Effects In Ovarian Cancer Cells Research Articles

Inhibition of CDK1 by RO-3306 Exhibits Anti-Tumorigenic Effects in Ovarian Cancer Cells and a Transgenic Mouse Model of Ovarian Cancer.

Ovarian cancer is the deadliest gynecological malignancy of the reproductive organs in the United States. Cyclin-dependent kinase 1 (CDK1) is an important cell cycle regulatory protein that specifically controls the G2/M phase transition of the cell cycle. RO-3306 is a selective, ATP-competitive, and cell-permeable CDK1 inhibitor that shows potent anti-tumor activity in multiple pre-clinical models. In this study, we investigated the effect of CDK1 expression on the prognosis of patients with ovarian cancer and the anti-tumorigenic effect of RO-3306 in both ovarian cancer cell lines and a genetically engineered mouse model of high-grade serous ovarian cancer (KpB model). In 147 patients with epithelial ovarian cancer, the overexpression of CDK1 was significantly associated with poor prognosis compared with a low expression group. RO-3306 significantly inhibited cellular proliferation, induced apoptosis, caused cellular stress, and reduced cell migration. The treatment of KpB mice with RO-3306 for four weeks showed a significant decrease in tumor weight under obese and lean conditions without obvious side effects. Overall, our results demonstrate that the inhibition of CDK1 activity by RO-3306 effectively reduces cell proliferation and tumor growth, providing biological evidence for future clinical trials of CDK1 inhibitors in ovarian cancer.

Knockdown of circMFN2 inhibits cell progression and glycolysis by miR-198/CUL4B pathway in ovarian cancer.

Circular RNA (circRNA) regulates malignant tumors, including ovarian cancer (OC). The present research study aimed to reveal the biological mechanism of circRNA mitofusin 2 (circMFN2) in OC. Cell biological behaviors were investigated using clonogenicity assay, EdU assay, transwell assay, and flow cytometry analysis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analysis were implemented to detect the levels of circMFN2, miR-198, Cullin 4B (CUL4B), and apoptosis-related proteins. Glycolysis was assessed by glucose assay kit, lactate assay kit, and ATP level detection kit. The relationships among miR-198, circMFN2, and CUL4B were verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. The xenograft mice model was used to analyze tumor growth in vivo. The expression of circMFN2 and CUL4B was increased, while miR-330-5p was decreased in OC tissues or cells. TheabsenceofCircMFN2hindered cell proliferation, migration, invasion, and glycolysisand promoted apoptosis in OC cells. We found that circMFN2 promoted CUL4B expression via sponging miR-198. MiR-198 depletion reversed circMFN2 knockdown-induced effects in OC cells. Furthermore, CUL4B overexpression overturned the inhibitory effect of miR-198 in OC cells. And the absence of circMFN2 inhibited tumor growth in vivo. CircMFN2 repressed OC progression by regulating the miR-198/CUL4B axis.

USP7 mediates TRAF4 deubiquitination to facilitate the malignant phenotype of ovarian cancer via the RSK4/PI3K/AKT axis.

Ubiquitin-specific peptidase 7 (USP7) is upregulated in multiple human cancers, including ovarian cancer; however, its functional role in the latter remains largely unknown. We conducted quantitative real-time PCR to detect the expression of USP7, TRAF4, and RSK4 in ovarian cancer cell lines. In addition, Western blotting served to determine USP7, TRAF4, RSK4, PI3K, and AKT (protein kinase B,PKB) protein levels and USP7 expression in the tissues was detected by immunohistochemical staining. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to evaluate cell viability, transwell assays to evaluate cell migration and invasion, and co-immunoprecipitation to evaluate TRAF4 ubiquitination. The results showed USP7 and TRAF4 upregulation, and RSK4 downregulation in ovarian cancer cell lines. Knocking down USP7 suppressed viability, migration, and invasion of ovarian cancer cells; TRAF4 knockdown and RSK4 overexpression had similar effects in ovarian cancer cells. TRAF4 is deubiquitinated and stabilized by USP7, whereas RSK4 is negatively regulated by TRAF4. A mouse xenograft model confirmed that knocking down USP7 suppressed ovarian tumor growth by regulating the TRAF4/RSK4/PI3K/AKT axis. Knocking down USP7 decreased the proliferation, migration, and invasion of ovarian cancer cells and suppressed ovarian tumor growth in mice. Mechanistically, USP7 increased TRAF4 ubiquitination, promoting its degradation and leading to RSK4 upregulation.

