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Ovarian cancer is a chemoresponsive tumor with very high initial response rates to standard therapy consisting of platinum/paclitaxel. However, most women eventually develop recurrence, which rapidly evolves into chemoresistant disease. Persistence of ovarian cancer stem cells (OCSCs) at the end of therapy has been shown to contribute to resistant tumors. In this study, we demonstrate that the long noncoding RNA HOTAIR is overexpressed in HGSOC cell lines. Furthermore, HOTAIR expression was upregulated in OCSCs compared with non-CSC, ectopic overexpression of HOTAIR enriched the ALDH+ cell population and HOTAIR overexpression increased spheroid formation and colony-forming ability. Targeting HOTAIR using peptide nucleic acid-PNA3, which acts by disrupting the interaction between HOTAIR and EZH2, in combination with a DNMT inhibitor inhibited OCSC spheroid formation and decreased the percentage of ALDH+ cells. Disrupting HOTAIR-EZH2 with PNA3 in combination with the DNMTi on the ability of OCSCs to initiate tumors in vivo as xenografts was examined. HGSOC OVCAR3 cells were treated with PNA3 in vitro and then implanted in nude mice. Tumor growth, initiation, and stem cell frequency were inhibited. Collectively, these results demonstrate that blocking HOTAIR–EZH2 interaction combined with inhibiting DNA methylation is a potential approach to eradicate OCSCs and block disease recurrence.

The majority of women diagnosed with epithelial ovarian cancer eventually develop recurrence, which rapidly evolves into chemoresistant disease. Persistence of ovarian cancer stem cells (OCSC) at the end of therapy may be responsible for emergence of resistant tumors. In this study, we demonstrate that in OCSC, the tumor suppressor disabled homolog 2–interacting protein (DAB2IP) is silenced by EZH2-mediated H3K27 trimethylation of the DAB2IP promoter. CRISPR/Cas9-mediated deletion of DAB2IP in epithelial ovarian cancer cell lines upregulated expression of stemness-related genes and induced conversion of non-CSC to CSC, while enforced expression of DAB2IP suppressed CSC properties. Transcriptomic analysis showed that overexpression of DAB2IP in ovarian cancer significantly altered stemness-associated genes and bioinformatic analysis revealed WNT signaling as a dominant pathway mediating the CSC inhibitory effect of DAB2IP. Specifically, DAB2IP inhibited WNT signaling via downregulation of WNT5B, an important stemness inducer. Reverse phase protein array further demonstrated activation of noncanonical WNT signaling via C-JUN as a downstream target of WNT5B, which was blocked by inhibiting RAC1, a prominent regulator of C-JUN activation. Coadministration of EZH2 inhibitor GSK126 and RAC1 inhibitor NSC23766 suppressed OCSC survival in vitro and inhibited tumor growth and increased platinum sensitivity in vivo. Overall, these data establish that DAB2IP suppresses the cancer stem cell phenotype via inhibition of WNT5B-induced activation of C-JUN and can be epigenetically silenced by EZH2 in OCSC. Targeting the EZH2/DAB2IP/C-JUN axis therefore presents a promising strategy to prevent ovarian cancer recurrence and has potential for clinical translation. Significance: These findings show that combining an epigenetic therapy with a noncanonical WNT signaling pathway inhibitor has the potential to eradicate ovarian cancer stem cells and to prevent ovarian cancer recurrence.

