IGROV-1 Cells Research Articles

Abstract 4791: Targeting ovarian cancer with novel 6-substituted pyrrolo [2,3-d] pyrimidine fluorinated antifolates via cellular uptake by folate receptor á and the proton-coupled folate transporter

Abstract The proton coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum, while folate receptor (FR) á delivers folate substrates into the cell via endocytosis. FRá and PCFT are highly expressed in ovarian cancer. PCFT transport is favored by acidic pH, including pHs associated with the acidic tumor microenvironment. We previously synthesized a novel class of 6-substituted pyrrolo[2,3-d]pyrimidine thiophene antifolates with 4- and 3-carbon bridge lengths, designated AGF117 and AGF150, targeted to FRá and PCFT, but not the ubiquitously expressed reduced folate carrier (RFC). To increase antitumor specificity and activity, reflecting multi-polar interactions with cellular targets, we synthesized fluorinated analogs of AGF117 and AGF150, designated AGF278 and AGF283. In the presence of a physiologic concentration of reduced folate, AGF278 and AGF283 potently inhibited proliferation of Chinese hamster ovary (CHO) cells engineered to express PCFT, but not RFC or FRá (R2/PCFT4) (IC50s of 6 and 1.5 nM, respectively), or FRá without PCFT or RFC (RT16) (IC50s of 4.8 and 5.4 nM). AGF278 and AGF283 also inhibited growth of IGROV1 (IC50s of 1.5 and 11 nM, respectively) and SKOV3 (IC50s of 37.8 and 30.2 nM, respectively) ovarian cancer cells. We measured [3H]methotrexate uptake in R2/PCFT4 CHO cells in the presence of AGF278 and AGF283; AGF278 and AGF283 potently inhibited PCFT transport with increased binding over AGF117 and AGF150, respectively, as reflected in Ki values. Analogous to AGF117 and AGF150, glycinamide ribonucleotide (GAR) formyltransferase (GARFTase), the first folate-dependent enzyme in de novo purine biosynthesis, was implicated as the cellular target for AGF278 and AGF283 as adenosine (10 ìM) and 5-aminoimidazole-4-carboxamide (320 ìM) protected IGROV1 cells from growth inhibition. GARFTase inhibition by AGF278 and AGF283 was confirmed by an in situ GARFTase assay which measures incorporation of [14C]glycine into formyl GAR, the GARFTase product, with potencies similar to those of the parent molecules. Preliminary in vivo studies with AGF278 using SKOV3 tumor xenografts in SCID mice established antitumor efficacy. Our results suggest that novel fluorinated 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates may offer significant promise for treating ovarian cancer, reflecting their selective cellular uptake by PCFT and FRá and potent inhibition of de novo purine biosynthesis. Additional utility of our fluorine-substituted analogs lies in their potential for positron emission tomography imaging of PCFT-expressing tumors. Citation Format: Mike R. Wilson, Manasa P. Ravindraa, Adrianne Wallace-Povirk, Lisa Polin, Zhanjun Hou, Aleem Gangjee, Larry H. Matherly. Targeting ovarian cancer with novel 6-substituted pyrrolo [2,3-d] pyrimidine fluorinated antifolates via cellular uptake by folate receptor á and the proton-coupled folate transporter. [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 4791.

Abstract 1359: Design, synthesis and evaluation of fluorinated antifolates for improved selectivity and potency against tumor cells

Abstract Folates are important single carbon transferring cofactors required for the biosynthesis of purine and pyrimidine nitrogen bases and DNA. Antifolates inhibit synthesis of these nitrogenous bases and function as antineopastic agents. The use of currently marketed antifolates such as methotrexate (MTX and pemetrexed (PMX) is limited by dose-limiting toxicity due to non-selective transport by the ubiquitously expressed reduced folate carrier (RFC). We previously reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine classical antifolates that are selectively transported by folate receptors (FR) and/or by the proton-coupled folate transporter (PCFT) over RFC and inhibit FR/PCFT expressing tumor cells (KB and IGROV1) at sub-nanomolar IC50 values. These antifolates are comparatively more potent and circumvent the important limitation of currently marketed clinical antifolates that lack selective uptake into tumors. As an extension of the SAR, we now report on the role of fluorine atom(s) in improving selectivity as well potency. Fluorine was incorporated into our 6-substituted pyrrolo[2,3-d]pyrimidine antifolates to improve pharmacodynamics by improving binding affinity to target proteins via a number of well documented electrostatic and hydrophobic interactions of fluorine. In addition, fluorines were strategically placed on the side chain of our targeted antifolates for improved potency and selectivity through induced conformational restriction due to a plausible intramolecular fluorine-hydrogen bonding interaction. The fluorinated analogs had IC50s in the subnanomolar range (IC50 = 0.33 to 0.29 nM) for KB tumor cells and nanomolar (IC50 = 2.77 to 3.47 nM) for IGROV-1 cells. In addition, these analogs showed 600- to 1000-fold selectivity for FRα expressing cells over RFC. Protection studies suggest that glycinamide ribonucleotide formyl transferase (GARFTase) is the intracellular targets. In vivo activity was established toward the SKOV3 tumor xenograft model. These preclinical studies suggest that these fluorinated analogs are candidates for possible clinical trial as antitumor agents. Citation Format: Aleem Gangjee, Manasa P. Ravindra, Mike R. Wilson, Adrianne W. Povrik, Lisa P. Hou, Carrie O’Connor, Zhanjun Hou, Larry H. Matherly. Design, synthesis and evaluation of fluorinated antifolates for improved selectivity and potency against tumor 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 1359.

