Ovarian Surface Epithelial Cells Research Articles

Stearoyl-CoA desaturase 1 inhibition induces ER stress-mediated apoptosis in ovarian cancer cells

Ovarian cancer is a leading cause of death among gynecologic tumors, often detected at advanced stages. Metabolic reprogramming and increased lipid biosynthesis are key factors driving cancer cell growth. Stearoyl-CoA desaturase 1 (SCD1) is a crucial enzyme involved in de novo lipid synthesis, producing mono-unsaturated fatty acids (MUFAs). Here, we aimed to investigate the expression and significance of SCD1 in epithelial ovarian cancer (EOC). Comparative analysis of normal ovarian surface epithelial (NOSE) tissues and cell lines revealed elevated SCD1 expression in EOC tissues and cells. Inhibition of SCD1 significantly reduced the proliferation of EOC cells and patient-derived organoids and induced apoptotic cell death. Interestingly, SCD1 inhibition did not affect the viability of non-cancer cells, indicating selective cytotoxicity against EOC cells. SCD1 inhibition on EOC cells induced endoplasmic reticulum (ER) stress by activating the unfolded protein response (UPR) sensors and resulted in apoptosis. The addition of exogenous oleic acid, a product of SCD1, rescued EOC cells from ER stress-mediated apoptosis induced by SCD1 inhibition, underscoring the importance of lipid desaturation for cancer cell survival. Taken together, our findings suggest that the inhibition of SCD1 is a promising biomarker as well as a novel therapeutic target for ovarian cancer by regulating ER stress and inducing cancer cell apoptosis.

Abstract 3077: Integrin expression during ovarian cancer progression

Abstract During peritoneal dissemination, ovarian cancer cells exfoliate from the primary tumor and aggregate in the peritoneal fluid. This elicits survival signals via changes in mitochondrial morphology and function. As spheroids adhere to secondary sites, mitochondrial function is restored in the form of recovery of mitochondrial morphology, increased mitochondrial membrane potential, glucose uptake and increased proliferation. These findings suggested a mitochondrial phenotypic switch from mitophagy in spheroids to upregulation of mitobiogenesis upon spheroids adhesion to secondary sites. Hence, our objective was to identify critical receptors that facilitate the adhesion of spheroids to secondary sites, focusing on integrins that facilitate cell-extracellular matrix (ECM) attachment and activate downstream signaling cascades. To investigate changes in integrin expression, we mimicked three major stages of ovarian cancer progression by developing adherent stage, spheroid stage and adherent spheroid stage using our mouse ovarian surface epithelial (MOSE) cell lines representing benign (MOSE-E) stage as well as slow-developing (MOSE-L) and fast-developing disease (MOSE-LTICv) in biologically relevant conditions. Using RT qPCR and Western blotting, we determined gene and protein expression levels of integrins and adhesion related genes. Confocal microscopy was used to determine protein localization during ovarian cancer progression. RT qPCR result indicated differential up-regulated expression of integrins in both spheroid and adherent spheroid stage, suggesting the role of different integrins during the stages of ovarian cancer progression. Specifically, we observed a decrease (integrin ɑ5, ɑ6) or increase (ɑ2𝛃;1) of integrins after aggregation and an upregulation of 𝛃;1, α2, αV, α3, α5 and immunomodulatory integrins such as αE and αM in adherent spheroids, indicating their role in mediating specific steps during progression of ovarian cancer from tumorigenesis to metastatic implantation. We also observed increased expression levels of extracellular matrix proteins and their modulators, and cell-to-cell adhesion genes during ovarian cancer progression that was more pronounced in the MOSE-LTICv cells which suggests their association with an aggressive cancer phenotype. Overall, this project will aid in the identification of adhesion targets to develop new anti-metastatic treatments that will reduce mortality in women with ovarian cancer. Citation Format: Nazia Bano, Eva M. Schmelz. Integrin expression during ovarian cancer progression [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 3077.