MiR-194-5p suppresses the warburg effect in ovarian cancer cells through the IGF1R/PI3K/AKT axis

Background: The Warburg effect is considered as a hallmark of various types of cancers, while the regulatory mechanism is poorly understood. Our previous study demonstrated that miR-194-5p directly targets and regulates insulin-like growth factor1 receptor (IGF1R). In this study, we aimed to investigate the role of miR-194-5p in the regulation of the Warburg effect in ovarian cancer cells. Methods: The stable ovarian cell lines with miR-194-5p overexpression or silencing IGF1R expression were established by lentivirus infection. ATP generation, glucose uptake, lactate production and extracellular acidification rate (ECAR) assay were used to analyze the effects of aerobic glycolysis in ovarian cancer cells. Gene expression was analyzed by quantitative polymerase chain reaction (qPCR) and western blot. Immunohistochemistry assays were performed to assess the expression of the IGF1R protein in ovarian cancer tissues. Results: Overexpression of miR-194-5p or silencing IGF1R expression in ovarian cancer cells decreases ATP generation, glucose uptake, lactate production, and ECAR and inhibits both the mRNA and protein expression of PKM2, LDHA, GLUT1, and GLUT3. While the knockdown of miR-194-5p expression led to opposite results. Overexpression of miR-194-5p or silencing IGF1R expression suppressed the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathway, whose activation can sustain aerobic glycolysis in cancer cells, and the knockdown of miR-194-5p expression promoted the activation of the PI3K/AKT pathway. Conclusion: Our results suggest that miR- 194-5p can inhibit the Warburg effect by negative regulation of IGF1R and further repression of the PI3K/AKT pathway, which provides a theoretical basis for further test of miR-194-5p as a target in the treatment of ovarian cancer.

Semaphorin 3A inhibits tumor progression via the downregulation of Lin28B in ovarian cancer.

Semaphorin 3A (Sema3A) has recently been proven to play an essential role in tumorigenesis. Here, the role of Sema3A in ovarian cancer is explored. The prognostic value of Sema3A was evaluated using the Kaplan-Meier plotter database, and stable expression cells were established by the delivery of lentivirus harboring SEMA3A cDNA or shRNA into OVCA433 and SKOV3 cells, respectively. Then CCK-8 assay, colony-formation assay, wound-healing assay, and Transwell assay were utilized to verify the effect of Sema3A on tumorigenesis. Co-cultures of ovarian cancer cells (OVCA433 and SKOV3) with a conditional medium collected from the established cells were further utilized to confirm the function of Sema3A. Then, the RNA-seq assay was adopted to explore the underlying mechanism. The results demonstrated that low expression of Sema3A was predictive of poor overall survival in patients with ovarian cancer. Functional experiments revealed that Sema3A inhibited proliferation, migration, and invasion in ovarian cancer cells. Secreted Sema3A in a conditioned culture medium also exhibited an anti-tumor effect in ovarian cancer cells. RNA-seq assay suggested that focal adhesion and Lin28B were involved in regulating Sema3A. Rescue assays further verified that Lin28B/ROCK1 axis was vital in the regulation of Sema3A and Lin28B significantly upregulated ROCK1 through let-7g microRNA. The presented data indicate that Sema3A inhibits proliferation and metastasis via the downregulation of Lin28B/ROCK1 in ovarian cancer.

Circular RNA pyridoxal kinase (circPDXK) involves in the progression of ovarian cancer and glycolysis via regulating miR-654-3p and hexokinase II

