bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2005: Modulation of tumor suppressor gene DNA-methylation by quercetin and dietary indoles
Authors: Samuel Temesgen , Fadlalla Khalda , Yehualaeshet Teshome , Turner Timothy
Published: July 2012

Epidemiological and preclinical research provide strong evidence for the anticancer properties of bioactive dietary compounds abundantly found in fruits, vegetables and other fiber-rich food stuff. However, the mechanisms by which nutritional intake of such dietary compounds prevent oncogenesis is largely unknown. We hypothesize that synergistic activities of different dietary compounds through epigenetic mechanisms underlie some of the chemopreventive functions of the dietary compounds. We undertook a preliminary study to examine the effects of Quercetin, Indole-3-Carbinol (I3C) and diindolylmethane (DIM) on promoter methylation profiles of selected genes known to be suppressed by methylation in colorectal cancer. Quercetin is a polyphenolic flavonoid, while I3C and DIM are dietary indoles, DIM being a derivative of I3C. The genes queried in this study were p16INK4a, E-cadherin (CDH1), and IGFBP7. p16INK4a is a cell cycle regulator silenced in up to 40-50% of colorectal carcinomas. E-cadherin is a tumor suppressor and metastasis regulator protein the loss of which promotes wnt-signaling, which is dysregulated in the majority of colorectal cancers. IGFBP7 has recently been identified as a p53-responsive gene frequently silenced in colorectal and gastric cancers. In this study, RKO colorectal cancer cells were treated with the three dietary compounds, vehicle (DMSO), or the demethylating compound 5-azacytidine. DNA was harvested and bisulfite converted. Methylation of the gene promoters was examined by methylation-specific PCR using primer sets that amplify only methylated DNA. Gel electrophoresis and Real-time PCR were used to assess the relative abundance of methylated DNA in control and treated samples. Preliminary results show that the dietary compounds can reverse the methylated-promoter phenotype, with the indole compounds having higher potency compared to quercetin especially in demethylating the CDH1 gene promoter. Further experiments will elucidate the molecular mechanisms through which the compounds modulate gene methylation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2005. doi:10.1158/1538-7445.AM2011-2005
Abstract 2007: Epigenetic reactivation of CCN5/WISP2 by combined effect of green tea polyphenol and epigenetic modulators in highly aggressive breast cancer cells
Authors: Chattopadhyay Indranil , Majumder Monami , De Archana , Banerjee Snigdha , Vanveldhuizen Peter , Banerjee Sushanta
Published: July 2012

CCN5/WISP-2 has been considered as an antiinvasive gene because CCN5/WISP-2 silencing augments the invasive phenotypes in vitro. One of the major functions of the CCN5 protein is to block aggressive behavior, such as the mesenchymal-to-epithelial transition (MET) of cancer cells. CCN5/WISP-2 is constitutively expressed in less aggressive human breast cancer cells, whereas its expression is undetected in the highly aggressive breast cancer cells. Dietary green tea polyphenol, (−)-epigallocatechin-3-gallate (EGCG), is believed to be an anticancer agent in part through its regulation of epigenetic processes. Our studies were aimed to address the epigenetic mechanisms of CCN5 reactivation by EGCG in highly aggressive breast cancer cells. In our current studies, we found that EGCG can reactivate CCN5 expression in highly aggressive breast cancer cell line (MDA-MB-231) and augments mesenchymal-epithelial transition process by modulating multiple EMT regulators. Combination studies using EGCG with the epigenetic modulators including the histone deacetylase (HDAC) inhibitor, trichostatin (TSA) and the DNA methyltransferase (DNMT) inhibitor such as 5-aza-2’-deoxycytidine (5- aza), revealed a synergistic effect of reactivation of CCN5 expression in highly aggressive breast cancer cells. Our findings help to assess the key mechanisms of EGCG chemoprevention and therapy by impacting epigenetic pathways toward aggressive breast tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2007. doi:10.1158/1538-7445.AM2011-2007
Abstract 2008: Effect of histone deacetylase inhibitor in combination with 5-fluorouracil on pancreas cancer and cholangiocarcinoma cell line
Authors: Yamada Shinichiro , Shimada Mitsuo , Utsunomiya Tohru , Morine Yuji , Imura Satoru , Ikemoto Tetsuya , Hanaoka Jun , Mori Hiroki , Kanamoto Mami , Iwahashi Shuichi , Saito Yu , Asanoma Michihito
Published: July 2012

Background: Histone deacetylase (HDAC) is well known to be associated with epigenetic regulation and carcinogenesis, and its inhibitors (HDACIs) induce the differentiation or apoptosis of cancer cells. Valproic acid (VPA) is one of the HDACIs, and it is clinically available as an oral antiepilepsy drug. We have reported that VPA augmented the anti-tumor effects of gemcitabine (GEM) in pancreas cancer cell lines and a cholangiocarcinoma cell line (2010 ASCO-GI), and we plan the clinical trial of combination therapy, S-1, that is a novel oral fluoropyrimidine derivative consisting of 5-fluorouracil (5-FU) prodrug tegafur, and VPA. We examined the augment effects of VPA with a combination of 5-FU in vitro.

