TOV-112D Cells Research Articles

TIMP-1 is Overexpressed and Secreted by Platinum Resistant Epithelial Ovarian Cancer Cells.

Epithelial Ovarian Cancer (EOC) is the most lethal gynecological cancer in developed countries, and the development of new strategies to overcome chemoresistance is an awaited clinical need. Angiogenesis, the development of new blood vessels from pre-existing vasculature, has been validated as a therapeutic target in this tumor type. The aim of this study is to verify if EOC cells with acquired resistance to platinum (PT) treatment display an altered angiogenic potential. Using a proteomic approach, we identified the tissue inhibitor of metalloproteinases 1 (TIMP-1) as the only secreted factor whose expression was up-regulated in PT-resistant TOV-112D and OVSAHO EOC cells used as study models. We report that TIMP-1 acts as a double-edged sword in the EOC microenvironment, directly affecting the response to PT treatment on tumor cells and indirectly altering migration and proliferation of endothelial cells. Interestingly, we found that high TIMP-1 levels in stage III–IV EOC patients associate with decreased overall survival, especially if they were treated with PT or bevacizumab. Taken together, these results pinpoint TIMP-1 as a key molecule involved in the regulation of EOC PT-resistance and progression disclosing the possibility that it could be used as a new biomarker of PT-resistance and/or therapeutic target.

Abstract 4800: Targeting mitochondrial and cytosolic one-carbon metabolism in epithelial ovarian cancer via folate receptor alpha

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Even though most patients initially respond to platinum-based therapy, the likelihood of disease reoccurrence is virtually 100%. Thus, there is an urgent need for new tumor-selective therapies for EOC. One such treatment option involves targeting tumors via folate receptor alpha (FRα) which is overexpressed in up to 90% of EOCs and shows increasing expression with stage and grade of disease. Our laboratory discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF291, AGF320, AGF347, AGF359 and AGF362) which inhibit mitochondrial one-carbon (C1) metabolism at serine hydroxymethyltransferase (SHMT) 2, with secondary inhibitions at cytosolic enzyme targets including those in de novo purine biosynthesis and SHMT1. Potent inhibition was seen toward isogenic Chinese hamster ovary (CHO) cell lines individually expressing FRα, the reduced folate carrier (RFC, ubiquitously expressed tissue folate transporter) and the proton-coupled folate transporter (PCFT, expressed in a limited number of normal tissues and inactive at normal pH, and several solid tumors at acidic pH including EOC), and with FRα-expressing tumor cells, including KB and IGROV1, a human EOC cell line. Inhibitory potencies were in order, AGF347 > AGF362 >> AGF291 = AGF320 = AGF359. Drug effects were substantially reduced with excess folic acid, confirming FRα-mediated drug uptake. Toward cisplatin resistant SKOV3, TOV112D and A2780 EOC cells, inhibition in the nanomolar range was detected with all compounds. Excess folic acid abrogated drug effects to varying degrees, suggesting significant uptake by the PCFT and/or the RFC, in addition to FRα. Short-term (5 minutes) cell uptake assays with the EOC cell lines and [3H]AGF347 confirmed transport by PCFT and RFC. With sustained exposures resulting in steady state AGF347 accumulations under both physiologic (pH 7.2) and acidic (pH 6.8, approximating the tumor microenvironment) conditions, FRα uptake predominated. [3H]AGF347 treatment of IGROV1 EOC resulted in substantial drug accumulation in both cytosol and mitochondria. Inhibition of cell proliferation for all analogs was reversed by addition of both glycine and adenosine, implicating C1 metabolism in mitochondria including SHMT2 and de novo purine biosynthesis in the cytosol as the targeted pathways; protection by 5-aminoimidazole-4-carboxamide (AICA) with or without glycine was incomplete, implying direct targeting of AICA ribonucleotide formyltransferase (AICARFTase), the second folate-dependent enzyme in purine biosynthesis. Our studies describe first-in-class FRα-selective compounds targeting mitochondrial and cytosolic C1 metabolism, with potent activity against EOC, including cisplatin resistant EOC. Citation Format: Adrianne Wallace-Povirk, Carrie O’Connor, Aamod Dekhne, Zhanjun Hou, Md. Junayed Nayeen, Khushbu Shah, Aleem Gangjee, Larry Matherly. Targeting mitochondrial and cytosolic one-carbon metabolism in epithelial ovarian cancer via folate receptor alpha [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4800.

