Serous Cell Lines Research Articles

Increase Paclitaxel Sensitivity to Better Suppress Serous Epithelial Ovarian Cancer via Ablating Androgen Receptor/Aryl Hydrocarbon Receptor-ABCG2 Axis.

Background: Epithelial ovarian cancer (EOC) is one of the most lethal gynecological malignancies and presents chemoresistance after chemotherapy treatment. Androgen receptor (AR) has been known to participate in proliferation. Yet the mechanisms of the resistance of this drug and its linkage to the AR remains unclear. Methods: To elucidate AR-related paclitaxel sensitivity, co-IP, luciferase reporter assay and ChIP assay were performed to identify that AR direct-regulated ABCG2 expression under paclitaxel treatment. IHC staining by AR antibody presented higher AR expression in serous-type patients than other types. AR degradation enhancer (ASC-J9) was used to examine paclitaxel-associated and paclitaxel-resistant cytotoxicity in vitro and in vivo. Results: We found AR/aryl hydrocarbon receptor (AhR)-mediates ABCG2 expression and leads to a change in paclitaxel cytotoxicity/sensitivity in EOC serous subtype cell lines. Molecular mechanism study showed that paclitaxel activated AR transactivity and bound to alternative ARE in the ABCG2 proximal promoter region. To identify AR as a potential therapeutic target, the ASC-J9 was used to re-sensitize paclitaxel-resistant EOC tumors upon paclitaxel treatment in vitro and in vivo. Conclusion: The results demonstrated that activation of AR transactivity beyond the androgen-associated biological effect. This novel AR mechanism explains that degradation of AR is the most effective therapeutic strategy for treating AR-positive EOC serous subtype.

Abstract B38: Synergistic effects of SHP2 and PI3K inhibitors in GAB2-overexpressing ovarian cancer

Abstract We previously found that the scaffold/adaptor growth factor receptor bound 2 (GRB2)-associated binding protein 2 (GAB2) is frequently amplified and overexpressed in primary high-grade serous ovarian cancers (HGSOCs) and cell lines. Ovarian cancer cell lines overexpressing GAB2 are dependent on GAB2 for activation of ERK1/2 and AKT signaling, consistent with its role in regulating the protein tyrosine phosphatase SHP2 (encoded by the PTPN11 gene) and the p85 regulatory subunit of phosphoinositide 3-kinase (PI3K). In this study, we investigated whether ovarian cancer cell lines overexpressing GAB2 are sensitive to combinatorial SHP2 and PI3K inhibition. Analysis of gene expression profiles reveals that primary HGSOCs overexpressing GAB2 are associated with increased ERBB, RAS, and MAPK activity signatures. Inhibition of SHP2 by an allosteric inhibitor SHP099 selectively inhibits ERK1/2 activity, proliferation, and survival of ovarian cancer cell lines with GAB2 amplification/overexpression. Treatment with SHP099 has a synergistic effect with multiple PI3K pathway inhibitors such as BKM120, a pan-class I PI3K inhibitor, at suppressing proliferation and survival of ovarian cancer cells overexpressing GAB2 in part through induction of apoptosis. Combinatorial SHP2 and PI3K inhibition more effectively activates both pro-apoptotic proteins BIM and BAD and inhibits expression of transcription factor c-MYC, a master regulator of cancer stem cells, compared with individual inhibitor, and sensitized cancer cells to chemotherapeutic drugs paclitaxel and cisplatin. Combinatorial SHP2 and PI3K inhibition also suppresses spheroid formation of ALDH-positive ovarian cancer stem cells. Together, our findings identify a previously unrecognized role of SHP2 in promoting proliferation and survival of ovarian cancer cells in which GAB2 is amplified and/or overexpressed, and suggest that combinatorial SHP2 and PI3K inhibition may be a promising therapeutic approach for HGSOCs with GAB2 amplification/overexpression. Citation Format: Hiu Wing Cheung. Synergistic effects of SHP2 and PI3K inhibitors in GAB2-overexpressing 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 B38.

Abstract PR03: Arginine deprivation as a potential targeted therapy for clear cell ovarian carcinoma

Abstract In this study, we explored the metabolic pathways of clear cell ovarian carcinoma (CCOC) and the therapeutic importance of aberrant arginine metabolism in this cancer. In 2017, an estimated 22,440 women will be diagnosed with epithelial ovarian carcinoma (EOC) in the United States. EOC is divided into subtypes based on histology and prognosis. Among them, CCOC is truly a unique entity. Histologically, CCOC is characterized by clear cytoplasm, which stains PAS positive, indicating aberrant cellular glycogen storage. Genomic studies in CCOC have identified recurrent mutations in the ARID1A and PIK3CA genes, both encoding proteins with crucial roles in cellular metabolism, which further supports CCOC being a metabolism-dependent malignancy. At late stage, CCOC is more aggressive and refractory to conventional platinum-based therapy, compared to other EOC subtypes. Despite the lack of efficacy, platinum-based chemotherapy is still the gold standard for treating all EOC subtypes. The lack of targeted therapy for CCOC paints a grim picture for the patients as they inevitably relapse. Using a mass spectrometry-based study, we characterized the whole proteome of 17 formalin-fixed, paraffin-embedded (FFPE) patient CCOC tumors. The CCOC cases separated into 2 distinct subgroups based on unsupervised hierarchical clustering. We identified the top 250 most differentially expressed proteins between these 2 groups using Protein Expression Control Analysis (PECA) and subsequent pathway analysis through KEGG. Of these 250 proteins, 56 were metabolism-related, including Argininosuccinate Synthase 1 (ASS1). ASS1 is a crucial enzyme in the cellular synthesis of arginine; a deficiency in the enzyme makes cancer cells dependent on extracellular arginine for survival. In ASS-1 deficient sarcomas, targeted small-molecule therapy depriving extracellular arginine results in cell death and sensitization to conventional chemotherapy. In transcriptomic analysis of 55 patient CCOC tumors and cell lines, 13 cases had low ASS1 RNA expression compared to others. Subsequently, we collected 97 CCOC cases from a local tissue bank and studied ASS1 protein expression using immunohistochemistry. In these cases, ASS1 expression ranges from strong to diffusely weak to null, confirming the differential expression discovered in the proteomic and transcriptomic study. To this end, ASS1 levels were assessed in CCOC, endometrioid, and high-grade serous cell lines. ASS1 was not expressed in a subset of CCOC cell lines and was low in others. We further demonstrate that a subset of CCOC cell lines are sensitive to arginine deprivation, indicating that there may be some CCOC tumors that would benefit from combined arginine deprivation in conjunction with the gold standard platinum-based therapy. This abstract is also being presented as Poster A13. Citation Format: Jennifer Xiao Ye Ji, Dawn R. Cochrane, Basile Tessier-Cloutier, Lien N. Hoang, Yikan Wang, Angela Cheung, Christine Chow, Shane Colborne, Christopher Hughes, Gregg B. Morin, David G. Huntsman. Arginine deprivation as a potential targeted therapy for clear cell ovarian carcinoma. [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 PR03.