Abstract 442: RASSF1A gene promoter methylation in primary tumors, adjacent morphologically normal tissues and plasma samples of patients with high grade serous ovarian cancer

Abstract Introduction: RASSF1A promoter methylation is frequent in high grade serous ovarian cancer (HGSC), the most common histological subtype. We examined RASSF1A promoter methylation in primary tumors, adjacent normal tissues and corresponding plasma samples of patients with HGSC, using real-time methylation specific PCR (real-time MSP) and methylation-sensitive high-resolution melting analysis (MS-HRMA). Materials and methods: A training group of 78 primary HGSC FFPEs was first analyzed using both real-time MSP and MS-HRMA. Our validation group consisted of 61 primary HGSC FFPEs, 58 adjacent tissues (analyzed using both real-time MSP and MS-HRMA), and 59 corresponding plasma samples (analyzed using real-time MSP). The specificity of both assays was evaluated by analyzing a group of 16 fallopian tube samples from healthy individuals. OVCAR29 and IGROV1 cell lines were used as positive controls. Results: In the training group, RASSF1A promoter methylation was detected in 33/78 (42.3%) by real-time MSP and in 33/78 (42.3%) by MS-HRMA (Agreement = 94.9%, P = 0.001, k = 0.895). The validation group results are shown in Table 1. According to the semi-quantitative MS-HRMA, RASSF1A promoter methylation was detected at significantly lower percentages in the adjacent morphologically normal tissues, compared to the paired primary tumors. In corresponding plasma samples, methylation was detected in 15/59 (25.4%) (Agreement with paired tumors = 60.7%, P = 0.259, k = 0.143). Both real-time MSP and MS-HRMA revealed no RASSF1A promoter methylation in the small group of normal fallopian tubes (n = 16). Conclusion: RASSF1A promoter is highly methylated in primary tumors and at lower percentages in the adjacent normal tissues. In all cases, MS-HRMA gave comparable results with real-time MSP. Evaluation of the clinical significance of RASSF1A promoter methylation in corresponding plasma requires further investigation. Table 1RASSF1A promoter methylationMethodPrimary tumor samples (validation group) N = 61Adjacent tissues N = 58Agreement (paired samples, N = 57)Real time MSP25/61 (41%)17/58 (29.3%)48/57(84.2%), P = 0.001, k = 0.652MS-HRMA28/61 (45.9%)21/58 (36.2%)51/57(89.5%), P = 0.001, k = 0.782Agreement (methods)58/61(95.1%), P = 0.001, Cohen's kappa = 0.90050/58(86.2%),P = 0.001, Cohen's kappa = 0.689 Citation Format: Lydia Giannopoulou, Issam Chebouti, Kitty Pavlakis, Sabine Kasimir-Bauer, Evi S. Lianidou. RASSF1A gene promoter methylation in primary tumors, adjacent morphologically normal tissues and plasma samples of patients with high grade serous 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 442.

Diolein Based Nanostructures as Targeted Theranostics.

Diolein based non-targeted theranostic nanoparticles (DO-NPs) containing 10%wt of the amphiphilic Gadolinium complex (C18)2DTPA(Gd), and targeted NPs, obtained by introducing growing amounts (3% wt, 6% wt or 10% wt) of (C18)2-Peg3000- FA in the sample composition, have been studied for their in vitro and in vivo properties. Cellular binding was studied by lCP-MS analysis of the Gadolinium content and by Surface Plasmon Resonance (SPR) assays. The best formulation in terms of selectivity towards IGROV-1 cells with respect to non-targeted DO-NPs, was that containing 3% (C18)2Peg3000- FA (P < 0.01). Cytotoxic studies and confocal microscopy analysis of IGROV-1 cells indicate high selective properties of the targeted doxorubicin (DOX) loaded NPs. Nanoparticles described here represent the first example in which a targeted carrier characterized by a stable foamy mesophase, provided by the Diolein component, combine the therapeutic effect due to the anticancer drug doxorubicin, with the imaging properties provided by paramagnetic gadolinium complexes for MRI. As evidenced by T(1w), and T(2w) MRI images and by the in vivo antitumor effect in IGROV-1 tumor-bearing mice, DO-NP3-FA/DOX provides very high therapeutic efficacy with a tumor growth regression of 80% and 50% higher as compared to the mice treated with saline solution and with Doxil, respectively.

Abstract A090: Exploring folate receptor α immunotherapy of breast carcinomas: Human monocytic cell-mediated killing triggered by IgG1 and IgE antibodies