Methods: A human pancreas cancer cell line (SUIT-2) and a cholangiocarcinoma cell line (HuCCT1) were used. The anticancer effects of VPA combined with 5-FU in SUIT-2 and HuCCT1. The cell viability was evaluated by MTT assay.

Results: Pancreas cancer cell line (SUIT-2): In 5-FU alone group, no effect of 5-FU was observed in dose of 1.0μM, and 17% and 30% of proliferation-inhibitory effects were observed in dose of 2.5 and 5.0μM, respectively. VPA (0.5mM) weakly suppressed cell viability by 13%. In combination of 5-FU and VPA, 19% of inhibitory effect was observed in dose of 5-FU 1.0μM/VPA 0.5mM, respectively.

Cholangiocarcinoma cell line: 5-FU (1.0μM) did not suppress the cell viability, 5-FU (2.5μM) suppressed by 23%. VPA (0.5mM) did not suppress the cell viability, while VPA (1.0mM) weakly decreased it by 11%. 5-FU (1.0μM) and VPA (0.5mM) reduced by 30%, which significantly augmented the anticancer effect of 5-FU alone or VPA alone (P<0.01).

Conclusion: VPA augmented the anti-tumor effect of 5-FU in both a pancreas cancer cell line and a cholangiocarcinoma cell line. Therefore, 5-FU plus VPA is suggested to be a promising therapeutic option for both pancreas cancer and cholangiocarcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2008. doi:10.1158/1538-7445.AM2011-2008
Abstract 2009: Histone deacetylase inhibitors (HDI) down-regulate Ron kinase and synergize with Ron knockdown or Ron monoclonal antibody 41A10to treat pancreatic cancer
Authors: Zou Yi , Howell Gillian , Brattain Michael G.
Published: July 2012

Receptor tyrosine kinase Ron (recepteur d'origine nantais) has been reported to be overexpressed in a panel of pancreatic cancer cells, as well as in tissue samples from pancreatic cancer patients and has been correlated to late stages of pancreatic cancer. Here we hypothesize that Ron kinase can be served as a potential therapeutic target. Our study showed that Ron-specific shRNA significantly altered oncogenic signaling as reflected by reduced pRon, pAkt and pERK in conjunction with reduced cell motility, as evidenced by wound healing assays and transwell assays. Ron knockdown also reduced HIF1α expression. HIF1α is constitutively expressed in many pancreatic cancer cells and has been reported to be a transcription factor for Ron and CXCR4 gene expression, indicating the reciprocal control for these two proteins. Targeting HIF1α and Ron may represent a novel therapeutic strategy for Ron-overexpressing pancreatic cancer patients. Histone deacetylase (HDAC) inhibitors have been reported to target HIF1α gene expression and alter the interaction of HSP70/90 with their client proteins through disruption of HSP70/HSP90 by HDAC6. We demonstrate here that HDAC inhibitors TSA and Panobinostat significantly reduced HIF1α, Ron kinase and CXCR4 expression in pancreatic cancer cells, which was associated with attenuation of pAkt and pERK. Thus our work indicates that HDAC inhibitor therapy targets both Ron and HIF1α. Our future work will demonstrate that Panobinostat can synergize with Ron knockdown or Ron monoclonal antibody to reduce cell growth by colony formation assay. Molecular mechanism will also be investigated. Ron, HIF1α and CXCR4 were reported to increase tumor metastasis. Whether HDI such as Panobinostat alone or in combination with Ron knockdown or Ron monoclonal antibody can reduce metastasis through downregulation of these metastasis related genes will be further investigated.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2009. doi:10.1158/1538-7445.AM2011-2009
Abstract 201: The expression of survivin in human oral squamous cell carcinoma and its relationship with clinical factors
Authors: Tamatani Tetsuya , Takamaru Natsumi , Ferdous Tarannum , Uchida Daisuke , Fujisawa Kenji , Nagai Hirokazu , Miyamoto Youji
Published: July 2012

[Background] Survivin is a member of the inhibitor of apoptosis protein family, which is a bifunctional protein that suppresses apoptosis and regulates cell division. Survivin is expressed in various malignant tumors. The overexpression of survivin has been reported to be a poorer prognostic factor in various malignancies. However, the prognostic value of survivin expression in patients with oral cancer is still controversial. Moreover, there are few comparative studies of survivin expression between the cytoplasm and nucleus of individual cells.

