Platinum-resistant Cells Research Articles

A metabolomic approach to identifying platinum resistance in ovarian cancer.

BackgroundAcquisition of metabolic alterations has been shown to be essential for the unremitting growth of cancer, yet the relation of such alterations to chemosensitivity has not been investigated. In the present study our aim was to identify the metabolic alterations that are specifically associated with platinum resistance in ovarian cancer. A global metabolic analysis of the A2780 platinum-sensitive and its platinum-resistant derivative C200 ovarian cancer cell line was performed utilizing ultra-high performance liquid chromatography/mass spectroscopy and gas chromatography/mass spectroscopy. Per-metabolite comparisons were made between cell lines and an interpretive analysis was carried out using the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic library and the Ingenuity exogenous molecule library.ResultsWe observed 288 identified metabolites, of which 179 were found to be significantly different (t-test p < 0.05) between A2780 and C200 cells. Of these, 70 had increased and 109 had decreased levels in platinum resistant C200 cells. The top altered KEGG pathways based on number or impact of alterations involved the cysteine and methionine metabolism. An Ingenuity Pathway Analysis also revealed that the methionine degradation super-pathway and cysteine biosynthesis are the top two canonical pathways affected. The highest scoring network of altered metabolites was related to carbohydrate metabolism, energy production, and small molecule biochemistry. Compilation of KEGG analysis and the common network molecules revealed methionine and associated pathways of glutathione synthesis and polyamine biosynthesis to be most significantly altered.ConclusionOur findings disclose that the chemoresistant C200 ovarian cancer cells have distinct metabolic alterations that may contribute to its platinum resistance. This distinct metabolic profile of platinum resistance is a first step towards biomarker development for the detection of chemoresistant disease and metabolism-based drug targets specific for chemoresistant tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13048-015-0140-8) contains supplementary material, which is available to authorized users.

The role of interleukin-8 (IL-8) and IL-8 receptors in platinum response in high grade serous ovarian carcinoma.

Platinum based drugs are the cornerstone of chemotherapy for ovarian cancer, however the development of chemoresistance hinders its success. IL-8 is involved in regulating several pro-survival pathways in cancer. We studied the expression of IL-8 and IL-8 receptors in platinum sensitive and resistant cell lines. Using qRT-PCR and immunohistochemistry, both platinum sensitive (PEA1, PEO14) and resistant (PEA2, PEO23) show increased expression of IL-8 and IL-8 receptors. IL-8RA shows nuclear and cytoplasmic expression, whilst IL-8RB is present solely in the cytoplasm. Knockdown of IL-8 increased sensitivity to cisplatin in platinum sensitive and reversed platinum resistance in resistant cell lines, decreased the expression of anti-apoptotic Bcl-2 and decreased inhibitory phosphorylation of pro-apoptotic Bad. IL-8 receptor antagonist treatment also enhanced platinum sensitivity. Nuclear localisation of IL-8RA was only detected in platinum resistant tumours. Inhibition of IL-8 signalling can enhance response in platinum sensitive and resistant disease. Nuclear IL-8RA may have potential as a biomarker of resistant disease.

Phase I study of azacitidine and oxaliplatin in patients with advanced cancers that have relapsed or are refractory to any platinum therapy.

BackgroundDemethylation process is necessary for the expression of various factors involved in chemotherapy cytotoxicity or resistance. Platinum-resistant cells may have reduced expression of the copper/platinum transporter CTR1. We hypothesized that azacitidine and oxaliplatin combination therapy may restore platinum sensitivity. We treated patients with cancer relapsed/refractory to any platinum compounds (3 + 3 study design) with azacitidine (20 to 50 mg/m2/day intravenously (IV) over 15 to 30 min, D1 to 5) and oxaliplatin (15 to 30 mg/m2/day, IV over 2 h, D2 to 5) (maximum, six cycles). Platinum content, LINE1 methylation (surrogate of global DNA methylation), and CTR1 expression changes (pre- vs. post-treatment) were assessed. Drug pharmacokinetics were analyzed.ResultsThirty-seven patients were treated. No dose-limiting toxicity (DLT) was noted at the maximum dose. The most common adverse events were anemia and fatigue. Two (5.4%) patients had stable disease and completed six cycles of therapy. Oxaliplatin (D2) and azacitidine (D1 and 5) mean systemic exposure based on plasma AUCall showed dose-dependent interaction whereby increasing the dose of oxaliplatin reduced the mean azacitidine exposure and vice versa; however, no significant differences in other non-compartmental modeled parameters were observed. Blood samples showed universal reduction in global DNA methylation. In tumor samples, hypomethylation was only observed in four out of seven patients. No correlation between blood and tumor demethylation was seen. The mean cytoplasmic CTR1 score decreased. The pre-dose tumor oxaliplatin levels ranged from <0.25 to 5.8 μg/g tumor. The platinum concentration increased 3- to 18-fold. No correlation was found between CTR1 score and oxaliplatin level, which was found to have a trend toward correlation with progression-free survival.ConclusionsOxaliplatin and azacitidine combination therapy was safe. CTR1 expression was not correlated with methylation status or tissue platinum concentration.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0065-5) contains supplementary material, which is available to authorized users.

