Human Ovarian Tumor Cells Research Articles

PGK1-AR axis: Benefits of a novel actor in PCOS pathology

PGK1-AR axis: Benefits of a novel actor in PCOS pathology

MiR‑132 is upregulated in polycystic ovarian syndrome and inhibits granulosa cells viability by targeting Foxa1.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine metabolic disorders characterized by hyperandrogenism, polycystic ovaries and ovulatory dysfunction. Several studies have suggested that the aberrant expression of microRNAs (miRNAs/miRs) plays an important role in the pathogenesis of PCOS; however, the role and underlying mechanisms of miR-132 in the development of PCOS remain unclear. In the present study, the expression of miR-132 in granulosa cells (GCs) derived from 26 patients with PCOS and 30 healthy controls was detected by reverse transcription-quantitative PCR (RT-qPCR). The apoptosis of GCs was examined using a TUNEL assay. The human ovarian granulosa-like tumor cell line, KGN, was cultured for Cell Counting Kit-8 assays following the overexpression or knockdown of miR-132. TargetScan was applied to identify the potential targets of miR-132, which was further verified by a luciferase assay, RT-qPCR and western blotting. The expression of miR-132 was decreased in GCs from patients with PCOS. Moreover, the GCs of patients with PCOS exhibited significantly increased apoptotic nuclei. Furthermore, the overexpression of miR-132 inhibited the viability of KGN cells. In addition, the results verified that miR-132 directly targeted forkhead box protein A1 (Foxa1), the knockdown of which suppressed KGN cell viability. On the whole, the findings of the present study demonstrated that miR-132 inhibited cell viability and induced apoptosis by directly interacting with Foxa1. Thus, miR-132 may be a potential target for the treatment of patients with PCOS.

OvCa-Chip microsystem recreates vascular endothelium–mediated platelet extravasation in ovarian cancer

AbstractIn ovarian cancer, platelet extravasation into the tumor and resulting metastasis is thought to be regulated mostly by the vascular endothelium. Because it is difficult to dissect complex underlying events in murine models, organ-on-a-chip methodology is applied to model vascular and platelet functions in ovarian cancer. This system (OvCa-Chip) consists of microfluidic chambers that are lined by human ovarian tumor cells interfaced with a 3-dimensional endothelialized lumen. Subsequent perfusion with human platelets within the device's vascular endothelial compartment under microvascular shear conditions for 5 days uncovered organ-to-molecular–level contributions of the endothelium to triggering platelet extravasation into tumors. Further, analysis of effluents available from the device's individual tumor and endothelial chambers revealed temporal dynamics of vascular disintegration caused by cancer cells, a differential increase in cytokine expression, and an alteration of barrier maintenance genes in endothelial cells. These events, when analyzed within the device over time, made the vascular tissue leaky and promoted platelet extravasation. Atorvastatin treatment of the endothelial cells within the OvCa-Chip revealed improved endothelial barrier function, reduction in inflammatory cytokines and, eventually, arrest of platelet extravasation. These data were validated through corresponding observations in patient-derived tumor samples. The OvCa-Chip provides a novel in vitro dissectible platform to model the mechanisms of the cancer-vascular-hematology nexus and the analyses of potential therapeutics.

Gene analysis of major signaling pathways regulated by gonadotropins in human ovarian granulosa tumor cells (KGN)†.

The female reproductive function largely depends on timing and coordination between follicle-stimulating hormone (FSH) and luteinizing hormone. Even though it was suggested that these hormones act on granulosa cells via shared signaling pathways, mainly protein kinases A, B, and C (PKA, PKB, and PKC), there is still very little information available on how these signaling pathways are regulated by each hormone to provide such differences in gene expression throughout folliculogenesis. To obtain a global picture of the principal upstream factors involved in PKA, PKB, and PKC signaling in granulosa cells, human granulosa-like tumor cells (KGN) were treated with FSH or specific activators (forskolin, SC79, and phorbol 12-myristate 13-acetate) for each pathway to analyze gene expression with RNA-seq technology. Normalization and cutoffs (FC 1.5, P≤0.05) revealed 3864 differentially expressed genes between treatments. Analysis of major upstream regulators showed that PKA is a master kinase of early cell differentiation as its activation resulted in the gene expression profile that accompanies granulosa cell differentiation. Our data also revealed that the activation of PKC in granulosa cells is also a strong differentiation signal that could control "advanced" differentiation in granulosa cells and the inflammatory cascade that occurs in the dominant follicle. According to our results, PKB activation provides support for PKA-stimulated gene expression and is also involved in granulosa cell survival throughout follicular development. Taken together, our results provide new information on PKA, PKB, and PKC signaling pathways and their roles in stimulating a follicle at the crossroad between maturation/ovulation and atresia.