[Purpose] The aims of present study were to evaluate the expression of survivin protein in oral squamous cell carcinoma (OSCC) and to elucidate the relationships among the survivin expression, clinical stages, histological differentiation and YK classification.

[Materials and Methods] Four human OSCC cell lines, B88, CAL27, HNt, and HSC3 cells were used in this study. Normal gingival epithelial cells served as control. Survivin expression of cultured cells was detected by western blot. Tissue specimens were obtains from 57 patients with OSCC after surgery or biopsy. Survivin expression was detected by an immunohistochemical method.

[Results] The expression of survivin was detected in cancer cells, but not in normal cells. Immunohistochemical analysis of 57 cases of OSCC showed that 50 (88%) cases expressed survivin. Moreover, analysis of 50 positive cases revealed 28 cases with survivin-positive staining both in the cytoplasm and the nucleus, and 22 cases with survivin expression in cytoplasm only. There was no relationship between survivin expression and clinical stages, or histological differentiation. There were significant differences between survivin expression of nucleus and YK classification, implicated invasion mode of cancer.

[Conclusions] These findings suggested that the expression of survivin in OSCC could be related to invasion mode.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 201. doi:10.1158/1538-7445.AM2011-201
Abstract 2011: Epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A against non-small cell lung cancer cells
Authors: Kikuchi Junko , Kinoshita Ichiro , Kikuchi Eiki , Shimizu Yasushi , Sakakibara-Konishi Jun , Oizumi Satoshi , Nishimura Masaharu , Dosaka-Akita Hirotoshi
Published: July 2012

Background: The polycomb group gene, EZH2, mediates the histone methyltransferase activity, and functions as transcriptional repressor involved in gene silencing. It is involved in the malignant transformation and biological aggressiveness of several human cancers. We have previously shown that high EZH2 expression is correlated with tumor aggressiveness and is an independent unfavorable prognostic factor in non-small cell lung cancers (NSCLCs). However, the role of EZH2 in lung cancer cells has not been fully determined.

Methods: In the present study, we evaluated the effect of a histone methyltransferase EZH2 inhibitor, 3-Deazaneplanocin A (DZNep), in NSCLC cell lines.

Results: DZNep depleted cellular levels of EZH2 and SUZ12, and inhibited histone H3 lysine 27 trimethylation. DZNep inhibited proliferation of NSCLC cells dose- dependently through apoptosis and G1 cell cycle arrest which was partially associated with cyclin A decrease and p27Kip1 accumulation.

Conclusion: Epigenetic therapy with the histone methyltransferase EZH2 inhibitor DZNep may constitute a novel approach for NSCLCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2011. doi:10.1158/1538-7445.AM2011-2011
Abstract 2012: Changes in gene expression and global histone marks in a human population exposed to nickel compounds
Authors: Arita Adriana G. , Wu Fen , Sun Hong , Kiok Kathrin , Kluz Thomas , Qu Qingshan , Zhao Najuan , Ruan Ye , Niu Jingping , Hayes Richard , Costa Max
Published: July 2012