BackgroundCircular RNA pyridoxal kinase (circPDXK; hsa_circ_0061893) is newly identified to be aberrantly expressed in ovarian cancer (OVCA); however, its functional role in OVCA cells remains to be expounded.MethodsReal-time quantitative polymerase chain reaction, western blotting, and immunohistochemistry quantified RNA and protein expression levels. MiRNA binding site prediction tools predicted direct interaction between two RNAs, and dual-luciferase reporter and RNA immunoprecipitation assays further confirmed that prediction. Cell-counting kit-8, colony formation, and 5-ethynyl-2ʹ-deoxyuridine assays measured cell growth; nude mice xenograft tumor experiment detected tumor growth. Transwell and Annexin V-fluorescein isothiocyanate/propidium iodide staining assays evaluated cell motility and apoptosis. Glycolysis process was determined by glucose uptake, lactate, and ATP assay kits.ResultsCircPDXK is highly expressed in OVCA patients’ tumor tissues and cells, concomitant with microRNA (miR)-654-3p downregulation and hexokinase II (HK2) upregulation. RNA interference of circPDXK could restrain cell viability, colony formation, DNA synthesis, migration, invasion, and glycolysis of OVCA cells, but also retard xenograft tumor growth. Allied with those are higher apoptosis rate, elevated Bax and E-cadherin levels, and depressed ki67 and HK2 levels. Compared to circPDXK inhibition, restoration of miR-654-3p functions analogical effects in OVCA cells in vitro. Mechanistically, there are direct interactions between miR-654-3p and circPDXK or HK2; moreover, miR-654-3p inhibition could weaken the functional roles of circPDXK interference in OVCA cells, and either HK2 ectopic expression abrogates the effects of miR-654-3p overexpression.ConclusionCircPDXK/miR-654-3p/HK2 axis could be a novel molecular mechanism of OVCA progression and glycolysis, and targeting circPDXK might overcome OVCA.

Abstract 5820: NF-κB signaling regulates miRNA expression in ovarian cancer

Abstract Epithelial ovarian cancer (EOC) remains the fifth leading cause of cancer related death in women worldwide, partly due to the survival of chemoresistant, stem-like tumor-initiating cells (TICs) that promote disease relapse. Previous studies described the role of the NF-kB pathway in promoting TIC chemoresistance and survival through the action of key transcription factors (TFs) like RelA and RelB. These TFs serve as key master regulators of genes important for the inflammatory response, which also overlap with the regulation of targets associated with cancer promotion. We previously showed that RelA and RelB exert different effects in ovarian cancer cells, with RelB relatively more important for tumor initiation. In this study, we hypothesize that NF-kB signaling regulates the expression of several microRNAs (miRNAs) differentially through RelA and RelB to promote TIC survival and proliferation. miRNAs comprise a subset of small, noncoding RNAs that regulate gene expression and present potential therapeutic targets for clinical use. Inducible shRNA stably was expressed in OV90 EOC cells to knockdown RelA or RelB; cells were subsequently analyzed by miR-seq to identify differentially expressed miRNAs in cells grown in TIC vs adherent (adh) conditions. To validate the miR-seq findings, we performed qPCR assays of the candidate miRNAs differentially expressed in TIC or adh conditions with RelA or RelB knocked down. We confirmed the decreased expression of oncogenic miRNAs hsa-miR-105-5p and hsa-miR-452-5p, while also confirming the increased expression of tumor suppressive miRNA hsa-miR-34a-5p observed in the miR-seq using OV90 cells. Ongoing work will validate these expression changes in additional cell lines and conduct functional assays to characterize the effect of mimicking or inhibiting these miRNAs of interest. The identification of miRNAs differentially expressed in TICs under the control of RelA or RelB will provide key insights in designing treatments against them to prevent disease relapse in patients with EOC. Citation Format: Rahul D. Kamdar, Brittney S. Harrington, Soumya Korrapati, Nathan Wong, Carrie D. House, Christina M. Annunziata. NF-κB signaling regulates miRNA expression in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5820.

Decreased EMILIN2 correlates to metabolism phenotype and poor prognosis of ovarian cancer.

This study aimed to explore the function and related mechanisms of elastin microfibril interfacer 2 (EMILIN2) in ovarian cancer. First, the expression level of EMILIN2 was detected in patient tissues and its correlation with overall survival rate was analysed. Then, EMILIN2 was overexpressed in ovarian cancer cell lines to observe its function and effect on Warburg effect. By detecting its promoter region methylation, the epigenetic regulatory role was explored. Finally, through the luciferase reporter assay and siRNA tools, the regulatory mechanism of p53 on EMILIN2 was investigated. It was detected in clinical samples that down-regulated EMILIN2 was associated with poor prognosis of ovarian cancer. It was further found that EMILIN2 regulated the metabolic phenotype of ovarian cancer cells. The expression of EMILIN2 was epigenetically regulated by its promoter methylation. Also, it was found that p53 regulated the expression of EMILIN2 and the p53/EMILIN2 axis regulated the Warburg effect in ovarian cancer cells. EMILIN2 was inhibited by methylation in ovarian cancer. In summary, p53 can promote and regulate its transcription by binding to the promoter region of EMILIN2, thereby affecting the Warburg effect and inhibiting tumours. Therefore, EMILIN2 might be a potential target for clinical diagnosis and treatment of ovarian cancer.