In vitro effects of FBXW7 mutation in serous endometrial cancer: Increased levels of potentially druggable proteins and sensitivity to SI-2 and dinaciclib.

Serous endometrial cancers (ECs) are clinically aggressive tumors that frequently harbor somatic mutations in FBXW7 (F-box and WD repeat domain-containing 7). The FBXW7 tumor suppressor is part of a SCF (complex of SKP1, Cullin 1, F-box protein) ubiquitin ligase complex which controls the degradation of numerous substrates that, if not properly regulated, can contribute to the initiation or progression of tumorigenesis. Despite reports that up to 30% of serous ECs include somatic mutations in FBXW7, the molecular effects of mutated FBXW7 in ECs have not been determined. Here, we used transient transfection and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) editing in serous EC cell lines to interrogate the molecular effects of six recurrent FBXW7 mutations. We show that FBXW7 mutations lead to increased Cyclin E1, steroid receptor coactivator 3 (SRC-3), c-MYC, Rictor, glycogen synthase kinase 3 (GSK3), P70S6 kinase, and protein kinase B (AKT) phosphorylated protein levels in serous EC cells. Furthermore, we demonstrate that CRISPR-edited FBXW7-mutant ARK1 serous EC cells exhibit increased sensitivity to SI-2 (a SRC inhibitor) and dinaciclib (a cyclin dependent kinase (CDK) inhibitor) compared to parental ARK1 cells. Collectively, our findings reveal biochemical effects of FBXW7 mutations in the context of EC and provide in vitro evidence of sensitivity to targeted inhibitors.

Abstract 3210: Targeting PTP4A3 phosphatase in ovarian cancer with the potent noncompetitive inhibitor JMS-631-053

Abstract PTP4A3 is a highly attractive molecular target for ovarian cancer (OvCa). Elevated levels of PTP4A3 mRNA and protein in human ovarian tumors correlate with disease progression, poor prognosis and poor survival. Genetic depletion of PTP4A3 in OvCa cell lines diminishes their ability to migrate and reduces their in vivo tumorigenicity while PTP4A3 overexpression increases tumor cell migration, invasion, and dissemination. Together, these data suggest PTP4A3 is a novel molecular target for OvCa; however, the lack of potent and selective PTP4A3 small molecule inhibitors has hindered PTP4A3’s definitive validation in OvCa and in other cancers. To drive the pharmacological validation of PTP4A3, we developed JMS-631-053 (7-imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione), a potent (Ki=3 nM in vitro), specific, noncompetitive, PTP4A3 inhibitor. JMS-631-053 inhibited cellular migration, invasion, and colony formation in soft agar. Potent OvCa cell-based effects were observed by profiling JMS-631-053 against a panel of 8 OvCa cell lines using a 2D drug susceptibility assay, with EC50 values as low as 600 nM. Likewise, JMS-631-053 killed OvCa 3D spheroids, including those derived from high grade serous OvCa cell lines, with EC50 values as low as 300 nM. The OvCa drug resistant cell lines (i.e., A2780CP20 and HeyA8MDR) retained responsiveness to JMS-631-053 (using 2D and 3D culturing conditions) suggesting that inhibition of PTP4A3 may be a viable therapeutic strategy for chemoresistant OvCa. Preliminary data also suggests that JMS-631-053 synergizes with cisplatin when used in combination studies. Hence, JMS-631-053 will be a valuable chemical tool for further validation of PTP4A3 as an OvCa target as well as provide potential leads for future drug discovery. Citation Format: John S. Lazo, Paula Pekic, Alex Cheung, Kelley McQueeney, Joseph Salamoun, Peter Wipf, Charles N. Landen, Elizabeth R. Sharlow. Targeting PTP4A3 phosphatase in ovarian cancer with the potent noncompetitive inhibitor JMS-631-053 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3210. doi:10.1158/1538-7445.AM2017-3210

Abstract 3221: Kevetrin induces p53-dependent and independent cell cycle arrest and apoptosis in ovarian cancer cell lines representing heterogeneous histologies

Abstract Ovarian cancer (OC) is a molecularly and histologically heterogeneous disease; however, standard treatment is the same for all subtypes. High-grade serous OC initially responds to chemotherapy; however, low-grade serous and clear cell OC are relatively chemoresistant. Limited treatment options are available upon recurrence. p53 mutations are found in over 90% of high-grade serous OC. Low-grade serous OC harbor wild type p53, but contain other mutations. During later stages of OC, tumors are a heterogeneous population of mutant cells; thus, development of a novel drug that addresses these molecular differences is highly desirable. Previously, we showed that Kevetrin stabilized wild type p53 and induced transcriptional targets in human lung carcinoma. We sought to validate Kevetrin as a potential treatment for OC with varied p53 status. Endometrioid carcinoma (A2780, wt p53), atypical non-serous clear cell (SKOV-3, deleted p53), and high-grade serous (OVCAR-3, mutant p53) OC cell lines were treated with Kevetrin. Kevetrin induced apoptosis in all three OC cell lines, as assayed by cleavage of PARP and caspase-3. Studies showed significant increases in p53 and p21 protein levels in A2780 cells in 24 to 48 hours; however, in OVCAR-3, Kevetrin downregulated oncogenic mutant p53. RNA levels of p53 and p21 were quantified by qRT-PCR 8 to 72 hours after treatment. No significant changes were observed in p53 mRNA, whereas an increase in p21 mRNA was observed in all three cell lines. In A2780, Kevetrin also induced levels of PUMA in a dose-dependent manner. To establish that Kevetrin mediates increased p21 expression requiring p53 in A2780 cells, p53 was depleted by siRNA. p53 depletion reduced p21 expression, as assayed by FACS, indicating p53 directs p21 expression in a dose-dependent manner after Kevetrin treatment. In an in vivo xenograft study in immunocompromised nude mice bearing established A2780 tumors, Kevetrin treatment inhibited tumor growth at well-tolerated doses. The mode of action in vivo also showed enhanced expression of p21 in tumor tissue, indicating p53 pathway activation in A2780 tumors. In contrast, in SKOV-3 cells and xenografts, a p53 independent increase in p21 expression was observed. In OVCAR-3 cells,Kevetrin altered the expression of three microRNAs (miRNA-27a, miRNA-1274b, miRNA-25), that are known to be dysregulated in OC, in a time-dependent manner. To gain further insight into the mechanism of action in cells with diverse p53 status, RNA-Seq is being performed in the three cell lines and tumor tissue from mice before and after Kevetrin exposure. In summary, Kevetrin has promise as an effective therapeutic agent for endometrioid, non-serous clear cell, and high-grade serous OC, with molecularly diverse p53 status. A Phase 1 clinical study was completed and a Phase 2 clinical study in ovarian cancer is scheduled to begin January 2017. Citation Format: Ashok Kumar, David P. Brennan, Karima Chafai-Fadela, Sylvia A. Holden, Siya Ram, Geoffrey I. Shapiro, Krishna Menon. Kevetrin induces p53-dependent and independent cell cycle arrest and apoptosis in ovarian cancer cell lines representing heterogeneous histologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3221. doi:10.1158/1538-7445.AM2017-3221