Novel Phenothiazine-Bridged Porphyrin-(Hetero)aryl dyads: Synthesis, Optical Properties, In Vitro Cytotoxicity and Staining of Human Ovarian Tumor Cell Lines.

We report here the synthetic procedure applied for the preparation of new AB3-type and trans-A2B2 type meso-halogenophenothiazinyl-phenyl-porphyrin derivatives, their metal core complexation and their peripheral modification using Suzuki–Miyaura cross coupling reactions with various (hetero)aryl (phenothiazinyl, 7-formyl-phenothiazinyl, (9-carbazolyl)-phenyl and 4-formyl-phenyl, phenyl) boronic acid derivatives. The meso-phenothiazinyl-phenyl-porphyrin (MPP) dyes family was thus extended by a series of novel phenothiazine-bridged porphyrin-(hetero)aryl dyads characterized by UV–Vis absorption/emission properties typical to the porphyrin chromophore, slightly modulated by increasing the size of peripheral substituents. Three phenothiazine-bridged porphyrin-heteroaryl dyads with fluorescence emission above 655 nm were selected as fluorophores in red spectral region for applications in cellular staining of human ovarian tumors. In vitro experiments of cell metabolic activity displayed a moderate toxicity on human ovarian tumor cell lines (OVCAR-3, cisplatin-sensitive A2780 and cisplatin-resistant A2780cis respectively). Visualization of the stained living cells was performed both by fluorescence microscopy imaging and by fluorescence lifetime imaging under two photon excitation (TPE-FLIM), confirming their cellular uptake and the capability of staining the cell nucleus.

Resveratrol protects mitochondrial quantity by activating SIRT1/PGC-1α expression during ovarian hypoxia.

PurposeResveratrol is a well‐known potent activator of sirtuin‐1 (SIRT1). We investigated the direct effects of hypoxia and resveratrol on SIRT1/ peroxisome proliferator‐activated receptor‐gamma coactivator 1α (PGC‐1α) pathways, vascular endothelial growth factor (VEGF), hypoxia‐inducible factor (HIF)‐1α, and mitochondrial quantity in a steroidogenic human ovarian granulosa‐like tumor cell line (KGN) cells.MethodsKGN cells were cultured with cobalt chloride (CoCl2; a hypoxia‐mimicking agent) and/or resveratrol. The mRNA and protein levels, protein secretion, and intracellular localization were assessed by real‐time PCR, Western blot analysis, ELISA, and immunofluorescence staining, respectively. Mitochondrial quantity was measured based on the mitochondrial DNA (mtDNA) copy number.ResultsCoCl2 simultaneously attenuated the levels of SIRT1 and mtDNA expression, and induced the levels of VEGF protein production. In contrast, resveratrol significantly increased the levels of SIRT1 and mtDNA copy number, but reduced VEGF production in normoxia. Resveratrol could recover CoCl2‐suppressed SIRT1 and mtDNA expression and antagonize CoCl2‐induced VEGF production. CoCl2 treatment resulted in a downregulation of PGC‐1α expression, and this effect was recovered by resveratrol. Resveratrol significantly suppressed the production of the CoCl2‐induced HIF‐1α and VEGF proteins.ConclusionThese results suggest that resveratrol improves mitochondrial quantity by activating the SIRT1/PGC‐1α pathway and inhibits VEGF induction through HIF‐1α under hypoxic conditions.

Demonstration of anti-tumour bystander killing with prodrug-preloaded suicide gene-engineered tumour cells: a potential improvement for cancer therapeutics

