Putative Binding Research Articles

Binding of phenothiazines into allosteric hydrophobic pocket of human thioredoxin 1.

Thioredoxins are multifunctional oxidoreductase proteins implicated in the antioxidant cellular apparatus and oxidative stress. They are involved in several pathologies and are promising anticancer targets. Identification of noncatalytic binding sites is of great interest for designing new allosteric inhibitors of thioredoxin. In a recent work, we predicted normal mode motions of human thioredoxin 1 and identified two major putative hydrophobic binding sites. In this work we investigated noncovalent interactions of human thioredoxin 1 with three phenotiazinic drugs acting as prooxidant compounds by using molecular docking and circular dichroism spectrometry to probe ligand binding into the previously predicted allosteric hydrophobic pockets. Our in silico and CD spectrometry experiments suggested one preferred allosteric binding site involving helix 3 and adopting the best druggable conformation identified by NMA. The CD spectra showed binding of thioridazine into thioredoxin 1 and suggested partial helix unfolding, which most probably concerns helix 3. Taken together, these data support the strategy to design thioredoxin inhibitors targeting a druggable allosteric binding site.

Abstract A02: CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: A new actionable pathway for ovarian cancer patients.

Abstract Epithelial Ovarian Cancers (EOCs) consist in a heterogeneous group of tumors. Late diagnosis and platinum-resistant recurrences make EOCs the most lethal gynecological malignancy. The central role played by CDKs (Cyclin-Dependent Kinase) in several cellular mechanisms, such as control of cell cycle progression, DNA repair and transcription and the association of their activity in different apoptotic pathways, make them attractive targets to overcome platinum-resistance in EOCs. We explored the possibility that the activity of one or more CDK was necessary to overcome the platinum-induced cell death in EOC cells. To this aim we used an unbiased functional genomic screening targeting, with specific shRNAs, all the 23 members of the CDKs family alone or in association with platinum. This screening identified CDK6 as the CDK most significantly involved in the regulation of platinum sensitivity in EOC cells since CDK6 silencing did not affect cell survival per se but strongly increased platinum-induced cell death. The effect of CDK6 silencing on the sensitivity to both carboplatinum and cisplatin was confirmed in a larger panel of EOC cell lines using multiple shRNAs. Next, the use of CDK6 dominant negative (CDK6D163N) and constitutively active (CDK6R31C) mutants demonstrated that CDK6 kinase activity was necessary to protect from platinum-induced death. Accordingly, an orally active CDK4/CDK6 inhibitor, PD0332991, was able to sensitize EOC cells to CBDCA or CDDP treatment both in vitro and in vivo, in a CDK6-dependent, CDK4-independent manner. To understand the molecular mechanism whereby CDK6 regulates platinum-induced cell death in EOC we focused on specific CDK6 phosphorylation targets and demonstrated that the transcription factor FOXO3a was a relevant downstream target of CDK6. Functional and biochemical analyses showed that upon platinum exposure, CDK6 binds and phosphorylates FOXO3a on a specific Serine residue. We identified the Cyclin D3/CDK6 complex as the one principally involved in FOXO3a phosphorylation and proved that FOXO3a phosphorylation by the Cyclin D3/CDK6 complex leaded to increased FOXO3a protein stability. Accordingly, FOXO3a downregulation sensitizes EOC cells to platinum while FOXO3a overexpression in CDK6 silenced cells was able to rescue the effect of CDK6 silencing on platinum-induced cell death. Mechanistically, we showed that the CDK6/FOXO3a axis was necessary to regulate the DNA damage response in EOC cells through the regulation of ATR/CHK1 pathway since, after platinum treatment, the knock-down of CDK6 or FOXO3a induced the downregulation of ATR and the decrease of CHK1 phosphorylation. Accordingly, we identified in the ATR promoter three putative FOXO3a binding sites and, using chromatin immunoprecipitation, we confirmed that ATR was a FOXO3a transcriptional target, in platinum treated EOC cells. Inhibition of CDK6, FOXO3a or ATR increased platinum dependent-cell death via the induction of the so-called Premature Chromosome Condensation (PCC), a lethal event that selectively kills cancer cells when the ATR/CHK1 pathway is inhibited in the presence of damaged DNA. Overall we demonstrated that in EOC platinum sensitivity could be increased by specific CDK6 inhibition also using a clinically available small molecule, the PD0332991. We are currently analyzing if in primary EOC the expression of CDK6, FOXO3a and/or Cyclin D3 could be used as biomarkers to select those patients that may benefit from a combination therapy based on platinum and CDK6 inhibition, further increasing the translational relevance of our work. Citation Format: Alessandra Dall'Acqua, Schiappacassi Monica, Gustavo Baldassarre. CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: A new actionable pathway for ovarian cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A02.

Abstract PR03: P53 inhibits the expression of the pyrimidine catabolic gene Dihydropyrimidine dehydrogenase (DPYD)