Abstract 394: Preclinical evaluation of ERX-208, a potent inducer of ER stress for the treatment of ovarian cancer

Abstract Background: Ovarian cancer (OCa) is the deadliest kind of gynecologic cancer in the United States. The long-term survival rate for OCa is less than 20% after five years. Intra-tumoral and inter-tumoral heterogeneity is implicated in tumor resistance to conventional therapies and the unsatisfactory clinical outcomes. Addressing these issues necessitates innovative therapies that target intrinsic common vulnerabilities within OCa. Recent studies have highlighted that high basal level of endoplasmic reticulum stress (ERS) in OCa as a critical vulnerability. We have identified a promising compound, ERX-208 that potently induces ERS in cancer cells. The objective of this study is to characterize the mechanisms and activity of ERX-208 using preclinical models. Methods: The biological activity of ERX-208 was examined across 17 distinct OCa cells, representing 5 diverse OCa subtypes. Mechanistic studies utilized Western blotting, immunohistochemistry (IHC), RNA-Seq analysis, and CRISPR/Cas9 knockouts (KO). Pharmacokinetics (PK) and toxicity studies were performed on C57BL/6 mice. Comprehensive preclinical assessments were carried out through cell line-derived xenografts (CDXs), patient-derived xenografts (PDXs), organoids (PDOs) and explants (PDEs). Results: ERX-208 demonstrated an IC50 of approximately 50-100 nM inducing ERS and reducing cell viability in OCa cells. Conversely, normal ovarian surface epithelial cells exhibited minimal effects from ERX-208. In vitro experiments revealed strong ERX-208-induced apoptosis in OCa cell lines. Mechanistic studies employing RNA sequencing, Western blotting, and RT-qPCR, confirmed activation of ERS pathways observed as early as 5 hours post-ERX-208 treatment. Our studies identified LIPA as a potential target, and KO of LIPA significantly diminished ERX-208 activity. Moreover, LIPA KO substantially impeded in vivo OCa tumor growth. ERX-208 up to a dose of 25 mg/kg showed no observable organ toxicity and had no effect on the mice's body weight. Dose range studies identified 10 mg/kg intraperitoneal as the minimal effective dose, achieving more than 50% tumor reduction. In preclinical models, ERX-208 inhibited the growth of OCa CDXs, PDXs, and ex vivo PDEs and PDO’s. IHC analyses indicated reduced proliferation (Ki-67) and increased activation of ERS markers, such as GRP78 and p-PERK. Conclusions: Collectively, our findings underscore the preclinical promise of ERX-208 as a potential therapeutic agent for treating OCa. Conflict: The patents surrounding ERX-208 are licensed to EtiraRx. Citation Format: Suryavathi Viswanadhapalli, Tae-Kyung Lee, Scott Elmore, Gaurav Sharma, Rahul Gopalam, Karla Parra, Tanner Reese, Michael Hsieh, Uday P. Pratap, Xue Yang, Behnam Ebrahimi, Henry Neal, Chia-Yuan Chen, Kara Kassees, Christian Cervantes, Zhenming Xu, Edward Kost, Gangadhara Reddy Sareddy, Rajeshwar R. Tekmal, Jung-Mo Ahn, Ganesh V. Raj, Ratna K. Vadlamudi. Preclinical evaluation of ERX-208, a potent inducer of ER stress for the treatment of 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 394.

Identification and characterization of multipotential stem cells in immortalized normal ovarian surface epithelial cells.

The ovarian surface epithelium (OSE) is a single layer of squamous-to-cuboidal epithelial cells that experience repetitive ovulatory rupture and subsequent repair. However, the characteristics of human immortalized ovarian surface epithelial cells (IOSE80) remain elusive. This study aims to determine whether IOSE80 cells have the characteristics of stem cell proliferation and multilineage differentiation and their application in regenerative medicine. IOSE80 cells are sequenced by high-throughput transcriptome analysis, and 5 sets of public data are used to compare the differences between IOSE80 cells and bone marrow mesenchymal stem cells, pluripotent stem cells, and oocytes in transcriptome profiling. The IOSE80 cells present a cobblestone-like monolayer and express the epithelial cell marker KRT18; the stem cell markers IFITM3, ALDH1A1, and VIM; lowly express stem cell marker LGR5 and germ cell markers DDX4 and DAZL. In addition, the GO terms "regulation of stem cell proliferation", "epithelial cell proliferation", etc., are significantly enriched ( P<0.05). IOSE80 cells have the potential to act as mesenchymal stem cells to differentiate into adipocytes with lipid droplets, osteoblasts, and chondroblasts in vitro. IOSE80 cells express pluripotent stem cell markers, including OCT4, SSEA4, TRA-1-60, and TRA-1-81, and they can be induced into three germ layers in vitro. IOSE80 cells also form oocyte-like cells in vitro and in vivo. In addition, IOSE80 cells exhibit robust proliferation, migration, and ovarian repair functions after in vivo transplantation. This study demonstrates that IOSE80 cells have the characteristics of pluripotent/multipotent stem cells, indicating their important role in tissue engineering and regenerative medicine.