Baicalein upregulates DDIT4 expression which mediates mTOR inhibition and growth inhibition in cancer cells

Baicalein is a natural flavone that exhibits anticancer properties. Using microarrays we found that DDIT4 was the highest transcript induced by baicalein in cancer cells. We confirmed in multiple cancer cell lines large, dose-related expression of DDIT4 by quantitative RT-PCR and immunoblot, which correlates with growth inhibition. Time course experiments demonstrate that DDIT4 is rapidly inducible, with high expression maintained for several days in vitro. Induction of DDIT4 expression is p53 independent based on evaluation of p53 knockout cells. Since DDIT4 is known to inhibit mTORC1 activity we confirmed that baicalein suppresses phosphorylation of mTORC1 targets. Using RNA interference we demonstrate that mTORC1 activity and growth inhibition by baicalein is attenuated by knockdown of DDIT4. We furthermore demonstrate suppression of established tumors by baicalein in a mouse model of breast cancer with increased DDIT4 expression in the tumors. Finally, we demonstrate that baicalein upregulates DDIT4 and causes mTORC1 and growth inhibition in platinum resistant cancer cells in marked contrast to platinum chemotherapy treatment. These studies demonstrate that baicalein inhibits mTORC1 through DDIT4 expression, and may be useful in cancer chemotherapy and chemoprevention.

Abstract 2317: SGI-110 alters ovarian cancer stem cells to prevent recurrent and chemoresistant ovarian cancer

Abstract Ovarian cancer stem cells (OCSCs) are associated with drug resistance and tumor relapse. Epigenetic aberrations, especially DNA methylation, result in silencing of tumor suppressor and differentiation-associated genes and regulate OCSCs' survival. To test the hypothesis that DNA hypomethylating agents can “reset” OCSCs towards differentiation, we investigated the effect of the DNA methytransferase inhibitor SGI-110 on OCSCs, defined by aldehyde dehydrogenase 1 (ALDH)(+) cells. We treated ALDH(+) cells from platinum-sensitive A2780 and platinum-resistant A2780-cp OC cells with SGI-110 (100nM, 72 hr) or with cisplatin (CDDP; 1.67M, 24hr). After a 4 day recovery period, %ALDH+ was analyzed using FACS, cell viability was determined, and ALDH(+) cells were grown as spheroids in culture or as xenografts in mice. The overall %ALDH+ cells was higher (P&amp;lt;0.05) at baseline in the SKOV3 (0.65%) and A2780-cp (1.07%) cells vs. A2780 (0.3%). SGI-110 inhibited (P&amp;lt;0.001) the growth and %ALDH+ of A2780, A2780-cp cells. SGI-110-CDDP inhibited cell growth vs. CDDP alone (p&amp;lt;0.001) and decreased (P&amp;lt;0.05) ALDH(+) cells in A2780 and A2780-cp. MTT assay showed A2780-cp-ALDH(+) cells were 3-fold more resistant than ALDH(-) cells to CDDP. SGI-110 decreased by 2 and 6-fold respectively the CDDP IC50 in A2780 and A2780-cp (P&amp;lt;0.05). SGI-110 induced both transient and prolonged inhibitory (P&amp;lt;0.05) effects on ALDH(+) cell migration and sphere and colony formation. DNA methylation and gene expression changes in the ALDH(+) spheres were assessed by pyrosequencing and qRT-PCR. HOXA10 and HOXA11 (differentiation-associated genes) were upregulated and the stemness markers ALDH1A1, Bmi1, Nanog, Notch3 and Oct4 were down regulated by SGI-110. ALDH+ cells treated with SGI-110 (100nM) showed prolonged tumor formation latency and reduced tumor growth in mice compared to ALDH+ cells alone, whereas ALDH- cells were non-tumorigenic. In an ip xenograft model derived from A2780 cells, CDDP decreased (P&amp;lt;0.001) tumor growth. The %ALDH(+) cells and spheroid numbers were increased (p&amp;lt;0.001) in the cell population dissociated from residual OC xenografts after CDDP treatment (3 weeks) compared to control (44% vs. 2.3%). Maintenance therapy with SGI-110 after maximal response induced by CDDP decreased recurrent tumor growth and metastases (P=0.05). DNA methylome assessed with Infinium 450K methylation arrays showed global demethylation of xenografts by SGI-110 vs. control (6% decrease in average β-value, p=0.05 and over 60,000 hypomethylated sites). In summary, our in vitro and in vivo data demonstrate that low dose SGI-110 or with CDDP, reduced the stemness capability of OCSCs and SGI-110 alone increased CDDP sensitivity and induced reexpression of differentiation-associated genes in ALDH+ cells. Therefore, SGI-110 in combination with cisplatin has the potential to alter OCSC in patients to prevent recurrent and chemoresistant OC. Citation Format: Yinu Wang, Horacio Cardenas, Fang Fang, Salvatore Condello, Pietro Taverna, Gavin Choy, Mohammad Azab, Kenneth Nephew, Daniela Matei. SGI-110 alters ovarian cancer stem cells to prevent recurrent and chemoresistant ovarian cancer. [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 2317. doi:10.1158/1538-7445.AM2014-2317

Anti-tumour activity of phosphoinositide-3-kinase antagonist AEZS-126 in models of ovarian cancer.