Abstract Background: Folate receptor α is a GPI-anchored cell surface protein that mediates the intracellular transport of folates. It is over-expressed in a number of malignancies, including ovarian carcinoma and mesothelioma and has limited normal tissue expression. More recently it has been found to be over-expressed in breast cancer, and particularly a less favourable prognostic form, known as ‘triple-negative’ breast cancer (TNBC). We examined a therapeutic antibody candidate recognising FRα; which we are preparing for Phase 1 clinical trials for its potential to target human immune effector cells against breast cancer cells. As part of this effort, we endeavoured to identify a breast cancer cell line model in which the ability for this antibody to induce Antibody-dependent Cell-mediated breast tumour cell death could be evaluated in vitro. Methods: Twenty-two breast cancer cell lines were evaluated by flow cytometry for surface expression of FRα using the anti-FRα antibody compared with IGROV1, a well characterised ovarian cancer cell line used as a positive control. We confirmed these findings with mRNA levels for cell lines available from the publically accessible Cancer Cell Line Encyclopaedia (CCLE). The two highest expressing breast cancer cell lines, T47D and CAL51 were then studied in a functional flow cytometric assay which can simultaneously measure antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP). The human monocytic U937 cells which express Fc receptors for IgG and IgE antibodies, served as effectors. We compared the capacity of the the anti-FRα antibodies (IgG1 and IgE isotypes) to activate monocytic cells against the FRα-expressing ovarian carcinoma IGROV1 cells, the breast carcinoma T47D and CAL51 cells and the FRα non-expressing breast cancer cell line, HCC1428. Results: There was wide variety in the levels of cell surface expression of FRα; by breast cancer cell lines, expressed as Mean Fluorescent Intensity (&amp;gt;than an isotype control). The highest expressing breast cancer cell lines, T47D and CAL51 had values of 1273 (SEM 13.6) and 1638 (SEM 24.7) respectively in comparison with the non-expressing cell line, HCC1428 with a value of 8 (SEM 0.54). Expression levels in the ovarian cancer cell line, IGROV1 were 16580 (SEM 2534). These values correlated well with mRNA levels from the CCLE database (Spearman's rank, r=0.6011, p=0.0065) which varied from 1x104-1x1011 amongst breast cancer cell lines but were still below those detected with IGROV1 ovarian cancer cells with an mRNA level of 7x1012. We detected no clear evidence of expressing/non-expressing cell lines clustering amongst any known molecular subtype of TNBC. In vitro functional assay assessments of breast carcinoma cell killing in the non-FRα expressing cell line, HCC1428, revealed that neither IgG nor IgE induced breast cancer cell cytotoxicity above that induced by a non-targeting isotype antibody control. In contrast anti-FRα IgG1 and IgE antibodies could induce 20-30% more ADCP and ADCC, respectively, of the FRα+ IGROV1 tumour cells than the isotype control. The breast carcinoma T47D cells, likewise, were killed by ADCP when exposed to anti-FRα IgG, while the anti-FRα IgE antibody induced high levels of ADCC of ~40% above isotype control. Furthermore, treatment of breast carcinoma cells with anti-FRα antibodies induced direct signalling effects that may lead to reduced growth and survival cascades. Conclusions: We have identified two breast cancer cell line models which could be candidates for further in vitro and in vivo assessment of the efficacy and mechanisms of the novel anti-FRα; IgE against breast carcinomas. We have demonstrated similar levels of human immune effector cell-mediated breast cancer cell killing in one of these cell lines, T47D, to a well characterised ovarian cancer cell line, IGROV1, not seen in the FRα; non-expressing cell line, HCC1428. This tumour antigen warrants further characterisation as a potential new immune therapy in TNBC, where immune infiltration is already known to be favourably prognostic. Citation Format: Matthew W. Fittall, Anthony Cheung, Gyula M. Petranyi, Diana Rodriguez-Dominguez, Heather J. Bax, Panagiotis Karagiannis, Kristina M. Ilieva, Andrew Tutt, Sophia N. Karagiannis. Exploring folate receptor α immunotherapy of breast carcinomas: Human monocytic cell-mediated killing triggered by IgG1 and IgE antibodies. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A090.

6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers as Targeted Antifolates for Folate Receptor α and the Proton-Coupled Folate Transporter in Human Tumors.

2-Amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine antifolate thiophene regioisomers of AGF94 (4) with a thienoyl side chain and three-carbon bridge lengths [AGF150 (5) and AGF154 (7)] were synthesized as potential antitumor agents. These analogues inhibited proliferation of Chinese hamster ovary (CHO) sublines expressing folate receptors (FRs) α or β (IC50s < 1 nM) or the proton-coupled folate transporter (PCFT) (IC50 < 7 nM). Compounds 5 and 7 inhibited KB, IGROV1, and SKOV3 human tumor cells at subnanomolar concentrations, reflecting both FRα and PCFT uptake. AGF152 (6) and AGF163 (8), 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine thiophene regioisomers, also inhibited growth of FR-expressing CHO and KB cells. All four analogues inhibited glycinamide ribonucleotide formyltransferase (GARFTase). Crystal structures of human GARFTase complexed with 5 and 7 were reported. In severe combined immunodeficient mice bearing SKOV3 tumors, 7 was efficacious. The selectivity of these compounds for PCFT and for FRα and β over the ubiquitously expressed reduced folate carrier is a paradigm for selective tumor targeting.

Abstract POSTER-BIOL-1309: Beta-catenin regulated ALDH1A1 is a target in ovarian cancer stem cells

