structure surrounding the distal oviduct and ovary. This, on one hand, prevents the direct influence of ovulatory follicular fluid (FF) on the exfoliated tumor cells. On the other hand, it hinders the seeding of exfoliated tumor cells into the ovary. Methods: In this study, we created a bursa-free mouse xenograft model to examine the effect of superovulation on peritoneal and ovarian metastases of transformed human tubal epithelial cells after intraperitoneal injection in NSG mice. Results: The bursa-free mouse model showed a better effect of ovulation on peritoneal metastasis. In this model, superovulation increased the number of transformed human tubal epithelial cell seedlings after intraperitoneal injection. Compared to the bursa-intact state, bursa-free ovaries were more vulnerable to external tumor seeding in either normal ovulation or superovulation state. Conclusions: This study provides the first in vivo evidence that intraperitoneal spreading of tubal HGSC cells is enhanced by ovulation. This study also demonstrated a mouse model for studying ovary-peritoneum interaction in cancer development.

Establishment and characterization of a novel ovarian high-grade serous carcinoma cell line\u2014IPO43

BackgroundEpithelial ovarian cancer (EOC) is an aggressive and lethal malignancy and novel EOC cell lines with detailed characterization are needed, to provide researchers with diverse helpful resources to study EOC biological processes and cancer experimental therapies.MethodsThe IPO43 cell line was established from the ascitic fluid of a patient with a diagnosis of high-grade serous carcinoma (HGSC) of the ovary, previously treated with chemotherapy.Cell immortalization was achieved in 2D cell culture and growth obtained in 2D and 3D cell cultures. The characterization of immortalized cells was done by immunocytochemistry, flow cytometry, cell proliferation, chromosomal Comparative Genomic Hybridization (cCGH), STR profile and Next Generation Sequencing (NGS).ResultsCharacterization studies confirmed that IPO43 cell line is of EOC origin and maintains morphological and molecular features of the primary tumor. cCGH analysis showed a complex profile with gains and losses of specific DNA regions in both primary ascitic fluid and cell line IPO43. The cell line was successfully grown in a 3D system which allows its future application in more complex assays than those performed in 2D models. IPO43 cell line is resistant to standard drug treatment in vitro.ConclusionsIPO43 is available for public research and we hope it can contribute to enrich the in vitro models addressing EOC heterogeneity, being useful to investigate EOC and to develop new therapeutic modalities.

Highly Expressed Progesterone Receptor B Isoform Increases Platinum Sensitivity and Survival of Ovarian High-Grade Serous Carcinoma.

Expression of the progesterone receptor (PR) has been reported to influence survival outcomes in patients with ovarian high-grade serous carcinoma (HGSC). In the present study, we attempted to investigate the association among PR and its isoforms' expression, platinum sensitivity, and survival in ovarian HGSC. This retrospective study reviewed ovarian HGSC patients who received surgery followed by adjuvant chemotherapy. We analyzed total PR and PR isoform-B (PR-B) expression by immunohistochemical staining and quantified using the H-score. Then, we compared platinum sensitivity and survival outcomes between those patients with weak and strong PR-B expression. Cisplatin viability assays were carried out in ovarian HGSC cell lines (OC-3-VGH and OVCAR-3) with different PR-B expression. Among 90 patients, 49 and 41 patients were considered to have platinum-sensitive and platinum-resistant disease, respectively. Pearson's correlation model showed that the H-score of total PR correlated positively with PR-B (r = 0.813). The PR-B H-score of tumors was significantly higher in the platinum-sensitive group (p = 0.004). Multivariate analysis revealed that the PR-B H-score and optimal debulking status were independent factors predicting platinum sensitivity. When compared with strong PR-B expression, patients with weak PR-B had significantly poorer progression-free (p = 0.021) and cancer-specific survival (p = 0.046). In a cell model, cisplatin-resistant OC-3-VGH cells expressed a lower level of PR-B than wild-type cells. Overexpression of PR-B or progesterone could increase cisplatin sensitivity in both OC-3-VGH and OVCAR-3 cells via the mechanism of promoting cisplatin-related apoptosis. When compared to weak PR-B, ovarian HGSC patients with a strong PR-B expression had a better chance of platinum sensitivity and survival, and this finding was compatible with our experimental results. Progesterone seemed to be a platinum sensitizer, but the value of adding progesterone in the treatment of ovarian HGSC should be further investigated.