Platinum resistance is the most crucial problem for treatment of ovarian cancer. There is a clinical need for new treatment strategies which overcome platinum resistance. Recently high level of AKT was shown to be involved in platinum resistance and furthermore in resistance against Natural-killer (NK)-cell mediated killing in ovarian cancer. Here, we investigate the ability of the PI3K/AKT inhibitor AEZS-126 alone and in combination with rapamycin to selectively target ovarian cancer cell proliferation and survival in vitro by MTT-assays and FACS based analysis. Furthermore the mechanism of cytotoxicity is analysed by FACS based assays. The NK-killing efficiency of ovarian cancer cells with and without pre-treatment with AEZS-126 was analysed. AEZS-126 showed good anti-tumour activity in in vitro models of ovarian cancer. Main mechanism of cytotoxicity seems to be necroptosis which could be abrogated by co-incubation with necrostatin-1. Furthermore pre-treatment of platinum resistant cells with AEZS-126 resulted in an increased accessibility of these tumour cells for killing by NK-cells. We demonstrated the highly efficient anti-tumour activity of AEZS-126 in in vitro models of ovarian cancer. Due to the good anti-tumour activity and the expected increase in NK-cell mediated killing even of platinum resistant tumour cells, AEZS-126 seems to be a promising candidate for clinical testing in ovarian cancer.

Annexin A4 induces platinum resistance in a chloride-and calcium-dependent manner.

Platinum resistance has long been a major issue in the treatment of various cancers. We previously reported that enhanced annexin A4 (ANXA4) expression, a Ca2+-regulated phospholipid-binding protein, induces chemoresistance to platinum-based drugs. In this study, we investigated the role of annexin repeats, a conserved structure of all the annexin family, responsible for platinum-resistance as well as the effect of knockdown of ANXA4. ANXA4 knockdown increased sensitivity to platinum-based drugs both in vitro and in vivo. To identify the domain responsible for chemoresistance, ANXA4 deletion mutants were constructed by deleting annexin repeats one by one from the C terminus. Platinum resistance was induced both in vitro and in vivo in cells expressing either full-length ANXA4 or the deletion mutants, containing at least one intact annexin repeat. However, cells expressing the mutant without any calcium-binding sites in the annexin repeated sequence, which is essential for ANXA4 translocation from the cytosol to plasma membrane, failed to acquire platinum resistance. After cisplatin treatment, the intracellular chloride ion concentration, whose channel is partly regulated by ANXA4, significantly increased in the platinum-resistant cells. These findings indicate that the calcium-binding site in the annexin repeat induces chemoresistance to the platinum-based drug by elevating the intracellular chloride concentration.

Novel combination of mitochondrial division inhibitor 1 (mdivi-1) and platinum agents produces synergistic pro-apoptotic effect in drug resistant tumor cells.

Overcoming platinum drug resistance represents a major clinical challenge in cancer treatment. We discovered a novel drug combination using cisplatin and a class of thioquinazolinone derivatives including mdivi-1 (mitochondrial division inhibitor-1), that induces synergistic apoptosis in platinum resistant tumor cells, including those from cisplatin-refractory endstage ovarian cancer patients. However, through study of the combination effect on Drp1 (the reported target of mdivi-1) knockout MEF cells and the functional analysis of mdivi-1 analogs, we revealed that the synergism between mdivi-1 and cisplatin is Drp1-independent. Mdivi-1 impairs DNA replication and its combination with cisplatin induces a synergistic increase of replication stress and DNA damage, causing a preferential upregulation of a BH3-only protein Noxa. Mdivi-1 also represses mitochondrial respiration independent of Drp1, and the combination of mdivi-1 and cisplatin triggers substantial mitochondrial uncoupling and swelling. Upregulation of Noxa and simultaneous mitochondrial swelling causes synergistic induction of mitochondrial outer membrane permeabilization (MOMP), proceeding robust mitochondrial apoptotic signaling independent of Bax/Bak. Thus, the novel mode of MOMP induction by the combination through the "dual-targeting" potential of mdivi-1 on DNA replication and mitochondrial respiration suggests a novel class of compounds for platinum-based combination option in the treatment of platinum as well as multidrug resistant tumors.

Differential outcome of MEK1/2 inhibitor-platinum combinations in platinum-sensitive and -resistant ovarian carcinoma cells

Deregulated pro-survival signalling plays a role in ovarian carcinoma drug resistance. Here, we show that cisplatin or oxaliplatin in combination with the MEK1/2 inhibitor CI-1040 resulted in a synergistic effect associated with enhanced apoptotic response in platinum-sensitive cells. The drug combinations were additive in platinum-resistant cells exhibiting increased phospho-ERK1/2, down-regulation of apoptosis-related factors (BAX, PUMA, FOXO1) and of phosphatases inhibiting ERK1/2 (DUSP5, DUSP6). Consistently, FOXO1 knockdown in sensitive cells reduced the efficacy of the combination treatment. Pharmacological targeting of ERK1/2 pathway increases cell sensitivity to platinum compounds by interfering with multiple events, ultimately favouring apoptosis induction in selected molecular backgrounds.