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&amp;lt;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.

Abstract A033: Anti-proliferative effects of lentinan, a beta-glucan from shiitake mushroom (lentinula edodes)

Abstract Cancer is one of the most significant health challenges worldwide. Lentinan, a β-glucan from shiitake mushroom, Lentinula edodes, has been demonstrated to have an inhibitory effect on selected cancer cells, but results are inconclusive. The aim of the present study was to investigate the anti-proliferative effect of lentinan on breast adenocarcinoma cells (MCF-7), prostate carcinoma (DU-145), and ovarian carcinoma (SKOV3) using MTS (3-(4,5-dimethylthiazol-2-yl)-5—(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and explore possible mechanisms of action of this compound. In the MCF-7 cells that were used as a positive control, lentinan without phenol red resulted in a significant inhibition of cell viability by 96% while in the presence of phenol red, the weak estrogen effect attributable to phenol red eliminated the significant inhibitory effect of lentinan. Treatment of DU-145 cells with different doses of lentinan resulted in up to a 42% inhibition in cell growth. In SKOV3 cells, lentinan induced a biphasic response where doses of 800 µg/ml or less increased cell viability by 76% – 148%; however, a dose of 1.6 mg/ml inhibited cell proliferation significantly by 87%. When SKOV3 cells were co-treated with lentinan (800 µg/ml or less) and laminarin (a Dectin-1 receptor antagonist), the proliferation-promoting effect of lentinan was dominant over the proliferation-inhibiting effect of laminarin. However, when lentinan was presented at significant higher levels (1600 µg/ml), the inhibitory effect of the two agents became additive in suppressing SKOV3 cell proliferation. Lentinan induced a biphasic response in human ovarian surface epithelial cells (HOSEpC) similar to SKOV3 cells, where the highest dose (1600 µg/ml) resulted in the inhibition of cell growth by 53%. Western blots after treatment of SKOV3 cells with 1600 µg/ml of lentinan demonstrated caspase-3 activation and a decreased anti-apoptotic protein Bcl-2 levels. Our findings reveal that lentinan can be considered as a potential anti-cancer drug for effective treatment of breast and prostate cancer. Citation Format: Titus Sombuor, Kenneth Shane Broughton, Michael Bergel. Anti-proliferative effects of lentinan, a beta-glucan from shiitake mushroom (lentinula edodes) [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 A033.

Replication stress and defective checkpoints make fallopian tube epithelial cells putative drivers of high-grade serous ovarian cancer

Clinical and molecular evidence indicates that high-grade serous ovarian cancer (HGSOC) primarily originates from the fallopian tube, not the ovarian surface. However, the reasons for this preference remain unclear. Our study highlights significant differences between fallopian tube epithelial (FTE) and ovarian surface epithelial (OSE) cells, providing the molecular basis for FTEs as site of origin of HGSOC. FTEs, unlike OSEs, exhibit heightened replication stress (RS), impaired repair of stalled forks, ineffective G2/M checkpoint, and increased tumorigenicity. BRCA1 heterozygosity exacerbates these defects, resulting in RS suppression haploinsufficiency and an aggressive tumor phenotype. Examination of human and mouse sections reveals buildup of the RS marker 53BP1 primarily in the fallopian tubes, particularly at the fimbrial ends. Furthermore, menopausal status influences RS levels. Our study provides a mechanistic rationale for FTE as the site of origin for HGSOC, investigates the impact of BRCA1 heterozygosity, and lays the groundwork for targeting early HGSOC drivers.