Ovarian cancer (OC) cells frequently metastasize to the omentum, and adipocytes play a significant role in ovarian tumor progression. Therapeutic interventions targeting aberrant DNA methylation in ovarian tumors have shown promise in the clinic, but the effects of epigenetic therapy on the tumor microenvironment are understudied. Here, we examined the effect of adipocytes on OC cell behavior in culture and impact of targeting DNA methylation in adipocytes on OC metastasis. The presence of adipocytes increased OC cell migration and invasion, and proximal and direct coculture of adipocytes increased OC proliferation alone or after treatment with carboplatin. Treatment of adipocytes with hypomethylating agent guadecitabine decreased migration and invasion of OC cells toward adipocytes. Subcellular protein fractionation of adipocytes treated with guadecitabine revealed decreased DNA methyltransferase 1 (DNMT1) levels even in the presence of DNA synthesis inhibitor, aphidicolin. Methyl-Capture- and RNA-sequencing analysis of guadecitabine-treated adipocytes revealed derepression of tumor-suppressor genes and epithelial–mesenchymal transition inhibitors. SUSD2, a secreted tumor suppressor downregulated by promoter CpG island methylation in adipocytes, was upregulated after guadecitabine treatment, and recombinant SUSD2 decreased OC cell migration and invasion. Integrated analysis of the methylomic and transcriptomic data identified pathways associated with inhibition of matrix metalloproteases and fatty acid α-oxidation, suggesting a possible mechanism of how epigenetic therapy of adipocytes decreases metastasis. In conclusion, the effect of DNMT inhibitor on fully differentiated adipocytes suggests that hypomethylating agents may affect the tumor microenvironment to decrease cancer cell metastasis. Implications: Epigenetic targeting of tumor microenvironment can affect metastatic behavior of ovarian cancer cells. Mol Cancer Res; 16(8); 1226–40. ©2018 AACR.

Purpose: PARP inhibitors (PARPi) are primarily effective against BRCA1/2-mutated breast and ovarian cancers, but resistance due to reversion of mutated BRCA1/2 and other mechanisms is common. Based on previous reports demonstrating a functional role for DNMT1 in DNA repair and our previous studies demonstrating an ability of DNA methyltransferase inhibitor (DNMTi) to resensitize tumors to primary therapies, we hypothesized that combining a DNMTi with PARPi would sensitize PARPi-resistant breast and ovarian cancers to PARPi therapy, independent of BRCA status. Experimental Design: Breast and ovarian cancer cell lines (BRCA-wild-type/mutant) were treated with PARPi talazoparib and DNMTi guadecitabine. Effects on cell survival, ROS accumulation, and cAMP levels were examined. In vivo, mice bearing either BRCA-proficient breast or ovarian cancer cells were treated with talazoparib and guadecitabine, alone or in combination. Tumor progression, gene expression, and overall survival were analyzed. Results: Combination of guadecitabine and talazoparib synergized to enhance PARPi efficacy, irrespective of BRCA mutation status. Coadministration of guadecitabine with talazoparib increased accumulation of ROS, promoted PARP activation, and further sensitized, in a cAMP/PKA-dependent manner, breast and ovarian cancer cells to PARPi. In addition, DNMTi enhanced PARP “trapping” by talazoparib. Guadecitabine plus talazoparib decreased xenograft tumor growth and increased overall survival in BRCA-proficient high-grade serous ovarian and triple-negative breast cancer models. Conclusions: The novel combination of the next-generation DNMTi guadecitabine and the first-in-class PARPi talazoparib inhibited breast and ovarian cancers harboring either wild-type– or mutant-BRCA, supporting further clinical exploration of this drug combination in PARPi-resistant cancers. Clin Cancer Res; 24(13); 3163–75. ©2018 AACR.

The transcription factor nuclear factor kappa B (NF-κB) and the long non-coding RNA (lncRNA) HOTAIR (HOX transcript antisense RNA) play diverse functional roles in cancer. In this study, we show that upregulation of HOTAIR induced platinum resistance in ovarian cancer, and increased HOTAIR levels were observed in recurrent platinum-resistant ovarian tumors vs. primary ovarian tumors. To investigate the role of HOTAIR during DNA damage induced by platinum, we monitored double-strand breaks and show that HOTAIR expression results in sustained activation of DNA damage response after platinum treatment. We demonstrate that ectopic expression of HOTAIR induces NF-κB activation during DNA damage response and MMP-9 and IL-6 expression, both key NF-κB target genes. We show that HOTAIR regulates activation of NF-κB by decreasing Iκ-Bα (NF-κB inhibitor) and establish that by inducing prolonged NF-κB activation and expression of NF-κB target genes during DNA damage, HOTAIR plays a critical role in cellular senescence and platinum sensitivity. Our findings suggest that a NF-κB-HOTAIR axis drives a positive-feedback loop cascade during DNA damage response and contributes to cellular senescence and chemotherapy resistance in ovarian and other cancers.

Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies. The persistence of quiescent OC stem cells (OCSCs) not eliminated by chemotherapy plays a key role in tumor relapse and metastasis. In the current study, we tested the hypothesis that targeting DAB2IP will inhibit OCSCs and prevent disease recurrence. Subpopulations of CSC and non-CSC were isolated from OC cell lines by fluorescence-activated cell sorting (FACS) based on aldehyde dehydrogenase (ALDH) activity, a consistent CSC marker. Expression of DAB2IP in ALDH(+) cells was lower (P 2) altered expression of 449 genes, including down-regulation of ALDH1A1, LGR5, PROM1 and TWIST1, markers strongly associated with CSC phenotypes. Ingenuity Pathway Analysis for upstream regulators of differentially expressed genes revealed WNT-signaling as a dominant pathway mediating the anti-OCSC effects of DAB2IP. Based on RNA-seq analysis, WNT5B expression decreased (P Citation Format: Xingyue Zong, Ali Ozes, Weini Wang, Fang Fang, Kenneth P. Nephew. Targeting EZH2/DAB2IP/C-JUN axis in ovarian cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 498.

Ovarian cancer (OC) is the fifth leading cause of cancer-related death among American women. Persistence of OC stem cells (OCSCs) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse. Long non-coding RNA HOXC transcript antisense RNA (HOTAIR) has been shown to be associated with chemoresistance and overexpressed in many types of cancers, including high-grade serous OC (HGSOC). Previously published work has demonstrated that NF-κB was activated by HOTAIR through I-κB inhibition via trimethylation of histone H3 lysine K27, which contributes to chemoresistance in HGSOC. NF-κB-medicated signaling pathways involved in maintaining characteristics of CSCs, such as targeting stem cell markers, CD44, CD133, and ALDH1 has been demonstrated but not well defined in OC. The goals of this study are to understand the mechanism of HOTAIR-mediated NF-κB signaling pathway in regulating OCSCs and develop novel strategies to target OCSCs and overcome OC recurrence and drug resistance. Quantitative RT-PCR analysis revealed that HOTAIR was overexpressed in OCSCs compared to non-OCSCs. In order to produce loss-of-function phenotypes of HOTAIR and investigate the function of this gene, we utilized the paired CRISPR guide RNA design to delete the functional sites of HOTAIR without affecting nearby protein-coding gene. Knockout of HOTAIR re-sensitized OC cells to platinum treatment and significantly decreased (P Citation Format: Weini Wang, Fang Fang, Ali R. Özeş, Kenneth P. Nephew. HOTAIR functions through NF-κB pathway in regulating ovarian cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1403.

Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies. Recent data have pointed to the persistence of quiescent ovarian cancer stem cells (OCSCs) not eliminated by chemotherapy and able to regenerate tumors as the main contributor to tumor relapse and metastasis. Downregulation of tumor suppressor DAB2IP significantly correlated with poor patient survival in OC. In the current study, we tested the hypothesis that DAB2IP is downregulated by EZH2 methylation in OC and targeting DAB2IP inhibits OCSCs, which would prevent disease recurrence. Subpopulations of CSC and non-CSC were isolated from OC cell lines by fluorescence-activated cell sorting (FACS) based on aldehyde dehydrogenase (ALDH) activity, a consistent CSC marker. Expression of DAB2IP in ALDH(+) cells was lower (P 2) altered expression of 449 genes, including downregulation of ALDH1A1, LGR5, PROM1, and TWIST1, markers strongly associated with CSC phenotypes. Ingenuity Pathway Analysis for upstream regulators of differentially expressed genes revealed Wnt-signaling as a dominant pathway mediating the anti-OCSC effects of DAB2IP. Based on RNA-seq analysis, WNT5B expression decreased (P Citation Format: Xingyue Zong, Ali Ozes, Weini Wang, Kenneth P. Nephew. Targeting EZH2/DAB2IP/Wnt axis in ovarian cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A81.