Peptide Targeted Gold Nanoplatform Carrying miR-145 Induces Antitumoral Effects in Ovarian Cancer Cells.

One of the recent attractive therapeutic approaches for cancer treatment is restoring downregulated microRNAs. They play an essential muti-regulatory role in cellular processes such as proliferation, differentiation, survival, apoptosis, cell cycle, angiogenesis, and metastasis, among others. In this study, a gold nanoplatform (GNPF) carrying miR-145, a downregulated microRNA in many cancer types, including epithelial ovarian cancer, was designed and synthesized. For targeting purposes, the GNPF was functionalized with the FSH33 peptide, which provided selectivity for ovarian cancer, and loaded with the miR-145 to obtain the nanosystem GNPF-miR-145. The GNPF-mir-145 was selectively incorporated in A2780 and SKOV3 cells and significantly inhibited cell viability and migration and exhibited proliferative and anchor-independent growth capacities. Moreover, it diminished VEGF release and reduced the spheroid size of ovarian cancer through the damage of cell membranes, thus decreasing cell viability and possibly activating apoptosis. These results provide important advances in developing miR-based therapies using nanoparticles as selective vectors and provide approaches for in vivo evaluation.

Iron Administration Overcomes Resistance to Erastin-Mediated Ferroptosis in Ovarian Cancer Cells.

ObjectivesDeveloping novel therapeutic approaches to defeat chemoresistance is the major goal of ovarian cancer research. Induction of ferroptosis has shown promising antitumor effects in ovarian cancer cells, but the existence of still undefined genetic and metabolic determinants of susceptibility has so far limited the application of ferroptosis inducers in vivo.MethodsErastin and/or the iron compound ferlixit were used to trigger ferroptosis in HEY, COV318, PEO4, and A2780CP ovarian cancer cell lines. Cell viability and cell death were measured by MTT and PI flow cytometry assay, respectively. The “ballooning” phenotype was tested as ferroptosis specific morphological feature. Mitochondrial dysfunction was evaluated based on ultrastructural changes, mitochondrial ROS, and mitochondrial membrane polarization. Lipid peroxidation was tested through both C11-BODIPY and malondialdehyde assays. VDAC2 and GPX4 protein levels were quantified as additional putative indicators of mitochondrial dysfunction or lipid peroxidation, respectively. The effect of erastin/ferlixit treatments on iron metabolism was analyzed by measuring intracellular labile iron pool and ROS. FtH and NCOA4 were measured as biomarkers of ferritinophagy.ResultsHere, we provide evidence that erastin is unable to induce ferroptosis in a series of ovarian cancer cell lines. In HEY cells, provided with a high intracellular labile iron pool, erastin treatment is accompanied by NCOA4-mediated ferritinophagy and mitochondrial dysfunction, thus triggering ferroptosis. In agreement, iron chelation counteracts erastin-induced ferroptosis in these cells. COV318 cells, with low baseline intracellular labile iron pool, appear resistant to erastin treatment. Notably, the use of ferlixit sensitizes COV318 cells to erastin through a NCOA4-independent intracellular iron accumulation and mitochondrial dysfunction. Ferlixit alone mimics erastin effects and promotes ferroptosis in HEY cells.ConclusionThis study proposes both the baseline and the induced intracellular free iron level as a significant determinant of ferroptosis sensitivity and discusses the potential use of ferlixit in combination with erastin to overcome ferroptosis chemoresistance in ovarian cancer.

The novel function of PMS2 mutation on ovarian cancer proliferation

The PMS2 gene encodes a protein that plays an essential role in repairing DNA. Recent studies have shown that the S815L PMS2 mutation, rarely found in ovarian cancer (3%), causes a reduction in the PMS2 protein’s mismatch repair activity in vitro. However, no studies have focused on how this mutation affects cancer progression. Although occurrences of patients with cancers associated with the PMS2 mutation are rare, the lack of studies published on this mutation still negatively impacts their treatment. Our study aimed to discover the novel effect of this mutation in ovarian cancer cells. We hypothesized that overexpressing the wild-type PMS2 gene would have tumor-suppressive effects while overexpressing S815L PMS2 mutant would negatively affect ovarian cancer cell proliferation. We tested three transfection conditions: pcDNA3 as a negative control variant, wild-type pcDNA3-PMS2, and pcDNA3-PMS2 mutated vectors. Using RT-PCR analysis, we optimized the transfection conditions for overexpression of both the wild-type PMS2 and mutant genes in OVCAR-3 cells. We acquired microscopic images to observe cell morphology and colonization. Also, we performed a Prestoblue assay to quantify the number of live cells in each condition. Results showed that the pcDNA3- PMS2 mutated vector has a higher cell proliferation than the wild-type vectors. As hypothesized, overexpressing the wild-type PMS2 gene had tumor-suppressive effects. This observation shows that the pcDNA3-PMS2 mutation has a tumor-progressive impact on the OVCAR-3 cells. Overall, we conclude that the PMS2 mutation has tumor-progressive effects in ovarian cancer cells while the unmutated version of the gene does not.