BackgroundTherapeutic approaches for cancer rely on careful consideration of finding the optimal way of delivering the pro-drug for cellular-based cancer treatment. Cell lines and cell cultures have been used in these studies to compare the in vitro and in vivo efficacy of autologous vs. allogeneic tumour cellular gene therapy. Here we have investigated and are reporting for the first time the effect of prodrug ganciclovir (GCV)-preloading (pre-treatment) in suicide gene therapy of cancer.MethodsThis study examines the effect of GCV-preloading (pre-treatment) on a range of tumour cell lines in conjunction with suicide gene therapy of cancer. To determine the efficacy of this modality, a series of in vitro and in vivo experiments were conducted using genetically modified and unmodified tumour cell lines.ResultsFollowing co-culture of herpes simplex virus thymidine kinase (HSV-TK) modified tumour cells and unmodified tumour cells both in vitro and in vivo, GCV-preloading (pre-treatment) of TK-modified human and mouse mesothelioma cells and ovarian tumour cells allowed them to mediate efficiently bystander killing of neighbouring unmodified tumour cells in vitro. In contrast, GCV-preloading of TK-modified human and mouse mesothelioma cells and ovarian tumour cells abolished their in vivo ability to induce bystander killing of unmodified tumour cells, although there was some tumour regression compared to control groups but this was not statistically significant. These results suggest that preloading TK modified tumour cells with GCV needs further study to define the most effective strategy for an in vivo application to retain their bystander killing potential after exposure to lethal doses of GCV in vitro.ConclusionsThis study highlights the promising possibility of improving the efficacy of pro-drug system to prevent any damage to the immune system and enhancing this type of suicide gene therapy of cancer, as well as the need for further studies to explore the discrepancies between in vitro and in vivo results.

Determination of Endogenous Radioresistance Biomarkers on HeLa Cells

The Genes such as carbonic anhydrase IX (CAIX), hypoxia inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) have been suggested as hypoxic biomarkers in cancer. Indeed, these endogenous biomarkers have been shown to have stronger prognostic value response after treatment by irradiation. However, inconsistent results suggest that factors other than oxygen influence their expression. This present study deciphers the level of expression of different radioresistance biomarkers in both normoxia and hypoxia conditions followed by irradiation of human ovarian tumor cell lines (uterine cervix squamous cell carcinoma (HeLa). HeLa cells were submitted to hypoxia (1% O2) conditions in a Thermo Scientific Heracell i CO2 incubator. The cells were subjected to two doses 4-10 Gy irradiation and re-incubate in their starting conditions for 4 hours, then fixed in 4% paraformaldehyde for 20 min. Protein expressions were assessed by immunocytochemistry staining and fluorescent images were captured by a Axio Imager Z1 fluorescence microscope with oil immersion lens at 63× magnification. In normoxia conditions there was no modification of the level of expression of the CAIX after irradiation. However, an increasing expression level of VEGF was noted. The level of expression of HIF-1 in normoxia was low compared to the other two proteins (CAIX and VEGF). Hypoxia conditions at 2% resulted in a low expression of CAIX and VEGF before and after irradiation at 10 Gy in HeLa cells. HIF-1 had a maximum expression level compared to CAIX and VEGF at 2% oxygen after irradiation in HeLa cells. As tumor hypoxia occurs in a deprived microenvironment, other environmental factors such as irradiation might interact with the effect of low oxygen concentration on gene expression. This study shows that irradiation of HeLa cells has a profound influence on the oxygen dependent induction of certain endogenous hypoxic markers as HIF-1, CAIX, and VEGF.

Abstract AP29: A SINGLE CELL RNA-SEQUENCING APPROACH TO UNCOVERING HUMAN OVARIAN TUMOR AND IMMUNE CELL HETEROGENEITY, AND THEIR RESPONSE TO MULLERIAN INHIBITING SUBSTANCE USING PATIENT ASCITES SAMPLES

Abstract Mullerian inhibiting substance (MIS) has been shown to inhibit the growth of a stem-like population of ovarian cancer cells both in vivo and in vitro. We have previously shown that its receptor, MISRII, is expressed in a majority of ovarian serous adenocarcinomas. However the determinants of response and the pathways elicited by MIS treatment have been difficult to elucidate due the heterogeneity of tumors both across the patient population and within each sample. To address this complexity we have developed an assay in which patient cancer cells and immune cells are isolated from ascites of recurrent chemoresistant ovarian cancer and incubated in cleared supernatant from autologous ascites for 24h in presence of drug (MIS 10ug/ml) or vehicle control (N=3). This protocol allows for short-term culture in conditions nearly identical to the peritoneal environment. A parallel experiment was conducted using matching pure primary cancer cell lines (derived from the ascites samples) treated in media (MIS 10ug/ml or vehicle for 24h), but lacking immune cells and soluble factors otherwise present in ascites. The effect of treatment on cell sates, and the interaction between immune cells and cancer cells was interrogated by clustering analysis of single cell RNA sequencing (inDROP). We uncovered a high degree of heterogeneity of expression of known and novel markers related to epithelial-mesenchymal states and stemness markers, both across patients and within patient samples, suggesting dynamically coexisting cancer cell states and hierarchies that are patient-specific. These markers were validated in vitro across a larger cohort of primary patient cells lines (N=12) grown as tumor spheroids (with and without MIS 10ug/ml for 24h). Additionally, we have identified a varying abundance of immune cell types (Macrophages, dendritic cells, NK cells, T cells, B cells), and an unexpected effect of MIS on gene expression in both macrophage and cancer cells. The knowledge gained on the response rate in patients, and the biomarkers identified in this study will be fundamental for the successful translation of MIS to the clinic, and further our understanding of the role of non-cell autonomous inhibition of cancer cells by immune components and patient heterogeneity. Citation Format: Raghav Mohan, Motohiro Kano, Hatice Saatcioglu, Nobuhiro Takahashi, LiHua Zhang, Nicholas Nagykery, Joy Poulo, Patricia K Donahoe, David Pepin. A SINGLE CELL RNA-SEQUENCING APPROACH TO UNCOVERING HUMAN OVARIAN TUMOR AND IMMUNE CELL HETEROGENEITY, AND THEIR RESPONSE TO MULLERIAN INHIBITING SUBSTANCE USING PATIENT ASCITES SAMPLES [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP29.