Comprehensive Comparative Study Using Ab Initio Computational Approaches on the Structures of Cisplatin, Oxaliplatin and BNP3029 (A Novel Substituted Cyano Ligand-based Platinum Analogue) and Activation Energy Barriers for the Attack of Nucleophiles on Cisplatin and BNP3029 and their Monoaquated Derivatives

Cisplatin is an important anti-cancer agent widely used in the clinic; however, it has several notable limitations. To develop novel platinum analogues, key characteristics were considered that may result in more effective platinum analogues. Herein results based on ab initio geometry optimizations (gas- and solution-phase) on cisplatin (1), oxaliplatin_1R_2R (2) and BNP3029 (3, a novel substituted cyano ligand-based platinum analogue, PtCl2[N≡C(CH2)3(C6H5)]2) using the recently published potentials and basis sets for platinum are presented. Optimized quantum mechanical derived geometries of the 3 platinum agents were in good agreement with available experimental geometries. The reactivity of BNP3029 was compared to cisplatin by computing the activation free energy barriers for the attack of various nucleophiles on both 1 and 3 and their monoaquated derivatives. Based on the activation energy barriers, it was determined that: (i) the reaction rate may be similar for the attack of water on cisplatin and BNP3029; (ii) the reaction rate for the attack of DNA bases was slower for monoaquated BNP3029 compared to monoaquated cisplatin; and (iii) the reaction rates for a thiol/thiolate attack on monoaquated cisplatin or monoaquated BN3029 were similar. BNP3029 demonstrated potent cytotoxic activity in a variety of human cancer cell lines in comparison to cisplatin and oxaliplatin and also had potent cytotoxic activity in several platinum resistant cell lines.

Comprehensive Comparative Analysis of the Morphological Changes in a 12-mer DNA Oligonucleotide upon Platination by Cisplatin, Oxaliplatin and BNP3029 (a Substituted Cyano ligand-based Platinum analogue) using Molecular Dynamics Simulation Studies

Cisplatin is an important anti-cancer agent widely used in the clinic; however, it has several notable limitations. To develop novel platinum analogues, key characteristics were considered that may result in more effective platinum analogues. In this study, we present comprehensive molecular dynamics simulation studies using a 12-mer DNA (5’-CCTCTggTCTCC-3’, gg= the site of platination) oligonucleotide which was platinated with cisplatin (1), oxaliplatin_1R_2R (2), and BNP3029 (3, a novel substituted cyano platinum analogue, PtCl2[N≡C(CH2)3(C6H5)]2), and analyzed the large data output using the Kolmogorov-Smirnov statistical analyses. In summary, data indicated that BNP3029-DNA had less A-like DNA morphology in comparison to cisplatin-DNA and oxaliplatin-DNA thus maintaining a more B-like DNA form. BNP3029 demonstrated more potent cytotoxic activity, relative to cisplatin and oxaliplatin, in a variety of human cancer cell lines, including several platinum-resistant cell lines.

Gallium phosphinoarylbisthiolato complexes counteract drug resistance of cancer cells

In cancer therapy the platinum-based drugs are used frequently with a good clinical outcome, but besides unwanted side effects which occur, the tumour cells subjected to treatment are prone to develop tolerance or even multidrug resistance (MDR). Metal compounds with a central atom other than platinum are efficient in targeting the chemoresistant cells, therefore the biological outcome of two recently synthesized gallium phosphinoarylbisthiolato complexes was studied, having the formula [X][Ga{PPh(2-SC6H4)2-κ(3)S,S',P}{PPh(2-SC6H4)2-κ(2)S,S'}] where [X] is either the NEt3H (1) or PPh4 (2) cation. Compounds 1 and 2 display in vitro cytotoxicity against both platinum-sensitive and platinum-resistant cell lines (A2780 and A2780cis). Morphological and ultrastructural evidence points toward their capacity to impair tumour cells survival. This behaviour is based on malignant cells capacity to selectively intake gallium, and to bind to the cellular DNA. They are able to cause massive DNA damage in treated cancer cells, focusing on 7-methylguanine and 8-oxoguanine sites and oxidizing the pyrimidine bases; this leads to early apoptosis of a significant percent of treated cells. The intrinsic and extrinsic apoptotic pathways are influenced through the modulation of gene expression following the treatment with complexes 1 and 2, which accompanies the negative regulation of P-glycoprotein 1 (Pgp-1), an important cellular ABC-type transporter from the multidrug resistance (MDR) family. The studied Ga(III) compounds demonstrated the capacity to counteract the chemoresistance mechanisms in the tumours defiant to standard drug action. Compound 2 shows a good anticancer potential and it could represent an alternative to platinum-based drugs especially in the situation of standard treatment failure.

Abstract A93: Targeting the increased invasive capability of non-small cell lung cancer platinum-resistant cells by histone deacetylase inhibitors.