Abstract Fluorouracil (5-FU) a widely used chemotherapeutic drug whose unpredictable pharmacokinetics is controlled by the pyrimidine catabolic gene dihydropyrimidine dehydrogenase (DPYD), that has recently also been shown to be a gatekeeper of the epithelial-to-mesenchymal transition (EMT) in breast cancer. Relatively little is known about the transcriptional control of DPYD and here we show for the first time an interaction between p53 and DPYD (involved in catabolism of pyrimidines as well as 5-FU) where p53 represses both the base-line expression of DPYD and that following 5-FU administration in vitro and in vivo. This mechanism affects the catabolic conversion of 5-FU to 5-FUH2 in mice in vivo. Using an in-silico approach we also identified several putative p53 binding sites (P53DBS) in and around ~20Kb upstream and downstream of the mouse DPYD gene. In-vivo ChIP from mice livers identified a key p53DBS binding site downstream (chr3: 119451237-11941257) of the gene to which p53 binds to at about 1.8 ± 0.05 fold over untreated control following a single IV bolus of 5-FU (150 mg/kg bw). Interestingly DPYD mRNA and protein levels were decreased by 1.8 and 1.5 fold respectively (P< 0.0005) in a p53-dependent manner in the liver. Similarly DPYD-repression was documented at both the mRNA and protein levels in the wild-type (WT) p53-expressing cancer cell lines H460, HCT-116, and A569. To further characterize the functional effect of DPYD repression in vivo, we utilized mice with a liver specific deletion of p53 (Albcre p53Δ/Δ) which showed ~2-fold (p≤0.03 wilcoxon rank-sum test) decrease plasma ratio of 5-FUH2/5-FU to mice with an intact allele of p53 in their livers (Albcre p53Δ/+). Increased plasma concentrations of 5-FU caused a significant tumor growth delay (TGD) in mouse colon cancer cells implanted in syngeneic Albcre p53Δ/+ versus Albcre p53Δ/Δ mice (TGD 11 vs 8.93 days). Our data also suggest a possible feedback mechanism of DPYD repression by p53 due to thymidylate synthase (TS) inhibition, a key mediator for supplying thymidine for DNA replication and itself a target for regulation by 5-FU. The addition of exogenous thymidine abrogated the p53-dependent repression of DPYD expression following siRNA-mediated targeting of TS. Further cross-validation experiments using the specific TS inhibitors raltitrexed and methotrexate showed similar effects. Furthermore treatment with the ATM (KU-55933) or DNA-PK (NU7026) inhibitors reversed the p53-dependent repression of DPYD-expression indicating a specific DNA damage feedback mechanism following TS inhibition. Taken together our data suggest that p53 plays an important role in controlling pyrimidine catabolism, particularly following metabolic stress imposed by nucleotide imbalances. In light of more recent discoveries of the role of DPYD in malignant progression, it is plausible that the relationship between p53 and DPYD can have implications beyond influencing the pharmacokinetics of 5-FU and may extend to control of EMT and metastasis. Thus our current efforts are focused on exploring short- and long-term consequences of p53-dependent DPYD-repression as part of p53⊠s tumor suppressor function as well as understanding cellular context for the observed link in vitro and in vivo. Citation Format: Prashanth Ravishankar Gokare, Niklas Finnberg, Jenny Dai, Wafik El-Deiry. P53 inhibits the expression of the pyrimidine catabolic gene Dihydropyrimidine dehydrogenase (DPYD). [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr PR03.

Abstract B63: CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: a new actionable pathway for ovarian cancer patients

Abstract Epithelial Ovarian Cancers (EOC) consist in a heterogeneous group of tumors. Late diagnosis and platinum-resistant recurrences make EOC the most lethal gynecological malignancies. The central role played by CDKs (Cyclin-Dependent Kinase) in some cellular mechanisms, like control of cell cycle progression, DNA repair and transcription and their activity in apoptotic pathways, make them attractive targets to overcome platinum-resistance in EOC. We explored if the activity of any CDK was necessary to overcome the platinum-induced cell death in EOC cells. We used an unbiased functional genomic screening targeting, with specific shRNAs, all the 23 members of the CDKs family alone or in association with platinum. This screening identified CDK6 as the most significantly involved in the regulation of platinum sensitivity since CDK6 silencing did not affect cell survival per se but strongly increased platinum-induced cell death. The effect of CDK6 silencing on the sensitivity to platinum drugs was confirmed in a larger panel of EOC cell lines. The use of CDK6 dominant negative (CDK6D163N) and constitutively active (CDK6R31C) mutants showed that CDK6 kinase activity was necessary to protect from platinum-induced death. Thus, an orally active CDK4/CDK6 inhibitor, PD0332991, was able to sensitize EOC cells to platinum treatment both in vitro and in vivo, in a CDK6-dependent CDK4-independent manner. To unveil the mechanism whereby CDK6 regulates platinum-induced cell death in EOC we focused on specific CDK6 phosphorylation targets and demonstrated that the transcription factor FOXO3a was a relevant downstream target of CDK6. Functional and biochemical analyses showed that upon platinum exposure, CDK6 binds and phosphorylates FOXO3a on a specific Serine residue. We identified the Cyclin D3/CDK6 complex as the one mainly involved in FOXO3a phosphorylation and proved that FOXO3a phosphorylation by the Cyclin D3/CDK6 complex leaded to increased FOXO3a protein stability. Moreover, FOXO3a downregulation sensitizes EOC cells to platinum while FOXO3a overexpression in CDK6 silenced cells was able to rescue the effect of CDK6 silencing on platinum-induced cell death. We showed that the CDK6/FOXO3a axis was necessary to regulate the DNA damage response in EOC cells through the regulation of ATR/CHK1 pathway, since after platinum treatment the silencing of both CDK6 or FOXO3a induced the downregulation of ATR and the decrease in CHK1 phosphorylation. We identified in the ATR promoter three putative FOXO3a binding sites and, using chromatin immunoprecipitation, we confirmed that ATR is a FOXO3a transcriptional target. Hence, inhibition of CDK6, FOXO3a or ATR increased platinum dependent-cell death via the induction of the so-called Premature Chromosome Condensation (PCC), a lethal event that selectively kills cancer cells when the ATR/CHK1 pathway is inhibited in the presence of damaged DNA. We demonstrated that in EOC, platinum sensitivity could be increased by specific CDK6 inhibition also using a clinically available small molecule, the PD0332991. We are currently analyzing if in primary EOC the expression of CDK6, FOXO3a and/or Cyclin D3 could be used as biomarkers to select patients that may benefit from a combination therapy based on platinum and CDK6 inhibition further increasing the translational relevance of our work. Citation Format: MONICA B. SCHIAPPACASSI, Alessandra Dall'acqua, Gustavo Baldassarre. CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: a new actionable pathway for ovarian cancer patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B63.