Polycomb repressive complex 2 (PRC2) member enhancer of zeste homolog 2 (EZH2) trimethylates histone H3 lysine 27 (H3K27me3), alters chromatin structure and contributes to epigenetic regulation of gene expression in normal and disease processes. Phosphorylation of EZH2 augmented EZH2 oncogenic activity in cancer but observations have been limited to threonine 350 (T350) and serine 21 (S21) residues by cyclin-dependent kinase 1 and protein kinase B, respectively. In addition, phosphorylation of the evolutionarily conserved T372 motif of EZH2 by p38 resulted in EZH2 interaction with Ying Yang 1 and promoted muscle stem cell differentiation. In the present study, we used epithelial ovarian cancer (OC) cells as a model to demonstrate that phosphorylation of EZH2 at T372 by protein kinase A (PKA) induced a dominant-negative EZH2 phenotype, inhibited OC cell proliferation and migration in vitro and decreased ovarian xenograft tumor growth in vivo. Phosphorylation of T372 by PKA enhanced the interaction between EZH2 and signal transducer and activator of transcription 3 (STAT3), and STAT3 binding to pT372-EZH2 reduced cellular levels of pSTAT3 and downregulated interleukin 6 receptor expression in OC. Furthermore, PKA-mediated pT372-EZH2 decreased ATP levels and altered mitochondrial gene expression, resulting in mitochondrial dysfunction and reduced OC cell growth. These findings demonstrate that PKA-mediated T372 phosphorylation reduces oncogenic EZH2 activity and reveal a novel role for pT372 in regulating EZH2 in OC and possibly other cancers.

© 2014 Nature America, Inc. All rights reserved. Many physiological functions of helicases are dependent on their ability to unwind nucleic acid duplexes in an ATP-dependent fashion. Determining the kinetic frameworks of these processes is crucial to understanding how these proteins function. We recently developed a fluorescence assay to monitor RNA duplex unwinding by DEAD-box helicases in real time. In this assay, two fluorescently modified short reporter oligonucleotides are annealed to an unmodified RNA loading strand of any length so that the fluorescent moieties of the two reporters find themselves in close proximity to each other and fluorescence is quenched. One reporter is modified with cyanine 3 (Cy3), whereas the other is modified with a spectrally paired black-hole quencher (BHQ). As the helicase unwinds the loading strand, the enzyme displaces the Cy3-modified reporter, which will bind to a capture or competitor DNA strand, permanently separating it from the BHQ-modified reporter. Complete separation of the Cy3-modified reporter strand is thus detected as an increase in total fluorescence. This assay is compatible with reagentless biosensors to monitor ATPase activity so that the coupling between ATP hydrolysis and duplex unwinding can be determined. With the protocol described, obtaining data and analyzing results of unwinding and ATPase assays takes ∼4 h.

Ovarian cancer (OC) recurrence after tumor eradication by chemotherapy invariably heralds poor outcome. Recent data point to persistence of quiescent cancer stem cells (CSCs) not eliminated by chemotherapy and able to regenerate tumors as the main contributor to tumor relapse. Downregulation of tumor suppressor gene (TSG) DAB2IP, a member of the Ras GTPase-activating protein family, significantly correlated with poor patient survival in high-grade serous (HGS) OC. Furthermore, loss of DAB2IP in prostate and colon cancer enriched CSC population, suggesting a key role for DAB2IP in modulating cancer stemness. In the current study, we tested the hypothesis that targeting DAB2IP would inhibit CSCs in OC and prevent disease recurrence. Subpopulations of CSC and non-CSC were isolated from Kuramochi, OVCAR3 and COV362 HGSOC cell lines by fluorescence-activated cell sorting (FACS) based on aldehyde dehydrogenase (ALDH) activity, a consistent CSC marker. It was previously demonstrated by us and others that ALDH(+) cells share characteristics of normal stem cells such as the ability to form anchorage-independent multicellular aggregates (spheroids), undergo membrane efflux, express stem cell restricted transcription factors, and generate tumors in vivo. DAB2IP was examined in the FACS-sorted cells using qPCR and western blot. Expression of DAB2IP in ALDH(+) cells was lower (P 2) altered expression of 449 genes, including markers strongly associated with CSC phenotypes, including down-regulation of ALDH1A1, LGR5, PROM1, TWIST1 and ATP-binding cassette transporters. Differentially expressed genes were subjected to Ingenuity Pathway Analysis (IPA) for upstream regulators. IPA identified Wnt-signaling pathways as top upstream regulators of these differentially expressed genes, suggesting that Wnt-signaling is a dominant pathway mediating the anti-OCSC effects of DAB2IP. In addition, treating cells with Wnt inhibitor decreased (P Citation Format: Xingyue Zong, Ali R Ozes, Kenneth P Nephew. TARGETING OVARIAN CANCER STEM CELLS THROUGH THE TUMOR SUPPRESSOR DAB2IP-MEDIATED WNT SIGNALING PATHWAY [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-062.