Hypoxia-inducible factor-1\u03b1 induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

BackgroundChemokines are involved in the homing of various cancer cells, including those of ovarian cancer (OvCa), to distant organs. They may also promote or inhibit cancer progression and metastasis. Hypoxia, a common phenomenon in malignant tumors, promotes cell proliferation regulated by HIF-1α. Hypoxia-induced genes are involved in metastasis-associated functions and in the epithelial-to-mesenchymal transition (EMT).ResultsTissue microarrays of human OvCa showed elevated expression of CX3CR1 and HIF-1α compared to normal cells, and their levels were higher in adenocarcinoma stages II and III. To substantiate these observations, we performed studies using OvCa cells. Following exposure to hypoxia, OVCAR-3, SW 626, and TOV-112D cells showed high expression of CX3CR1, a transmembrane protein involved in the adhesion and migration of leukocytes, causing an increased chemotactic response to CX3CL1, the ligand for CX3CR1. As determined by flow cytometry, immunofluorescence, RT-PCR, and western blots, there were higher expressions of CX3CR1 and HIF-1α in OvCa cell lines exposed to hypoxia. Further, OvCa cells expressing CX3CR1 were sensitive to the CX3CL1 ligand. Chemotaxis based on chemokine receptors was influential in elevating the expression of EMT markers and matrix metalloproteinases, which are involved in the progression and metastasis of cancer cells.ConclusionsIn OvCa cells, CX3CR1 was upregulated in a process involving hypoxia-mediated regulation of HIF-1α. The elevated levels of CX3CR1, which were sensitive to CX3CL1, increased EMT markers that led to the progression and metastasis of OvCa. Thus, CX3CR1 and HIF-1α are suitable targets for treatment of OvCa.

Identification of lncRNA-miRNA-mRNA regulatory network associated with epithelial ovarian cancer cisplatin-resistant.

To construct a long noncoding RNA (lncRNA)-microRNA (miRNA)-messenger RNA (mRNA) regulatory network related to epithelial ovarian cancer (EOC) cisplatin-resistant, differentially expressed genes (DEGs), differentially expressed lncRNAs (DELs), and differentially expressed miRNAs (DEMs) between MDAH and TOV-112D cells lines were identified. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted to analyze the biological functions of DEGs. Downstream mRNAs or upstream lncRNAs for miRNAs were analyzed at miRTarBase 7.0 or DIANA-LncBase V2, respectively. A total of 485 significant DEGs, 85 DELs, and 5 DEMs were identified. Protein-protein interaction (PPI) network of DEGs contrains 81 nodes and 141 edges was constructed, and 25 hub genes related to EOC cisplatin-resistant were identified. Subsequently, a lncRNA-miRNA-mRNA regulatory network contains 4 lncRNAs, 4 miRNAs, and 35 mRNAs was established. Taken together, our study provided evidence concerning the alteration genes involved in EOC cisplatin-resistant, which will help to unravel the mechanisms underlying drug resistant.

Abstract B31: Tumor-intrinsic B7-H3 regulates drug resistance, metabolism, and pathogenesis in ovarian cancer

Abstract B7-H3 (CD276) is a cell surface glycoprotein of B7 family of checkpoint blockade proteins that has been considered a valuable novel biomarker and a promising target for cancer treatment. B7-H3 is overexpressed in a wide variety of human cancers and is associated with tumor progression, metastasis, and poor patient outcome. In addition to its function in cancer immunity, B7-H3 also has tumor-intrinsic functions related to drug resistance, metabolic reprogramming, migration, and invasion of cancer cells. B7-H3 overexpression in the ovarian tumors and tumor vasculature has been reported and B7-H3 monoclonal antibodies have shown inhibitory effects in ovarian cancer xenograft studies. Nonetheless, the molecular pathways regulated by B7-H3 in ovarian cancer remain largely unknown. Here we explored the role of tumor intrinsic B7-H3 in ovarian cancer cells by genetic modulation of B7-H3 expression levels in ovarian cancer cells, using CRISPR/Cas9-mediated B7-H3 knockout and B7-H3 overexpression using retrovirus system for cell transduction. Knockout of B7-H3 in OV-90 cells, which showed high endogenous expression of B7-H3, resulted in decreased glycolysis and oxidative phosphorylation through downregulation of HIF1α, and its downstream target glycolytic proteins LDHA and PDK1. Furthermore, OV-90 B7-H3 KO cells showed decreased phosphorylation of the prosurvival protein STAT3, reduced clonogenic and migratory capability, and increased sensitivity to chemotherapeutic agent carboplatin. Overexpression of B7-H3 in TOV112D cells, which has low endogenous expression of B7-H3, resulted in the opposite effects, leading to increased glycolysis and mitochondrial respiration, increased STAT3 phosphorylation, increased migration, and increased resistance to carboplatin. We also performed RNA-seq analysis of OV-90 B7-H3 WT and KO clones and found several signaling pathways involved in cell migration, adhesion, and inflammatory response. Finally, we found that the growth of OV-90 B7-H3 KO cells was dramatically defective in xenograft models using both athymic nude and SCID immunocompromised mice. These results suggest that tumor-intrinsic B7-H3 plays a critical role in the growth of ovarian cancer and warrants further investigation of therapeutic approaches blocking B7-H3 functions for the treatment of this deadly malignancy in women. Citation Format: Holly Taylor, Jaroslav Slamecka, Alla Musiyenko, Elaine Gavin, Tiffany S. Norton, Ileana Aragon, Taylor Young, Jennifer Scalici, Rodney P. Rocconi, Ming Tan, Luciana Madeira da Silva. Tumor-intrinsic B7-H3 regulates drug resistance, metabolism, and pathogenesis in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B31.