The Effects of Hsp90α1 Mutations on Myosin Thick Filament Organization.

Heat shock protein 90α plays a key role in myosin folding and thick filament assembly in muscle cells. To assess the structure and function of Hsp90α and its potential regulation by post-translational modification, we developed a combined knockdown and rescue assay in zebrafish embryos to systematically analyze the effects of various mutations on Hsp90α function in myosin thick filament organization. DNA constructs expressing the Hsp90α1 mutants with altered putative ATP binding, phosphorylation, acetylation or methylation sites were co-injected with Hsp90α1 specific morpholino into zebrafish embryos. Myosin thick filament organization was analyzed in skeletal muscles of the injected embryos by immunostaining. The results showed that mutating the conserved D90 residue in the Hsp90α1 ATP binding domain abolished its function in thick filament organization. In addition, phosphorylation mimicking mutations of T33D, T33E and T87E compromised Hsp90α1 function in myosin thick filament organization. Similarly, K287Q acetylation mimicking mutation repressed Hsp90α1 function in myosin thick filament organization. In contrast, K206R and K608R hypomethylation mimicking mutations had not effect on Hsp90α1 function in thick filament organization. Given that T33 and T87 are highly conserved residues involved post-translational modification (PTM) in yeast, mouse and human Hsp90 proteins, data from this study could indicate that Hsp90α1 function in myosin thick filament organization is potentially regulated by PTMs involving phosphorylation and acetylation.

Analysis of L1-chimeric transcripts derived from bidirectional promoter of human-specific L1

LINE-1s (L1s) have contributed to gene structure variation and they can affect the expression of nearby genes in the human genome. Here, we collected 35 human-specific L1s that may play a role as alternative promoters. In addition, we identified 54 L1-chimeric transcripts generated from these L1s using bioinformatics’ tools and carried out reverse transcription-PCR to analyze their expressional pattern in 20 human normal tissues and 9 human cancer tissues. Consequently, 30 L1-chimeric transcripts were experimentally confirmed. Most L1-chimeric transcripts were broadly expressed. Interestingly, we found that EST CD709363 derived from C14orf37 gene was expressed in trachea only among normal tissues, but it was expressed in several cancer tissues including brain, lung, skin, and esophagus. We also newly identified three alternative transcripts, which were not in the UCSC genome database. One alternative transcript was derived from RABGAP1L gene and the other two transcripts were from CAMK4 gene. In addition, we analyzed putative transcription binding sites within the four L1s located in the promoter region. These had several transcription factor binding sites related to promoters. Our results show that human-specific L1s could contribute to human transcriptome diversity and transcriptional gene expression in different types of human tissues.

Abstract B33: Regulation and function of Nrf2-associated long noncoding RNA

Abstract We hypothesized that the long non coding RNA (lncRNA), Loc344887, is induced by Nrf2 activation and acts as a transcriptional co-activator of the Nrf2 target gene NQO1. Nrf2 is a transcription factor that is important in oxidative stress responses. Upon oxidative stress, Nrf2 is released from its binding partner Keap1. Nrf2 translocates to the nucleus, partners with small Maf proteins, binds to antioxidant response elements (ARE) and induces detoxifying and metabolizing genes such as NQO1, HMOX1, and GSTs. We propose that Loc344887 is acting as a co-activator of Nrf2, either directly in the Nrf2-Maf complex, or indirectly by some other downstream mechanism, in order to regulate NQO1 expression. Loc344887 has significantly reduced expression in human colon tumors when compared to normal colon tissue (TCGA database). Human HCT116 colon cancer cells and CCD841 non-cancer colonic epithelial cells replicate this expression difference between tumor and non-transformed. We used these cell lines to study the regulation and function of Loc344887. HCT116 and CCD841 cells were treated with 15 μM sulforaphane (SFN), a dietary isothiocyanate known to have chemopreventative properties due to Nrf2 activation. Loc344887 increased 40-fold in HCT116 cells, but was not induced in CCD841 cells. Loc344887 was also induced by oltipraz and tert-butylhydroquinone, known Nrf2 activators. Keap1 knockdown upregulated Loc344887, and inducibility of Loc344887 by SFN was lost when Nrf2 was knocked down using RNAi. The gene promoter of Loc344887 contains putative AREs, and chromatin immunoprecipitation assays identified putative Nrf2 and MafK binding regions. Because Loc344887 is regulated by Nrf2, we hypothesized that Loc344887 may be involved in NRF2 gene activation. Knockdown of Loc344887 caused a loss of NQO1 induction by both SFN and Keap1 knockdown; however, this effect was not seen for HMOX1. Time-course assays showed that Loc344887 is induced as early as 1 h post-SFN treatment and peaks at ~8 h, similar to HMOX1. NQO1, however, was induced much slower, peaking after 24 h. This suggests that other factor(s), such as Loc344887, needs to be present for full induction to occur. This study shows that Loc344887 is an Nrf2 target gene and is required for complete NQO1 induction, but not for HMOX1 activation. Thus, there is a differential response for Nrf2 target genes that depends, in part, on lincRNAs as co-regulators. Further studies are required to access the mechanism of NQO1 regulation. We hypothesize that once Loc344887 is induced, it acts as a scaffold for MafK, Nrf2 and/or other transcription factors on the NQO1 promoter. Loc344887 localizes primarily to the nucleus, and preliminary RNA immunoprecipitation experiments show that Loc344887 binds directly to MafK. Further studies will also test whether Loc344887 is required for regulation of other Nrf2 target genes, or if other lncRNAs are involved in Nrf2 target gene induction. Studies supported in part by Chancellor's Research Initiative funding from Texas A&M University, and by NIH grants CA090890, CA122959, P30 ES00210, P30 ES023512. Citation Format: Gavin Johnson, Laura Beaver, David E. Williams, Emily Ho, Roderick H. Dashwood. Regulation and function of Nrf2-associated long noncoding RNA. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B33.