Abstract 3419: Uncovering the functional relevance of FBXW7 mutations in endometrial cancer

Abstract The goal of this study is to elucidate the functional consequences of FBXW7 mutations in the context of endometrial carcinoma (EC). EC is a heterogeneous disease, consisting of multiple histological subtypes associated with distinct clinical outcomes. Endometrioid endometrial cancer is the most commonly diagnosed subtype and is associated with an overall favorable prognosis. Less common but more clinically aggressive subtypes that contribute disproportionately to patient deaths include clear cell and serous ECs. We previously reported that FBXW7 is somatically mutated in 10% of endometrioid, 13% of clear cell and 29% of serous ECs. FBXW7 functions as a substrate-recognition protein within a SKP/CUL1/F-Box ubiquitin ligase complex, which targets numerous proteins for ubiquitin-mediated proteosomal degradation. Although FBXW7 is a known tumor suppressor, the functional consequences of these mutations have not yet been fully elucidated in the context of EC. As a means to this end, we utilized shRNA and Western blotting to identify proteins that are upregulated following FBXW7 depletion in ARK1 serous EC cells. Among proteins that were consistently upregulated were cyclin E1 and cMYC. We treated EC cells harboring endogenous FBXW7 mutations and those with wildtype FBXW7 with a proteasome inhibitor to confirm that EC cells regulate cyclin E1 and cMYC through proteasome degradation. Furthermore, we identified three EC cell lines that harbor endogenous mutations in FBXW7 and show that these cell lines exhibit delayed degradation of these substrates compared to EC cells expressing wildtype FBXW7. Finally, we generated expression constructs for wildtype FBXW7α and six recurrent mutants found in EC. We show that transient exogenous overexpression of five out of six recurrent FBXW7 mutant constructs in two different serous EC cell lines resulted in increased levels of phosphorylated cMYC and cyclin E1 compared to wildtype FBXW7α. These results suggest that not all recurrent FBXW7 mutations are functionally equivalent. Our findings begin to reveal the molecular consequences of FBXW7 mutations in EC and ongoing studies are aimed at elucidating additional functional consequences of these aberrations in the context of EC. Citation Format: Mary Ellen Urick, Bo Hong, Meghan L. Rudd, Daphne W. Bell. Uncovering the functional relevance of FBXW7 mutations in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3419. doi:10.1158/1538-7445.AM2017-3419

Abstract 1197: Endothelin-1 receptor/β-arrestin1 is an actionable node that regulates YAP/TAZ signaling and chemoresistance in high-grade ovarian cancer

Abstract The widespread activation of YAP and TAZ transcriptional regulators has been shown to promote tumor progression and resistance to anti-cancer therapies. Extensive studies have identified different upstream regulators of YAP/TAZ, including G-protein coupled receptor (GPCR). However how GPCR signals regulate YAP/TAZ puzzles have remained elusive. Among the GPCR, we reveal that the endothelin-1 (ET-1) receptor activates YAP/TAZ pathway in high-grade serous ovarian cancer (HG-SOC) sensitive and resistant cell lines and primary cultures. In HG-SOC cells, the aberrant activation of ET-1 receptor (ET-1R), promotes an enhanced nuclear shuttling of dephosphorylated YAP and TAZ in a time-dependent manner, through the scaffold protein β-arrestin1 (β-arr1). Interestingly, ET-1-induced YAP/TAZ nuclear accumulation is further improved in platinum-resistant cells and is prevented by expressing a mutant β-arr1 incapable of nuclear distribution. Moreover, the ET-1/ETAR axis activation induces the co-localization of β-arr1, YAP and TAZ in the nucleus. Mechanistically, in sensitive and to a greater extent in resistant cells, ET-1 induces the formation of a complex β-arr1/Trio, a Rho-Guanine nucleotide exchange factor family member, which dephosphorylates YAP, inducing its nuclear accumulation and the transcription of YAP/TAZ target genes. β-arr1, RhoA or Trio depletion, as well as the treatment with the dual ETAR/ETBR antagonist macitentan, significantly reduces RhoA GTPase activity and YAP nuclear translocation, suggesting that ET-1/ETAR axis regulates YAP/TAZ activity by driving β-arr1/Trio/RhoA pathway. According with these results, the ET-1-induced upregulation of YAP/TEAD target genes, such as CYR61, CTGF, ANKRD1 and EDN1, as well as TEAD transcriptional activity and cell invasion, are inhibited upon β-arr1, Trio, or YAP silencing, or macitentan treatment, demonstrating that β-arr1-mediated intersections are required for ET-1-induced YAP signaling. At the chromatin level, the activation of ET-1 axis promotes the recruitment of YAP, TEAD and β-arr1 on TEAD binding sites of CTGF, ANKRD1 and EDN1 promoters, an effect inhibited by macitentan treatment. These results indicate that β-arr1 can act as a transcription co-activator that bind TEAD thereby activating transcription of EDN1, which in turn, can sustain persistent YAP/TAZ activity through an autocrine loop. In murine orthotopic model of patient-derived xenograft (PDX) of HG-SOC, ET-1R blockade by macitentan, inhibits tumor growth, enhances the sensitivity to chemotherapy and reduces YAP and TEAD transcriptional activity. Altogether our results establish for the first time YAP/TAZ as critical downstream effectors of ET-1R/β-arr1 signaling, providing mechanistic insights that targeting ET-1R signaling overcomes YAP/TAZ driven platinum-based therapy failure in HG-SOC. Citation Format: Piera Tocci, Roberta Cianfrocca, Laura Rosanò, Rosanna Sestito, Valeriana Di Castro, Giovanni Blandino, Anna Bagnato. Endothelin-1 receptor/β-arrestin1 is an actionable node that regulates YAP/TAZ signaling and chemoresistance in high-grade ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1197. doi:10.1158/1538-7445.AM2017-1197