Correction: Ruxolitinib sensitizes ovarian cancer to reduced dose Taxol, limits tumor growth and improves survival in immune competent mice.

[This corrects the article DOI: 10.18632/oncotarget.21541.].

Ruxolitinib sensitizes ovarian cancer to reduced dose Taxol, limits tumor growth and improves survival in immune competent mice.

BackgroundChemotherapy initially reduces the tumor burden in patients with ovarian cancer. However, tumors recur in over 70% of patients, creating the need for novel therapeutic approaches.MethodsWe evaluated Ruxolitinib, an FDA-approved JAK 1/2 kinase inhibitor, as a potential adjunctive therapy for use with low-dose Taxol (Paclitaxel) by assessing the impact on in vitro proliferation and colony formation of ID8 cells or human TOV-112D ovarian cancer cells, as well as flow cytometric measurement of surface markers associated with cellular stress and stemness by ID8 cells. The syngeneic ID8 murine model of ovarian cancer was used to assess the impact of Ruxolitinib and Taxol, individually and in combination, on tumor initiation and growth, as well as capacity to extend survival.ResultsRuxolitinib (≤10 μM) sensitized both ID8 and TOV-112D cells to low concentrations of Taxol (≤5 nM), limiting cell proliferation and colony formation in vitro. Mechanistically, we demonstrated that Taxol induced expression of stress and stemness markers including GRP78 and CD133 was significantly reduced by addition of Ruxolitinib. Finally, we demonstrated that a single administration of a low-dose of Taxol (10 mg/Kg) together with daily Ruxolitinib (30 mg/Kg; which is equivalent to plasma concentrations of ∼ 0.01 μM steady-state) limited ID8 tumor growth in vivo and significantly extended median survival up to 53.5% (median 70 v 107.5 days) as compared to control mice.ConclusionTogether, these data support the use of Ruxolitinib in combination with low-dose Taxol as a therapeutic approach with the potential for improved efficacy and reduced side effects for patients with recurrent ovarian cancer.

Abstract A28: Casein kinase 1ε (CSNK1ε) is a synthetic lethal target in MYC-driven ovarian cancer

Abstract The MYC family of oncogenes encodes transcription factors that are master regulators of cell proliferation and is a central driver in many human cancers. Without obvious druggable domains, direct inhibition of MYC by small molecules has remained a challenge. An alternative strategy is to exploit the concept of synthetic lethality, where specific combinations of mutations lead to cell death, while each mutation alone is not essential for survival. Targeting these synthetic lethal interactions provides an opportunity to minimize toxicity by sparing normal cells without mutations. Given that MYC amplification is reported in approximately 40% of ovarian cancers and is a leading cause of gynecologic cancer-related deaths, identifying novel therapeutic targets for this disease is of urgent clinical importance. We previously utilized a high-throughput functional genomics approach to identify 149 synthetic lethal candidates with cMYC overexpression using isogenic human foreskin fibroblast cells. In this study, 45 genes were selected for further study based on druggability, involvement in cancer pathways, and differential toxicity. We focused on 23 genes based on their expression pattern in ovarian cancers, of which 9 genes were identified to exhibit strong synthetic lethality in ovarian cancer cells with cMYC overexpression. Casein kinase 1ε (CSNK1ε) was one of the top candidates and was therefore selected for subsequent validation and further analysis. Knock-down of CSNK1ε had minimal toxicity in human fibroblasts, suggesting the possibility of a good therapeutic window. Using qPCR and immunohistochemistry analysis of 55 frozen samples from ovarian cancer patients, we showed significant correlation of MYC and CSNK1ε expression. Moreover, analysis of publically available microarray data showed that patients with high CSNK1ε expression are associated with poorer survival. Next, we performed functional validation in two sets of ovarian cancer cell lines with either high-MYC expression (IGROV-1 and TOV112D) or low-MYC expression (CaOV3 and DOV13). Using RNAi and small molecule inhibitors (IC261, PF-670462, PF-4800567), we demonstrated that CSNK1ε inhibition reduced cell viability and attenuated the WNT and SHH target pathways specifically in cells with high-MYC expression. Treatment with the CSNK1ε specific inhibitor, PF-4800567, further revealed that upregulation of CSNK1δ could be a potential mechanism of resistance. Finally, the growth of TOV112D cells (high MYC) in a tumor xenograft model was significantly inhibited by daily treatment with IC61. These findings reveal a new strategy to target the undruggable MYC oncogene through synthetic lethal interactions in MYC-driven ovarian cancer. Targeting CSNK1ε offers a novel therapeutic approach to complement traditional chemotherapies currently used to treat this disease in the clinic. Citation Format: Goldie Y. L. Lui, Franz Schaub, Shaifali Agrawal, Kazuyuki Kitatani, Christopher J. Kemp, Masafumi Toyoshima, Carla Grandori. Casein kinase 1ε (CSNK1ε) is a synthetic lethal target in MYC-driven ovarian cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A28.