Abstract Spheroids are 3D multi-cell aggregates formed in non-adherent culture conditions. In ovarian cancer (OC), they serve as a vehicle for cancer cell dissemination in the peritoneal cavity and protect cells from extracellular stress-induced anoikis. We hypothesized that spheroids are enriched in cancer stem cells and investigated pathways activated under these conditions. Affymetrix-based gene expression profiling of OC cells grown as spheroids vs. monolayers identified β-catenin pathway being upregulated in 3D cultures. Increased expression of β-catenin and target genes (cyclin D1, c-myc) was demonstrated in spheroids vs. monolayers and in successive spheroid generations using OC cell lines and primary cultures. B-catenin function measured as TCF/LEF1 reporter activity was augmented in spheroids vs. monolayers and siRNA mediated β-catenin knock down decreased the number of spheroids (p&amp;lt;0.001). Along with β-catenin, the expression level of ALDH1A1, an OC stem cell marker, was increased in successive spheroid generations. The percentage of ALDH+ cells was significantly higher in spheroids vs. monolayers in IGROV1, A2780, SKOV3, and primary OC cells. B-catenin knock-down decreased ALDH1A1 expression, suggesting that it is a β-catenin target. The percentage of ALDH+ cells and the number of spheroids formed were increased in the cell population dissociated from OC xenografts treated with carboplatin compared to control (44% vs. 2.3%), supporting that ALDH1A1 is a stem cell marker. A37 is a novel ALDH1A1 enzymatic inhibitor (Ki of 5nM). A37 decreased cell viability and spheroid formation by IGROV1 cells (p&amp;lt;0.001). A37 also decreased the percentage of ALDH+ cells under 3D culture conditions. These data support the role of β-catenin regulated ALDH1A1 in the maintenance of OC spheroids and of a stem cell phenotype and propose new ALDH1A1 inhibitors targeting this cell population. (Funding: US Department of Veterans Affairs and Ovarian Cancer Research Fund) Citation Format: Salvatore Condello, Sarbajeet Nagdas, Cynthia Morgan, John Turek, Thomas Hurley, Daniela Matei. Beta-catenin regulated ALDH1A1 is a target in ovarian cancer stem cells [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-BIOL-1309.

Abstract POSTER-BIOL-1352: Notch3 promotes anoikis resistance in ovarian cancer

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer. Recently, a number of genome-wide studies in ovarian cancer patient samples have postulated that the course of disease can influenced by the Notch3 signaling pathway. In one study, 11% of human tumor samples showed mutation or amplification of the Notch3 receptor alone. Subsequent papers have correlated Notch3 overexpression in human samples with shorter progression-free survival as well as overall survival. In vitro studies have shown Notch3 to be involved in apoptosis resistance and chemoresistance, as well as cell cycle alterations. Together, these human studies have implicated Notch3 expression and amplification with decreased outcomes in ovarian cancer patients. To test the potential mechanism by which Notch3 over-expression promotes ovarian cancer, we examined several molecular and cellular functions of Notch3 in ovarian cancer cells. Decreasing the levels of Notch3 in ovarian cancer cells increases the susceptibility of these cells to apoptosis, which is consistent with previous studies. Using an effective siRNA-mediated knockdown of Notch3 combined with transfer to ultra-low attachment plates revealed a significant decrease in the ability of Notch3-knockdown IGROV-1 cells to remain viable in anchorage-independent conditions over 24 hours. Our data show that not only does Notch3 prevent apoptosis, but specifically promotes anoikis resistance. Further examining the mechanism underlying this novel finding revealed the positive roles of extracellular matrix proteins and FAK in promoting the anchorage-independent growth of IGROV-1 cells. Strikingly, mRNA and protein levels of col4α2 are reduced when Notch3 levels are decreased and exogenous collagen IV supplementation can largely reverse the anoikis sensitivity. Reduction of col4α2 expression by siRNA induces cell death on its own and high Notch3 expression levels correlate with higher col4α2 expression in human ovarian tumor samples. Together, these studies have revealed that Notch3 promotes anoikis resistance through col4α2 expression and may partly explain its positive correlation with poor survival in ovarian cancer patients. Citation Format: Caitlin W. Brown, Alexander S. Brodsky, Richard N. Freiman. Notch3 promotes anoikis resistance in ovarian cancer [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-BIOL-1352.

Abstract 4483: Impact of loss of folate receptor alpha on antitumor effects of novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with substrate activities for both folate receptors and the proton-coupled folate transporter

Abstract Major folate uptake mechanisms include the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT), and folate receptors α and β. Antifolates such as methotrexate or pemetrexed are excellent substrates for human RFC (hRFC) and variously for human PCFT (hPCFT) and human folate receptor α (hFRα). Recent attention has focused on developing tumor-targeted folate-based therapies, reflecting tumor-selective uptake by hFRα and/or hPCFT. Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers AGF94 (2′,5′) and AGF154 (2′,4′) were synthesized and characterized as potent inhibitors of cell proliferation of engineered Chinese hamster ovary (CHO) cells expressing hPCFT or hFRα, but not for CHO cells expressing hRFC. In 41 ovarian cancer specimens from patients, hFRα, hPCFT and hRFC were all expressed. FRα transcript levels increased with stage of disease, although hPCFT levels were constant. IGROV1 is a clinically relevant ovarian cancer cell line that expresses both hFRα and hPCFT. IGROV1 cells were highly sensitive to both AGF94 and AGF154, as reflected in inhibition of cell proliferation in vitro. Growth inhibition by AGF94 or AGF154 in IGROV1 cells was partially reversed by excess folic acid, likely reflecting drug uptake by hFRα, as well as by hPCFT. To evaluate the impact of loss of hFRα on AGF94 and AGF154 in vitro inhibition of IGROV1 cell proliferation, FRα was knocked down (&amp;gt;95%) by lentivirus shRNAs. Clonal isolates were generated (n = 17) and profiled by RT-PCR for expression of the folate transporters and key enzymes likely to impact antifolate sensitivities (e.g., folylpolyglutamyl synthetase, β-glycinamide ribonucleotide formyltransferase), relative to a non-targeted control (NTC) cells. Gene expression profiles were similar for IGROV1 hFRα knockdown (KD) clones and NTC cells. Two representative hFRα KD clones were assayed for inhibition of cell proliferation by AGF94 and AGF154, with IC50s for IGROV1 KD clones of 26±1.8 and 26±9 nM for AGF94, and 65±1.2 and 81±2.4 nM for AGF154. For comparison, IC50s in the IGROV1 NTC cells expressing both hFRα and hPCFT were 0.6±0.1 nM for AGF94 and 2.2±0.8 nM for AGF154. In contrast, the hFRα-specific antifolate, AGF102 showed IC50s of &amp;gt; 1000 nM and ∼2.5 nM for KD and NTC cells. The preservation of substantial inhibitory activity for AGF94 and AGF154, accompanying the nearly complete loss of FRα expression in the KD clones is likely due to the sustained uptake of these novel antifolates by hPCFT. For AGF154, this was confirmed by direct uptake assays with [3H]-AGF154. Our results suggest that 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with substantial substrate activities for hFRα and hPCFT may offer significant advantages in treating tumors that express both these transport systems. Citation Format: Zhanjun Hou, Steve Orr, Christina George, Adrianne Wallace, Larry H. Matherly, Lei Wang, Si Yang, Aleem Gandjee. Impact of loss of folate receptor alpha on antitumor effects of novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with substrate activities for both folate receptors and the proton-coupled folate transporter. [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 4483. doi:10.1158/1538-7445.AM2015-4483