All of the studies on the global and selective gene promoter changes in epigenetic marks induced by carcinogenic Nickel compounds have been mostly done in tissue culture model systems. Here we report the results of the first study to evaluate the global changes in epigenetic marks and changes in gene expression that occur in the peripheral blood lymphocytes of a human population occupationally exposed to Nickel compounds. Nickel (II) is a toxic nonessential transition metal used in modern industry with other metals to form alloys to produce coins, jewelry, stainless steel, Ni-Cd batteries, and carbon nanoparticles. Nickel is of great environmental concern because occupational exposure to Ni compounds has been associated with an increased risk for lung and nasal cancers. Using an ELISA method, a decrease in both H3K4me3 and H3K9me2 histone marks were found with increasing levels of Nickel in the urine of subjects recruited to the study from a nickel refinery in Jinchang, China. Changes in global levels of other epigenetic marks are also being measured in the samples of subjects recruited to the study. Inter- and intra-individual variability of H3K4me3 and H3K9me2 and other epigenetic marks will also be evaluated in primary lymphocytes of this particular Chinese population. Additionally, changes in gene expression patterns between individuals with low exposure to Ni and those with higher exposure will be compared in order to identify a possible biomarker of Ni exposure. Studies on the effect of Ni compounds to human health are very important since Nickel is a classified human carcinogen. Here we are studying for the first time the mechanisms of nickel-associated changes in gene regulation and global histone epigenetic marks found in the primary lymphocytes of a human population occupationally exposed to Nickel compounds.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2012. doi:10.1158/1538-7445.AM2011-2012
Abstract 2014: Modulation of DNA methyltransferases in leukemia cells by curcumin and its associated anti-leukemia activities
Authors: Liu Zhongfa , Wang Hongyan , Wu Lai-chu , Xie Zhiliang , Du Liping , Mo Xiaokui , Chiu Ming , Perrotti Danilo , Grever Michael , Byrd John C. , Liu Shujun , Marcucci Guido , Chan Kenneth K.
Published: July 2012

Purpose: Curcumin, a natural product of curcuma longa and a safe and widely consumed spice, has been shown to exhibit strong in-vitro and in-vivo anti-tumor activity through the inhibition of NF-κB activity, increase of reactive oxygen species (ROS) levels and decrease of the global DNA methylation levels in leukemia cells. We investigated here the molecular mechanisms of down-regulation of DNA Methyltransferases in leukemia cells by curcumin and its associated anti-leukemia activities.

Method: Leukemia cell lines MV4-11, Kasumi-1 and K562 cells and primary leukemia cells from patient with AML were treated with 1-30 μM of curcumin and MV4-11 xenografted nu/nu athymic mice was given i.p. curcumin at 100 mg/kg, and the transcription and protein levels of DNMT1, DNMT3a, DNMT3b, Sp1, p15INK4B and p21 were assessed by either RT-PCR or western blot. The inhibition of NF-κB was determined using Electrophoretic Mobility Shift Assays and the disruption of Sp1-NF-κB/DNMTs promoter complex was investigated by anti-body gel supershifts. The hypomethylating activity of curcumin on the promoter of p15INK4B in MV4-11 cells in-vitro was assessed by a LC-MS/MS method for regional DNA methylation. The anti-proliferative activity of curcumin and cell cycle distribution and apoptosis were evaluated using MTS assay and flow cytometry.

Results: In addition to the pre-defined direct inhibition of the enzymatic activity of DNA methyltransferase 1 (DNMT1), curcumin disrupts Sp1/NF-κB complex on DNMT1, DNMT3a and DNMT3b promoter thereby decreasing the transactivation activity of these complexes and down-regulating their mRNA and protein expression levels in AML cells in vitro and in vivo. Consistent with these findings, we showed that promoter hypermethylation of silenced TSGs i.e., p15INK4B decreased while their expression was up-regulated. This results in the increase of protein function, as we showed that in association with p15INK4B re-expression, curcumin induced cell cycle arrest in the S-phase and increase in SubG1 cell fraction, thereby resulting in significant anti-leukemia activity.

Conclusion: Different from nucleoside analogs (e.g., azanucleosides), curcumin is an effective S-phase independent DNA methylation inhibitor. Further evaluation of curcumin as a single agent and in combination with other epigenetic targeting compounds in human disease is warranted. Supported by NIH-NCI-R21 CA135478 [ZL], NIH-NCI-RO1 CA102031 [GM], and the BioMedical Mass Spectrometry Laboratory [KC & ZL].

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2014. doi:10.1158/1538-7445.AM2011-2014
Abstract 2017: Demethylation of HIC1 gene sensitizes thyroid cancer cells to TRAIL-induced apoptosis
Authors: Siraj Abdul K. , Al-Rasheed Maha , Husain Azhar , Bavi Prashant , Nuaim Abdul Rahman Al , Alsobhi Saif Al-Deen S. , Al-Dayel Fouad Al-Dayel Fouad , Uddin Shahab , Al-Kuraya Khawla S.
Published: July 2012

Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration. It serves as an alternative to genetic defects in the inactivation of tumor suppressor and other genes in human cancers. Several genes have been shown to be epigenetically inactivated in a wide range of tumors and most neoplasms show hypermethylation of one or more genes. Concurrent methylation status of the CpG islands of 24 genes were determined in 40 PTC cases and in a panel of PTC cell lines, using methylation specific PCR. Seven genes demonstrated a relatively high frequency of aberrant methylation: RASF1 (95%), HIC1 (55%), GSTP1 (26%), P14 (25%), TMS1 (24%), DCR1 (17%) and SERF (15%). Five genes (BRACA1, P15INK4B, MGMT, BLU and APC) showed a low frequency (2-6%) of methylation, and no methylation was detected for the remaining twelve genes (P16INK4A, APAF1, FANCF, FAS, MINT25, P73, MLH1, DAPK, RARB, CASP8, E-Cadherin and SOC1.