Osteopontin-c isoform levels are associated with SR and hnRNP differential expression in ovarian cancer cell lines.

Osteopontin-c splicing isoform activates ovarian cancer progression features. Imbalanced expression of splicing factors from serine/arginine -rich and heterogeneous ribonucleoproteins families has been correlated with the generation of oncogenic splicing isoforms. Our goal was to investigate whether there is any association between the transcriptional patterns of these splicing factors in ovarian cells and osteopontin-c expression levels. We also aimed to investigate the occurrence of these splicing factors binding sites inside osteopontin exon 4 and adjacent introns. To test associations between osteopontin-c and splicing factors expression patterns, we used an in vitro model in which OVCAR-3 cells overexpressing osteopontin-c (OVCAR-3/OPNc++) presented higher transcriptional levels of osteopontin-c than two other ovarian carcinoma cells (TOV-112D, SKOV-3) and ovarian non-tumoral cell lines (IOSE 364 and IOSE 385). The transcriptional levels of osteopontin-c, serine/arginine-rich, and hnRNP factors were evaluated using real-time polymerase chain reaction. Human Splice Finder software was used to search for putative splicing factor binding sites in osteopontin genomic regions. OVCAR-3/OPNc++ cells presented higher transcriptional levels of hnRNP than serine/arginine-rich when compared to TOV-112D, SKOV-3, and IOSE cells. TOV-112D and SKOV-3 cells also overexpressed hnRNP in relation to serine/arginine-rich transcripts. Putative binding sites for these splicing factors have been predicted on osteopontin exon 4 and their upstream and downstream intronic regions. Our data showed that higher osteopontin-c expression levels are associated with a predominance of hnRNP in relation to serine/arginine-rich transcripts and that osteopontin exon 4 and adjacent intronic sequences contain predicted binding sites for some of these tested splicing factors. In conclusion, differential expression of these splicing factors in ovarian cancer cells could be one of the putative mechanisms leading to aberrant splicing of the osteopontin primary transcript. Future work, aiming to control ovarian cancer progression by downregulating osteopontin-c levels, could include strategies that also regulate heterogeneous ribonucleoproteins and serine/arginine-rich expression levels in order to modulate osteopontin splicing.