Abstract 3817: Suicidal autophagy induced by immunotherapy targeting folate receptor in ovarian cancer

Abstract Objective Cancer cells depend on folate metabolism for DNA replication, making folate receptor alpha (FRα) an important therapeutic target. Blocking FRα with a humanized anti-FRα monoclonal antibody, MORAB-003 offers a potent clinical opportunity; however, the mechanisms underlying its efficacy were not well understood. We investigated the functional mechanisms underlying MORAB-003 induced tumor inhibition in ovarian cancer. Methods Using FACS analysis and real-time PCR, FRα expression was determined in an array of human ovarian cancer cells. Next, we examined the effects of MORAB-003 monotherapy and combination with docetaxel in tumor growth using the xenograft mouse models. In vitro, we analyzed ovarian cancer cells with 3-dimensional (3-D) spheroid-like architecture. We next performed systems-based analysis including reverse-phase protein and genomic microarrays, and discovered an array of factors/genes involved in autophagy was upregulated by MORAB-003. Functional assays including FACS with AO staining, tandem mRFP/mGFP-LC3 fluorescence microscopy, transmission electron microscopy (TEM) and siRNAs knockdown were performed to further investigate MORAB-003-induced autophagy in tumor inhibition. Results IGROV1 and SKOV3ip1 cells express high-level FRα and A2780 cells express low-level FRα. In vivo, MORAB-003 monotherapy or a combination of MORAB-003 and docetaxel led to significantly decreased tumor growth in IGROV1 and SKOV3ip1 models, but not in A2780 model. Immunohistochemistry analysis on tumor sections indicated that MORAB-003 significantly reduced proliferative cancer cells, but had minimum effects on tumor cell apoptosis or angiogenesis. Network analysis on protein and gene expression profiles in IGROV1 and SKOV3ip1 tumors revealed that MORAB-003 significantly upregulated a number of factors/genes involved in autophagy, including LC3II, ATG3, ATG4B, S116-PEA-15 and BECN1. Furthermore, S116-PEA-15 and Beclin-1 were required for caspase-3-activated cell death in 3-D IGROV1 and SKOV3ip1 spheroids treated with MORAB-003. Results from autophagic flux assays indicated that MORAB-003 treatment led to an abundant accumulation of late-stage autophagic vacuoles. Blocking the autophagic vacuolization or fusion using bafilomycin A1 or hydroxychloroquine reversed the MORAB-003 induced reduction of cell viability in 3-D IGROV1 and SKOV3ip1 spheroids. Furthermore, queries from the Cancer Genome Atlas data (TCGA) revealed that copy-number gains of the FOLR1 gene significantly correlated with shorter disease-free survival in patients with the immunoreactive subtype of ovarian serous carcinoma and uterine carcinoma. Conclusion MORAB-003 inhibits tumor growth via a non-canonical autophagy associated with programmed cell death. This finding has substantial implications for clinical development of anti-FRα therapies, especially with regard to novel combinations and predictive biomarkers. Citation Format: Yun-Fei Wen, Anil K. Sood. Suicidal autophagy induced by immunotherapy targeting folate receptor in 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 3817. doi:10.1158/1538-7445.AM2015-3817

Abstract 2428: Translational study of F14512, a novel vectorised epipodophyllotoxin, which demonstrates a marked activity on ovarian cancer models from patients

Abstract Epithelial ovarian cancer is the fourth cause of death among cancer-bearing women, frequently associated with carboplatin resistance, underlining the need of more efficient and targeted therapies. F14512 is a novel concept of drug vectorization that we propose here to investigate in ovarian cancer models. F14512 is an epipodophyllotoxin-core linked to a spermine chain, which enters selectively tumor cells via the polyamine transport system (PTS), currently in clinical phase II evaluation in AML. We compared the effects of F14512 versus etoposide against OVCAR-3, IGROV-1, SKOV-3, A2780S and A2780R cancer cell lines. Using the F17073 PTS fluorescent probe, we determined the PTS activity of these cells and of 18 clinical samples. As results, F14512 displayed strong anti-proliferative and pro-apoptotic activities in carboplatin-resistant models, being increased in cell lines with high levels of PTS. Consistently, at a dose of 1.25 mg/kg, F14512 significantly inhibited tumor growth in cisplatin-resistant SKOV-3 xenograft model. Interestingly, ex vivo analysis indicated that 15 patients sample out of 18 presented a higher F17073 fluorescent probe incorporation into CD326-positive tumor cells as compared to normal cells, even in the case of 2 platinum-refractory patients. Therefore, F14512 is a targeted drug with a potent anti-tumor efficacy regardless the status of cisplatin resistance. The high PTS activity detected in fresh clinical samples highlights the potential of F14512 as a new therapy for PTS-positive and platinum resistant ovarian cancer patients. Citation Format: Benoît Thibault, Gregoire Zorza, Samuel Meignan, Nicolas Guilbaud, Christian Bailly, Jean-Pierre Delord, Bettina Couderc, Anna Kruczynski, Pierre Ferre, Jean-Philippe Annereau. Translational study of F14512, a novel vectorised epipodophyllotoxin, which demonstrates a marked activity on ovarian cancer models from patients. [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 2428. doi:10.1158/1538-7445.AM2015-2428