IFNγ induces JAK1/STAT1/p65 NFκB-dependent interleukin-8 expression in ovarian cancer cells, resulting in their increased migration.

Interferon-γ (IFNγ) is a pleiotropic cytokine that has a crucial role in immune response and tumor immunity. Because of its anti-tumor effects, IFNγ has been used in cancer treatment. However, IFNγ also has tumor-promoting functions that are less well understood. Here, we show that IFNγ induces expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) in ovarian cancer (OC) cells. The IFNγ-induced IL-8 expression is dependent on JAK1, STAT1, and p65 NFκB, and is associated with an increased occupancy of K314/315 acetylated p65 NFκB and Ser-727 phosphorylated STAT1 at the IL-8 promoter. Neutralization of IL-8 using anti-IL-8 antibody reduces IFNγ-induced migration of OC cells, and their invasion ability in 3D spheroids. Together, these findings identify IL-8 as a novel target induced by IFNγ/JAK1/STAT1/p65 NFκB signaling, and indicate that the IFNγ-induced IL-8 contributes to IFNγ pro-tumorigenic effects in ovarian cancer cells.

The FGFR Family Inhibitor AZD4547 Exerts an Antitumor Effect in Ovarian Cancer Cells.

The dysregulation of fibroblast growth factor (FGF) signaling has been implicated in tumorigenesis, tumor progression, angiogenesis, and chemoresistance. The small-molecule AZD4547 is a potent inhibitor of FGF receptors. This study was performed to investigate the antitumor effects and determine the mechanistic details of AZD4547 in ovarian cancer cells. AZD4547 markedly inhibited the proliferation and increased the apoptosis of ovarian cancer cells. AZD4547 also suppressed the migration and invasion of ovarian cancer cells under nontoxic conditions. Furthermore, it attenuated the formation of spheroids and the self-renewal capacities of ovarian cancer stem cells and exerted an antiangiogenic effect. It also suppressed in vivo tumor growth in mice. Collectively, this study demonstrated the antitumor effect of AZD4547 in ovarian cancer cells and suggests that it is a promising agent for ovarian cancer therapy.

Tumor extracelluar hyroxyapatite: a potential biomarker for imaging ovarian cancer

<h3>Objectives:</h3> Identifying new strategies to improve response to poly ADP-ribose polymerase inhibitors (PARPi) in women with homologous recombination (HR)-proficient ovarian cancer is a key clinical challenge. The nuclear orphan receptor NR4A1 has pro-tumor effects in ovarian cancer cells through transcriptional regulation of key genes promoting growth, survival and HR DNA repair. NR4A1 is also expressed in immune cells in the tumor microenvironment and may play a role in inhibiting re-polarization of pro-tumor M2-like tumor-associated macrophages (TAMs) to a more effective anti-tumor M1-like TAM phenotype. Our group has shown that the NF-kappaB (NF-ĸB) pathway is a key regulator of TAM phenotype, with upregulated NF-ĸB signaling promoting M1-like functions. Our goal therefore was to determine whether inhibition of NR4A1 can improve responses to PARPi both by increased tumor DNA damage and apoptosis, and by promoting an anti-tumor M1 phenotype in TAMs by modulating NF-ĸB activity. <h3>Methods:</h3> Cultured HR-proficient ID8 mouse ovarian cancer cells, mouse ovarian TAMs and mouse bone marrow-derived macrophages were treated with the established NR4A1 antagonist C-DIM-pPHOH (C-DIM) and/or the PARPi olaparib (OLA) for 24-72 hours. C57BL/6 mice with established intra-peritoneal ID8 tumors were treated with vehicle, C-DIM, OLA or combined C-DIM/OLA for 3 weeks. Standard techniques were used to determine cell growth (Sulforhodamine B assay), HR efficiency (<i>BRCA1</i> and RAD51 foci formation), NF-ĸB activity (nuclear p65, luciferase reporter activity), and expression of markers of DNA damage (pH2AX), apoptosis (cleaved PARP), proliferation (PCNA), and macrophage M1 (CCL3, iNOS) and M2 (CD206, arginase-1) polarization. Statistical significance (p<0.05) between group means was determined by Student's t test for <i>in vitro</i> studies and Mann-Whitney test for mouse studies. <h3>Results:</h3> In ID8 cells, co-treatment with C-DIM and OLA significantly reduced cell growth and HR efficiency, and increased DNA-damage-induced apoptosis, compared to OLA treatment alone. NR4A1 was highly expressed in macrophages, and C-DIM increased NF-ĸB activity and induced M1 polarization alone and when combined with olaparib. Similar effects of combined treatment were observed <i>in vivo.</i> Compared to mice treated with OLA alone, there was significantly reduced ascites volume and omental tumor mass in C-DIM/OLA mice at the time of sacrifice. Combined treatment also reduced proliferation and increased DNA damage and apoptosis in harvested tumors, and increased M1 polarization of TAMs in ascites fluid, compared to OLA mice. <h3>Conclusions:</h3> NR4A1 inhibition sensitizes ovarian tumors to PARPi though direct effects on tumors by targeting the HR pathway, and by modulating TAM function towards an anti-tumor phenotype. These findings have important implications for expanding the use of PARPi in women with HR-proficient ovarian tumors, which would benefit a substantial number of women with this devastating disease.