Ovarian cancer research: Examining ovarian function and dysfunction

Ovarian cancer research: Examining ovarian function and dysfunction Ovarian cancer is largely a misnomer because the cells giving rise to malignant tissue are not derived from either follicular or luteal cells. Instead, ovarian cancer cells evolve from ovarian surface epithelial cells (OSEs) that cover the surface of the ovary and fallopian tube and separate these tissues from the abdominal peritoneal cavity. Unlike other cancers that metastasize through the vascular system, ovarian cancer cells detach from the ovarian surface, disseminate and grow in the peritoneal cavity. What are the causes of ovarian cancer? The answers to this question are complex. There is not just one, but many underlying factors. However, the most common cause that initiates cancer involves mutations in specific genes, such as tumor protein 53 (TP53), BRAC1/2, KRAS, PTEN, that lead to abnormal cell proliferation and evasion of cell death. Many of these genes are involved in DNA repair, essential for maintaining normal gene expression; others are involved in metabolic/ growth regulatory pathways.

Abstract 354: The progression of changes in cardiac structure and function in mice with epithelial ovarian cancer

Abstract Introduction: Cancer cachexia, characterized by the progressive loss of muscle mass (i.e., atrophy), has a significant impact on patient prognosis and quality of life. While skeletal muscle atrophy and weakness are well characterized in cancer patients, the progression of cancer-induced cardiac atrophy has received little attention. Preliminary work from our lab has shown that cardiac dysfunction, measured by invasive hemodynamic assessment, precedes the development of metastatic disease in a mouse model of epithelial ovarian cancer (EOC). Yet, the time course of changes in cardiac structure and function throughout the progression of ovarian cancer remain unknown. Thus, we non-invasively assessed changes in cardiac structure and function through the progression of EOC. Methods: To investigate the impact of advanced ovarian cancer on cardiac structure and function, we used an orthotopic, syngeneic mouse model of EOC. This model was generated by injecting transformed murine ovarian surface epithelial cells (ID8; 1.0 × 106) or saline (surgical sham) directly under the ovarian bursa of syngeneic mice. Echocardiography was used to assess changes in cardiac structure and function throughout the progression of EOC at days 45, 60, 75, and 90 post-tumor induction (PTI). Morphometric assessments at each timepoint were used to determine the presence of cardiac atrophy. Results: Echocardiography showed that EOC mice had preserved left ventricular (LV) function (i.e., ejection fraction) with no change in structural dimensions at days 45, 60 and 75 PTI. By day 90 PTI, LV wall thickness and end-systolic dimension decreased, suggesting evidence of cardiac atrophy. Consistent with echocardiographic data, morphometric assessment showed profound atrophy at 90 days PTI, but preserved cardiac mass at all other timepoints compared to shams. Conclusion: Together, this work demonstrates that when non-invasive techniques are used to measure changes in cardiac structure, atrophy is not evident until advanced stage ovarian cancer (i.e., 90 days PTI). Despite this, preliminary work from our lab has shown that cardiac dysfunction is present in EOC mice by 45 days PTI when assessed by invasive hemodynamics. Although echocardiography is a widely used technique for measuring indices of cardiac structure and function, our work demonstrates it lacks sensitivity to detect cardiac deficits until the overt stages of disease that accompany cancer cachexia. Thus, it is important to consider that subclinical cardiac dysfunction may exist in the early stages of cancer progression, however, these changes may go undetected unless more sensitive hemodynamic assessments are employed. Citation Format: Bridget Coyle-Asbil, Leslie M. Ogilvie, Madison Pereira, Kathy Matuszewska, Luca J. Delfinis, Shivam Gandhi, Keith R. Brunt, Christopher G. Perry, Jim Petrik, Jeremy A. Simpson. The progression of changes in cardiac structure and function in mice with epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 354.