Abstract NTOC-085: PPARγ AGONISTS AUGMENT ANTICANCER EFFECTS OF XIAP INHIBITION ON HUMAN GRANULOSA CELL TUMOR–DERIVED CELLS

Abstract BACKGROUND: Ovarian granulosa cell tumors (GCT) are hormonally active cancers characterized by indolent growth and late, invasive relapse. GCT are unusual in that they have an unexplained propensity for late recurrence. ~80% of patients with aggressive or recurrent tumors die from their disease. Aside from surgery the therapeutic options are very limited. We have identified the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ) and the X-linked inhibitor of apoptosis protein (XIAP) as potential specific therapeutic targets. PPARγ is a transcription factor that impedes proliferation and promotes terminal differentiation in granulosa cells (GC), is highly expressed in GCT. Its activity however is transrepressed by constitutive activity of the critical pro-survival NF-κB signaling pathway. Constitutive NF-κB signaling is potentially a consequence of a positive feed-forward loop involving XIAP, which is also highly expressed in GCT. As a potent inhibitor of apoptosis, XIAP is an attractive therapeutic target. We have shown in vitro that inhibiting XIAP releases NF-κB transrepression, and together with PPARγ activation, induces apoptosis. We thus hypothesize that XIAP antagonism sensitizes GCT to pro-apoptotic strategies such as PPARγ activation. METHODS AND RESULTS: We have used stable isotope labeling with amino acids in cell culture (SILAC), a proteomic approach to identify differentially expressed proteins after combined PPARγ activation (rosiglitazone) and XIAP inhibition (Smac mimetic). Using two GCT-derived cell lines, KGN and COV434, we identified 52 proteins which were differentially expressed (by 1.5 fold) with the combined treatment. Upregulated proteins included several involved in metabolic processes including acyl-CoA desaturase (4.50-fold), phosphoglycerate kinase 1 (2.87-fold) and α-enolase (1.75-fold). As PPARγ plays a pivotal role in lipid and glucose metabolism, upregulation of these proteins is a consequence of PPARγ activity being restored. Additionally, we observed downregulation of fascin (-1.65 fold), the main actin-bundling protein associated with cell motility and migration. Overexpression has been observed in metastatic tumors and correlated with clinically aggressive phenotypes, poor prognosis and shorter survival. We used the xCELLigence RTCA system to monitor invasion of KGN cells through a Matrigel® basement membrane in real-time. KGN cells treated either with Vehicle, RGZ/RA or SM alone or combined RGZ/RA/SM were plated in the upper chamber of a CIM-Plate 16 and cell invasion through the Matrigel® membrane was monitored. When compared to vehicle treated cells, we observed that the RGZ/RA/SM-treated KGN cells were (i) 30% less invasive and (ii) demonstrated a delayed onset of invasion by 17 hours towards serum-containing media with and without drugs in the lower chamber, respectively. CONCLUSION: We are currently investigating the mechanism involved in XIAP inhibition abrogating transrepression of PPARγ in GCT-derived cells. This study will improve our understanding of the molecular mechanisms in GCT pathophysiology as well as enable identification of new targets for therapeutic strategies. These observations may also translate to ovarian epithelial cell cancers (EOC) as increased apoptosis was observed in serous EOC-derived cell lines expressing both XIAP and PPARγ. We propose that a combination therapy involving the abrogation of XIAP may be of greater efficacy for the treatment of GCT and potentially other ovarian cancer subtypes. Citation Format: Simon Chu, Dilys TH Leung, Maria Alexiadis and Peter J Fuller. PPARγ AGONISTS AUGMENT ANTICANCER EFFECTS OF XIAP INHIBITION ON HUMAN GRANULOSA CELL TUMOR–DERIVED CELLS [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-085.

Abstract A55: Human endogenous retroviruses differentially regulate immune checkpoints and modulation of immune responses in serous ovarian carcinoma.