Glucocorticoid receptor activation inhibits chemotherapy-induced cell death in high-grade serous ovarian carcinoma

ObjectivesTo test the hypothesis that glucocorticoid receptor (GR) activation increases resistance to chemotherapy in high-grade serous ovarian cancer (HGS-OvCa) and that treatment with a GR antagonist will improve sensitivity to chemotherapy. MethodsGR expression was assessed in OvCa cell lines by qRT-PCR and Western blot analysis and in xenografts and primary human tumors using immunohistochemistry (IHC). We also examined the effect of GR activation versus inhibition on chemotherapy-induced cytotoxicity in OvCa cell lines and in a xenograft model. ResultsWith the exception of IGROV-1 cells, all OvCa cell lines tested had detectable GR expression by Western blot and qRT-PCR analysis. Twenty-five out of the 27 human primary HGS-OvCas examined expressed GR by IHC. No cell line expressed detectable progesterone receptor (PR) or androgen receptor (AR) by Western blot analysis. In vitro assays showed that in GR-positive HeyA8 and SKOV3 cells, dexamethasone (100nM) treatment upregulated the pro-survival genes SGK1 and MKP1/DUSP1 and inhibited carboplatin/gemcitabine-induced cell death. Concurrent treatment with two GR antagonists, either mifepristone (100nM) or CORT125134 (100nM), partially reversed these effects. There was no anti-apoptotic effect of dexamethasone on chemotherapy-induced cell death in IGROV-1 cells, which did not have detectable GR protein. Mifepristone treatment alone was not cytotoxic in any cell line. HeyA8 OvCa xenograft studies demonstrated that adding mifepristone to carboplatin/gemcitabine increased tumor shrinkage by 48% compared to carboplatin/gemcitabine treatment alone (P=0.0004). ConclusionsThese results suggest that GR antagonism sensitizes GR+ OvCa to chemotherapy-induced cell death through inhibition of GR-mediated cell survival pathways.

Folic acid-conjugated europium complexes as luminescent probes for selective targeting of cancer cells.

We report the synthesis of three optical probes (Eu(3+)⊂1, Eu(3+)⊂2, and Eu(3+)⊂3) having a luminescent Eu complex (signaling unit) bonded in different positions to folic acid (FA), the folate receptor (FR) targeting unit. The structures of the two regioisomers Eu(3+)⊂1 and Eu(3+)⊂2 were assigned by mass spectrometric experiments. The optical properties and stability of these probes were assessed in phosphate-buffered saline, cell culture medium, rat serum, and cellular lysate, and results indicated that they are chemically and photophysically stable. Cytotoxicity was studied with ovarian cancer cells having high (SKOV-3), intermediate (OVCAR-3), low (IGROV-1), or null (A2780) expression of FRs. The internalized probe, evaluated in SKOV-3, IGROV-1, and A2780 cells, was in the order Eu(3+)⊂2 > Eu(3+)⊂1 > Eu(3+)⊂3. No internalization was observed for A2780 cells. Such results, together with those obtained in competition experiments of FA versus Eu(3+)⊂2 and FA or Eu(3+)⊂2 versus (3)H-FA, indicate that internalization is receptor-mediated and that Eu(3+)⊂2 shows high selectivity and specificity for FR.

Lysophosphatidic acid (LPA) stimulates invasion and metastatic colonization of ovarian cancer cells through Rac activation

To investigate the mechanisms of lysophosphatidic acid (LPA) in stimulating invasion and metastatic colonization of ovarian cancer cells. The metastatic ability in vivo of ovarian cancer SK-OV3, HEY, OVCAR3, and IGROV1 cells was determined in tumor-bearing nude mouse models. Matrigel assay was used to detect the changes of response in vitro of ovarian cancer cells to LPA after Rac(-) or Rac(+) adenovirus treatment. LPA-induced Rho GTPase activation was detected by GST-fusion protein binding assay. The peritoneal metastatic colonization assay showed overt metastatic colonization in mice receiving SK-OV3 and HEY cell inoculation, indicating that they are invasive cells. Metastatic colonization was not detected in animals receiving OVCAR3 and IGROV1 cells, indicating that these cells are non-invasive cells. In the matrigel invasion assay, exposure to LPA led to a notably greater migratory response in metastatic SK-OV3 and HEY cells (Optical density: SK-OV3 cells: 0.594±0.023 vs. 1.697±0.049, P<0.01; HEY cells: 0.804±0.070 vs. 1.851±0.095, P<0.01). But LPA did little in the non-metastatic OVCAR3 and IGROV1 cells (Optical density A: OVCAR3 cells: 0.336±0.017 vs. 0.374±0.007, P>0.05; IGROV1 cells: 0.491±0.036 vs. 0.479±0.061, P>0.05). LPA migratory responses of ovarian cancer cells were closely related to their metastatic colonization capabilities (r = 0.983, P<0.05). Rac(-) blocked the LPA response of invasive SK-OV3 and HEY cells (LPA-induced fold increase of cell migration: SK-OV3 cells: 2.988±0.095 vs. 0.997±0.100,P=0.01; HEY cells: 2.404±0.059 vs. 0.901±0.072, P=0.01). But Rac(+) confered the non-invasive cells with LPA response and invasion capability (LPA-induced fold increase of cell migration: OVCAR3 cells: 1.072±0.080 vs. 1.898±0.078, P<0.01; IGROV1 cells: 1.002±0.044 vs. 2.141±0.057, P<0.05). Among Rho GTPases, only Rac activation was different between ovarian cancer cell lines with different metastatic capability after LPA stimulation: Cdc42 could not be activated in both the invasive and non-invasive cell lines. RhoA could be activated in both the invasive and non-invasive cell lines. Rac could be activated by LPA in the invasive ovarian cancer cell lines. However, Rac could not be activated in the non-invasive cell lines. Lysophosphatidic acid stimulates invasion and metastasis of ovarian cancer cells through Rac activation.