Long noncoding RNA PCAT-1 knockdown prevents the development of ovarian cancer cells via microRNA-124-3p.

Long noncoding RNA prostate cancer-associated transcript 1 (PCAT-1) is overexpressed in human malignancies and its silence abates the exaggeration of cancers. Whereas, the activity of PCAT-1 silence in ovarian cancer (OC) remains elusive. Here, our study was designed to corroborate the function of PCAT-1 silence in cellular activities and the molecular mechanisms. PCAT-1 in human ovarian tumor tissue specimens and cell lines (A2780 and SKOV3) were quantified by real-time quantitative reverse polymerase chain reaction (qRT-PCR). Reinforced silence of PCAT-1 and microRNA (miR)-124-3p was established by transfection and identified by qRT-PCR. The viability, apoptosis as well as migration and invasion were examined. Western blot was exploited for analysis of proteins involved in proliferation, apoptosis, migration and invasion, and signaling transduction. OC tissues showed the accumulation of PCAT-1. Silencing PCAT-1 caused the impediment of proliferation, migration, and invasion with the increase in apoptosis. PCAT-1 knockdown repressed the expression of cyclin D1, CDK6, p53, Bax, cleaved caspase-3, metallopeptidases, and vimentin with the restoration of miR-124-3p. However, the roles of PCAT-1 silence were weakened in the absence of miR-124-3p. PCAT-1 silence caused decrease in Wnt3a, β-catenin, and phosphorylation of protein kinase B and mechanistic target of rapamycin was abolished by miR-124-3p inhibitor. The tumor-suppressive role of PCAT-1 silence was mediated by miR-124-3p.

MiRNA-21 inhibition suppresses the human epithelial ovarian cancer by targeting PTEN signal pathway.

ObjectTo explore the role of microRNA-21 in human epithelial ovarian cancer (EOC).MethodsWe used RT-PCR to test the expressions of miRNA-21 in EOC cells and normal ovarian epithelial cells, as well as the tumor samples and the tumor-adjacent normal tissues. The vector of LV3 pGLV-H1-GFP-miR-21 was used to decrease the level expression of endogenous miR-21 in cells. Further, we investigated how miR-21 affected the biological events of EOC through determining the changes in proliferation, cycle and invasion of EOC cells, and measured the tumorigenesis in xenograft models. The association between phosphatase and tensin homolog deleted on chromosome ten (PTEN) and miR-21 were tested by RT-PCR. Next, siRNA was used to knockdown PTEN gene which help us to assess the functional association between miR-21 and PTEN in vivo and in vitro.ResultsIn EOC cell lines and human epithelial ovarian tumor cells, we found that miR-21 altered the biological features of EOC cells, including suppression of proliferation and invasion and arrest of cell cycle, and also resulted in a decrease in tumorigenesis in the in vitro xenograft models. The association between PTEN and miR-21 was confirmed in previous research. From our results, the down-regulation of PTEN gave rise to the miR-21 decrease, regardless of the cells or tissues.ConclusionThe suppression of microRNA-21 inhibits the progression of EOC profoundly. In EOC, miR-21 is negatively correlated with the expression of PTEN gene.