Abstract P168: ChIP-seq Analysis of Genomic Binding Sites for the Transcription Factor Fosl2 in Kidney Collecting Duct Cells

Kidney collecting duct principal cells play a key role in blood pressure regulation and water balance in part through the action of vasopressin. We integrated data from prior proteomics and transcriptomics studies of cultured mouse mpkCCD cells using Bayes’ Rule to rank transcription factors (TFs) likely to be involved in vasopressin-mediated transcriptional regulation. The top ranked TF was the AP-1 component Fos-like 2 (Fosl2) and the third ranked TF was Sox4. We carried out ChIP-seq analysis (HiSeq 2000 sequencer) to identify genomic sites of Fosl2 binding using an antibody successfully employed for ChIP-seq in the Mouse ENCODE Project. mpkCCD cells treated with the vasopressin analog dDAVP or vehicle were separately analyzed. The success of the immunoprecipitation of Fosl2 was confirmed by western blotting and LC-MS/MS of the immuno-precipitated material (ChIP-MS). ChIP-MS identified two different Fosl2 peptides, but there were no peptides corresponding to other AP-1 proteins. ChIP-MS also identified Bub1b, a nuclear protein kinase known to phosphorylate histones, as a Fosl2-interacting protein. Fosl2-binding sites successfully mapped to genes by ChIP-seq included a Fosl2 binding site, centered at 87 bp upstream from the transcription start site [TSS] of Sox4, that was strongly downregulated in response to dDAVP, n=2; confirmed by ChIP-PCR. TF binding site prediction (Genomatix) identifies putative Sox4 binding sites in vasopressin-regulated genes beta-ENaC (-822 bp from TSS) and aquaporin-2 (-515 bp from TSS). These data provide an initial step in identification of the vasopressin-regulated transcriptional network in renal collecting duct cells.

Abstract 2208: Interferon Consensus Sequence-Binding Protein (ICSBP) regulates the transforming growth factor-beta type I receptor expression in osteosarcoma cells

Abstract Transforming growth factor-beta (TGF-β), known as a tumor suppressor, may exert tumor promoting activity in the advanced stage of cancer. Previously, we reported that interferon consensus sequence-binding protein (ICSBP), also known as interferon regulatory factor-8, up-regulates TGF-β type I receptor (TGF-βR I), and augments growth in HL-60 leukemic cells. In the current study, we demonstrate that ICSBP expression up-regulates TGF-βR I and increase in cell growth in U2OS human osteosarcoma cells. To further investigate the mechanism of TGF-βR I up-regulation by ICSBP, we tested the possibility that ICSBP regulates promoter activity of TGF-βR I gene. We analyzed the promoter region of the human TGF-βR I gene in U2OS cells to find putative transcription elements regulated by ICSBP. Luciferase reporter gene assays indicated that ICSBP expression could enhance TGF-βR I gene expression, and by promoter deletion analysis we identified a functional ICSBP-dependent region located at nucleotides -220 to +5 in the TGF-βR I promoter. We also performed EMSA (Electro Mobility Shift Assay) using probe and found that there are putative ICSBP binding sites between -222 to -204 regions. In conclusion, ICSBP increased TGF-βR I expression by binding to TGF-βR I promoter (-220/218) regions in U2OS cells. Note: This abstract was not presented at the meeting. Citation Format: Jee Young Sung, Hyeryeong Kim, Kyung-Sil Yoon, Yong-Nyun Kim, Byung-Kiu Park. Interferon Consensus Sequence-Binding Protein (ICSBP) regulates the transforming growth factor-beta type I receptor expression in osteosarcoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2208. doi:10.1158/1538-7445.AM2015-2208

The multicopy sRNA LhrC controls expression of the oligopeptide-binding protein OppA in Listeria monocytogenes

Listeria monocytogenes is the causative agent of the foodborne disease listeriosis. During infection, L. monocytogenes produces an array of non-coding RNAs, including the multicopy sRNA LhrC. These five, nearly identical sRNAs are highly induced in response to cell envelope stress and target the virulence adhesin lapB at the post-transcriptional level. Here, we demonstrate that LhrC controls expression of additional genes encoding cell envelope-associated proteins with virulence function. Using transcriptomics and proteomics, we identified a set of genes affected by LhrC in response to cell envelope stress. Three targets were significantly down-regulated by LhrC at both the RNA and protein level: lmo2349, tcsA and oppA. All three genes encode membrane-associated proteins: A putative substrate binding protein of an amino acid ABC transporter (Lmo2349); the CD4+ T cell-stimulating antigen TcsA, and the oligopeptide binding protein OppA, of which the latter 2 are required for full virulence of L. monocytogenes. For OppA, we show that LhrC acts by direct base paring to the ribosome binding site of the oppA mRNA, leading to an impediment of its translation and a decreased mRNA level. The sRNA-mRNA interaction depends on 2 of 3 CU-rich regions in LhrC allowing binding of 2 oppA mRNAs to a single LhrC molecule. Finally, we found that LhrC contributes to infection in macrophage-like cells. These findings demonstrate a central role for LhrC in controlling the level of OppA and other virulence-associated cell envelope proteins in response to cell envelope stress.