Homologous recombination plays an important role in the high-fidelity repair of DNA double-strand breaks. A central player in this process, RAD51, polymerizes onto single-stranded DNA and searches for homology in a duplex donor DNA molecule, usually the sister chromatid. Homologous recombination is a highly regulated event in mammalian cells: some proteins have direct enzymatic functions, others mediate or overcome rate-limiting steps in the process, and still others signal cell cycle arrest to allow repair to occur. While the human BRCA2 protein has a clear role in delivering and loading RAD51 onto single-stranded DNA generated after resection of the DNA break, the mechanistic functions of the RAD51 paralogs remain unclear. In this study, we sought to determine the genetic interactions between BRCA2 and the RAD51 paralogs during DNA DSB repair. We utilized siRNA-mediated knockdown of these proteins in human cells to assess their impact on the DNA damage response. The results indicate that loss of BRCA2 alone imparts a more severe phenotype than the loss of any individual RAD51 paralog and that BRCA2 is epistatic to each of the four paralogs tested.

Long non-coding RNAs (lncRNAs) play key roles in human diseases, including cancer. Functional studies of the lncRNA HOTAIR (HOX transcript antisense RNA) provide compelling evidence for therapeutic targeting of HOTAIR in cancer, but targeting lncRNAs in vivo has proven to be difficult. In the current study, we describe a peptide nucleic acids (PNA)-based approach to block the ability of HOTAIR to interact with EZH2 and subsequently inhibit HOTAIR-EZH2 activity and resensitize resistant ovarian tumors to platinum. Treatment of HOTAIR-overexpressing ovarian and breast cancer cell lines with PNAs decreased invasion and increased chemotherapy sensitivity. Furthermore, the mechanism of action correlated with reduced nuclear factor-kappaB (NF-κB) activation and decreased expression of NF-κB target genes matrix metalloprotease 9 and interleukin 6. To deliver the anti-lncRNA to the acidic (pH approximately 6) tumor microenvironment, PNAs were conjugated to pH-low insertion peptide (pHLIP). Treatment of mice harboring platinum-resistant ovarian tumor xenografts with pHLIP-PNA constructs suppressed HOTAIR activity, reduced tumor formation and improved survival. This first report on pHLIP-PNA lncRNA targeting solid tumors in vivo suggests a novel cancer therapeutic approach.

Ovarian cancer is the leading cause of death from gynecologic malignancies. Failure to eliminate ovarian cancer stem cells (OCSCs) by the end of conventional chemotherapy contributes to tumor relapse and metastasis. Understanding the molecular features of OCSCs may allow for effective targeting and eradicating these cells and tumor remission. We reported tumor-initiation and expansion capacity by ALDH+ OCSCs after platinum-based treatment of ovarian cancer xenografts. In addition, loss of DAB2IP in prostate and colon cancer promoted CSC-like features, suggesting a critical role in modulating CSC maintenance. DAB2IP, a novel member of Ras GTPase-activating (GAP) protein family, was identified as a potent tumor suppressor that interfered with many aspects of cancer progression and was frequently downregulated in tumors. In the current study, we aimed to elucidate the tumor-suppressor role of DAB2IP in ovarian cancer in the context of OCSCs and explore epigenetic approaches to upregulate its expression. OCSCs and non-CSCs subpopulations were isolated from multiple high-grade serous ovarian cancer cell lines (Kuramochi, OVCAR3, and COV362). Reduced (p Citation Format: Xingyue Zong, Ali R. Ozes, Kenneth P. Nephew. Targeting DAB2IP in ovarian cancer stem cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B07.