Estrogen regulates forkhead transcription factor 2 to promote apoptosis of human ovarian granulosa-like tumor cells

Granulosa cell tumors of the ovary (GCTs) are the predominant form of ovarian stromal tumors and can lead to abnormally secreted estrogen hormones. Studies have reported that forkhead transcription factor 2 (FOXL2) inhibits estrogen synthesis and its gene mutation can lead to GCTs. We unexpected found that estrogen also regulates the expression level of FOXL2. High-dose estrogen increased the expression of FOXL2 in ovarian-like granulosa (KGN) cells at both the mRNA and protein levels. However, no research has reported on the molecular regulatory mechanism and function between estrogen and FOXL2 in the development of GCTs. In this research, FOXL2 was highly expressed in KGN cells and ovarian stromal tumor tissues. Deletion of FOXL2 increased the estrogen secretion in KGN cells. In turn, high-dose estrogen increased the FOXL2 expression levels. FOXL2 was phosphorylated by GPR30 (G protein coupled receptor)-Protein kinase C (PKC) signaling pathway upon estrogen stimulation. Estrogen inhibited cell migration and proliferation, while promoting cell apoptosis. Deletion of FOXL2 inhibited the influence of estrogen on cell proliferation, migration, and apoptosis. Results suggest that estrogen via regulating FOXL2 suppresses cell proliferation and induces cell apoptosis.

Adiponectin receptor agonist AdipoRon induces apoptotic cell death and suppresses proliferation in human ovarian cancer cells.

We tested the hypothesis that stimulation of adiponectin receptors with the synthetic agonist AdipoRon suppresses proliferation and induces apoptotic death in human high grade serous ovarian tumor cell lines and in ex vivo primary tumors, mediated by activation of 5' AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR). We determined the effect of AdipoRon on high grade serous ovarian tumor cells lines (OVCAR3, OVCAR4, A2780) and ex vivo primary tumor tissue. Western blotting analysis was performed to examine changes in activation of AMPK and mTOR signaling and flow cytometry was utilized to examine changes in cell cycle progression. Immunofluorescence of cleaved caspase-3 positive cells and flow cytometry of annexin V positive cells were used to determine changes in apoptotic response. The CyQUANT proliferation assay was used to assess cell proliferation. AdipoRon treatment increased AMPK phosphorylation (OVCAR3 P = 0.01; A2780 P = 0.02) but did not significantly alter mTOR activity. AdipoRon induced G1 cell cycle arrest in OVCAR3 (+ 12.1%, P = 0.03) and A2780 (+ 12.0%, P = 0.002) cells. OVCAR3 and OVCAR4 cells treated with AdipoRon underwent apoptosis based on cleaved caspase-3 and annexin V staining. AdipoRon treatment resulted in a dose dependent decrease in cell number versus vehicle treatment in OVCAR3 (-61.2%, P < 0.001), OVCAR4 (-79%, P < 0.001), and A2780 (-56.9%, P < 0.001). Ex vivo culture of primary tumors treated with AdipoRon resulted in an increase in apoptosis measured with cleaved caspase-3 immunohistochemistry. AdipoRon induces activation of AMPK and exhibits an anti-tumor effect in ovarian cancer cell lines and primary tumor via a mTOR-independent pathway.

New findings on the antiproliferative activity of the silver(I) complex with 5-fluorouracil against human multi-resistant NCI/ADR-RES ovarian tumor cells

Metal complexes with antitumor activities have been studied as an alternative to overcome tumor resistance to current pharmaceuticals. Recently, we described the synthesis of a silver(I) complex with 5-fluorouracil (Ag-5fu) with an effective activity in vitro against human multi-resistant ovarian tumor cells (NCI/ADR-RES) when compared to 5-fluorouracil (5fu) and cisplatin. Therefore, for a better understanding of the effect of Ag-5fu and its precursors 5fu and silver(I), the compounds were evaluated by colony formation capacity and flow cytometry assays to analyze cell cycle and cell death induction [phosphatidylserine residues (PS) exposition, multicaspases activation, production of reactive oxygen species (ROS) and mitochondrial membrane depolarization] on NCI/ADR-RES tumor cells. As observed for 5fu, Ag-5fu was able to promote G1 phase arrest and to totally inhibit colony formation. Besides, as observed to AgNO3, Ag-5fu promoted a potent PS externalization and multicaspases activation with loss of plasmatic membrane integrity. None of the compounds induced reactive oxygen species (ROS) generation. The Ag-5fu promoted mitochondrial membrane depolarization over time. The results suggest that Ag-5fu may induce regulated cell death in NCI/ADR-RES cells probably by intrinsic apoptosis. Silver(I) and 5fu play different roles on the effect of Ag-5fu in NCI/ADR-RES cells, and the activity of the Ag-5fu complex seems to be more than a simple combination of the activities of free 5fu and silver(I) ions.