Statistical analysis demonstrated that frequency of methylated and unmethylated HIC1 was significant (p <0.05). Further clinical analysis demonstrated significant association of only HIC1 methylation with: age of patients, stage of disease and expression of HIC1 IHC data (p = <0.0001, <0.0173 and < 0.0448; respectively). HIC1 has been shown to be hypermethylated in different cancers and confers bad prognosis. HIC1 is a tumor suppressor gene which encodes a transcriptional repressor with five Kruppel-like Cys2-His2 zinc fingers in the C terminus and a protein-protein interaction domain called the BTB/POZ domain at the N terminus.

Analysis of BCPAP-1 and TPC1 (papillary thyroid cell lines) revealed that they are completely methylated for HIC1 gene. Treatment of methylated thyroid cell line with the methyltransferase inhibitor, 5-aza-2’deoxy-cytidine, resulted in partial demethylation and re-expression of HIC1 gene, suggesting the role of methylation in silencing of the gene. We further evaluated the role of HIC1 gene after demethylation for apoptosis. After demethylation of HIC1 gene with 5-aza-2’deoxy-cytidine we treated our PTC cell lines with increasing doses of TRAIL and evaluated its response by Annexin/PI dual staining. We found that after demethylation of HIC1 there was increase in percentage apoptosis following increasing doses of TRAIL suggesting that HIC1 gene plays a major role in inducing apoptosis in PTC.

In conclusion, HIC1 methylation may be a promising molecular marker to predict patient outcome in PTC, and may be used as therapeutic demethylating target.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2017. doi:10.1158/1538-7445.AM2011-2017
Abstract 2019: An epigenetic reactivation screen identifies pathways important in pleomorphic liposarcomas
Authors: Pascarelli Kara , Plantinga Matthew , Doyle Katherine , Roberts Chad , Zhou Yan , von Mehren Margaret , Lev Dina , Broccoli Dominique
Published: July 2012

Treatment of soft tissue sarcomas with chemotherapy has met with limited success as few agents have demonstrated clinical efficacy, with standard therapies having a 25-30% response rate in large multi-institutional trials with current standard therapy of doxorubicin and ifosfamide. Pleomorphic liposarcomas have complex karyotypes, hindering identification of characteristic genome changes amenable to targeted treatment. Aberrant epigenetic silencing of genes is observed in cancers and is an established mechanism to abrogate tumor suppressor pathways. To gain insight into the biology of pleomorphic liposarcomas, we carried out an epigenetic reactivation screen to identify genes silenced by methylation of CpG dinucleotides. Three cell lines derived from pleomorphic liposarcomas were treated with 5-aza-2-deoxycytidine for 2 cell doublings prior to extraction of RNA. Total RNA from duplicate cultures of treated and untreated cells was hybridized to the Affymetrix U133plus2 expression chip. Initial filtering for only those genes affected in all three cell lines identified 201 genes with altered expression, 59 of which are down regulated and 142 of which are up regulated. We validated candidate genes by sequencing sodium bisulfite modified genomic DNA isolated from the three cell lines used in the reactivation screen. We chose candidates reported as being affected by promoter hypermethylation in other systems, stratifin (SFN); aka 14-3-3-σ, clusterin (CLU) and e-cadherin (CDH1). The data confirm that all candidates exhibit methylation of CpG islands located within 400 bp of the 5’ start site. To further validate candidates we tested the methylation status of SFN, CLU, and CDH1 in DNA isolated from12 pleomorphic liposarcomas, as well as DNA isolated from adipocytes, preadipocytes and adult adipose derived stem cells. Gene interaction networks were generated through the use of Ingenuity Pathway Analysis (Ingenuity® Systems, www.ingenuity.com). Conditional hypergeometric tests were done for testing the association of Gene Ontology using GOstats package in Bioconductor. Top scoring networks identified in this analysis included cancer, lipid metabolism, and cell cycle/death. In conclusion, the pathways identified here may form the basis for future development of targeted therapy against pleomorphic liposarcomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2019. doi:10.1158/1538-7445.AM2011-2019
Abstract 2021: Mechanism of the coordinate regulation of liver cancer by nuclear receptor CAR and beta catenin
Authors: Dong Bingning , Moore David
Published: July 2012