PURPOSE: Most women diagnosed with advanced-stage ovarian cancer develop chemo-resistant tumor recurrence, which is uniformly fatal. Long non-coding RNAs (lncRNAs) play key roles in human diseases, including cancer. Functional studies of the lncRNA HOTAIR (HOX transcript antisense RNA) provide compelling evidence for therapeutic targeting of HOTAIR in ovarian cancer to impact chemoresistance, but targeting lncRNAs in vivo has proven to be difficult. EXPERIMENTAL DESIGN: In the current study, we describe a peptide nucleic acids (PNA)-based approach to effectively block the ability of HOTAIR to interact with EZH2 and subsequently inhibit oncogenic HOTAIR-EZH2 activity. In order to deliver the anti-lncRNA to the acidic (pH approximately 6) microenvironment of solid tumors in vivo, PNAs were conjugated to pH-low insertion peptide (pHLIP). RESULTS: Treatment of HOTAIR-overexpressing ovarian cancer cell lines with PNAs decreased cancer cell invasion and increased chemotherapy sensitivity. The mechanism of action of the anti-lncRNA agent correlated with reduced nuclear factor-kappaB (NF-κB) activation and decreased expression of key NF-κB target genes including matrix metalloprotease 9 and interleukin 6. PNA treatment of ALDH1A1 positive/ovarian tumor initiating cells overexpressing HOTAIR markedly decreased ALDH1A1 activity and reduced the ovarian cancer stem cell population. In mice harboring ovarian tumor xerographs and treated with pHLIP-PNA constructs, HOTAIR oncogenic activity was suppressed, ovarian tumors were resensitized to platinum, and a significant (64%) survival improvement was observed. CONCLUSIONS: Toward developing new therapeutic approaches against chemoresistant ovarian cancer, we designed a PNA-targeting strategy for HOTAIR that effectively blocks the ability of HOTAIR to interact with EZH2 and inhibits oncogenic HOTAIR-EZH2. This first report on pHLIP-PNA targeting HOTAIR in vivo offer pre-clinical proof-of-concept and represents a possible treatment alternative for chemotherapy-resistant ovarian cancer. Citation Format: Ali R. Özeş, Yinu Wang, Fang Fang, and Kenneth P. Nephew. THERAPEUTIC TARGETING USING TUMOR SPECIFIC PEPTIDES INHIBITS LONG NON–CODING RNA HOTAIR ONCOGENIC ACTIVITY IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP14.

Eukaryotic initiation factor 4A (eIF4A) is a DEAD-box helicase that stimulates translation initiation by unwinding mRNA secondary structure. The accessory proteins, eIF4G, eIF4B, and eIF4H enhance the duplex unwinding activity of eIF4A, but the extent to which they modulate eIF4A activity is poorly understood. Here, we use real time fluorescence assays to determine the kinetic parameters of duplex unwinding and ATP hydrolysis by these initiation factors. To ensure efficient duplex unwinding, eIF4B and eIF4G cooperatively activate the duplex unwinding activity of eIF4A. Our data reveal that eIF4H is much less efficient at stimulating eIF4A unwinding activity than eIF4B, implying that eIF4H is not able to completely substitute for eIF4B in duplex unwinding. By monitoring unwinding and ATPase assays using identical conditions, we demonstrate that eIF4B couples the ATP hydrolysis cycle of eIF4A with strand separation, thereby minimizing non-productive unwinding events. Using duplex substrates with altered GC contents, but with similar predicted thermal stabilities, we further show that the rate of formation of productive unwinding complexes is strongly influenced by the local stability per base pair in addition to the stability of the entire duplex. This finding explains how a change in the GC content of a hairpin while maintaining overall predicted thermal stability is able to influence translation initiation.