Synthesis and Preliminary Anticancer Evaluation of 6-Mercaptopurine –Methotrexate Conjugate as Possible Mutual Prodrug

Small molecules drug conjugate mutual prodrug design (SMDC) composed of folate and lethal agent conjugate, rigidly bonded via hydrophilic bridge and self immolative disulfide bond ; represent new interesting approaches for cancer treatment , the component of SMDC intended for targeting folate receptor , along with greater conservation of component until reaching the target tumor tissue . The designing and synthesis of compound VI and VIII derived from 6-Mercaptopurine (6-MP) and Methotrexate ( MTX) conjugate altogether as mutual prodrugs were processed forward successfully by multistep reaction procedures , and by Thin Layer Chromatography (TLC) for preliminary detection of products and their intermediates, along with their purity. The structures of two final compounds and their intermediates were proclaimed by melting point measurement, infrared spectrometry and ¹HNMR analysis given results greatly correspond with theoretical proposed chemical structure of synthesized compounds. Furthermore, cytotoxic activity evaluation on cell line level had been done for two final compounds against human breast tumor cell (MCF-7) and human ovarian tumor cell (SKO-3) types of cancer cell line and the results were confirmed which show greater cytotoxic tumor activity of two final compounds, while compound VI possess optimal activity proportional with increased number of 6-MP molecules.

Next Generation Potent Cell‐active Inhibitors of the Oncogenic PTP4A3 Phosphatase

High grade serous ovarian cancer and triple negative breast cancer respond poorly to existing drugs and, thus, demand new therapies. The protein tyrosine phosphatase PTP4A3 (Phosphatase of Regenerating Liver‐3, PRL‐3) is overexpressed in these diseases. Moreover, PTP4A3 promotes cancer cell migration and invasion and is believed to be the most oncogenic of all tyrosine phosphatases. We recently synthesized the novel iminothienopyridone, JMS‐053, and demonstrated that it is a specific, reversible, allosteric, and cell active small molecule PTP4A3 inhibitor with an in vitro IC50 for PTP4A3 of 30 nM. The water solubility of JMS‐053 is limited, however. Therefore, we synthesized twenty next generation analogs to improve aqueous solubility and to aid in determining the structure activity relationship for the chemotype, the PTP4A3 binding site, and the mechanism of inhibition. Seventeen analogs retained potent in vitro PTP4A3 inhibition with IC50 values &lt;100 nM. Four compounds, EJR‐866‐36, EJR‐866‐68, EJR‐855‐81, and NRT‐892‐04, had in vitro PTP4A3 IC50values of &lt;40 nM. EJR‐866‐75 and NRT‐870‐59 were computationally more drug‐like than JMS‐053. EJR‐855‐81, NRT‐870‐59, EJR‐866‐75, and NRT‐870‐59 were cytotoxic to human Hs578T breast cancer and OVCAR4 ovarian cancer 3D spheroid tumor cells grown in vitro with IC50 values below 20 mM. To further expand the mechanistic activity of this class of inhibitors, we have synthesized four third‐generation analogs of JMS‐053, which combine the iminothienopyridone motif with a chemical moiety capable of binding a E3 ubiquitin ligase that will accelerate the intracellular degradation of PTP4A3. One of these novel compounds, EJR‐887‐35, had a PTP4A3 in vitro IC50 value of 107 nM and retained cytotoxicity to Hs578T and OVCAR4 cells with IC50 values below 20 mM in 3D spheroid tumors. These results suggest the iminothienopyridone chemotype could be valuable for the development of the first potent, cell active PTP4A3 phosphatase inhibitors directed against breast and ovarian cancer.Support or Funding InformationDepartment of Defense (Award W81XWH‐18‐1‐0011, BC170507), NIH Grant P30 CA044579 and S10 OD021723, and the Fiske Drug Discovery Fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulation of double-stranded RNA pattern recognition receptor signaling in ovarian cancer cells promotes inflammatory queues.