Inhibition of c-Myc using 10058-F4 induces anti-tumor effects in ovarian cancer cells via regulation of FOXO target genes

Ovarian cancer, characterized by rapid growth and asymptomatic development in the early stage, is the fifth common cancer in women. The deregulated expression of c-Myc in more than 50% of human tumors including ovarian cancer makes this oncogenic master transcription factor a potential therapeutic target for cancer treatment. In the present study, we evaluated the anti-tumor effects of 10058-F4, a small molecule c-Myc inhibitor, on ovarian cancer cells. We found that 10058-F4 not only inhibited the proliferation and clonal growth of ovarian cancer cells but also enhanced the cytotoxic effects of chemotherapeutic drugs. Our results also revealed that c-Myc inhibition using 10058-F4 increased the intracellular reactive oxygen species production coupled with suppressed expression of hTERT. RT-qPCR analysis indicated that 10058-F4 enhanced the mRNA levels of the forkhead box O (FOXO) family of transcription factors, including FOXO1, 3, and 4. Moreover, 10058-F4 induced G1 cell cycle arrest in 2008C13 ovarian cancer cells, along with increased expression of some key targets of FOXOs involved in the regulation of cell cycle such as p15, p21, p27, and GADD45A. The results of our study also showed that the 10058-F4-induced apoptosis in 2008C13 cell line was associated with the upregulation of FOXO downstream genes, including PUMA, Bim, and FasL. In conclusion, our results, for the first time, suggest that the anti-tumor effects of 10058-F4 in ovarian cancer cells might be mediated through upregulation of FOXO transcription factors and their key target genes involved in G1 cell cycle arrest, apoptosis, and autophagic cell death.

Abstract 1213: PDE10A as a novel target to suppress Wnt/β-catenin signaling and other oncogenic pathways in ovarian cancer