Abstract Purpose: The first aim is to investigate the activation of 7 immune checkpoints at different time points following transfection of three diverse ERV envelope (env) genes in serous ovarian carcinoma cells. The second aim is to determine if the ERV-W1 Syncytin-1 env and ERV-3 protein are expressed in primary serous OvCa tumors and ascites fluids to explore a possible role in the tumor microenvironment. Introduction: The overall survival of serous OvCa has not greatly improved. We and others have demonstrated up-regulation of interferon (IFN) type I signaling pathway viral defense genes including IFN stimulated genes (ISGs), in a variety of human tumors (ovary, breast, lung) but also in ovarian and colon carcinoma cell lines treated with the DNA methyltransferase inhibitor Aza-C. Aza-C treatment of serous OvCa cell lines leads to an induction of PD-L1 expression as mediated downstream signaling via IFNg. We recently demonstrated that activated ERVs function at the RNA level to ignite the IFN response. Exogenous retroviruses like HIV have evolved to suppress the immune response by up-regulation of PD-L1 on target cells. Tumors mimic regulatory mechanisms involving immunosuppression and inflammation, which we propose involves ERVs. Experimental Procedures: ERV-3, Syncytin-1 and ERV-W2 env CMV overexpression vectors and a control EGFP-vector were transfected into OvCa serous cell lines (TykNu, A2780 and Hey) and RNA expression of 7 immune checkpoint molecules (LGALS9, PDL-2, CD80, CD276, PD-L1, VTCN1 and PD1) were monitored at days 3 and 7 post transfection using real time PCR. Immunohistochemistry (IHC) and Western analyses using antibodies for Syncytin-1 and ERV-3 were performed with primary serous OvCa tumors. Western and ELISA analyses of serous OvCa ascites were executed to detect Syncytin-1 protein levels. Syncytin-1 protein concentrations were quantified in ascites with two different antibodies using ELISA and correlated to a standard curve based on purified Syncytin-1 levels. Results: Three serous OvCa cell lines following ERV env transfection showed differences of immune checkpoint activation. TykNu demonstrated the highest fold-levels of induction for LGALS9 (73.1x), PD-L1 (15.2x), PD-L2 (9.8x) and PD1 (6.1x) at day 3 for all ERV expression vectors, which decreased by day 7 except for PD1. A2780 showed increased levels for LGALS9 (7.2x) and PD1 (5.2x) with all ERV expression vectors at day 3, which decreased by day 7. In contrast, Hey cells showed no significant increase of the analyzed immune checkpoint genes. Syncytin-1 and ERV-3 protein was highly expressed in primary OvCa epithelial cells using IHC, compared to patient matched control ovarian tissues. Western analyses of ascites from OvCa patients showed high levels of Syncytin-1 in the supernatant, supporting protein secretion. Absolute quantification using real time PCR of Syncytin-1 and ERV-3 RNA with fractionated OvCa ascites cells demonstrated high amounts of molecules. ELISA results confirmed high levels of Syncytin-1 (98.6 - 525.8 microg/ml) in ascites of primary OvCa and in one relapse patient (413.4 microg/ml), but were virtually undetectable in an ascites from a gastro-intestinal carcinoma and control peritoneal fluids (2.7 microg/ml). Conclusions: Our findings that various immune checkpoints are differentially regulated following ERV activation in serous OvCa cell lines endorses that analyses of immune checkpoints prior to treatment could help stratify patient therapy. ERVs like exogenous retroviruses not only play an essential functional role at the RNA level via TLR3, but also possibly at the protein level via TLR4 modulation of immune / inflammatory responses. Citation Format: Pamela L. Strissel, Katherine B. Chiappinelli, Johanna Strehl, Franziska MB Würfel, Matthias W. Beckmann, Stephen B. Baylin, Reiner Strick. Human endogenous retroviruses differentially regulate immune checkpoints and modulation of immune responses in serous ovarian carcinoma.. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A55.

Abstract 2504: Assessing the efficacy of the anti metabolic drug, CPI-613, for targeting of ovarian cancer stem cells

Abstract Cancer stem cells (CSC) are believed to contribute to the recurrent and often platinum resistant ovarian cancer (OvCa). Moreover, it has been suggested that some anticancer treatments result in an increased frequency of CSC relative to non-CSC. More recently, we have determined that the PARP inhibitor olaparib increased the frequency of CD133, CD117 and ALDH positive cells in the established serous OvCa cell lines OVCAR4 and CaOV4 and in primary cell lines derived from serous carcinomas obtained with IRB approval at the time of initial surgery. We also showed that olaparib treatment increased sphere forming capacity, expression of stemness genes and CSC proliferation suggesting that olaparib has the unintended effect of stimulating CSC function. Previous work has shown that the metabolic inhibitor CPI-613 preferentially targets ovarian CSCs due to their altered metabolism. Our objective here was to determine whether CPI-613 could negate the effect of olaparib on CSC function. OVCAR4 and CaOV4 cells were treated with 50 μM CPI-613 and the relative frequency of CD133+ and CD177+ cells was assessed at 72 hours post treatment by flow cytometry with cell viability analyzed by MTT. Additionally, the sphere forming capacity of CPI-613 treated cells relative to that of vehicle treated cells was determined in an extreme limiting dilution assay. Finally, the expression of stemness genes in the CPI-613 treated and vehicle treated cells was evaluated by RT-PCR. Treatment of OVCAR4 and CaOV4 cells with CPI-613 decreased the frequency of CD133+ and CD117+ positive cells relative to untreated cells (p <0.001) without affecting cell viability. Additionally, sphere forming capacity was reduced in the CPI-613 treated cells. The decrease in both CD133+ and CD117+ cell frequency and sphere forming capacity suggests that CPI-613 is targeting the CSC population in these cells. Dual treatment with olaparib and CPI-613 blunted the increase in CD133+ and CD117+ cell frequency induced by single agent olaparib. Parallel treatment of CSC-rich spheres resulted in a considerable decrease in cell viability and frequency of CD133+ and CD117+ cells Clinical trials with olaparib in OvCa have resulted in increased progression free survival but no significant difference in overall survival has been observed. This raises the question of whether treatment with olaparib selects for the recurrence of more resistant disease, driven in part by the proliferation of CSCs. Our results suggest that CPI-613-mediated disruption of the mitochondrial respiratory pathway preferentially targets CSC and suppresses olaparib's effects on CSC function. In conclusion, targeting CSCs with non-traditional strategies could augment clinical outcomes and CPI-613 represents a promising candidate for combination with FDA approved olaparib treatment in OvCa. Citation Format: Chiara Bellio, Rosemary Foster, Whitfield B. Growdon, Bo R. Rueda. Assessing the efficacy of the anti metabolic drug, CPI-613, for targeting of ovarian cancer stem cells. [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 2504.

The prognostic significance of specific HOX gene expression patterns in ovarian cancer.

HOX genes are vital for all aspects of mammalian growth and differentiation, and their dysregulated expression is related to ovarian carcinogenesis. The aim of the current study was to establish the prognostic value of HOX dysregulation as well as its role in platinum resistance. The potential to target HOX proteins through the HOX/PBX interaction was also explored in the context of platinum resistance. HOX gene expression was determined in ovarian cancer cell lines and primary EOCs by QPCR, and compared to expression in normal ovarian epithelium and fallopian tube tissue samples. Statistical analysis included one-way ANOVA and t-tests, using statistical software R and GraphPad. The analysis identified 36 of the 39 HOX genes as being overexpressed in high grade serous EOC compared to normal tissue. We detected a molecular HOX gene-signature that predicted poor outcome. Overexpression of HOXB4 and HOXB9 was identified in high grade serous cell lines after platinum resistance developed. Targeting the HOX/PBX dimer with the HXR9 peptide enhanced the cytotoxicity of cisplatin in platinum-resistant ovarian cancer. In conclusion, this study has shown the HOX genes are highly dysregulated in ovarian cancer with high expression of HOXA13, B6, C13, D1 and D13 being predictive of poor clinical outcome. Targeting the HOX/PBX dimer in platinum-resistant cancer represents a potentially new therapeutic option that should be further developed and tested in clinical trials.

Folic acid-targeted iron oxide nanoparticles as contrast agents for magnetic resonance imaging of human ovarian cancer.