Abstract NTOC-105: RUXOLITINIB SENSITIZES OVARIAN CANCER CELLS TO LOW–DOSE PACLITAXEL THERAPY

Abstract In ovarian cancer chemotherapy initially reduces tumor burden; however, >70% of patients experience tumor recurrence, creating a need for additional therapies. We evaluated the ability of Ruxolitinib, a FDA approved JAK 1/2 kinase inhibitor, as a potential adjunctive therapy for paclitaxel treatment of ovarian cancer. Using murine ID8 and human TOV-112D ovarian cancer cells we tested the ability of Ruxolitinib alone and in combination with low-dose paclitaxel to limit cell proliferation and colony formation in vitro. Additionally, using flow cytometry we assessed the capacity of paclitaxel to induce the expression of cell surface markers associated with stemness and the cellular stress response. We next determined if Ruxolitinib could reduce paclitaxel-induced expression of stemness and stress markers. Finally daily administration of Ruxolitinib, both alone and in combination with a single low-dose administration of paclitaxel, was tested for the capacity to reduce tumor growth and extend survival using the syngeneic ID8 luciferase-expressing model of ovarian cancer in C57Bl/6 mice. Our in vitro data demonstrate that Ruxolitinib can sensitize both murine ID8 and human TOV-112D cells to low concentrations of paclitaxel, as evidenced by a significant reduction of cell proliferation and colony formation. We find that pre-incubation with Ruxolitinib is sufficient to sensitize cells to subsequent incubation with paclitaxel. Paclitaxel-induced expression of stemness and stress markers (SCA-1, GRP78 and CD133) is reduced by co-incubation with Ruxolitinib. In vivo, Ruxolitinib plus administration of a single low-dose paclitaxel treatment is sufficient to limit tumor growth and significantly extend median survival from in mice bearing ID8 ovarian cancer tumors (Control 59days vs Ruxolitinib-paclitaxel 77 days). Together, these data indicate that Ruxolitinib can increase the susceptibility of both murine and human ovarian cancer cells to reduced concentrations paclitaxel chemotherapy in vitro and that combination therapy with Ruxolitinib and a single low-dose paclitaxel administration extends survival in mice. Our data provide important pre-clinical data to support the use of Ruxolitinib to enhance the efficacy of paclitaxel therapy for patients with ovarian cancer. Citation Format: Patrick Reeves PhD, Mojgan Abbaslou MD, Mark C. Poznansky MD PHD. RUXOLITINIB SENSITIZES OVARIAN CANCER CELLS TO LOW–DOSE PACLITAXEL THERAPY [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-105.

Functional role of the Tau protein in epithelial ovarian cancer cells.

AimThe microtubule‐associated Tau protein is a marker of paclitaxel sensitivity in ovarian cancer. The aim of the present study was to elucidate the function of the Tau protein in epithelial ovarian cancer.MethodsThe correlation between Tau protein expression and the response to paclitaxel by using several ovarian cancer cell lines was investigated.ResultsA Western blot showed that the expression level of the Tau protein was the highest in the TOV112D cells. A cell‐counting kit showed that the proliferation rates were more inhibited in the cells with down‐regulated Tau protein than in the control cells, both with and without paclitaxel treatment. The proliferation rates of the control cells and the TOV112D cells also were compared with Tau protein overexpression. The level of cell proliferation was more inhibited in the cells that overexpressed the Tau protein, compared to the control cells, both with and without paclitaxel treatment. It was shown that both the down‐regulation and the overexpression of the Tau protein were related to the inhibition of TOV112D cell proliferation. Early and late apoptosis of the TOV112D cells that were transfected with Tau cDNA plasmid construct or Tau small interfering RNA significantly increased.ConclusionThese findings suggest that the molecular targeting of the Tau protein could be a potential treatment for ovarian cancer.

Abstract 3007: Evaluating novel 4-aminoquinoline inhibitors of NQO2 in ovarian cancer

Abstract Introduction: NRH quinone oxidoreductase 2 (NQO2) is one of the mammalian enzymes responsible for the two-electron reduction of quinones to hydroquinones. NQO2 can use a variety of dihydronicotinamide analogues as co-factors. In particular, the co-factor, EP0152R has been used in Phase I clinical trials to support evaluation of the NQO2-mediated reductive activation of CB1954. As well as being a prodrug-activating enzyme, there is evidence to show that NQO2 may be involved in cancer progression as a consequence of its ability to act as a “nanny” protein, alter cyclin D expression and modulate the activity of NF-κB. Thus, we have synthesized a series of novel 4-aminoquinolines to study their ability to inhibit NQO2 in ovarian cancer cells (OVCs). We then propose to compare the results of pharmacological inhibition with those obtained by genetic down-regulation of NQO2 level. Methods: NQO2 level in a panel of OVC cell lines was determined by using western blot analysis and Cytochrome C based spectrophotometric enzyme activity assay. Novel compounds have been synthesized and assessed for their inhibitory potency against recombinant human NQO2 (rhNQO2), using DCPIP spectrophotometric assay. MTT cytotoxicity assay was utilized for several purposes: Firstly, to determine the toxicity of known and novel NQO2 inhibitors in the SKOV3 cell line; and secondly, to evaluate the sensitivity of OVC cell lines to CB1954 in the presence of EP0152R, and subsequently measure the intracellular NQO2 inhibitory potency of novel compounds, by combining them with CB1954 and EP0152R. Results and conclusion: In the OVCs, NQO2 protein level and enzymatic activity showed an excellent correlation; however, expression and activity differed widely among the OVC cell lines. The NQO2 levels were highest in SKOV3 and lowest in the TOV112D cell lines. The sensitivity of OVCs to CB1954 was significantly increased when combined with EP0152R; supporting the notion that NQO2 mediates the toxicity of CB1954. This was confirmed when a strong correlation was observed between the cellular NQO2 activity and the responsiveness of the OVC cell lines to CB1954. Further, overexpressing NQO2 in TOV112D cells resulted in a significant enhancing of its responsiveness to treatment with CB1954. The novel 4-aminoquinolines inhibited the activity of recombinant NQO2 when used in the nano-molar range. The inherent cytotoxicity of the inhibitors (as measured by the IC50 following 24 and 96 hours exposure to SKOV3 cells) ranged between 1.7 to >100μM. There was no relationship between toxicity and the ability of the compounds to inhibit the activity of rhNQO2. However, some of these compounds showed functional activity as NQO2 inhibitors in cells; this was revealed by their ability to inhibit the toxicity of CB1954. Several compounds were able to do this at sub-μM, non-toxic concentrations and therefore they may be able to act as molecular probes for the (multi-) functional activity of NQO2 in cells. Citation Format: Balqis A. Ikhmais, Buthaina Hussein, Ian Stratford, Sally Freeman, Joaana Neill. Evaluating novel 4-aminoquinoline inhibitors of NQO2 in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3007.