Aberrant activation of β-catenin is a critical contributor to many different cancers, including up to 34% of human hepatocellular carcinogenesis. Liver-specific activation of β-catenin is not sufficient to drive tumorigenesis in mice. However, nearly all of the tumors produced in the widely used diethylnitrosamine (DEN; carcinogen) plus phenobarbital (PB; promoter) mouse liver cancer model have activating mutations in β-catenin. The nuclear receptor CAR, a primary regulator of drug metabolism and detoxification, is the target of PB in the DEN-PB model, suggesting a synergistic functional interaction of these two transcription factors. Thus, we hypothesize that β-catenin and CAR synergistically induce liver tumorigenesis. We induced CAR and full β-catenin activation by TC treatment (CAR agonist) of liver specific-Cre/catnb lox/lox(ex3) mice (homozygous β-catenin activation by knockout of phosphorylation sites in exon3). Consistent with previous studies, activation of either CAR or β-catenin only induces mild hepatomegaly but not tumorigenesis in one month. However, the combined activation of both CAR and β-catenin causes uncontrolled liver growth (up to 25% liver/body weight ratio). Ki67 staining reveals significant hepatocyte proliferation in the double activation group, but not in any of the single activation groups. This indicates that CAR and β-catenin coordinately regulates liver growth. Uncontrolled liver growth leads to mouse death, which prevents us studying tumorigenesis. Accordingly, we also treated liver specific-Cre/catnb wt/lox(ex3) mice (heterozygous β-catenin activation) with TC. After 8 month, HCC were observed in 13 out of 15 experimental treated mice whereas no tumor was observed in the control mice (either TC treated catnb wt/lox(ex3) mice or CRE treated catnb wt/lox(ex3) mice, suggesting a cooperative and synergistic role of CAR and β-catenin in HCC formation. Bioinformatic gene array analysis shows that tumors generated in our mouse model share similar expression signature with human HCC carrying β-catenin mutation. Thus, the study on the mechanism of our mouse model will provide new therapeutic approaches in liver cancer and its prevention.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2021. doi:10.1158/1538-7445.AM2011-2021
Abstract 2023: Isolation of breast tumor initiating cells (TICs) by exogenous delivery of the OCT4 transcription factor
Authors: Beltran Adriana S. , Richarson Bryan , Rivenbark Ashley , Casbas-Hernandez Patricia , Yang Xinni , Blancafort Pilar
Published: July 2012

Tumor Initiating cells (TICs) are being extensively studied for their role in tumor etiology, maintenance and resistance to treatment. Herein, we describe a novel approach to generate TICs by transduction of human primary breast cell preparations with exogenous expression of the OCT4 transcription factor. The OCT4 transduced breast colonies (OTBCs) underwent an immortalization process, as reflected by loss of p16 and enhanced telomerase expression. OTBCs exhibited cancer stem cell antigens, such as CD44high/CD24low/-. Consistently, these cells generated subcutaneous tumors in nude mice with colonization capabilities. As little as 1-50 cells were sufficient to induce tumor formation and thereby these cells had TIC properties. Histological analysis of these tumors revealed poorly differentiated breast carcinomas. RNAi-mediated knock-down of OCT4 and downstream embryonic targets of OCT4, resulted in suppression of the self-renewal ability, suggesting that this phenotype was dependent on OCT4. Genome-wide microarray analysis supported the finding that OCT4 over-expression in these clones was molecularly associated with Epithelial-to-Mesenchymal Transition (EMT) gene signatures and strongly correlated with the claudin-low intrinsic subtype of breast cancer. Our experimental approach provides a novel model system to dissect genes, small RNAs and small molecules that could be used to effectively target breast cancer self-renewal and tumor initiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2023. doi:10.1158/1538-7445.AM2011-2023
Abstract 2027: CBX7 protein, whose expression is decreased in human carcinomas, positively regulates E-cadherin expression by interacting with the HDAC2 protein
Authors: Federico Antonella , Pallante Pierlorenzo , Bianco Mimma , Monti Maria , Cozzolino Marianna , Leone Vincenza , Pucci Pietro
Published: July 2012