Characterization and cloning of an 11S globulin with hemagglutination activity from Murraya paniculata.

A ~56 kDa protein having hemagglutination activity was purified and characterized from the Murraya paniculata seeds. The gel electrophoresis studies demonstrated that protein is primarily of two different subunits, molecular weight ~ 35 and 21 kDa held together by disulfide-linkages and predominantly by secondary forces. The cloning and sequence analysis revealed that the protein exhibited a substantial sequence identity to seed storage 11S globulin family proteins. The sequence analysis of Murraya paniculata globulin (MPG) demonstrated higher and lower molecular weight polypeptides to be acidic (α) and basic (β) respectively. The sequence analysis further showed that it possesses a characteristic bi-cupin motif and a putative metal binding pocket. CD analysis revealed that the MPG was a β/α protein with a slightly higher content of the former. Conformational changes in protein have been studied by fluorescence spectrometry by using various chemical treatments. The results demonstrated that MPG belongs to 11S globulin family and exhibit's hemagglutination activity, which implicates it to be possessing lectin-like property.

Abstract B07: Estrogen-related receptor alpha regulates S6K1 expression and tamoxifen sensitivity in breast cancer

Abstract Estrogen related receptor alpha (ERRα) is an orphan nuclear receptor that plays important role in the metabolic activities and proliferation of cancer cells. We investigated the role of ERRα in regulating the expression of S6K1 in breast cancer cells. First, using RT-qPCR and immunoblotting we found that ERRα overexpression in estrogen receptor (ER) positive MCF7 cells results in decreased S6K1 expression and activity. Conversely, downregulation of ERRα expression resulted in increased S6K1 expression and activity in ER-negative MDA-MB-231 cells. Second, overexpression of ERRα in MCF7 cells also resulted in decreased transcriptional activity of the RPS6KB1 gene promoter as revealed by luciferase reporter assay. On the other hand, ERRα knockdown in MDA-MB-231 cells resulted in increased promoter activity. Third, because the proximal 1 kilobase (Kb) promoter region of S6K1 contains several putative estrogen receptor DNA binding elements (EREs), we investigated whether ERRα modulates S6K1 expression by direct promoter binding using chromatin immunoprecipitation (ChIP) technique. We detected two binding peaks approximately 300 and 600 kb away from the transcription start site (TSS). In addition, we showed that downregulation of ERRα caused increased cell proliferation and sensitivity to the mTOR inhibitor rapamycin. Suprisingly, tamoxifen treatment increased the proliferative capacity of these cells. Interestingly, cytoplasmic and nuclear staining of ERRα in tumor specimens derived from patients in the Stockholm breast cancer trial of adjuvant tamoxifen revealed that (1) in triple-negative tumors, the low-expressing cytoplasmic ERRα group had a worse prognosis, whereas a clear improvement in recurrence-free survival was found in the group with high ERRα-expressing tumors; and (2) in the ERα-negative subgroup, high ERRα rendered a clear tamoxifen benefit, which is very interesting, as this group is not receiving tamoxifen today and may constitute a subgroup which could gain from tamoxifen. Overall, our data indicate that ERRα regulates S6K1 expression and affects the sensitivity to rapamycin tamoxifen. Our long-term goal is to establish ERRα as a marker for endocrine and rapamycin therapy response, as well as a novel target in combination therapy approaches. Citation Format: Subrata Manna, Josefine Bostner, Anya Alayev, Olle Stal, Marina K. Holz. Estrogen-related receptor alpha regulates S6K1 expression and tamoxifen sensitivity in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B07.

De-novo design, synthesis and evaluation of novel 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivatives as HIV-1 reverse transcriptase inhibitors.

BackgroundAcquired Immune Deficiency Syndrome (AIDS) is the advanced stage of infection caused by Human Immunodeficiency Virus (HIV). HIV/AIDS had a great impact on society, both as an illness and as a source of discrimination. Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) are structurally diverse group of compounds which binds to Reverse Transcriptase (RT) enzyme of HIV. Like other anti-HIV drugs, long-term clinical effectiveness of approved NNRTIs has been hampered due to the rapid development of drug resistance. So, there is an urgent need to discover the NNRTIs, which can be effective against the drug sensitive as well as drug resistant strains of HIV-1 RT.ResultsTwo series of novel thirty, 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline analogues (5a-o) and (8a-o) were designed and synthesized as inhibitor of HIV-1 reverse transcriptase. All the synthesized compounds were characterized by infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, mass spectroscopy and evaluated for in-vitro RT inhibitory activity. Among the tested compounds, eighteen compounds exhibited more than 50 % inhibition at tested 100 μM concentration, in which two compounds 8h and 8l showed promising inhibition (74.82 and 72.58 %) respectively. The preliminary structure–activity relationship (SAR) of the test compounds and docking studies of the two significantly active compounds 8h and 8l were performed to examine their putative binding with HIV-RT. Predicted physiochemical parameters of the synthesized compounds were within the acceptable range of drugable properties.ConclusionThe results obtained from this investigation revealed that, the synthesized compounds (5a-o) and (8a-o) showed moderate to promising HIV-1 RT inhibition activity. The overall SAR studies can help in identification of further lead as well as in designing of newer potential inhibitor of HIV-1 RT.Graphical Best docked pose of compound 8h inside the non-nucleoside inhibitory binding pocket of 3MEE enzyme.