Efficient mRNA recruitment to the human ribosome requires a region of single stranded RNA close to the cap structure. The eukaryotic initiation factor 4A (eIF4A) is a DEAD box helicase that is essential for unwinding any stable secondary structure that would inhibit this step. Although studied for many years, the majority of helicase assays involving eIF4A and its associated proteins have failed to rigorously analyze the kinetic events that occur during mRNA unwinding. To provide this kinetic understanding we have developed a continuous fluorescence-based assay to measure RNA duplex unwinding events. This assay utilizes an RNA oligonucleotide modified with cyanine 3 (Cy3) annealed to a complimentary strand modified with black hole quencher (BHQ). Separation of the RNA duplex region significantly enhances the Cy3 fluorescence, enabling us to measure RNA helicase activity in real time by fluorescence spectroscopy.Data will be presented to show how we are using this assay to determine the kinetic role of accessory factors on the helicase activity by eIF4A. Moreover, we show how this data combined with a continuous coupled ATPase assay is enabling us to determine the relationship of ATP hydrolysis to the unwinding of duplex RNA by this DEAD box helicase.We will also present data employing RNA duplexes of different lengths, allowing us to understand how the processivity of eIF4A is influenced by other initiation components.This study will provide us with the foundation to begin understanding the kinetic framework of mRNA recruitment to the human ribosome.

Histone methyltransferases (HMT) are chromatin modifiers that regulate the transcriptomic landscape in normal development as well as in diseases such as cancer.1 Enhancer of Zeste 2 (EZH2), a component of the polycomb repressive complex 2 (PRC2), trimethylates histone 3 lysine 27 (H3K27me3), resulting in a more compact chromatin structure known to repress gene activation.2 Overexpression of EZH2 has been strongly implicated in oncogenesis of many different cancers, including ovarian cancer (OC),2 and increased EZH2 activity has been linked to OC resistance to platinum-based chemotherapy, presumably by inhibiting crucial tumor suppressors and genes involved in OC metastasis such as integrins.2,3 Although HMT inhibitors (HMTI) that target EZH2 specifically or HMTs in general are promising anticancer therapeutics,1 the mechanism(s) underlying EZH2 regulation is incompletely understood, and the use of clinically relevant model systems to better understand the translational potential of this important class of epigenetic modifiers is clearly an important area of investigation. In the July 1, 2013 issue of Cell Cycle, Amatangelo and colleagues investigated the effect of a specific inhibitor of EZH2, GSK343, on human epithelial ovarian cancer.4 After GSK343 treatment of ovarian cancer (OC) cells grown on a 2D monolayer, a 90% decrease in total H3K27me3 was observed, demonstrating drug specificity to EZH2, and EZH2 protein levels remained unchanged, corroborating the mechanism of GSK343 action as an inhibitor of EZH2 HMT activity. Despite this, no effect on growth was observed on OC cells in 2D culture, although previous studies have reported that reducing the HMT activity of EZH2 markedly alters cell physiology in OC and other cancers. For example, phosphorylation of EZH2 by protein kinase B (AKT) on serine 21 (S21) reduced EZH2 enzymatic activity and as well as integrin α2 expression.5 In addition, EZH2 phosphorylation on residues threonine 350 and 487 (T350 and T487) by cyclin-dependent kinase 1 and 2 (CDK1 and CDK2), respectively, inhibited cell proliferation, migration, and anchorage-independent growth, indicating a role for EZH2 HMT activity in migration and invasion.6,7 To further investigate the effect of GSK343-mediated EZH2 inhibition on OC, Amatangelo and colleagues4 extended the scope of their study by using a 3D cell culture system composed of a matrigel extracellular matrix (ECM). It is well known that the tumor microenvironment is both heterogeneous in nature, composed stromal fibroblasts, immune cells, and vascular endothelial cells, and can significantly impact both the metastatic potential of cancer cells as well as their ability to resist chemotherapy. For these reasons, the ECM closely recapitulates the tumor microenvironment, representing a more clinically relevant model system compared with monolayer culture conditions on plastic. Taking this approach, Amatangelo and colleagues observed that GSK343 treatment inhibited OC cell growth and invasion in a 3D culture and correlated with apoptosis induction in OC cells.4 The results indicate regulation of EZH2 by the OC-ECM interaction as well as improved efficacy of GSK343 to suppress the ability of OC to remodel the ECM and metastasize (Fig. 1). Figure 1. Comparing the sensitivity of epithelial ovarian cancer cells to the histone methyltransferase inhibitor (HMTI) GSK343 in 2D (i.e., plastic, upper) and 3D (i.e., matrigel extracellular matrix, lower). No effect of GSK343 on ovarian cancer ... As epigenetic therapies for OC are in the clinical arena8 (and see SGI-110 in Combination With Carboplatin in Ovarian Identifier: NCT01696032, CancerClinicalTrials.gov), the exciting new study by the Zhang lab provides compelling evidence for using 3D culture systems to gain key insight into the biological roles of EHZ2 in OC and the sensitivity of this cancer to EZH2-specific inhibitors. Furthermore, as suggested by the Zhang lab,4 the numerous discrepancies observed between the efficacy of inhibitors in 2D culture and in vivo animal systems may be better explained by using the third dimension.