Abstract 1989: Multiple functional implications of MYB in ovarian cancer

Abstract Ovarian cancer (OC) is the most common and deadliest gynecologic malignancies in the United States, with an estimated 21,290 new cases and 14,180 deaths in 2015. Therefore, it is highly imperative to identify novel molecular targets driving OC progression, so that an effective treatment strategy can be developed against this devastating disease. In the present study, we investigated the role of MYB in OC pathogenesis. MYB/c-MYB is a cellular progenitor of v-MYB oncogenes, which encodes for a transcription factor, and confers its oncogenic activity by regulating the expression of several target genes. First, we examined the expression of MYB in clinical specimens and OC cell lines by immunohistochemical and immunoblot analysis, respectively. We observed intense staining of MYB in the tissues of all the subtypes of OC, while no or negligible staining was observed in normal ovarian tissues. Moreover, we found MYB to be expressed in all the OC cell lines examined, with its heightened expression in highly aggressive (TOV112D, SKOV3-ip) and chemo-resistant (A2780-Cip) OC cell lines. To examine the pathological significance of MYB in OC, we silenced its expression in TOV112D, SKOV3-ip and A2780-Cip OC cell lines. Functional studies demonstrate that silencing of MYB led to decrease in growth and clonogenic ability in TOV112D, SKOV3-ip and A2780-Cip cells, as compared to their scrambled sequence-transfected (Scr) control cells. Furthermore, MYB-silencing reduced motility and invasion, as well as resulted in the loss of mesenchymal and gain of epithelial markers. In addition, our data showed that TOV112D-shMYB, SKOV3-ip-shMYB and A2780-Cip-shMYB were more sensitive to cisplatin-cytotoxicity as compared to their respective controls. Altogether, our studies provide the first experimental evidence for a functional role of MYB in altered growth, malignant behavior and chemoresistance of ovarian cancer. Citation Format: Sanjeev Kumar Srivastava, Seema Singh, Arun Bhardwaj, James E. Carter, Rodney P. Rocconi, Jennifer Scalici, Ajay P. Singh. Multiple functional implications of MYB in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1989.

In vitro effects of a new fused azaisocytosine-like congener on relative cell proliferation, necrosis and cell cycle in cancer and normal cell cultures.

The study was aimed at describing the mode of action of an innovative drug-like congener of fused azaisocytosine-EIMTC (ethyl 8-(4-methoxyphenyl)-4-oxo-6,7-dihydroimidazo[2,1-c][1,2,4]triazine-3-carboxylate)-on cancer cells in early in vitro oncology-related bioassays. Micromolar concentrations of EIMTC were effective at inhibiting the growth of two types of malignant multiple myeloma cells (including cells resistant to thalidomide) while having less cytotoxic effect on normal HSF cells. Furthermore, EIMTC was disclosed as capable of producing the statistically significant decrease in the number of cells in the S phase (in HeLa, TOV112D, T47D and Vero cells) and in the G2/M phase (in TOV112D cells) as well as evoking the distinctly higher necrosis rates in malignant than normal cells of the same epithelial origin. These results are promising in the sense that the bicyclic nucleobase-like structure related to azaisocytosine may target epithelial cancer cells and inhibit their growth while having less effect on normal cells. This may be due to induction of necrosis.

Ribosomal S6 kinase 4 (RSK4) expression in ovarian tumors and its regulation by antineoplastic drugs in ovarian cancer cell lines.