BackgroundImproved methods for the early and specific detection of ovarian cancer are needed.MethodsIn this experimental study, we used folic acid (FA)-targeted iron oxide (Fe3O4) nanoparticles (NPs) as a T2-negative contrast agent for magnetic resonance (MR) imaging to accurately detect ovarian cancer tissues in an intraperitoneal xenograft tumor model. Human serous ovarian cell line (Skov-3), with overexpressed FA receptors, was chosen as the targeted tumor cell mode. For in vivo experiments, the cells were injected intraperitoneally into nude mice to produce intraabdominal ovarian cancers. FA-targeted and non-targeted Fe3O4 NPs were prepared.ResultsFA-targeted Fe3O4 NPs with a mean size of 9.2 ± 1.7 nm have a negligible cytotoxicity to human serous ovarian cell line (Skov-3). Importantly, the results of cellular uptake suggested that FA-targeted Fe3O4 NPs have a targeting specificity to Skov-3 cells overexpressing FA receptors. FA-targeted Fe3O4 NPs could be specifically localized by magnetic resonance (MR) imaging to the intraperitoneal human ovarian carcinoma tissues, as documented by a statistically significant difference (p = 0.002, n = 3) in T2 signal intensities of xenograft tumor tissues when injected with FA-targeted and non-targeted Fe3O4 NPs at 4 h post-injection.ConclusionFA-targeted Fe3O4 NPs appear to be promising agents for the detection of human ovarian carcinoma by MR imaging, and possibly also for the hyperthermal treatment of the tumors.

Abstract B05: Assessment of TP53 mutation status in primary high-grade serous ovarian cancer and cell line models: Comparison between immunohistochemistry and next-generation sequencing.

Abstract Mutations in TP53 have been shown to occur in around 96% of high-grade serous ovarian cancer (HGSOC). Different TP53 mutations can lead to accumulation of mutant p53 in the cell that support gain of function effects, or loss or abrogation of both wild-type and mutant p53. Success of new therapeutic strategies for HGSOC that target mutant p53 will likely depend on detailed knowledge of the type of TP53 mutation present and its influence on mutant p53 levels in the cell. The aim of this study was to identify the spectrum of TP53 mutations in a cohort of primary HGSOCs and compare this with p53 immunostaining patterns. In addition, we sought to identify TP53 mutations in the previously uncharacterised HGSOC cell line models OV167 and OV202, as well as the clear cell adenocarcinoma line OV207. DNA from fresh frozen tumors and cell line samples were processed for next generation sequencing using The Fluidigm Access TP53 Array System run on the Fluidigm Biomark HD™ Real-Time PCR fluidics system (Fluidigm Corporation). Amplicon libraries were pooled and analyses performed in a single massively parallel sequencing run using the MiSeq Sequencing System (Illumina Inc.). Unaligned BAM files were analyzed within the Broad Institute's Genome Analysis Toolkit (GATK) best practice pipeline. The ANNOVAR software tool was used to generate an annotated variants list. Variants were further filtered based on their frequency in the 1000 Genome Database and predicted SIFT scores. Mutations were visualized using the Broad Institute's Integrative Genomics Viewer. All samples in which a mutation was identified underwent Sanger sequencing using primers recommended in the International Agency for Research on Cancer TP53 database. Immunohistochemistry for p53 was performed on formalin fixed paraffin embedded tissue sections using a commercially available mouse monoclonal anti-human antibody (Protein Clone DO-7, cat. #M7001, Dako) on an automated staining platform (Leica BOND-III™ autostainer). The percentage of cells showing positive nuclear staining for p53 was reported. A mutation in TP53 was identified in 94% (68 of 72) of HGSOC. The majority of mutations identified (62%, 42 of 68) were missense, with all except one of these mutations located in the p53 DNA binding domain. Overall, 82% (56 of 68) of mutations occurred within the DNA binding domain. Frameshift, stop or splice mutations (38%, 26 of 68) were identified in exons or flanking intronic sequence between exons 4 – 10. The presence or location of a TP53 mutation did not influence overall survival. Sanger sequencing was unable to reliably detect variants at an allele frequency less than 25%. TP53 mutations were identified in all cell lines at a mutant allele frequency ≥99%. Based on percent of nuclei positive for p53, tumors were immunohistochemically graded as High, Intermediate or Low for p53. All missense mutations scored High for expression of the p53 protein. Considerable variability was observed in p53 staining levels in samples containing stop, frameshift or splice mutations, with samples in all of High, Intermediate and Low categories. Furthermore, a single sample shown to be wild-type for TP53 was graded as High for p53, raising the possibility that p53 had been activated in this tumor via an alternative mechanism. The level of p53 did not affect overall survival. Taken together, these results suggest that caution needs to be exercised when using p53 immunohistochemistry as a surrogate marker for either presence or type of TP53 mutation. A combination of next generation sequencing and p53 immunohistochemistry is required to robustly define both mutation type and effect on mutant p53 levels in the cell. Citation Format: Alexander J. Cole, Trisha Dwight, Ying Zhu, Anthony J. Gill, Kristie-Ann Dickson, Adele Clarkson, Gregory B. Gard, Jayne Maidens, Susan Valmadre, Roderick J. Clifton-Bligh, Deborah J. Marsh. Assessment of TP53 mutation status in primary high-grade serous ovarian cancer and cell line models: Comparison between immunohistochemistry and next-generation sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B05.

Abstract A23: The dual roles of microRNA-200: From inclusion cyst formation to cell migration.