The Polycomb Repressive Complex 2 (PRC2) has been implicated in cancer and a role for PRC2 during DNA damage response (DDR) has recently been reported. To examine changes in gene expression with DDR in cisplatin (cddp)-resistant ovarian cancer (OC), we performed whole transcriptome RNA-seq analysis of isogenic cddp-sensitive and -resistant OC cell lines (A2780 and A2780-cp). Differential expression of PRC components (SUZ12, EZH1, SIRT1, PHC1 &2) (P Citation Format: Ali R. Ozes, Dave F. Miller, Yunlong Liu, Kenneth P. Nephew. Non-coding RNA HOTAIR connects DNA damage signaling to NF-κB activation in cisplatin resistant 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 3551. doi:10.1158/1538-7445.AM2014-3551

The Polycomb Group (PcG) Repressive Complexes 1 and 2 (PRC1 and PRC2) maintain gene expression patterns by epigenetically modifying chromatin. PRC2 suppresses transcription by catalyzing the trimethylation of histone H3 at lysine 27 (H3K27me3), a hallmark of repressed chromatin. Although overexpression of PRC2 components has been implicated in cancer initiation, progression and metastasis how PRC2 is recruited to its target genes remains poorly understood. Recent work suggests the interaction of PRC2 with the long-non coding (lnc) RNA HOX transcript antisense intergenic RNA (HOTAIR) could serve as a potential guide to silence target genes, but the mechanism has yet to be defined. In this study, we performed whole transcriptome RNA-seq analysis of isogenic cisplatin- sensitive and -resistant A2780 ovarian cancer (OC) cell lines. Of the lncRNA examined, HOTAIR was markedly induced in the cisplatin resistant cells (5-fold upregulation compared to the cisplatin-sensitve A2780 parental line). shRNA depletion of HOTAIR resensitized the drug-resistant cells to cisplatin. Based on analysis of the HOTAIR promoter using luciferase assays, we identified two putative NF-κB binding sites and three E-box binding sites. To further investigate whether these sites contributed to HOTAIR expression, platinum-sensitive cells were treated with different growth factors and cytokines (0 to 48hrs) and analyzed for changes in HOTAIR expression. Treatment with TNF-α, a well-known inflammatory cytokine and established activator of NF-κB, resulted in 16 fold-induction of HOTAIR. In accord with these results, we observed significant enrichment of both NF-κB and cMYC on the HOTAIR promoter using ChIP assay. Pharmacological inhibition of cMYC and NF-κB reduced HOTAIR expression by 2-fold, and siRNA depletion of the NF-κB p65 subunit or cMYC similarly reduced (P Citation Format: Ali R. Ozes, Dave Miller, Cong Guo, Anurag Bhattrai, Yunlong Liu, Kenneth P. Nephew. The transcriptional regulation of the long non-coding RNA HOTAIR in ovarian cancer. [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 5189. doi:10.1158/1538-7445.AM2013-5189