Abstract 4248: Epithelial ovarian cancer causes cardiac dysfunction and myofilament dysregulation

Abstract Introduction: The intersections between cancer and cardiovascular disease (CVD) have become increasingly relevant in the care of both patient populations. To date, the cardio-oncology field has largely focused on how cancer treatment influences long-term cardiac health. Indeed, many cancer therapeutics show various toxic effects to the cardiovascular system. However, studies have also shown cardiac deficits in therapy-naïve cancer patients. This suggests that these two disease states interact through similar signaling pathways, yet the mechanisms underlying cancer-induced cardiac dysfunction remain unknown. Thus, we investigated cardiac structure and function throughout the progression of epithelial ovarian cancer (EOC). Methods: We used an orthotopic, syngeneic mouse model of ovarian cancer. Briefly, transformed murine ovarian surface epithelial cells from C57BL/6 mice (ID8; 1.0 × 106), or saline for surgical controls, were injected under the ovarian bursa of syngeneic mice. In this model, 60 days after tumor induction, mice form large ovarian masses, numerous peritoneal lesions, and develop abdominal ascites - consistent with the clinical features of stage III (advanced) EOC. To determine whether cardiac abnormalities precede the presence of advanced disease, at 45 days post-tumor induction, mice were anesthetized, and left ventricular (LV) structure and function were assessed by echocardiography and invasive hemodynamics, respectively. Myofilament activity was measured with an actomyosin MgATPase assay and protein phosphorylation was determined by SDS-PAGE with Pro-Q Diamond phosphoprotein staining. Results: EOC mice showed systolic (i.e., reduced contraction rate) and diastolic dysfunction (i.e., reduced relaxation rate, elevated diastolic filling pressure), with no evidence of hypertrophy or atrophy (i.e., no change in LV wall thickness or dimension). While there were no observed differences in myofilament ATPase activity, phosphorylation of regulatory myosin light chain 2 (MYL2) and cardiac myosin binding protein C (cMyBP-C) decreased, and tropomyosin (Tpm) phosphorylation increased in EOC mice. Conclusion: Together, these data demonstrate that ovarian cancer impairs cardiac function and alters the regulation of myofilament proteins critical for contraction and relaxation. Interestingly, the observed dephosphorylation of MYL2 and cMyBP-C and hyperphosphorylation of Tpm are consistent with features of heart failure. The findings build upon our current understanding of the interactions between cancer and CVD and show that deficits in the heart precede the development of advanced malignancy. Future work further will investigate the tumor-heart axis to identify how tumor development disrupts normal cardiac physiology. Citation Format: Leslie M. Ogilvie, Madison Pereira, Kathy Matuszewska, Bridget Coyle-Asbil, Luca J. Delfinis, Shivam Gandhi, Keith R. Brunt, Christopher G. Perry, W. Glen Pyle, Jim Petrik, Jeremy A. Simpson. Epithelial ovarian cancer causes cardiac dysfunction and myofilament dysregulation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4248.

IVM Advances for Early Antral Follicle-Enclosed Oocytes Coupling Reproductive Tissue Engineering to Inductive Influences of Human Chorionic Gonadotropin and Ovarian Surface Epithelium Coculture.

In vitro maturation (IVM) is not a routine assisted reproductive technology (ART) for oocytes collected from early antral (EA) follicles, a large source of potentially available gametes. Despite substantial improvements in IVM in the past decade, the outcomes remain low for EA-derived oocytes due to their reduced developmental competences. To optimize IVM for ovine EA-derived oocytes, a three-dimensional (3D) scaffold-mediated follicle-enclosed oocytes (FEO) system was compared with a validated cumulus-oocyte complex (COC) protocol. Gonadotropin stimulation (eCG and/or hCG) and/or somatic cell coculture (ovarian vs. extraovarian-cell source) were supplied to both systems. The maturation rate and parthenogenetic activation were significantly improved by combining hCG stimulation with ovarian surface epithelium (OSE) cells coculture exclusively on the FEO system. Based on the data, the paracrine factors released specifically from OSE enhanced the hCG-triggering of oocyte maturation mechanisms by acting through the mural compartment (positive effect on FEO and not on COC) by stimulating the EGFR signaling. Overall, the FEO system performed on a developed reproductive scaffold proved feasible and reliable in promoting a synergic cytoplasmatic and nuclear maturation, offering a novel cultural strategy to widen the availability of mature gametes for ART.