CBX7 is a chromobox family protein encoding a novel Polycomb protein whose expression shows a progressive reduction, well related with the malignant grade of the thyroid neoplasias. Indeed, CBX7 protein levels decreased in an increasing percentage of cases going from benign adenomas to papillary, follicular and anaplastic thyroid carcinomas. To elucidate the function of CBX7 in carcinogenesis, we searched for CBX7 interacting proteins by a proteomic analysis. By this approach, we identified several proteins among which we selected Histone deacetylase 2 that is well known to play a key role in neoplastic cell transformation and to downregulate E-cadherin expression, whose loss is a critical event in the Epithelial-Mesenchymal Transition (EMT). We confirmed by co-immunoprecipitation that CBX7 physically interacts with the Histone deacetylase 2 protein and is able to inhibit its activity. Then, we showed that both these proteins bind the E-cadherin promoter, and that CBX7 is able to upregulate E-cadherin expression. Consistent with these data we found a positive statistical correlation between CBX7 and E-cadherin expression in human thyroid carcinomas. Finally, we demonstrated that the expression of CBX7 increases the acetylation status of the histones H3 and H4 on the E-cadherin promoter. Therefore, the ability of CBX7 to positively regulate E-cadherin expression by interacting with Histone deacetylase 2 and inhibiting its activity on the E-cadherin promoter would account for the correlation between the loss of CBX7 expression and a highly malignant phenotype in thyroid cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2027. doi:10.1158/1538-7445.AM2011-2027
Abstract 2030: A novel role for the 185delAG BRCA1 mutant protein, BRAT, in ovarian cancer pathology
Authors: Woolery Kamisha T. , Linger Rebecca J. , Kruk Patricia A.
Published: July 2012

Objective: Familial history is the strongest risk factor for developing ovarian cancer (OC) and is associated with BRCA1 truncation mutations, such as the founder mutation 185delAG. Studying these risk-associated mutations may reveal signaling pathways important in OC etiology. Normal human ovarian surface epithelial (HOSE) cells expressing the 185delAG mutant, BRAT, exhibit phenotypic changes due, in part, to up-regulation of maspin, a tumor suppressor important in apoptosis, invasion, and metastasis that exhibits unique up-regulation in some cancer types. In the current study, we determined BRAT's impact on expression of Interleukin-1beta (IL1β), another key player in invasion and metastasis.

Methods: HOSE cells transiently and stably expressing the 185delAG mutation and HOSE cells with endogenous 185delAG were analyzed for differential target gene expression by semi-quantitative, real time PCR, western blot; as well as, IL1β bioactivity. Western blot was used to identify cell signaling targets of BRAT mediated IL1β expression via MAPK pathways. Breast epithelial and breast cancer cells transiently and stably expressing BRAT were evaluated by real time PCR to determine whether BRAT induced IL1β is tissue specific.

Results: BRAT-expressing HOSE cells exhibited enhanced IL1β mRNA expression. Higher levels of pro-IL1β protein were detected by Western blotting of cell lysate and conditioned media. Increased phosphorylation of MAPK pathway constituents were seen in BRAT-expressing HOSE cells as detected by Western blotting of cell lysate. However, expression of BRAT in normal breast and breast cancer cell lines failed to significantly alter IL1β expression levels.

Conclusion: Normal HOSE cells expressing the BRAT mutation exhibit increased expression of IL1β. Further investigation is warranted to elucidate the mechanism of tissue-specific BRAT-mediated IL1β expression since increased IL1β expression may represent an early step of malignant transformation contributing to ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2030. doi:10.1158/1538-7445.AM2011-2030
Abstract 2032: Regulation of MUC1 expression by activated EGFR and PPARγ in human uterine and pancreatic cancer cell lines
Authors: Dharmaraj Neeraja , Carson Daniel D.
Published: July 2012