Inflammation and cancer are inter-related, and both pro- and anti-tumorigenic effects are possible in different contexts, highlighting the importance of characterizing specific inflammatory pathways in distinct tumor types. Malignant cells and non-cancerous cells such as fibroblasts, infiltrating leukocytes (i.e., dendritic cells [DC], macrophages, or lymphocytes) and endothelial cells, in combination with the extracellular matrix, constitute the tumor microenvironment (TME). In the last decades, the role of the TME in cancer progression has gained increased attention and efforts directed at abrogating its deleterious effects on anti-cancer therapies have been ongoing. In this context, we investigated the potential of mouse and human ovarian cancer cells to produce inflammatory factors in response to pathogen recognition receptor (PRR) signaling, which might help to shape the biology of the TME. We determined that mouse ovarian tumors generate chemokines that are able to interact with receptors harbored by tumor-associated DCs. We also found that dsRNA triggers significant pro-inflammatory cytokine up-regulation in both human and mouse ovarian tumor cell lines, and that several PRR can simultaneously contribute to the stimulated inflammatory response displayed by these cells. Thus, dsRNA-activated PRRs may not only constitute potentially relevant drug targets for therapies aiming to prevent inflammation associated with leukocyte recruitment, or as co-adjuvants of therapeutic treatments, but also might have a role in development of nascent tumors, for example via activation of cancer cells by microbial molecules associated to pathogens, or with those appearing in circulation due to dysbiosis.

Abstract A57: Adoptive T cell therapy for ovarian cancer: Application of a surgically relevant model

Abstract Ovarian cancer is the most lethal gynecologic cancer, with more than 20,000 new diagnoses and 15,000 cancer-related deaths annually. Current standard therapy for advanced ovarian cancer incorporates upfront surgical cytoreduction followed by cytotoxic chemotherapy. But even with advances in surgical technique, chemotherapeutics and targeted delivery methods, the overall five-year survival rate remains ~50%, highlighting the need for novel treatment strategies. We have focused our efforts on immunotherapy by adoptive transfer of engineered T cells that can target proteins uniquely overexpressed in ovarian cancer. Recent work has identified mesothelin (MSLN) as a potential target, as this protein is overexpressed in malignant ovarian tissue, promotes the oncogenic phenotype, and has limited expression in healthy cells. Preclinical studies have demonstrated that mouse and human T cells engineered to express T cell receptors (TCR) with high-affinity for MSLN kill respectively murine and human ovarian tumor cells in vitro. Furthermore, in vivo studies in mice treating metastatic disease developing after intraperitoneal introduction of mouse ID8 ovarian cancer cells have demonstrated that engineered, adoptively transferred T cells preferentially accumulate in ID8 ovarian cancers, reduce tumor burden and prolong survival. However, our results also revealed that the tumor microenvironment (TME) can limit the persistence and killing capacity of engineered T cells. Immunosuppressive cells, inhibitory ligands that reduce T cell function, and cell death-inducing ligands are abundant within ID8 tumors, and cellular and molecular analyses of human ovarian cancer specimens showed that human T cell therapy will face similar TME-mediated obstacles. The ovarian cancer TME is also a nutrient- and oxygen-deprived milieu, and the adaptive metabolic responses required for survival by infiltrating T cells have protean effects on T cell function. Thus, strategies that modulate T cell responses to inhibitory signals, including metabolic pathways, have the potential to influence activity in the TME, and to enhance T cell function and improve anti-tumor efficacy. Ongoing studies exploring strategies to overcome elements common to the human and murine TME, including both direct modulation of the microenvironment and engineering of T cells to overcome critical components of immune evasion by solid tumors and to promote T cell survival and function, will be discussed. Building upon this work, we have been developing a novel model that more directly reflects the clinical setting requiring intervention. Following orthotopic injection of ID8 ovarian cancer cells directly beneath the ovarian bursa, tumorigenesis occurs initially within the ovary/fallopian tube microenvironment, and is followed by development of small volume metastatic disease. This orthotopic model, which results in formation of intraperitoneal metastatic implants originating from the ovary, replicates the development and patterns of metastatic spread of human ovarian cancer. Following intervention with primary surgical cytoreduction that includes hysterectomy and bilateral salpingo-oophorectomy, mice are being treated by adoptive transfer of antigen-specific T cells to target minimal residual disease in the peritoneal cavity, which models the clinical situation of patients requiring treatment after optimal surgical cytoreduction. Ongoing studies will be discussed that have been designed to modulate the function and persistence of transferred, MSLN-specific T cells and assess the impact on in vivo tumor development and survival in this clinically relevant model of ovarian cancer treatment. Citation Format: Christopher B. Morse, Kristin G. Anderson, Breanna M. Bates, Edison Y. Chiu, Nicolas M. Garcia, Philip D. Greenberg. Adoptive T cell therapy for ovarian cancer: Application of a surgically relevant model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A57.

Cadmium induces ovarian granulosa cell damage by activating PERK-eIF2α-ATF4 through endoplasmic reticulum stress.