Zinc Finger Protein 90 Knockdown Promotes Cisplatin Sensitivity via Nrf2/HO-1 Pathway in Ovarian Cancer Cell.

Our study discussed the role of Zfp90 in ovarian cancer (OC) cell lines' sensitivity to cisplatin. We used two OC cell lines, SK-OV-3 and ES-2, to evaluate their role in cisplatin sensitization. The protein levels of p-Akt, ERK, caspase 3, Bcl-2, Bax, E-cadherin, MMP-2, MMP-9 and other drug resistance-related molecules, including Nrf2/HO-1, were discovered in the SK-OV-3 and ES-2 cells. We also used a human ovarian surface epithelial cell to compare the effect of Zfp90. Our outcomes indicated that cisplatin treatment generates reactive oxygen species (ROS) that modulate apoptotic protein expression. The anti-oxidative signal was also stimulated, which could hinder cell migration. The intervention of Zfp90 could greatly improve the apoptosis pathway and block the migrative pathway to regulate the cisplatin sensitivity in the OC cells. This study implies that the loss of function of Zfp90 might promote cisplatin sensitization in OC cells via regulating the Nrf2/HO-1 pathway to enhance cell apoptosis and inhibit the migrative effect in both SK-OV-3 and ES-2 cells.

Elevated Levels of Lamin A Promote HR and NHEJ-Mediated Repair Mechanisms in High-Grade Ovarian Serous Carcinoma Cell Line

Extensive research for the last two decades has significantly contributed to understanding the roles of lamins in the maintenance of nuclear architecture and genome organization which is drastically modified in neoplasia. It must be emphasized that alteration in lamin A/C expression and distribution is a consistent event during tumorigenesis of almost all tissues of human bodies. One of the important signatures of a cancer cell is its inability to repair DNA damage which befalls several genomic events that transform the cells to be sensitive to chemotherapeutic agents. This genomic and chromosomal instability is the most common feature found in cases of high-grade ovarian serous carcinoma. Here, we report elevated levels of lamins in OVCAR3 cells (high-grade ovarian serous carcinoma cell line) in comparison to IOSE (immortalised ovarian surface epithelial cells) and, consequently, altered damage repair machinery in OVCAR3. We have analysed the changes in global gene expression as a sequel to DNA damage induced by etoposide in ovarian carcinoma where lamin A is particularly elevated in expression and reported some differentially expressed genes associated with pathways conferring cellular proliferation and chemoresistance. We hereby establish the role of elevated lamin A in neoplastic transformation in the context of high-grade ovarian serous cancer through a combination of HR and NHEJ mechanisms.

VSELs and OSCs together sustain oogenesis in adult ovaries and their dysfunction results in age-related senescence, PCOS, POI and cancer

Multiple studies using single-cell RNA sequencing (scRNAseq) have failed to detect stem cells in adult ovaries. We have maintained that two populations of ovarian stem cells including pluripotent, very small embryonic-like stem cells (VSELs) and tissue-committed ‘progenitors’ termed ovarian stem cells (OSCs) can easily be detected in Hematoxylin and Eosin-stained ovary surface epithelial (OSE) cells smears prepared from both mice and human ovaries. Most likely the stem cells never get subjected to scRNAseq since they pellet down only by centrifuging cells suspension at 1000 g while cells for scRNAseq were invariably prepared by centrifuging at 200-400 g. A recent article provided further explanation for the failure of scRNAseq to detect ovarian stem cells. Extensive reanalysis of data (generated by scRNAseq) using an advanced software successfully detected OSCs and meiotic markers supporting neo-oogenesis in adult human ovaries. But this article remained critical on the biological relevance of VSELs and their relationship with OSCs. By carefully studying the OSE cells smears (which hold VSELs, OSCs and germ cell nests GCNs), prepared by partial trypsin digestion of intact mice ovaries during different stages of estrus cycle, we have successfully delineated novel functions of VSELs/OSCs in vivo under physiological conditions. VSELs undergo asymmetrical divisions to self-renew and give rise to slightly bigger OSCs which in turn undergo symmetrical divisions and clonal expansion to form GCNs, regular neo-oogenesis and follicle assembly. GCNs have been earlier described in fetal ovaries and during OSE cells culture (from adult ovaries) in response to FSH treatment. Dysfunction of VSELs/OSCs (which express ERα, ERβ, FSHR) due to neonatal exposure to endocrine disruption results in ovarian insufficiency and polycystic ovaries. VSELs have also been implicated in ovarian cancer. Age-related ovarian senescence/menopause is also due to dysfunction and blocked differentiation of VSELs/OSCs. These novel findings in vivo along with abundant in vitro and lineage tracing studies data in published literature provides huge scope for further research, offers novel avenues to manage ovarian pathologies and calls for re-writing of textbooks.