Abstract Background: Historically, metaplasia of ovarian surface epithelial (OSE) cells in the cortical inclusion cysts is associated with ovarian cancer development. Recent studies have also suggested fallopian tube epithelium as another origin of ovarian cancer cells. MicroRNA-200 (miR200) family is related to epithelial-mesenchymal transition (EMT) and overexpressed in ovarian cancer. However, the potential involvement of miR200 in ovarian pathogenesis is largely unknown. Objective: To study any morphological and signaling differences between immortalized normal human ovarian surface epithelial (OSE) cells and ovarian cancer cells with changes in miR200 expression and control cells growing in three-dimensional (3D) cultures. Methods: OSE cells with stable ectopic expression of miR200 and ovarian cancer cells with stable miR200 knockdown were established using lentiviral agents. The resulting cells and control cells were grown as 3D cultures first in Matrigel to form the cyst-like spheroids and then transferred to Collagen I matrix for cell migration study. Immunofluorescence and confocal microscopy was applied to study the morphologies of the 3D structures, and in conjunction with Western blot analysis, to study changes in signaling pathways in response to changes of miR200 expression and in the presence of a variety of pharmacologic inhibitors. Clinical samples were examined for miR200 expression to evaluate the results from 3D in vitro cultures. Results: In comparison with the wild-type control counterparts, OSE cells with ectopic expression of miR200 had elevated E-cadherin expression, showed more compact and prolonged cyst stabilization in both Matrigel and Collagen I 3D cultures; whereas the ovarian cancer cells with miR200 knockdown showed increased TGFβ expression and multiple lumens with enhanced cell death in Matrigel culture, and change from collective migration mode to single cells in Collagen I culture. Both acetylated tubulin and Histone H3 staining of mitotic cells in Matrigel culture demonstrated ovarian cancer cells with miR200 knockdown had abnormal spindle orientation and defective cell cycle division. Application of TGFβ inhibitors reversed the single cell migration phenotype of miR200 knockdown cancer cells to collective movement. However, the downstream pathways responsible for the switching of migration phenotype were not the same between the serous and mucinous cell lines. For the mucinous cell line, miR200 controlled the switching through TGFβ-ROCK pathway, whereas the downstream pathway for the switching in the serous cell line remained unknown. qRT-PCR analysis of micro-dissected cells from clinical samples verified the elevated expression of miR200 in endosalpingiosis cysts and tumor tissues when compared with normal ovarian surface epithelia and fallopian tube epithelia. Conclusion: The qRT-PCR validation study verifies the findings of our 3D in vitro culture study and demonstrates that miR200 is important for inclusion cyst formation and collective movement of ovarian cancer cells during tumor invasion. Perturbation of this pathway in cancer cells will accelerate cell death through abnormal cell division. Hence, miR200 may be an interesting target in ovarian cancer therapy. Citation Format: Pui Wah Choi, Junzheng Yang, Wing Ping Fong, Ross S. Berkowitz, William R. Welch, Gregory J. Goodall, Shu Wing Ng. The dual roles of microRNA-200: From inclusion cyst formation to cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A23.

Overexpression of GAB2 in ovarian cancer cells promotes tumor growth and angiogenesis by upregulating chemokine expression.

We previously found that the scaffold adapter GRB2-associated binding protein 2 (GAB2) is amplified and overexpressed in a subset of primary high-grade serous ovarian cancers and cell lines. Ovarian cancer cells overexpressing GAB2 are dependent on GAB2 for activation of the phosphatidylinositol 3-kinase (PI3K) pathway and are sensitive to PI3K inhibition. In this study, we show an important role of GAB2 overexpression in promoting tumor angiogenesis by upregulating expression of multiple chemokines. Specifically, we found that suppression of GAB2 by inducible small hairpin RNA in ovarian cancer cells inhibited tumor cell proliferation, angiogenesis and peritoneal tumor growth in immunodeficient mice. Overexpression of GAB2 upregulated the secretion of several chemokines from ovarian cancer cells, including CXCL1, CXCL2 and CXCL8. The secreted chemokines not only signal through endothelial CXCR2 receptor in a paracrine manner to promote endothelial tube formation, but also act as autocrine growth factors for GAB2-induced transformation of fallopian tube secretory epithelial cells and clonogenic growth of ovarian cancer cells overexpressing GAB2. Pharmacological inhibition of inhibitor of nuclear factor kappa-B kinase subunit β (IKKβ), but not PI3K, mechanistic target of rapamycin (mTOR) or mitogen-activated protein kinase (MEK), could effectively suppress GAB2-induced chemokine expression. Inhibition of IKKβ augmented the efficacy of PI3K/mTOR inhibition in suppressing clonogenic growth of ovarian cancer cells with GAB2 overexpression. Taken together, these findings suggest that overexpression of GAB2 in ovarian cancer cells promotes tumor growth and angiogenesis by upregulating expression of CXCL1, CXCL2 and CXCL8 that is IKKβ-dependent. Co-targeting IKKβ and PI3K pathways downstream of GAB2 might be a promising therapeutic strategy for ovarian cancer that overexpresses GAB2.

Abstract POSTER-TECH-1111: High-throughput drug sensitivity and resistance testing of ovarian cancer cell lines provides useful strategy for assessing drug repositioning and therapeutic possibilities of emerging drugs

Abstract As almost all therapeutic approaches for ovarian cancer (OC) are organ-based therapies that have been tested in large-scale clinical studies, no precision medicine approach to OC currently exists. There is an urgent need to develop a better understanding of the drug responsiveness in OC, particularly in the clear cell and mucinous subtypes, which tend to be resistant to the commonly used platinum-based regimens. This would help to improve survival and facilitate precision medicine therapy approaches. In order to discover previously unsuspected anti-cancer effects of approved and emerging drugs, we applied Drug Sensitivity and Resistance Testing (DSRT) technology (Pemovska et al., Cancer Discovery, 2013) to determine detailed dose-response effects of 306 drugs in 29 established OC cell lines (12 clear cell, 6 mucinous, 1 endometrioid, and 10 serous and other OC cell lines). The panel covered 137 approved drugs as well as emerging, investigational and pre-clinical oncology compounds such as kinase and non-kinase inhibitors. All drugs were tested at 5 different concentrations, covering a 10,000-fold concentration range by using Labcyte nano-dispensing technology, in order to generate detailed dose-response curves for each drug in each cell line. The area under the curve was estimated from the dose-response curve to obtain the drug sensitivity score. Bioinformatic processing of the drug response data from OC cell lines resulted in several key observations. First, the clusters did not depend on the histological origin of the OC cell lines but were clustered according to drug categories. Second, our result showed that many emerging or non-approved drugs were sensitive in subgroups of OC cell lines. For example, MEK inhibitors and mTOR inhibitors show high sensitivity in many OC cell lines. They can be candidates for drug repositioning. Third, we confirmed reproducibility of our findings. In summary, as per the data from direct testing of drug efficacy, several emerging anti-cancer drugs show potential responses in subsets of OC cell lines. DSRT technology provides a powerful strategy for assessing drug response in OC cell models, and it could also help asses drug responses in patient-derived ex vivo model systems of OC. Analysis of correlations of DSRT data with genomic data of the cell lines is underway and will yield new translational and pharmacogenomic opportunities for treating OC. Citation Format: Akira Hirasawa, Astrid Murumägi, Mariliina Arjama, Bhagwan Yadav, John Patrick Mpindi, Krister Wennerberg, Tero Aittokallio, Daisuke Aoki, Olli Kallioniemi. High-throughput drug sensitivity and resistance testing of ovarian cancer cell lines provides useful strategy for assessing drug repositioning and therapeutic possibilities of emerging drugs [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1111.