Abstract Objective: A leading theory for ovarian carcinogenesis proposes that the inflammation associated with incessant ovulation is a driver of oncogenesis. Consistent with this theory, epidemiological studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease ovarian cancer risk. Previous studies suggest, however, that the antineoplastic activity of NSAIDs does not require the traditional cyclooxygenase (COX) enzyme inhibition, and rather may be exerted through phosphodiesterase (PDE) inhibition. PDEs represent a potentially unique chemopreventive target for ovarian cancer given that ovulation is regulated by cyclic nucleotide signaling. This study evaluates the effects of phosphodiesterase 10A (PDE10A) inhibition as a novel chemopreventive and therapeutic approach for ovarian cancer. Methods: We investigated the effects of PDE10A small molecule inhibitors, Pf-2545920 and MCI-030 (a novel non-COX inhibitory NSAID-derivative), and PDE10A knockout by CRISPR/Cas9 gene editing in various ovarian cancer cell lines using in vitro assays that measured cell proliferation, cell viability, cell cycle arrest, apoptosis, cell migration and invasion. Downstream signaling pathways affected by PDE10A inhibition and gene knockout (KO) were assessed by western-blotting, confocal microscopy, and RNA sequencing. Results: Analysis of The Cancer Genome Atlas (TCGA) ovarian cancer database and an institutional cohort of ovarian cancer patients revealed that high PDE10A mRNA expression was associated with significantly worse overall survival. PDE10A expression was also positively correlated with oncogenic and inflammatory signaling pathways in the TCGA dataset. PDE10A inhibition with Pf-2545920 or MCI-030 decreased ovarian cancer cell proliferation, while inducing cell cycle arrest and apoptosis. Using inhibitors to block PKA (H89) and PKG (KT5823) kinase activity, we demonstrated that the pro-apoptotic effects of PDE10A inhibition were mediated by activation of cGMP/PKG and cAMP/PKA signaling. SKOV3 and OV-90 PDE10A KO cells showed decreased colony formation, cell proliferation, and migration and invasion properties compared to their wild-type (WT) counterparts. Moreover, PDE10A inhibition decreased Wnt-induced β-catenin nuclear translocation as well as decreased EGF-mediated activation of RAS/MAPK and AKT pathways in ovarian cancer cells. RNA sequencing of SKOV3 PDE10A KO clones revealed that pathways associated with cancer, Wnt and TGF-β signaling were downregulated compared to WT control cells. Conclusions: Our data demonstrate that PDE10A has pro-tumorigenic effects in ovarian cancer cells and its high expression in ovarian cancer patients is associated with poor prognosis. Altogether, our results warrant future studies of PDE10A as a novel target for ovarian cancer chemoprevention and/or treatment. Citation Format: Rebecca M. Barber, Elaine Gavin, Alla Musiyenko, Wito Richter, Kevin J. Lee, Annelise Wilhite, Joel F. Andrews, Steve McClellan, Ileana Aragon, Antonio Ward, Xi Chen, Adam Keeton, Kristy Berry, Gary A. Piazza, Jennifer M. Scalici, Luciana Madeira da Silva. PDE10A as a novel target to suppress Wnt/β-catenin signaling and other oncogenic pathways in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1213.

OTX015 Epi-Drug Exerts Antitumor Effects in Ovarian Cancer Cells by Blocking GNL3-Mediated Radioresistance Mechanisms: Cellular, Molecular and Computational Evidence.

Simple SummaryThe outcome for women diagnosed with ovarian cancer (OC), the most aggressive gynecological tumor worldwide, remains very poor. Encouraging therapeutic impact of epigenetic drugs has been suggested in a wide range of human solid tumors, including OC. The present study assessed the in vitro cytostatic and cytotoxic effects of OTX015, a pan Bromodomain and Extra-Terminal motif inhibitor, in human OC cells, both as single treatment and in combination with radiotherapy. Cellular, molecular and computational network analyses indicated the centrality of GNL3 downregulation in mediating the OTX015-related antitumor efficacy that blocks disease progression/maintenance and radioresistance acquisition. Our preclinical results confirm that targeted and combinatorial treatments represent effective anticancer strategies to be translated in the clinical research for improving OC patient care.Ovarian cancer (OC) is the most aggressive gynecological tumor worldwide and, notwithstanding the increment in conventional treatments, many resistance mechanisms arise, this leading to cure failure and patient death. So, the use of novel adjuvant drugs able to counteract these pathways is urgently needed to improve patient overall survival. A growing interest is focused on epigenetic drugs for cancer therapy, such as Bromodomain and Extra-Terminal motif inhibitors (BETi). Here, we investigate the antitumor effects of OTX015, a novel BETi, as a single agent or in combination with ionizing radiation (IR) in OC cellular models. OTX015 treatment significantly reduced tumor cell proliferation by triggering cell cycle arrest and apoptosis that were linked to nucleolar stress and DNA damage. OTX015 impaired migration capacity and potentiated IR effects by reducing the expression of different drivers of cancer resistance mechanisms, including GNL3 gene, whose expression was found to be significantly higher in OC biopsies than in normal ovarian tissues. Gene specific knocking down and computational network analysis confirmed the centrality of GNL3 in OTX015-mediated OC antitumor effects. Altogether, our findings suggest OTX015 as an effective option to improve therapeutic strategies and overcome the development of resistant cancer cells in patients with OC.

Small extracellular vesicles ameliorate peripheral neuropathy and enhance chemotherapy of oxaliplatin on ovarian cancer.