Upregulation of ryanodine receptor-1 dependent calcium signaling contributes to the malignance of high-grade serous ovarian cancer (HGSOC).

Ca2+ signaling plays crucial roles in cancer metastasis and progression. We analyzed RNAseq data from TCGA database and our patient samples; and identified over-expression of ryanodine receptor 1 (RYR1), the target of the FDA-approved drug dantrolene, is associated with poor survival of HGSOC patients. RYR1 expression is also markedly higher in several HGSOC cell lines compared to normal human ovarian surface epithelial cells (HOSE) and fallopian tube epithelial cells. Suppression of RYR1 expression/activity by shRNA or dantrolene significantly inhibited proliferation and migration of HGSOC cells (OVCA432, OVCA433, OV90). Ca2+ fluorescence measurement showed that the resting [Ca2+] and Ca2+ release triggered by the RYR-agonist 4-chloro-m-cresol (4-cmc) were significantly higher in HGSOC cells than the HOSE cells. Store-operated Ca2+ entry (SOCE) induced by 4-cmc was also enhanced in HGSOC cells. The elevated resting [Ca2+], the 4-cmc-induced Ca2+ release and SOCE in the HGSOC cells were reduced to the control level by shRNA against RYR1; and were significantly potentiated by over-expression of RYR1. RYR-dependent endoplasmic reticulum (ER)-mitochondrial Ca2+ transfer was examined by expressing the ER specific Ca2+ biosensor G-CEPIA1er and mitochondria Ca2+ biosensor CEPIA2mt in HGSOC cells. Activation of RYR by 4-cmc triggered a large reduction in ER [Ca2+], which was associated with a robust increase in mitochondrial [Ca2+]. Activation of RYRs by photorelease of caged cADP-ribose in a subcellular region caused an immediate transient local reduction in ER [Ca2+] and a fast transient local increase in mitochondria [Ca2+]. In contrast, RyR-dependent ER-mitochondrial Ca2+ transfer was not observed in the HOSE cells. These observations are the first direct evidence of enhanced RYR-mediated ER-mitochondrial Ca2+ transfer in ovarian cancer cells, and upregulation of RYR1-dependent Ca2+-signaling pathway contributes to the malignant phenotypes of HGSOC cells.

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 (&amp;lt;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.

Potent and Targeted Sindbis Virus Platform for Immunotherapy of Ovarian Cancer.

Our laboratory has been developing a Sindbis viral (SV) vector platform for treatments of ovarian and other types of cancers. In this study we show that SV.IL-12 combined with an agonistic OX40 antibody can eliminate ovarian cancer in a Mouse Ovarian Surface Epithelial Cell Line (MOSEC) model and further prevent tumors in mice rechallenged with tumor cells after approximately 5 months. Treatment efficacy is shown to be dependent upon T-cells that are transcriptionally and metabolically reprogramed. An influx of immune cells to the tumor microenvironment occurs. Combination of sequences encoding both IL-12 and anti-OX40 into a single SV vector, SV.IgGOX40.IL-12, facilitates the local delivery of immunoregulatory agents to tumors enhancing the anti-tumor response. We promote SV.IgGOX40.IL-12 as a safe and effective therapy for multiple types of cancer.