Abstract Cellular resistance to antitumor drugs involves multiple changes including modulation of pro-angiogenic and invasive properties. Since epigenetic alterations have been implicated in the aggressive behavior of lung cancer, a disease for which platinum-based therapy is available, we examined the capability of SAHA [a pan-histone deacetylase (HDAC) inhibitor) and ST3595 (a hydroxamate-based HDAC inhibitor) to interfere with proliferative as well as invasive potential of non-small cell lung cancer (NSCLC) cell systems. Cellular, biochemical and molecular approaches were employed including growth inhibition and invasion assays, western blot analyses and proteome profilers, quantitative RT-PCR including TaqMan arrays, and gain of function assays by cDNA transfection. We used two experimental systems of NSCLC, H460, and A549 cell lines, and generated the cisplatin-resistant variants (H460/Pt, A549/Pt), which were found to exhibit increased invasive abilities as compared to parental cells. These features were associated with a wide modulation of the levels of angiogenesis- and invasion-related factors in the cell conditioned media. In the H460/Pt model, several bioactive molecules (e.g., angiogenin, endothelin-1, ADAMTS1) were upregulated compared to the parental counterpart. The levels of uPA, IL8, and MIF were found to be increased in the conditioned media from both H460/Pt and A549/Pt cells. SAHA and ST3595 produced a marked antiproliferative effect in both platinum-sensitive and -resistant cell lines and induced a strong inhibition of cell invasive properties, that was more marked after ST3595 exposure. The analysis of the modulation of the mRNAs of genes involved in the metastatic process indicated that both SAHA and ST3595 up-regulated the metastasis suppressor KiSS1. Quantitative RT-PCR analysis of TXNIP, which has been reported to be an up-stream regulator of KiSS1 suggested that TXNIP might be involved in KiSS1 induction. Up-modulation of this metastasis suppressor by cell exposure to HDAC inhibitors was evident also at the protein level. Forced expression of KiSS1 resulted in significant decrease of invasive capability, whereas no change in cisplatin sensitivity was found. Such findings indicate that HDAC inhibitors overcome platinum resistance in NSCLC cell systems and counteract the invasive potential of resistant cells through mechanisms involving KiSS1. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A93. Citation Format: Valentina Zuco, Giuliana Cassinelli, Giacomo Cossa, Laura Gatti, Enrica Favini, Franco Zunino, Nadia Zaffaroni, Cinzia Lanzi, Paola Perego. Targeting the increased invasive capability of non-small cell lung cancer platinum-resistant cells by histone deacetylase inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A93.

Annexin A4-conferred platinum resistance is mediated by the copper transporter ATP7A

Although platinum drugs are often used for the chemotherapy of human cancers, platinum resistance is a major issue and may preclude their use in some cases. We recently reported that enhanced expression of Annexin A4 (Anx A4) increases chemoresistance to carboplatin through increased extracellular efflux of the drug. However, the precise mechanisms underlying that chemoresistance and the relationship of Anx A4 to platinum resistance in vivo remain unclear. In this report, the in vitro mechanism of platinum resistance induced by Anx A4 was investigated in endometrial carcinoma cells (HEC1 cells) with low expression of Anx A4. Forced expression of Anx A4 in HEC1 cells resulted in chemoresistance to platinum drugs. In addition, HEC1 control cells were compared with Anx A4-overexpressing HEC1 cells in xenografted mice. Significantly greater chemoresistance to cisplatin was observed in vivo in Anx A4-overexpressing xenografted mice. Immunofluorescence analysis revealed that exposure to platinum drugs induced relocation of Anx A4 from the cytoplasm to the cellular membrane, where it became colocalized with ATP7A, a copper transporter also well known as a mechanism of platinum efflux. ATP7A expression suppressed by small interfering RNA had no effect on HEC1 control cells in terms of chemosensitivity to platinum drugs. However, suppression of ATP7A in Anx A4-overexpressing platinum-resistant cells improved chemosensitivity to platinum drugs (but not to 5-fluorouracil) to a level comparable to that of control cells. These results indicate that enhanced expression of Anx A4 confers platinum resistance by promoting efflux of platinum drugs via ATP7A.

Abstract B38: The role of TWIST1 in chemoresistance and metastasis in ovarian cancer – potential therapeutic applications