Survival rate in ovarian cancer depends on the stage of the disease. RSK4, which has been considered as a tumor suppressor factor, controls cells invasion due to its antiinvasive and antimetastatic properties. Modulation of RSK4 expression could be an important event to increase the survival rate in ovarian cancer patients. Thus, the goal of the present study was to establish the differences in RSK4 expression among normal, benign and malignant ovarian tissues and to determine whether antineoplastic drugs regulate its expression in SKOV3 and TOV-112D cells. RSK4 levels in 30 malignant ovarian tumors, 64 benign tumors and 36 normal ovary tissues were determined by reverse transcription polymerase chain reaction and Western blot. Modulation of RSK4 expression by two antineoplastic drugs (cisplatin and vorinostat) was also studied in the SKOV3 and TOV-112D ovarian cancer cell lines using the same techniques. RSK4 mRNA and protein levels were decreased in malignant ovarian tumors as compared to benign tumors and normal tissue. These low-RSK4 levels were significantly associated with advanced stages of ovarian cancer. RSK4 expression was increased after incubation of SKOV3 and TOV-112D cell lines with cisplatin and vorinostat for 24h. The combination of these antineoplastic drugs did not produce a synergistic or additive effect. These results suggest that RSK4 is expressed at low levels in malignant ovarian tumors, which correlates with advanced stages of the disease. Additionally, RSK4 expression is regulated by cisplatin and vorinostat in two ovarian cancer cell lines.

Role of NSC319726 in ovarian cancer based on the bioinformatics analyses.

AimThis study aimed to explore the molecular mechanisms of NSC319726 in ovarian cancer by bioinformatics analyses.MethodsGene expression profile GSE35972 was downloaded from the Gene Expression Omnibus database. The data set contains six samples, including three samples of TOV112D cells untreated and three samples of TOV112D cells treated with NSC319726. The differentially expressed genes (DEGs) between untreated and treated samples were analyzed using the limma package. Kyoto Encyclopedia of Genes and Genomes pathway analysis was performed using the signaling pathway impact analysis package, followed by the functional annotation of DEGs and protein–protein interaction network construction. Finally, the subnetwork was identified, and Gene Ontology functional enrichment analysis was performed on the DEGs enriched in the subnetwork.ResultsA total of 120 upregulated and 126 downregulated DEGs were identified. Six Kyoto Encyclopedia of Genes and Genomes pathways were significantly perturbed by DEGs, and the pathway of oocyte meiosis was identified as the most perturbed one. Oocyte meiosis was enriched by eight downregulated DEGs, such as ribosomal protein S6 kinase, 90 kDa, and polypeptide 6 (RPS6KA6). After functional annotation, eight transcription factors were upregulated (such as B-cell CLL/lymphoma 6 [BCL6]), and three transcription factors were downregulated. Seven tumor suppressor genes, such as forkhead box O3 (FOXO3), were upregulated. Additionally, in the protein–protein interaction network and subnetwork, cyclin B1 (CCNB1) and cell division cycle 20 (CDC20) were hub genes, which were also involved in the functions related to mitotic cell cycle.ConclusionNSC319726 may play an efficient role against ovarian cancer via targeting genes, such as RPS6KA6, BCL6, FOXO3, CCNB1, and CDC20, which are involved in oocyte meiosis pathway.

Abstract 356: Mutaome-based magnolin sensitivity in ovarian cancer cells

Abstract Signaling pathways mediated through Ras/Raf/MEK/ERK and/or PI3K/AKT/mTOR signaling pathways play a key role in cell transformation and cancer development. During carcinogenesis, genomic alterations, accumulation of mutations and amplification of growth signaling, induce cellular phenotype changes including escaping aging, metabolic change of energy source and resistance against therapeutic agents. Recently, our research group has identified a natural compound, magnolin, that inhibits ERK1 and ERK2 kinase activities with about 87 uM and 16.5 uM of IC50, respectively. However, the effect of magnolin in cancer cells has been poorly understood. To explore the effect of magnolin in cancer cells, we examined anti-proliferation effect of magnolin in pancreatic, colon and ovarian cancer cells. We found that striking differences of cancer cell proliferation was observed in ovarian cancer cells, TOV112D and SKOV3, by magnolin treatment. In TCGA mutaome data-base analysis, we found that TOV112D and SKOV3 harbor the different mutations in different genes which are known to involve in chemo-resistance. In cell cycle analysis, we found that TOV112D reduced S-phase of cell cycle and increased G1/G0-phase of cell cycle. Importantly, activation protein profiles in TOV112D and SKOV3 cells by Western blotting showed that phosphorylation of p90RSK including RSK2 was higher in SKOV3 cells compared with TOV112D cells. These results strongly suggested that alteration of p90RSK signaling may play an important role in expression of chemo-resistance in ovarian cancer cells. Citation Format: Ji-Hong Song, Woo-Young Kim, Yong-Yeon Cho. Mutaome-based magnolin sensitivity in ovarian cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 356. doi:10.1158/1538-7445.AM2015-356

MRI reveals the in vivo cellular and vascular response to BEZ235 in ovarian cancer xenografts with different PI3-kinase pathway activity.