Distinct niche structures and intrinsic programs of fallopian tube and ovarian surface epithelial cells

Epithelial ovarian cancer (EOC) can originate from either fallopian tube epithelial (FTE) or ovarian surface epithelial (OSE) cells, but with different latencies and disease outcomes. To address the basis of these differences, we performed single cell RNA-sequencing of mouse cells isolated from the distal half of fallopian tube (FT) and surface layer of ovary. We find at the molecular level, FTE secretory stem/progenitor cells and OSE cells resemble mammary luminal progenitors and basal cells, respectively. An FT stromal subpopulation, enriched with Pdgfra + and Esr1 + cells, expresses multiple secreted factor (e.g., IGF1) and Hedgehog pathway genes and may serve as a niche for FTE cells. In contrast, Lgr5 + OSE cells express similar genes largely by themselves, raising a possibility that they serve as their own niche. The differences in intrinsic expression programs and niche organizations of FTE and OSE cells may contribute to their different courses toward the development of EOCs.

Exosomal circNFIX promotes angiogenesis in ovarian cancer via miR-518a-3p/TRIM44 axis.

Ovarian cancer (OC) is a gynecological cancer with high mortality. OC-derived exosomal circRNAs can regulate angiogenesis. This study aims to explore the role and mechanism of exosomal circRNA nuclear factor I X (CircNFIX) derived from OC cells in angiogenesis. Quantitative real-time polymerase chain reaction was employed to evaluate the levels of circNFIX, miR-518a-3p, and tripartite motif protein 44 (TRIM44) in OC and adjacent tissues. Exosomes from the ovarian surface epithelial cell (HOSEpiC) and OC cells (SKOV3 or OVCAR3) were isolated by differential centrifugation. Exosomes were cocultured with the human umbilical vein endothelial cells (HUVECs). The angiogenesis capacity was analyzed by Tube formation assay. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and Transwell assays were used to determine the cell viability and migration ability. The dual-luciferase report, RNA immunoprecipitation (RIP), and RNA pull-down assays were applied to validate the gene's interaction. CircNFIX and TRIM44 expression were higher and miR-518a-3p was lower in OC tissues than in the adjacent tissues. Upregulated circNFIX and TRIM44 were significantly correlated with the tumor size and International Federation of Gynecology and Obstetrics (FIGO) stage of OC patients. HUVECs treated OC-derived exosomes had higher proliferation, migration, and angiogenesis capacities than the control group. While OC-derived exosomal circNFIX silencing restrained HUVECs' proliferation, migration, and angiogenesis, compared with the OC-derived exosomes group. OC-derived exosomal circNFIX positively regulated TRIM44 expression by targeting miR-518a-3p in HUVECs. OC-derived exosomal circNFIX promoted angiogenesis by regulating the Janus-activated kinase/signal transducer and activator of transcription 1 (JAK/STAT1) pathway via miR-518a-3p/TRIM44 axis in HUVECs.

Telomerase inhibitor MST-312 and quercetin synergistically inhibit cancer cell proliferation by promoting DNA damage

Quercetin is a natural flavonoid with well-established anti-proliferative activities against a variety of cancers. Telomerase inhibitor MST-312 also exhibits anti-proliferative effect on various cancer cells independent of its effect on telomere shortening. However, due to their low absorption and toxicity at higher doses, their clinical development is limited. In the present study, we examine the synergistic potential of their combination in cancer cells, which may result in a decrease in the therapeutic dosage of these compounds. We report that MST-312 and quercetin exhibit strong synergism in ovarian cancer cells with combination index range from 0.2 to 0.7. Co-treatment with MST-312 and quercetin upregulates the DNA damage and augments apoptosis when compared to treatment with either compound alone or a vehicle. We also examined the effect of these compounds on the proliferation of normal ovarian surface epithelial cells (OSEs). MST-312 has a cytoprotective impact in OSEs at lower dosages, but is inhibitory at higher doses. Quercetin did not affect the OSEs proliferation at low concentrations while at higher concentrations it is inhibitory. Notably, combination of MST-312 and quercetin had no discernible impact on OSEs. These observations have significant implications for future efforts towards maximizing efficacy in cancer therapeutics as this co-treatment specifically affects cancer cells and reduces the effective dosage of both the compounds.