Abstract 2548: Metformin and everolimus act synergistically with paclitaxel against ovarian cancer

Abstract Background: Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, mostly due to the diagnosis of the disease at advanced stages and to chemoresistance to platinum- and taxane-based standard therapy. EOC comprises a heterogeneous group of diseases classified based on morphologic and molecular-genetic features. High grade serous ovarian cancer is the most prevalent EOC subtype, being followed by the clear cell subtype. Both EOC subtypes are vastly aggressive and frequently acquire the chemoresistant phenotype, thus challenging researchers and clinicians. Notably, hyperactivation of PI3K and MAPK pathways are frequently related to resistance to standard therapies in EOC, thus, metformin (MET) and everolimus (EVE) have emerged as novel therapeutic options against EOC. The present work aimed to investigate the action of MET and EVE, in vitro, in ES-2 (clear cell) and A2780 (serous) cell lines. Methods: Metabolic cell viability (MCV) assays were conducted after the treatment of the cells with MET or EVE in monotherapy and in association with paclitaxel (PAC) by the MTT method. Cell cycle progression was evaluated by flow cytometry/propidium iodide. Apoptosis was analyzed by the annexin V-FITC assay. Western blot was performed to investigate MEK/ERK and PI3K/AKT pathways. Findings: MCV of EOC lineages was reduced by MET and EVE in different concentrations. The combination of MET, at low and safe concentration (10uM), with PAC 100nM reduced the MCV in 56.55% for ES-2, and 71.38% for A2780. Similarly, EVE 0.06 nM and PAC 100 nM decreased the VCM in 66.4% and 73.38% for ES-2 and A2780, respectively. MET 10μm and EVE 0.06nM associated with PAC 12.5nM kept the MCV at levels comparable to PAC 100nM. These findings are of extreme clinical relevance as they reveal that the synergism between MET and EVE with PAC likely maintains the drug citotoxicity at lower concentrations than those associated to its severe and limiting side effects, as neurotoxicity. Annexin-FITC assay was conducted in ES-2 lineage treated with PAC alone and combined with MET or EVE. Low percentage of apoptotic/necrotic cells was observed, which suggests the occurrence of additional processes, such as autophagic cell death. EVE induced ES-2 cell cycle arrest; however, MET, at low concentration, did not cause the same effect. There were no significant cell cycle changes due to the combined treatment of ES-2 with PAC and MET or EVE. Western blot analysis demonstrated that the combination of EVE and PAC increased, whereas MET alone or in combination with PAC decreased the phosphorylation of ERK1/2 and AKT in ES2 cells. Conclusions: Our data suggest that MET or EVE exerts synergistic effect with PAC against EOC, thus opening an avenue to explore novel strategies against this highly lethal disease. Ongoing experiments are sought to elucidate the cellular pathways modulated by MET and EVE alone or in combination with PAC in EOC lineages, as well as the elicited cellular events and mechanisms. Citation Format: Nayara G. Tessarollo, Isabella S. Guimaraes, Taciane Ladislau, Ian Victor Silva, Leticia B. A. Rangel. Metformin and everolimus act synergistically with paclitaxel against ovarian cancer. [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 2548. doi:10.1158/1538-7445.AM2015-2548

Transcriptome profile of OVCAR3 cisplatin-resistant ovarian cancer cell line

BackgroundThe NIH:OVCAR-3 is a cisplatin refractory cell lineestablished from malignant ascites of a patient with pro-gressive adenocarcinoma of the ovary after combinationchemotherapy with cyclophosphamide, Adriamycin, andcisplatin [1]. Thus, OVCAR3 serves as a model cell linefor drug resistance in ovarian cancer. Here, we perform acomparative transcriptome analysis from the US NationalCancer Institute human tumor cell line anticancer drugscreen (NCI60) dataset [2]. Our results indicate a specificgene transcription profile of OVCAR3 genes relative tonon-cancerous Human Ovarian Surface Epithelial cells(HOSE) and drug sensitive Serous Ovarian CancerEpithelial Samples (CEPI) and SKOV3 cell lines. Pathwayenrichment analysis from OVCAR3 unique transcriptswas conducted using KEGG; Disease and Drug termenrichment used the PharmGKB [3] databases.Materials and methodsDatasets from the NCI60 were obtained from the GeneExpression Omnibus (GEO) of NCBI [4] (OVCAR3 andSKOV3 from series GSE2003, OSE and CEPI from series

VIP regulates CFTR membrane expression and function in Calu-3 cells by increasing its interaction with NHERF1 and P-ERM in a VPAC1- and PKCε-dependent manner.

Vasoactive intestinal peptide (VIP) is a topical airway gland secretagogue regulating fluid secretions, primarily by stimulating cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride secretion that contributes to the airways innate defense mechanism. We previously reported that prolonged VIP stimulation of pituitary adenylate cyclase-activating peptide receptors (VPAC1) in airway cells enhances CFTR function by increasing its membrane stability. In the present study, we identified the key effectors in the VIP signaling cascade in the human bronchial serous cell line Calu-3. Using immunocytochemistry and in situ proximity ligation assays, we found that VIP stimulation increased CFTR membrane localization by promoting its colocalization and interaction with the scaffolding protein Na(+)/H(+) exchange factor 1 (NHERF1), a PDZ protein known as a positive regulator for CFTR membrane localization. VIP stimulation also increased phosphorylation, by protein kinase Cε of the actin-binding protein complex ezrin/radixin/moesin (ERM) and its interaction with NHERF1 and CFTR complex. On the other hand, it reduced intracellular CFTR colocalization and interaction with CFTR associated ligand, another PDZ protein known to compete with NHERF1 for CFTR interaction, inducing cytoplasmic retention and lysosomal degradation. Reducing NHERF1 or ERM expression levels by specific siRNAs prevented the VIP effect on CFTR membrane stability. Furthermore, iodide efflux assays confirmed that NHERF1 and P-ERM are necessary for VIP regulation of the stability and sustained activity of membrane CFTR. This study shows the cellular mechanism by which prolonged VIP stimulation of airway epithelial cells regulates CFTR-dependent secretions.