Crystal structure of the MazG-related nucleoside triphosphate pyrophosphohydrolase from Thermotoga maritima MSB8.

The MazG family proteins, which are highly conserved in bacteria, are nucleoside triphosphate pyrophosphohydrolases that hydrolyze all canonical nucleoside triphosphates, and are also involved in removing noncanonical nucleoside triphosphates to prevent their incorporation into DNA or RNA. The primary structure of TM0360 from Thermotoga maritima MSB8 suggested that TM0360 is a MazG-related nucleoside triphosphate pyrophosphohydrolase. The crystal structure of the TM0360 protein was determined by the MAD technique at 2.0Å resolution. The asymmetric unit contains an intact dimer molecule. The overall structure of TM0360 is similar to the known structures of the dimeric MazG protein and dUTPases. The putative NTP binding pocket in TM0360, identified by considering the probable NTP-interacting residues and structural features, suggested that TM0360 resembles the C-terminal domain of Escherichia coli MazG, although TM0360 may be a truncated paralog of the N-terminal domain of T. maritima MazG (TM0913), according to its primary structure. The putative function of TM0360 is discussed, based on structural homology.

Biological Property of Recombinant Hemagglutinin-Neuraminidase Protein of Avian Paramyxovirus Type 6 Expressed by Recombinant Baculovirus

Hemagglutination inhibition (HI) test employing whole virus antigen is a prescribed serological test for serotyping, diagnosis and surveillance for avian paramyxoviruses (APMVs). For use as alternative to the virus antigen, hemagglutininneuraminidase (HN) protein gene of the wild duck isolate APMV-6/WB12-163FS of APMV serotype 6 (APMV-6) was amplified, cloned and expressed in Spodoptera frugiperda insect cells. The HN gene of 1,842 bps in length showed nucleotide and amino acid homology of 93.4% and 97.1%, respectively with that of APMV-6 prototype strain. Putative sialic acid binding motif and potential N-linked glycosylation sites were conserved. In Western blot analysis, the expressed protein had a molecular mass of 66 kDa and reacted specifically with antiserum to APMV-6. In addition, the recombinant HN protein showed biological properties such as hemagglutination (HA) and elution. The recombinant HN protein produced from infected cells showed high HA titers (approximately 2 13 HA unit/ml). The HA activity of the recombinant HN protein was inhibited by antisera to APMV-6. In cross HA inhibition test, the recombinant HN protein had the highest titers with antisera to homologous APMV serotype, although there was weak cross reaction with some of antisera to other APMV serotypes. Our results indicated that recombinant APMV-6 HN protein would have the potential as alternative to the APMV-6 antigen in HI assays.

Abstract B40: Blocking Ras chaperons for cancer therapy

Abstract We have previously developed a new concept and a new class of Ras inhibitors that affect specifically the active forms of Ras. This concept is based on the knowledge that the farnesyl moiety common to all Ras proteins might not only serve as a lipophilic lipid anchor but also confer functional specificity on Ras. This raised the possibility that the farnesyl group may act as part of a recognition unit for specific anchorage lipids or protein(s) that interact with Ras in the cell membrane. Such binding partners of Ras have been identified where it was shown that galectin-1 and galectin-3 discovered first as lectin binding proteins, act respectively as specific binding partners of farnesylated active H-Ras and K-Ras. Recently Nucleoline was identified as a specific chaperon of active N-Ras. The galectins possess a putative hydrophobic binding pocket-the putative farnesyl binding site. Galectin-1 was shown to drive H-Ras.GTP nanocluster formation and to be an integral component of the H-Ras-GTP nanocluster, a site at which Raf is activated. Similarly, galectin-3 drives K-Ras.GTP nanoclustring and robust K-Ras signaling. Ras signaling to ERK is strongly diminished in Galectin -3 knockout MEFS and in thyroid cancer cells that lack Galectin - 3. Together these findings mark the farnesyl-binding pockets as outstanding targets for Ras directed therapy, assuming that compounds that would block such sites will act as Ras inhibitors. The Ras inhibitor, S-trans-trans farnesylthiosalicylic acid (Salirasib, FTS) was indeed designed to mimic the farnesyl moiety in the carboxy terminal of Ras and was found to induce accelerated dislodgement of Ras from the cell membrane and subsequently degradation of the protein. FTS was also found to be highly efficient in inhibiting growth of many types of cancer cell lines that exhibit high Ras.GTP levels both, in vitro and in vivo. More recent phase 2 clinical trials showed that oral Salirasib treatment of pancreatic and of non-small-lung cancer patients increased significantly survival of the patients with no toxic effects. We propose to use Salirasib for the treatment of pancreatic and of non-small-lung carcinoma. Note: This abstract was not presented at the conference. Citation Format: Yoel Kloog, Roni Haklai, Galit Elad-Sfadia. Blocking Ras chaperons for cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B40. doi: 10.1158/1557-3125.RASONC14-B40

Abstract A19: A Ras-Arf-Egr-C/EBPβ axis underlying oncogene-induced senescence and cancer