Background:The phosphoinositide-3 kinase (PI3K) pathway is an attractive therapeutic target. However, difficulty in predicting therapeutic response limits the clinical implementation of PI3K inhibitors. This study evaluates the utility of clinically relevant magnetic resonance imaging (MRI) biomarkers for noninvasively assessing the in vivo response to the dual PI3K/mTOR inhibitor BEZ235 in two ovarian cancer models with differential PI3K pathway activity.Methods:The PI3K signalling activity of TOV-21G and TOV-112D human ovarian cancer cells was investigated in vitro. Cellular and vascular response of the xenografts to BEZ235 treatment (65 mg kg−1, 3 days) was assessed in vivo using diffusion-weighted (DW) and dynamic contrast-enhanced (DCE)-MRI. Micro-computed tomography was performed to investigate changes in vascular morphology.Results:The TOV-21G cells showed higher PI3K signalling activity than TOV-112D cells in vitro and in vivo. Treated TOV-21G xenografts decreased in volume and DW-MRI revealed an increased water diffusivity that was not found in TOV-112D xenografts. Treatment-induced improvement in vascular functionality was detected with DCE-MRI in both models. Changes in vascular morphology were not found.Conclusions:Our results suggest that DW- and DCE-MRI can detect cellular and vascular response to PI3K/mTOR inhibition in vivo. However, only DW-MRI could discriminate between a strong and weak response to BEZ235.

The role of S100A14 in epithelial ovarian tumors

S100A14 is an EF-hand calcium-binding protein that has been reported to be involved in the progression of many malignancies. However, its role in ovarian cancer has not yet been clarified. In this study, we investigated the significance of S100A14 expression in epithelial ovarian cancers (EOCs) as well as it's mechanism of action. On both RNA and protein levels, S100A14 was overexpressed in transformed cells. Immunohistochemical staining demonstrated that S100A14 expression was associated with advanced stage (P<0.001) and poor tumor grade (P<0.001). Moreover, S100A14 overexpression was an independent prognostic factor for overall survival (HR = 4.53, P = 0.029). We also investigated S100A14's functional role by employing lentiviral-mediated overexpression and knockdown in EOC cells. S100A14 overexpression promoted cell proliferation, tumorigenesis, migration, and invasion, whereas S100A14 knockdown inhibited these properties. TOV112D cells that overexpressed S100A14 also exhibited greater tumor growth potential in xenografted mice. S100A14 promoted such a malignant phenotype in EOC cells through the PI3K/Akt pathway. Taken together, our data indicate that S100A14 has a crucial role in EOC progression, and its overexpression is associated with poor prognosis. Further study of S100A14's molecular mechanisms may lead to the development of a novel therapeutic target for ovarian cancer.

ATL

ARHI is a maternally imprinted tumor suppressor gene that is responsible for initiating programmed cell death and inhibiting cancer cell growth. However, the influence of ARHI on epithelial ovarian cancer cell death and the underlying mechanisms behind how ARHI regulates cancer cells still require further studies. Epithelial ovarian cancer cells TOV112D and ES-2 were used in this in vitro study. Cell proliferation, apoptosis, and autophagy activities were compared in TOV112D and ES-2 cells transfected with ARHI vectors or control vectors. Bcl-2 siRNA was transfected into TOV112D cells to investigate the roles of Bcl-2 played in regulating apoptosis and autophagy. ARHI expression was reduced in TOV112D and ES-2 cells compared with normal epithelial ovarian cells (NOE095 and HOSEpiC). Overexpressed ARHI inhibited cancer cell proliferation, whereas induced forced cell apoptosis and excessive formation of autophagosomes inhibited promoted cell death. Furthermore, we found that Bcl-2 expression moderately declined in response to ARHI overexpressing in ES-2 and TOV112D cells; meanwhile, more apoptotic cells and higher LC3 level presented after silence of Bcl-2 in TOV112D cells. Reduced Bcl-2-Beclin 1 complex were observed in ARHI overexpressing cells. Moreover, modulation of ARHI to Bcl-2 expression could be ascribed partially to the activation of PI3k/AKT pathway. The addition of LY294002 enabled to suppress Bcl-2 expression and cell proliferation. The silence of ARHI expression in vitro seems to accelerate the malignant transformation of healthy ovarian cells by restraining apoptosis and autophagy. The overexpressed ARHI in TOV112D cancer cells suppresses the activation of PI3K/AKT and reduces the expression of Bcl-2, leading to enhanced cell apoptosis and autophagic cancer cell death.