There are no effective treatments for chemotherapy induced peripheral neuropathy (CIPN). Small extracellular vesicles (sEVs) facilitate intercellular communication and mediate nerve function and tumour progression. We found that the treatment of mice bearing ovarian tumour with sEVs derived from cerebral endothelial cells (CEC‐sEVs) in combination with a chemo‐drug, oxaliplatin, robustly reduced oxaliplatin‐induced CIPN by decreasing oxaliplatin‐damaged myelination and nerve fibres of the sciatic nerve and significantly amplified chemotherapy of oxaliplatin by reducing tumour size. The combination therapy substantially increased a set of sEV cargo‐enriched miRNAs, but significantly reduced oxaliplatin‐increased proteins in the sciatic nerve and tumour tissues. Bioinformatics analysis revealed the altered miRNAs and proteins formed two distinct networks that regulate neuropathy and tumour growth, respectively. Intravenously administered CEC‐sEVs were internalized by axons of the sciatic nerve and cancer cells. Reduction of CEC‐sEV cargo miRNAs abolished the effects of CEC‐sEVs on oxaliplatin‐inhibited axonal growth and on amplification of the anti‐cancer effect in ovarian cancer cells, suggesting that alterations in the networks of miRNAs and proteins in recipient cells contribute to the therapeutic effect of CEC‐sEVs on CIPN. Together, the present study demonstrates that CEC‐sEVs suppressed CIPN and enhanced chemotherapy of oxaliplatin in the mouse bearing ovarian tumour.

Berberine inhibits the Warburg effect through TET3/miR-145/HK2 pathways in ovarian cancer cells

Background: Berberine, as an alkaloid, has a significant antitumor effect, but its mechanism in tumor metabolism, especially the Warburg effect has not been elucidated.Objectives: To study the molecular mechanism of berberine regulating the Warburg effect in ovarian cancer cells.Methods: Treatment by berberine in SKOV3 and 3AO cells or inhibited by miR-145 inhibitor transfection in berberine-treated cells to examine the changes in HK2 expression, glucose consumption and lactate production. The methylation status in the promoter region of pre-miR-145 gene was examined by bisulfite sequencing. Dual-luciferase reporter assay was conducted to verify the direct binding of miR-145 to HK2. Finally, the expression of TET3 in ovarian cancer was investigated by quantitative real-time PCR and immunohistochemistry.Results: We found berberine inhibited the Warburg effect by up-regulating miR-145, miR-145 targeted HK2 directly. Berberine increased the expression of miR-145 by promoting the expression of TET3 and reducing the methylation level of the promoter region of miR-145 precursor gene. We further found that TET3 expression was negatively correlated with clinical stage and pathological grade.Conclusions: Our results revealed berberine increased the TET3-mediated demethylation and promoted the suppression of miR-145 on HK2 to antagonize the Warburg effect of ovarian cancer cells.

Upregulation of MicroRNA-34a Sensitizes Ovarian Cancer Cells to Resveratrol by Targeting Bcl-2.

PurposeResveratrol (REV), a natural compound found in red wine, exhibits antitumor activity in various cancers, including ovarian cancer (OC). However, its potential anti-tumor mechanisms in OC are not well characterized. Here, we tried to elucidate the underlying mechanisms of REV in OC cells.Materials and MethodsThe anti-proliferative effects of REV against OC cells were measured using CCK-8 assay. Apoptosis was measured using an Annexin V-FITC/PI apoptosis detection kit. The anti-metastasis effects of REV were evaluated by invasion assay and wound healing assay. The miRNA profiles in REV-treated cells were determined by microarray assay.ResultsOur results showed that REV treatment suppresses the proliferation, induces the apoptosis, and inhibits the invasion and migration of OV-90 and SKOV-3 cells. miR-34a was selected for further study due to its tumor suppressive roles in various human cancers. We found miR-34a overexpression enhanced the inhibitory effects of REV on OC cells, whereas miR-34a inhibition had the opposite effect in OC cells. In addition, we verified that BCL2, an anti-apoptotic gene, was found directly targeted by miR-34a. We also found that REV reduced the expression of Bcl-2 in OC cells. Further investigations revealed that overexpression of Bcl-2 significantly abolished the anti-tumor effects of REV on OC cells.ConclusionOverall, these results demonstrated that REV exerts anti-cancer effects on OC cells through an miR-34a/Bcl-2 axis, highlighting the therapeutic potential of REV for treatment of OC.