Abstract In normal cells, persistent oncogenic Ras activation triggers senescence, an intrinsic tumor suppression mechanism that protects cells from malignant transformation. Oncogene-induced senescence (OIS) is dependent on tumor suppressors such as p19/p14Arf, and loss of those genes facilitates transformation by oncogenic Ras. We previously reported that C/EBPβ, a transcription factor that has both pro- and anti-oncogenic functions, contributes to OIS in primary fibroblasts. Notably, C/EBPβ is down-regulated in H- or K-RasV12-transformed NIH 3T3 cells in a manner that requires loss of p19Arf. The potential role of C/EBPβ down-regulation in RasV12-induced transformation prompted us to investigate the mechanisms of Cebpb gene transcription. Here we show that Cebpb is an early growth factor-induced gene whose induction is suppressed by H-RasV12 in NIH 3T3 cells and p19Arf-/- MEFs. The early growth response (Egr) transcription factors are also down-regulated by H-RasV12 in these cells. Over-expression of any of Egr proteins (Egr1-4) restored C/EBPβ levels in 3T3Ras cells. Interestingly, Egrs induce growth arrest and senescence in 3T3Ras cells in a manner that is only partially dependent on reactivation of C/EBPβ. Egr1-4 proteins stimulated transcription from a promoter-reporter construct containing ∼3 kb of the Cebpb 5' flanking region. The proximal Cebpb promoter contains several putative Egr binding motifs, and mutating three of these sites disrupted Egr-mediated transactivation of the Cebpb promoter. Chromatin immunoprecipitation assays showed that ectopically expressed and endogenous Egrs bind to the Cebpb proximal promoter. Furthermore, microarray data from Oncomine show a strong correlation between CEBPB and EGR expression in human tumor samples, regardless of whether C/EBPβ levels increase or decrease in tumors compared to normal tissue. Since CEBPB and EGRs are down-regulated in lung adenocarcinomas and their elevated expression is associated with a better prognosis in lung cancer patients (Prognoscan), we over-expressed Egrs in A549 lung adenocarcinoma cells, which carry a KRAS mutation. Ectopic Egr2-4 induced CEBPB mRNA expression and dramatically suppressed cell proliferation. Collectively, our data indicate that decreased Egr levels account for oncogenic Ras-induced down-regulation of C/EBPβ in certain transformed cells, and restoration of Egrs induces growth arrest and C/EBPβ expression. Our findings identify the Egr-C/EBPβ axis as an important pathway in determining the cellular response (transformation or senescence) to oncogenic Ras-Arf signaling. Citation Format: Jacqueline Salotti, Krisada Sakchaisri, Peter F. Johnson. A Ras-Arf-Egr-C/EBPβ axis underlying oncogene-induced senescence and cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A19. doi: 10.1158/1557-3125.RASONC14-A19

Abstract 2310: Epigenetic silencing of a potential tumor suppressor NR4A3 by aberrant JAK/STAT signaling predicts prognosis in gastric cancer

Abstract Gastric cancer is the second leading cause of cancer worldwide. Epigenetic silencing of tumor-suppressors has emerged as an important underlying mechanism in the gastric carcinogenesis. Previous studies showed that infection of H. pylori activates JAK/STAT3 signaling pathway in gastric cancer. However, the role of this aberrant signaling remains unclear. We hypothesized that activation of JAK/STAT signaling leads to epigenetic silencing of STAT3 target genes in gastric cancer. To test this hypothesis, a constitutively activated mouse STAT3 mutant (STAT3c) was transfected into MKN28 gastric cancer cells in which the JAK/STAT signaling pathway is inactive. STAT3c stable transfectant (S16) showing hyperphosphorylation of STAT3 demonstrated increased cell proliferation as compared to vector control (C9). Integrative expression microarray coupled with bioinformatic analysis identified putative STAT3 targets, NR4A3 that are down-regulated in S16 cells. In association with up-regulation of DNMT1, NR4A3 exhibited increased promoter methylation in S16 but not C9 cells as demonstrated by bisulphite sequencing and demethylation treatment. Interestingly, NR4A3 was also found to be epigenetically silenced in AGS cells where JAK/STAT signaling is constitutively activated. ChIP-PCR experiment revealed that STAT3 bound to the putative STAT3 binding site in NR4A3 promoter of AGS cells. Depletion of STAT3 by lenti-viral knockdown restored NR4A3 expression in this cell. Interestingly, luciferase reporter assay using the NR4A3 promoter containing putative STAT3 binding site exhibited a further 1.6 fold increment after deleting the STAT3 binding region (P < 0.005). Ectopic expression of NR4A3 in AGS cells reduced cancer cell growth in colony formation assay (P < 0.001). In clinical specimens, quantitative MSP demonstrated a significant correlation between the degree of NR4A3 methylation and STAT3 nuclear translocation in 72 gastric tumor samples (P < 0.05). Importantly, methylation of NR4A3 was significantly associated with patients with shorter survival (P < 0.05). In conclusion, our result demonstrated that aberrant JAK/STAT3 signaling confers epigenetic silencing of a potential tumor suppressor, NR4A3 in gastric cancer. Methylation of NR4A3 may be able to serve as a prognostic indicator in gastric cancer patients. Citation Format: Michael W. Y. Chan, Li-Han Zeng, Liang-Yu Chang, Claudia Dittner, Jian-Liang Chou, Yao-Ting Huang, Alfred S.L. Cheng, Jiayuh Lin, Kun-Tu Yeh. Epigenetic silencing of a potential tumor suppressor NR4A3 by aberrant JAK/STAT signaling predicts prognosis in gastric 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 2310. doi:10.1158/1538-7445.AM2014-2310

Abstract 1385: Orphan nuclear receptor estrogen-related receptor alpha (ERRα) characterized as a novel transcriptional regulator of the oncogenic fusion gene TMPRSS2:ERG in prostate cancer