Abstract Introduction: Epithelial Ovarian Cancer (EOC) is a leading cause of death among women with gynecologic cancers. Most patients respond to primary treatment with platinum-based therapies, but eventually develop platinum-resistant metastatic disease. Novel treatment strategies to address both metastasis and platinum-resistance are therefore urgently needed. A promising target for such therapy is TWIST1, a basic helix-loop-helix transcription factor which is aberrantly reactivated in many cancers, and which acts as a master regulator of epithelial to mesenchymal transition (EMT), a process by which epithelial cancer cells gain the ability to migrate and metastasize. A growing body of literature suggests EMT as a chemoresistance mechanism, though little research has focused on the role of TWIST1 in chemoresistance, or as potential therapeutic target in EOC. Purpose: To a. characterize the relationship between TWIST1 and platinum-resistance in EOC, and b. to develop dendrimer delivery of RNA-based TWIST1 inhibitors to prevent metastatic spread and platinum- resistance in EOC. Methods: TWIST1 expression was evaluated via Western Blot in A2780 and A2780R cells, which are platinum-sensitive and platinum-resistant EOC cell lines, respectively. TWIST1 was knocked down via siRNA against TWIST1 complexed with poly(amidoamine) dendrimers, or using shRNA (without dendrimer). Cellular uptake of siRNA-dendrimer complexes was assayed via fluorescent microscopy of AlexaFluor tagged siRNA. Expression of TWIST1 was quantified by Western blotting and quantitative real-time RT-PCR. To assay chemoresistance, the platinum-resistant EOC cells, A2780R, were treated with logarithmically increasing doses of cisplatin (CDDP), and cytotoxicity assayed by sulforhodamine B staining, and compared with a platinum-sensitive A2870 EOC cells, as well as A2780R knockdown of TWIST1. Lastly, TWIST1 expression was correlated with EOC progression, in twenty-two EOC patient samples (eighteen Stage III/IV, four Stage I/II, obtained at City of Hope) via immunohistochemistry (IHC) using anti-Twist polyclonal antibody (Glackin 14-2). Results: TWIST1 was significantly expressed in A2780R cells, with little to no protein expression in A2780 cells. Development of siRNA-dendrimer complexes has resulted in effective, long-term TWIST1 silencing in EOC cells, with peak efficacy up to one week following transfection A2780 cells. TWIST1 knockdown in platinum-resistant EOC cells results in reversal of resistance, with sensitivity approaching that of parental, platinum-sensitive cells. A CDDP dose of 10 μM produced 40% cell death in resistant cells with TWIST1 knockdown, compared to 0% cell death in untreated resistant cells. By comparison, treatment of sensitive cells with the same dose yielded approximately 60% cell death. TWIST1 expression in human EOC tissues by IHC shows strong nuclear expression in advanced stage III/IV cancers, compared to low nuclear expression in early stage I/II disease. Interestingly, stage I/II cancers displayed TWIST1 expression in stromal cells surrounding the tumor as opposed to strong nuclear expression in advanced stage samples, possibly as a result of an inflammatory response. Conclusions: TWIST1 is a promising novel therapeutic target in advanced EOC given its role in metastasis and chemoresistance. It is predominantly expressed in advanced stage disease and platinum-resistant EOC cell lines. Long-term silencing of this transcription factor is feasible with dendrimer complexing, and results in significant reversal of platinum resistance in vitro. Preclinical mouse models to confirm these in vitro results will pave the way for clinical applications to silence EMT transcriptional suppressors and potentially reverse chemoresistance and prevent metastasis. Citation Format: Cai Roberts, Ernest S. Han, Gina Lowe, James Finlay, Sharon Wilczynski, Lucy Ghoda, Carlotta Glackin, Thanh H. Dellinger. The role of TWIST1 in chemoresistance and metastasis in ovarian cancer – potential therapeutic applications. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B38.

Abstract PR04: DNA-PK inhibition resensitizes ovarian cancer cells to cisplatin in vivo and is associated with modulation of AKT pathway signaling

Abstract Sustained proliferative signaling and resisting cell death are two fundamental hallmarks of cancer. The AKT pathway is a frequently aberrant, highly interconnected, intracellular signaling network that connects a wide variety of cell stimuli to proliferative and apoptotic cellular control. We have shown that inappropriate control of the AKT pathway underlies failure of chemotherapy in ovarian cancer and furthermore have shown that DNA-PKcs, a component of the non-homologous-end-joining DNA repair pathway, phosphorylates AKT on serine residue 473 in the nucleus of platinum resistant ovarian tumor cells, but not sensitive cells from the same patient. DNA-PK mediated AKT activation in chemoresistant cells results in inhibitory phosphorylation of the pro-apoptotic protein BAD and stabilization of the anti-apoptotic Bcl-2 protein. We report that inhibition of DNA-PK restores response to cisplatin in chemoresistant ovarian cancer cells in vivo. SKOV3 tumor xenografts were implanted subcutaneously into the flanks of Balb/c nu/nu mice and treated with 7.5mg/kg or 10mg/kg DNA-PKcs inhibitor, NU7441 daily (5days on/2 days off) for 2 weeks alone or in combination with 1mg/kg cisplatin twice weekly. DNA-PK inhibition or cisplatin alone were ineffective however in combination decreased tumor growth at 14 days by 89% (7.5mg/kg NU7441) and 90% (10mg/kg NU7441), relative to platinum only treatment. Tumor xenografts were recovered and analyzed by reverse phase protein array (RPPA). Analysis has revealed AKT pathway signaling alterations that underlie the enhanced response to cisplatin on addition of NU7441. DNA-PK inhibition is an attractive therapeutic strategy for resensitizing resistant tumors to platinum based therapy, directly linking the DNA damage caused by platinum to the pro-survival AKT pathway. This abstract is also presented as Poster A50. Citation Format: Azadeh Cheraghchi-Bashi-Astaneh, Camila H. De Sousa, Elaina Maginn, Yan Dai, Harpreet Wasan, Gordon B. Mills, Hani Gabra, Euan A. Stronach. DNA-PK inhibition resensitizes ovarian cancer cells to cisplatin in vivo and is associated with modulation of AKT pathway signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR04.

Abstract IA17: Targeting the methylome for epigenetic resensitization of ovarian cancer