High LIN28A Expressing Ovarian Cancer Cells Secrete Exosomes That Induce Invasion and Migration in HEK293 Cells.

Epithelial ovarian cancer is the most aggressive and deadly form of ovarian cancer and is the most lethal gynecological malignancy worldwide; therefore, efforts to elucidate the molecular factors that lead to epithelial ovarian cancer are essential to better understand this disease. Recent studies reveal that tumor cells release cell-secreted vesicles called exosomes and these exosomes can transfer RNAs and miRNAs to distant sites, leading to cell transformation and tumor development. The RNA-binding protein LIN28 is a known marker of stem cells and when expressed in cancer, it is associated with poor tumor outcome. We hypothesized that high LIN28 expressing ovarian cancer cells secrete exosomes that can be taken up by nontumor cells and cause changes in gene expression and cell behavior associated with tumor development. IGROV1 cells were found to contain high LIN28A and secrete exosomes that were taken up by HEK293 cells. Moreover, exposure to these IGROV1 secreted exosomes led to significant increases in genes involved in Epithelial-to-Mesenchymal Transition (EMT), induced HEK293 cell invasion and migration. These changes were not observed with exosomes secreted by OV420 cells, which contain no detectable amounts of LIN28A or LIN28B. No evidence was found of LIN28A transfer from IGROV1 exosomes to HEK293 cells.

SILAC-based quantitative proteomics identified lysosome as a fast response target to PDT agent Gd-N induced oxidative stress in human ovarian cancer IGROV1 cells.

Biological systems have developed an intact network and strategies in response to various environmental pressures such as irradiation, viral invasion and oxidative stress. Therefore, elucidation of the cellular response mechanism toward oxidative stress can contribute to the knowledge of redox regulation. By using a newly developed gadolinium based photodynamic therapy (PDT) agent Gd-N and SILAC quantified proteomic analysis, we observed 485 proteins dysregulated in expression, 106 in phosphorylation and 1050 in oxidation. Interestingly, lysosome was discovered as the main organelle affected by Gd-N induced singlet oxygen, along with the down regulation of a majority of lysosomal acid hydrolases and proton pump complex ATP6V/TCIRG1. Besides, phosphorylation sites with sequence patterns "TP" or "SP" were enriched in dysregulated phosphoproteins. Protein oxidation also shows sequence patterns in target proteins with "M.D" or "KM" taking methionine as the central residue. Oxidized proteins were most enriched in the pathways of Parkinson's disease, an oxidative stress closely related neurodegenerative disease. In conclusion, our study reveals new insights into the cellular mechanism to oxidative stress and may contribute to the discovery of new targets and development of novel PDT agents.

Anionic glycolipids related to glucuronosyldiacylglycerol inhibit protein kinase Akt.

New glucuronosyldiacylglycerol (GlcADG) analogues based on a 2-O-β-D-glucopyranosyl-sn-glycerol scaffold and carrying one or two acyl chains of different lengths have been synthesized as phosphatidylinositol 3-phosphate (PI3P) mimics targeting the protein kinase Akt. The Akt inhibitory effect of the prepared compounds was assayed using an in vitro kinase assay. The antiproliferative activity of the compounds was tested in the human ovarian carcinoma IGROV-1 cell line in which we found that two of them could inhibit proliferation, in keeping with the target inhibitory effect.

Antioxidant, 5-lipoxygenase inhibitory and cytotoxic activities of compounds isolated from the Ferula lutea flowers.