Abstract The expression of androgen-responsive TMPRSS2:ERG fusion gene, which results in aberrant expression of the oncogenic transcription factor ERG under the regulation of androgen receptor (AR), is characterized as the predominant factor responsible for the initiation and also advanced progression of prostate cancer (PCa). However, its upstream transcriptional regulation in androgen-independent PCa is still not fully understood. Recent studies show that the orphan nuclear receptor estrogen-related receptor alpha (ERRα/NR3B1) plays certain regulatory roles in the advanced progression of prostate cancer, such as its cross-talk with AR signaling. Based on this, we hypothesize that besides AR, ERRα could also regulate or target to the promoter of fusion gene TMPRSS2:ERG in prostate cancer cells. Here we showed that treatment with an ERRα-specific inverse agonist (XCT790) or knockdown of ERRα could decrease the TMPRSS2:ERG expression in AR-positive VCaP prostate cancer cells. Besides, the expression levels of TMPRSS2:ERG could be elevated by ectopic ERRα expression, with further elevation by co-expression with a genetically modified ERRα-specific coactivator PGC1α(2X9), in AR-negative NCI-H660 prostate cancer cells. By ChIP and luciferase reporter analysis, three putative ERRα transactivation DNA binding sites were identified in the promoter region of TMPRSS2:ERG fusion gene. Besides, in vitro phenotype characterization studies showed that ectopic expression of TMPRSS2:ERG, using an ERRα-responsive and TMPRSS2:ERG fusion gene promoter-driven expression plasmid in AR-negative prostatic cell lines, including PC-3 prostate cancer cells and BPH-1 immortalized prostatic epithelial cells, could promote their invasion capacity. Moreover, both ERRα and TMPRSS2:ERG showed elevated expressions in a xenograft model of castration-resistant prostate cancer cells (VCaP-CRPC). Besides, luciferase reporter assays showed that both TMPRSS2:ERG promoter and ERRα-binding element (ERRE) transcriptional activities were elevated in VCaP-CRPC cells as compared to the VCaP parental cells; and their transcriptional activities could be suppressed by XCT790 or sh-ERRα. Based on these results, we conclude that ERRα could directly transactivate the TMPRSS2:ERG fusion gene in prostate cancer and may contribute to its transactivation in AR-negative prostate cancer cells and the advanced castration-resistant prostate cancer. This study is supported by a General Research Fund from the Research Grants Council of Hong Kong (Project code: 2140629). Citation Format: Zhenyu Xu, Shan Yu, Franky Leung Chan. Orphan nuclear receptor estrogen-related receptor alpha (ERRα) characterized as a novel transcriptional regulator of the oncogenic fusion gene TMPRSS2:ERG in prostate 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 1385. doi:10.1158/1538-7445.AM2014-1385

Abstract 2191: Identification of functional risk alleles in colorectal cancer-associated regions in African Americans

Abstract Background. There are disparities in both colorectal cancer (CRC) incidence and survival between African Americans (AAs) and Non-Hispanic Whites (NHWs). Genetic susceptibility factors are believed to contribute to CRC development but have been inadequately studied in AAs. Our previous work showed both shared and independent CRC-associated risk alleles are present in some gene regions identified in genome-wide association studies (GWAS) in NHWs. In the present work, using a functional bioinformatics approach, we searched for risk alleles in these regions in AAs. Methods. 10 chromosomal regions (8q23.3, 8q24.21, 10p14, 11q23.1, 14q22.2, 15q13.3, 16q22.1, 18q21.1, 19q13.11, 20p12) from COGENT consortium meta-analysis were selected for DNA sequencing. Three additional high-interest regions were included. The region surrounding each associated SNP was analyzed using r2 plots, based on European-ancestry genotype data from HapMap Phase 3. DNAs from 63 individuals (25 AA CRC cases and 38 AA controls) were sequenced using Agilent SureSelect enrichment arrays and Illumina HiSeq platform technology. Variants from the targeted sequencing were combined with 1000 genomes variant data for a total of 16,193 variants. Potentially functional variants were identified based on the following criteria: the variant is (i) in a coding region, (ii) an expression quantitative trait locus (eQTL) or a DNAseI eQTL (DsQTL), (iii) in a miRNA binding site, and (iv) in a transcription factor binding site. Golden Helix software was used to process the data. Using the Sequenom Massarray platform, we genotyped variants in discovery and replication sets for a total of 1177 AA CRC cases and 1264 AA controls. We calculated odds ratios (OR) and 95% confidence intervals using logistic regression, assuming an additive genetic model and adjusting for age, sex, and African ancestry. Results. We identified 81 variants in coding sequences, 82 eQTLs, 5 dsQTLs, 38 variants in putative microRNA binding sites, 33 variants in possible transcription factor binding sites, and 6 variants that are in two or more of the preceding categories, for a total of 245 variants. After assay development and quality control, 163 SNPs were available for genetic analysis. Nominal associations (p<0.05) were detected between CRC and 17 variants. We have completed genetic analysis in the replication set for 8 of the 17 SNPs. The SNPs rs1862748 (OR, 0.73; P = 0.001) in the CDH1 region and 16888699 (OR, 1.39; P = 0.0006) in the EIF3H region were statistically significant in both the discovery and replication series. rs1862748 has previously been associated with AA CRC and was identified as a eQTL for ZFP90. rs16888699 was identified as an eQTL for UTP23. Conclusion. Our results suggest that rs1862748 and rs16888699 are CRC risk alleles, validating the bioinformatics approach. Functional studies will are now needed to characterize the genetic mechanisms underlying risk. Citation Format: Fabio Pibiri, Ricky Rechenmacher, Rosa Xicola, Rick Kittles, Robert Sandler, Temitope Keku, Xavier Llor, John Carpten, Sonia Kupfer, Nathan Ellis. Identification of functional risk alleles in colorectal cancer-associated regions in African Americans. [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 2191. doi:10.1158/1538-7445.AM2014-2191