Abstract Women with advanced stage ovarian cancer (OC) have a five-year survival rate of less than 25%. Although most patients respond to platinum-based chemotherapy, relapses are common, leading to platinum-resistant OC, which is uniformly fatal. OC progression is associated with accumulation of epigenetic alterations. In particular, deoxycytosine methylation of CpG islands in promoter regions of tumor suppressor genes (TSGs) plays a prominent role in the development and progression of drug-resistant epithelial OC. We recently demonstrated for the first time in a clinical trial that therapeutic interventions targeting the OC methylome reverse drug resistance and induce meaningful clinical responses. We showed that repetitive low-dose decitabine reactivated silenced genes and restored sensitivity to carboplatin, providing strong clinical and biological support for further study of hypomethylating agents in heavily pre-treated, platinum-resistant ovarian cancer patients. While the FDA-approved demethylating agent decitabine is prone to deamination by cytidine deaminase, SGI-110 (Astex Pharmaceuticals, Inc.), a dinucleotide analogue of decitabine, is more stable, less toxic, and a promising alternative to restoring silenced TSG expression in cancer cells by reversal of DNA methylation. Our preclinical evaluation demonstrated that SGI-110 resensitized platinum-resistant OC cell lines to cisplatin (CDDP) (3-fold reduction in IC50) and reduced the CDDP IC50. SGI-110 treatment induced significant demethylation and subsequent transcriptional derepression of tumor suppressors and differentiation-associated genes in OC cells. SGI-110 alone or in combination with CDDP was well tolerated in non-tumor bearing mice. Significant antitumor activity was observed in mice harboring subcutaneous OC tumors and treated with single SGI-110 and SGI-110 + CDDP treatment in both a biweekly and daily (QD5) regimen. In addition to reducing tumor growth in xenografts, SGI therapy was effective in causing global as well as TSG demethylation and gene reexpression in vivo. Furthermore, the antitumor activity of SGI-110 was associated with reduced chromatin compaction, allowing greater CDDP intercalation into DNA, as assessed by increased DNA platinum adducts in SGI-treated OC cells. The results of our preclinical study support our recently activated clinical trial NCT01696032 using SGI-110 in combination with carboplatin in patients with recurrent, platinum-resistant OC. Clinical specimens (tumor and plasma samples) will be analyzed for epigenetic biomarker changes. We seek to bring forward the new concept of epigenetic targeting in platinum resistant OC by priming the tumors with SGI-110 and set the stage for interventions targeting the OC epigenome, as well as guide and impact the design of future clinical investigations in OC. Citation Format: Kenneth P. Nephew, Daniela Matei, Pietro Taverna, Fang Fang, Jessica Tang, Gavin Choy, John Lyons, Mohammad Azab, Jay Pilrose, John Turchi. Targeting the methylome for epigenetic resensitization of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA17.

Abstract A17: FBxW7 duality in ovarian cancer: Novel insight into ovarian cancer pathogenesis

Abstract Rationale: FBxW7 is a tumor suppressor gene that has been shown to be linked to ubiquitin-dependent proteolysis and regulation of the G1-S cell cycle checkpoint. FBxW7 mutations have been implicated in the impaired degradation of NOTCH, JUN, and cMYC, all of which have been demonstrated as key players in the ovarian cancer pathobiology. In this study we characterized the functional and prognostic significance of FBxW7 in ovarian cancer. Methods: Platinum-sensitive (A2780) and resistant (C200 and SKOV3) ovarian cancer cell lines were utilized, as well as cell lines derived from normal ovarian epithelium (IOSE). Ovarian cancer cell lines were transfected with FBxW7 constructs using Lipofectamine 2000tm. qPCR was performed to determine levels of gene and microRNA expression. Western blot analysis was performed to assess protein levels. Cell proliferation assays and FACS were performed determine cell proliferation and viability. A tissue microarray was used for immunohistochemical staining using FBxW7 antibody. Tissue stains were scored manually. Staining intensities were correlated with patient outcomes to calculate the overall survival and progression-free survival using Kaplan Meier methods and log-rank tests. Results: Upregulation of FBxW7 in platinum-sensitive cell lines decreases the substrates of FBxW7 ubiquitination, such as cMYC, NOTCH, and JUN. However, similar levels of FBxW7 expression did not decrease these substrate levels in platinum-resistant cell lines. microRNAs associated with stemness and chemoresistance also had differential expression profiles with FBxW7 transient transfection. Cell proliferation assays also highlighted differences between these sensitive and resistant cell lines. For tissue array analysis, 516 samples were used for FBxW7 IHC staining where 206 (40.2%) of samples were with high-grade serous histology. The majority of samples had FBxW7 staining of high intensity (273; 52.9%). There was no relationship between overall survival or progression-free survival and FBxW7 tissue staining in the total group. However, when analyzing only the high-grade serous histology, there was a trend of increased expression of FBxW7 being associated with better progression-free survival (p=0.051). Conclusions: Our studies suggest that FBxW7 have a dual role in ovarian cancer, having less effect on those cell lines associated with chemoresistance and stemness. In ovarian tumor tissues, there was a trend for elevated levels of FBxW7 expression and better progression free survival. Further studies on FBxW7 and its role in ovarian cancer pathogenesis are warranted. Citation Format: Elizabeth Louise Dickson, Lihua Li, Samuel Leung, Christine Chow, Rachel Isaksson Vogel, David Huntsman, C. Blake Gilks, Subbaya Subramanian. FBxW7 duality in ovarian cancer: Novel insight into ovarian cancer pathogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A17.

Abstract 5550: The flavone baicalein increases DDIT4 protein expression and inhibits mTOR activity in platinum-resistant epithelial ovarian cancer cells.