This study aimed to investigate whether cadmium induces ovarian granulosa cell damage by activating protein kinase R-like endoplasmic reticulum kinase (PERK)-eIF2α-ATF4 through endoplasmic reticulum (ER) stress and to elucidate the underlying regulation mechanism. Two models of cadmium exposure were established. In one model, ovarian granulosa cells isolated from 21-day-old female Sprague Dawley rats were cultured in vitro for 36 h and exposed to CdCl2 (0, 5, 10, and 20 μM), and in another model, a human ovarian granulosa tumor cell line (COV434) was used to construct the binding immunoglobulin protein (BIP)-knockdown cell line sh-BIP and exposed to 0 and 20 μM CdCl2. After exposure to cadmium for 12 h, the expression mRNA and protein levels of BIP, p-PERK, and p-eIF2α were determined in the two models. miRNAs related to BIP were also detected in granulosa cells after cadmium exposure. We found that mRNA and protein levels of all factors were upregulated in each cadmium-dose group, except for BIP mRNA expression in the 5 μM Cd group. The BIP gene was knocked down in COV434 cells before exposure to cadmium. All factors were upregulated in COV434 cells exposed to Cd, and the expression of the p-eIF2α protein was downregulated in sh-BIP cells exposed to Cd. In addition, no differences in BIP-related miRNAs were detected in cadmium-exposed rat ovarian granulosa cells versus the control group. Cadmium induces ovarian granulosa cell damage by inducing ER stress.

Abstract 2555: Engineering adoptive T cell therapy to co-opt Fas ligand-mediated death signaling in solid tumors

Abstract Over 20,000 women are diagnosed with ovarian cancer annually, and over half will die within 5 years. This rate has changed little in the last 20 years, highlighting the need for therapy innovation. A promising new strategy with the potential to control tumor growth without toxicity to healthy tissues employs immune T cells engineered to target proteins uniquely overexpressed in tumors. Mesothelin (MSLN) contributes to the malignant and invasive phenotype in ovarian cancer, and has limited expression in healthy cells, making it a candidate immunotherapy target in these tumors. Using patient-derived cell lines and the ID8 mouse cell line, we found that T cells engineered to express a human or mouse MSLN-specific high-affinity T cell receptor (TCRMSLN) can kill respectively human or murine ovarian tumor cells in vitro. In a disseminated ID8 tumor model, adoptively transferred TCRMSLN T cells preferentially accumulated within established tumors, delayed ovarian tumor growth, and significantly prolonged mouse survival. However, our data also revealed that elements in the tumor microenvironment (TME) limit engineered T cell persistence and ability to kill cancer cells. To identify immunosuppressive features active in the ovarian TME in both human and murine disease, we performed gene expression analyses on whole tumor tissue (Thermo Fisher) or sorted cell populations (Nanostring). Deep transcriptome profiling confirmed the expression of similar gene signatures in human cancers and the preclinical ID8 model, including immunosuppressive pathways. RNA sequencing revealed consistently high expression of Fas ligand (FasL), notable because FasL signaling can play a significant role in the generation and persistence of tumors. Fas/FasL signaling can mediate T cell death, including activation-induced cell death, an apoptotic mechanism responsible for regulating T cell expansion, suggesting tumor cells may upregulate FasL for protection from tumor-infiltrating lymphocytes. By flow cytometry and immunohistochemistry, we and others previously detected FasL in the tumor vasculature and TME of human ovarian cancer. To overcome this potential T cell evasion mechanism, we generated a panel of immunomodulatory fusion proteins (IFP) containing the Fas receptor (Fas) extracellular binding domain fused to a CD28 or 4-1BB co-stimulatory domain rather than the natural death domain. Relative to T cells modified with only TCRMSLN, T cells engineered to express both TCRMSLN and a Fas IFP better infiltrate tumors, preferentially expand/persist, and retain function in the TME when transferred into tumor-bearing mice. Moreover, adoptive immunotherapy with IFP+ T cells significantly prolonged survival in tumor-bearing mice, relative to TCRMSLN T cells lacking an IFP. As many solid tumors overexpress FasL, the use of IFPs may provide an opportunity to enhance engineered adoptive T cell therapy in many other malignancies. Citation Format: Kristin Anderson, Shannon Oda, Breanna Bates, Edison Chiu, Christopher Morse, Nicolas Garcia, Philip Greenberg. Engineering adoptive T cell therapy to co-opt Fas ligand-mediated death signaling in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2555.