A phytochemical investigation of the Ferula lutea (Poir.) Maire flowers has led to the isolation of a new compound, (E)-5-ethylidenefuran-2(5H)-one-5-O-β-d-glucopyranoside (1), designated ferunide, 4-hydroxy-3-methylbut-2-enoic acid (2), reported for the first time as a natural product, together with nine known compounds, verbenone-5-O-β-d-glucopyranoside (3), 5-O-caffeoylquinic acid (4), methyl caffeate (5), methyl 3,5-O-dicaffeoylquinate (6), 3,5-O-dicaffeoylquinic acid (7), isorhamnetin-3-O-α-l-rhamnopyranosyl(1→6)-β-d-glucopyranoside, narcissin (8), (−)-marmesin (9), isoimperatorin (10) and 2,3,6-trimethylbenzaldehyde (11). Compounds 3–10 were identified for the first time in Ferula genus. Their structures were elucidated by spectroscopic methods, including 1D and 2D NMR experiments, mass spectroscopy and X-ray diffraction analysis (compound 2), as well as by comparison with literature data. The antioxidant, anti-inflammatory and cytotoxic activities of isolated compounds were evaluated. Results showed that compound 7 exhibited the highest antioxidant activity with IC50 values of 18 ± 0.5 µmol/L and 19.7 ± 0.7 µmol/L by DPPH radical and ABTS radical cation, respectively. The compound 6 exhibited the highest anti-inflammatory activity with an IC50 value of 5.3 ± 0.1 µmol/L against 5-lipoxygenase. In addition, compound 5 was found to be the most cytotoxic, with IC50 values of 22.5 ± 2.4 µmol/L, 17.8 ± 1.1 µmol/L and 25 ± 1.1 µmol/L against the HCT-116, IGROV-1 and OVCAR-3 cell lines, respectively.

Abstract 2278: The SMAC-mimetic birinapant regulates autocrine TNF production by caspase-8:RIPK1 complex via p38MAPK pathway

Abstract Birinapant (TL32711), a SMAC-mimetic currently in clinical trials, antagonizes multiple members of the inhibitor of apoptosis (IAP) protein family to promote apoptosis in cancer cells through the formation of a caspase-8:RIPK1 complex and induction of autocrine tumor necrosis factor (TNF). Evidently, birinapant-resistant cells do not produce TNF upon drug treatment. However, the mechanism by which birinapant-sensitive cells produce TNF is not clear. Owing to the fact that mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes, we explored the role of MAPKs in birinapant-induced TNF production. Western blot analyses on a panel of birinapant-sensitive (SK-OV-3, EVSA-T, TOV-21G: all with an IC50 &amp;lt; 200nM) and birinapant-resistant (IGROV-1, HCT116, WIDR &amp; SK-MEL-28: all with an IC50 &amp;gt; 10μM) cell lines showed that birinapant induced rapid and robust phosphorylation of p38MAPK in sensitive cells, which corresponded to increased TNF production as assessed by ELISA. However, resistant cells were not affected at multiple time points. The p38MAPK inhibitor, LY2228820, reduced the levels of birinapant-induced TNF (2-fold with 5nM birinapant after 24h) in birinapant-sensitive cancer cells (SK-OV-3), whereas inhibitors of Erk1/2 (PD98059) and JNK (SP600125) had no effect on TNF production. Interestingly, RNAi-mediated inhibition of p38α (MAPK14) isoform significantly inhibited birinapant-induced TNF production (∼3-fold with 5nM birinapant after 24h) in SK-OV-3 cells while inhibition of p38β (MAPK11) isoform upregulated TNF production (∼3-fold), suggesting that there may be a balance between p38MAPK isoforms to regulate TNF production. Pharmacological and RNAi-mediated inhibition of RIPK1 and caspase-8 activities in SK-OV-3 cells resulted in reduced birinapant-induced p38MAPK phosphorylation and TNF production. Furthermore, commercially available pooled siRNAs targeting different isoforms of cFLIP, a catalytically-inactive homologue of caspase-8, upregulated p38MAPK phosphorylation (∼4-fold) and TNF production (∼8 to 26-fold) in birinapant-resistant cancer cell lines (T24, HT1376 &amp; IGROV-1), and conferred differential sensitivity to the drug treatment (T24-IC50: 7nM; HT1376-IC50: &amp;gt;10nM; IGROV-1-IC50: &amp;gt;1μM). Experiments involving cFLIP long (cFLIP-L) and short (cFLIP-S) isoform-specific siRNAs showed that inhibition of cFLIP-L, but not cFLIP-S, in T24 &amp; HT1376 cells induced p38MAPK phosphorylation and TNF production, and conferred sensitivity to birinapant. However, the combined inhibition of cFLIP-L and cFLIP-S in IGROV-1 cells was required to induce p38MAPK phosphorylation, TNF production and apoptosis. Collectively, these observations indicate that birinapant-induced autocrine TNF production is triggered by the activation of the caspase-8/RIPK1/p38MAPK axis, and that cFLIP isoforms may confer a context-dependent negative regulatory effect. Citation Format: Gurpreet Singh Kapoor, Christopher A. Benetatos, Yasuhiro Mitsuuchi, Eric M. Neiman, Guangyao Yu, Mark A. Mckinlay, Jennifer Burns, John Silke, Stephen M. Condon, Srinivas K. Chunduru. The SMAC-mimetic birinapant regulates autocrine TNF production by caspase-8:RIPK1 complex via p38MAPK pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2278. doi:10.1158/1538-7445.AM2014-2278

MicroRNA-370 suppresses proliferation and promotes endometrioid ovarian cancer chemosensitivity to cDDP by negatively regulating ENG

MicroRNAs (miRNAs) are a class of non-coding RNAs that post-transcriptionally inhibit gene expression. In this study, we discovered that microRNA-370 (miR-370) was down-regulated in endometrioid ovarian cancer cells. In IGROV1 and TOV112D endometrioid ovarian cancer cells, miR-370 suppressed cellular viability and colony formation. miR-370 also enhanced endometrioid ovarian cancer cell chemosensitivity to cDDP. Endoglin (ENG) was directly and negatively regulated by miR-370. In addition, hypermethylation was a potential mechanism of miR-370 epigenetic silencing. We conclude that miR-370 acts as a tumor suppressor in endometrioid ovarian cancer via ENG regulation.