Abstract OBJECTIVES: Baicalein is a natural flavone and an active component in traditional Chinese medicine. We have recently found that epithelial ovarian cancer cells treated with baicalein increases DDIT4 gene expression, a mediator of DNA damage response. Our objectives are to compare the effects of baicalein to carboplatin and gemcitabine on platinum sensitive and resistant ovarian cancer cell lines. Specifically we examined DDIT4 protein expression, effect on mTOR activity, and growth inhibition. METHODS: The ovarian cancer cell line A2780 and the derivative platinum resistant cell line A2780R were treated with carboplatin, gemcitabine, and baicalein with appropriate controls and growth was assessed at 72 hours by MTT assay. Alternatively cells were treated at identical concentrations for 24 hours and western blots were performed for DDIT4 protein expression, mTOR activity as reflected by phosphorylation of S6K1 (pS6K1) and S6 (pS6), as well as unphosphorylated controls and actin. RESULTS: MTT assay demonstrated that carboplatin and gemcitabine do not inhibit the growth of the A2780R cells at concentrations that inhibit A2780 while baicalein inhibits both cell lines nearly equally (2.5 to 20 μM). DDIT4 protein expression increased in a dose dependent fashion in both cell lines treated with baicalein. Furthermore, the mTOR targets S6K1 and S6 demonstrate a dose dependent decrease in phosphorylation, reflecting inhibition of mTOR activity (Figure 1). In contrast, DDIT4 protein expression was not altered in carboplatin and gemcitabine treated A2780R cells with minimal effect on mTOR activity, whereas A2780 cells displayed increased DDIT4 protein with increasing doses as well as corresponding decreases in the phosphorylation of the mTOR targets. CONCLUSIONS: Baicalein can inhibit growth of both platinum sensitive and resistant ovarian cancer cells. The inhibitory effects of baicalein correlate with both DDIT4 protein expression and inhibition of mTOR activity as reflected by phosphorylation of the mTOR targets S6K1 and S6. Baicalein may be a novel treatment option for patients with ovarian cancer, especially those that are platinum resistant. DDIT4 may mediate growth inhibition through inhibition of mTOR. Interestingly, DDIT4 expression and inhibition of mTOR activity may also play a role in the antitumor effects of the chemotherapeutics carboplatin and gemcitabine in sensitive ovarian cancer cells. Citation Format: Ernest S. Han, Yujun Wang, Quanhua Xing, Jin Yan, Richard Yip, Mark Wakabayashi, John Yim. The flavone baicalein increases DDIT4 protein expression and inhibits mTOR activity in platinum-resistant epithelial ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5550. doi:10.1158/1538-7445.AM2013-5550

Abstract 957: Aldehyde dehydrogenase isoform 1A1 mediates platinum resistance in ovarian cancer through the KLF4/p21 cell cycle arrest pathway.

Abstract Background: Aldehyde dehydrogenase isoform 1A1 (ALDH1A1) activity has been reported in numerous cancers to have a role in chemoresistance. However, the previously proposed resistant mechanism of protecting DNA via free radical scavenger and thus promoting cell survival within a chemically hostile environment has yet to be elicited in cancer. Considering the heterogeneity of cancer, we propose an alternative mechanism of ALDH-mediated platinum-resistance in ovarian cancer must exist. Methods: Isogenic ovarian cancer cell lines (A2780: platinum sensitive &amp; A2780/CP70: platinum resistant) were evaluated for potential mechanisms of platinum resistance. ALDEFLUOR kit was utilized to measure ALDH activity as well as sort cell lines into ALDH(+) &amp; ALDH(-) phenotypes for experiments. An ALDH1A1 shRNA knockdown model was established to determine its effect on cancer stem cell properties, downstream signaling, and platinum resistance. RTQ, Western blot and PCR microarrays were utilized to evaluate potential mechanisms of ALDH-mediated platinum resistance, with select downstream siRNA mediated downregulation of KLF4. Results: Compared to Platinum-sensitive A2780 cells, platinum-resistant CP70 cells demonstrated a 100-fold increase in ALDH1A1 activity (0.2 vs. 24%, respectively (p&amp;lt; 0.001). ALDH(+) cells demonstrated enhanced malignant characteristics of 2-fold increase of tumorigenicity and invasion. A 3-fold increase in cancer stem cell transcriptional factor Kruppel-like factor-4 (KFL4) was shown in ALDH(+) cells (p=0.007). Increased KFL4, upregulated cell-cycle regulator p21, thereby causing G1 arrest and allows for cancer cell repair and survival. Compared to control, shRNA-ALDH knockdown demonstrated expected decreases in KFL4, with subsequent 5-fold decrease in p21 levels and its associated mediator CDK4. Cell cycle kinetics demonstrated a significant decrease in G1-associated cell cycle arrest with decreases in apoptotic threshold, which allows DNA damage to induce apoptosis confirmed with 4-fold increase in apoptotic factor BAX and cell death. Platinum sensitivity was restored in shRNA-ALDH cells, with significant increases in response rates to carboplatin with a 60% reduction of carboplatin dose needed to achieve IC50. Conclusion: ALDH mediates platinum resistance via a specific KFL4/p21 cell cycle arrest pathway. Inhibition of this pathway resulted in reversal of platinum resistant ovarian cancer cells and could be utilized for possible targeting for inducing platinum sensitivity. Citation Format: Erhong Meng, Aparna Mitra, Steven McClelan, Lalita Shevde, Rodney P. Rocconi. Aldehyde dehydrogenase isoform 1A1 mediates platinum resistance in ovarian cancer through the KLF4/p21 cell cycle arrest pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 957. doi:10.1158/1538-7445.AM2013-957 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.