Epigenetic Silencing of miR-33b Promotes Peritoneal Metastases of Ovarian Cancer by Modulating the TAK1/FASN/CPT1A/NF-κB Axis.

Simple SummaryOmental metastasis and peritoneal dissemination are frequently observed in ovarian cancer peritoneal metastases and are associated with high mortality and poor prognosis. The tumor microenvironment is known to influence cancer epigenomics, which plays an essential role in promoting tumor development and metastatic progression. Therefore, investigation of the epigenetic mechanisms underlying the growth of ovarian cancer cells in the omental metastatic microenvironment is of great importance. Here, we report that miR-33b is significantly silenced by DNA hypermethylation in metastatic ovarian cancer cells to adapt to a lipid-rich microenvironment. Restoration of miR-33b was shown to impair lipid metabolic activities and reduce the oncogenic properties of ovarian cancer cells by negatively regulating the TAK1/FASN/CPT1A/NF-κB pathway, indicating that targeting this signaling cascade may be a molecular therapeutic choice for ovarian cancer metastatic progression.Peritoneal metastases are frequently found in high-grade serous carcinoma (HGSOC) patients and are commonly associated with a poor prognosis. The tumor microenvironment (TME) is a complex milieu that plays a critical role in epigenetic alterations driving tumor development and metastatic progression. However, the impact of epigenetic alterations on metastatic ovarian cancer cells in the harsh peritoneal microenvironment remains incompletely understood. Here, we identified that miR-33b is frequently silenced by promoter hypermethylation in HGSOC cells derived from metastatic omental tumor tissues. Enforced expression of miR-33b abrogates the oncogenic properties of ovarian cancer cells cocultured in omental conditioned medium (OCM), which mimics the ascites microenvironment, and in vivo tumor growth. Of note, restoration of miR-33b inhibited OCM-upregulated de novo lipogenesis and fatty acid β-oxidation in ovarian cancer cells, indicating that miR-33b may play a novel tumor suppressor role in the lipid-mediated oncogenic properties of metastatic ovarian cancer cells found in the omentum. Mechanistic studies demonstrated that miR-33b directly targets transforming growth factor beta-activated kinase 1 (TAK1), thereby suppressing the activities of fatty acid synthase (FASN) and carnitine palmitoyltransferase 1A (CPT1A) in modulating lipid metabolic activities and simultaneously inhibiting the phosphorylation of NF-κB signaling to govern the oncogenic behaviors of ovarian cancer cells. Thus, our data suggest that a lipid-rich microenvironment may cause epigenetic silencing of miR-33b, which negatively modulates ovarian cancer peritoneal metastases, at least in part, by suppressing TAK1/FASN/CPT1A/NF-κB signaling.

Differential expression of estrogen receptor subtypes in ovarian high-grade serous carcinoma and clear cell carcinoma.

PurposeTo investigate the role of estrogen receptors (ERs) in high‐grade serous carcinoma (HGSC) and clear cell carcinoma (CCC) of the ovary and evaluate ERs as prognostic biomarkers for ovarian cancer.MethodsThis study included 79 patients with HGSC (n = 38) or CCC (n = 41) treated at our institution between 2005 and 2014. Immunohistochemistry examined protein expression of ERα, ERβ, and G protein‐coupled estrogen receptor‐1 (GPER‐1); relationships between ERα, ERβ, and GPER‐1 with patient survival were evaluated. Additionally, cell proliferation assay and phosphokinase proteome profiling were performed.ResultsIn HGSC patients, expression of ERα, cytoplasmic GPER‐1, or nuclear GPER‐1 was associated with poor progression‐free survival (PFS) (P = .041, P = .010, or P = .013, respectively). Cytoplasmic GPER‐1 was an independent prognostic factor for PFS in HGSC patients (HR = 2.83, 95% CI = 1.03‐9.16, P = .007). ER expressions were not associated with prognosis in CCC patients. GPER‐1 knockdown by siRNA reduced the cells number to 60% of siRNA‐control‐treated cells (P < .05), and GPER‐1 antagonist, G‐15 inhibited two HGSC cell lines proliferation (KF and UWB1.289) in a dose‐dependent manner. Phosphoprotein array revealed that GPER‐1 silencing decreased relative phosphorylation of glycogen synthase kinase‐3.ConclusionsHigh GPER‐1 expression is an independent prognostic factor for PFS in HGSC patients, and GPER‐1 may play a role in HGSC cell proliferation.