Many epithelial tumors including endometrial and pancreatic cancers, overexpress the high molecular weight, transmembrane mucin, MUC1, on their cell surface. Overexpression and aberrant glycosylation of MUC1 makes tumor cells poorly adherent, increases drug resistance and promotes cancer cell metastasis. MUC1 has three distinct domains: an extracellular domain (ECD) composed of a tandem repeat region rich in serine, threonine and proline; a transmembrane domain (TM) and; a short cytoplasmic tail domain (CT) involved in a variety of signal transduction events. Previous studies (Wang et al. 2010. Mol. Endocrinol. 24: 1368-1379) demonstrated that the Peroxisome Proliferator Activated Receptor γ (PPARγ) agonist, rosiglitazone severely inhibits progesterone stimulated MUC1 expression. Two independent approaches were used to determine if rosiglitazone as well as inhibitors of Epidermal Growth Factor Receptor (EGFR) tyrosine kinase activity reduced MUC1 expression in the human uterine epithelial cell line, HES, the uterine adenocarcinoma cell line, HEC-1A, and in the human pancreatic adenocarcinoma cell lines, HPAFII and CAPAN2. EGFR activation by Epidermal Growth Factor (EGF) stimulated (1.5-2 fold) MUC1 protein expression 1.5-2.0 fold in both uterine and pancreatic cancer cells, while treatment with EGFR tyrosine kinase inhibitor, AG1478, reduced both basal and stimulated MUC1 protein expression. Furthermore, the PPARγ agonist, rosiglitatzone, markedly (> 90%) reduced EGFR-driven MUC1 expression detected by antibodies directed against the MUC1 ECD and CT. A combination of biochemical and molecular biological assays have been used to identify the transcriptional and signal transduction pathways activated by EGFR leading to increased MUC1 expression and the impact of AG1478 and rosiglitazone on these events. Understanding the molecular basis of how these agents reduce MUC1 expression may offer novel therapeutic avenues to improve cancer chemotherapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2032. doi:10.1158/1538-7445.AM2011-2032
Abstract 204: p53-induced gene 3 activity is glutathione peroxidase 3 dependent
Authors: Yu Yan P. , Wang Hui , Luo Katherine L. , Luo Jian-Hua
Published: July 2012

The expression of glutathione peroxidase 3 (GPx3) was found widely altered in a variety of human malignancies. Methylation and deletion of GPx3 were reported in several types of human cancers. Recently, GPx3 characterized as a tumor suppressor gene. However, the mechanism of the tumor suppressor activity of GPx3 is not clear. In this report, we demonstrated that GPx3 interacts with p53-induced gene 3 (PIG3) in both prostate cancer cells and immortalized prostate epithelial cells. The PIG3 binding motif is located in amino acid 2-114 of GPx3. Expression of GPx3 resulted in apopotosis and necrotic cell death. Interaction of GPx3 and PIG3 led to dramatic increase of reactive oxygene species (ROS) inside the cells and activation of caspase-3. Knocking down of PIG3 or mutation of PIG3 binding motif in GPx3 abrogated ROS generation activity induced by GPx3. These results suggest that PIG3 activity is GPx3-dependent, and reveal a novel signaling pathway that leads to GPx3 mediated tumor suppression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 204. doi:10.1158/1538-7445.AM2011-204
Abstract 2047: Betulinic acid exerts anti-angiogenic activity via regulation of HIF-1 alpha and STAT3 in PC-3 prostate cancer cells under hypoxia
Authors: Kim Bong I. , Shin Jimin , Jung Deok B. , Park Moon Y. , Ko Hyun Suk , Song Hyo Sook , Koh Wonil , Jung Ji Hoon , Kwon Tae-Rin , Han Ihn , Kim Ji-Hyun , Yun Sun-Mi , Rhee Yun-Hee , Cho Sung Yun , Lee Hyo-Jung , Lee Hyo-Jeong , Jeong Soo-Jin , Lee Eun-Ok , Kim Sung Hoon
Published: July 2012

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor constitutively overexpressed in several human cancer cells and related to various cellular processes such as cell survival, angiogenesis and proliferation. In the present study, we examined whether betulinic acid (BA), a triterpene from the bark of white birch, could inhibit hypoxia-mediated activation of STAT3 and HIF-1 alpha in androgen independent human prostate cancer PC-3 cells. Our results show that BA inhibited the transcriptional activities and nuclear accumulation of both HIF-1 alpha and STAT3 under hypoxia. In addition, BA significantly reduced cellular and secreted levels of hypoxia-induced vascular endothelial growth factor (VEGF), a critical angiogenic factor, in PC-3 cells. BA prevented in vitro capillary tube formation in human umbilical vein endothelial cells (HUVECs) maintained in conditioned medium of hypoxia-induced PC-3 cells. Moreover, silencing HIF 1 alpha and STAT3 by siRNA transfection augmented the suppression of VEGF expression by BA under hypoxia. Mechanistically, BA inhibited the recruitment of HIF-1 alpha and STAT3 on the VEGF promoter. Taken together, these findings suggest that BA may exert angio-prevention via inactivation of hypoxia-induced STAT3 and HIF-1 alpha in PC-3 cells. The in vivo anti-angiogenic and anti-PCa efficacy are being evaluated.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2047. doi:10.1158/1538-7445.AM2011-2047
Abstract 2048: USF2 is HIF2α specific co-transcriptional activator
Authors: Pawlus Matthew , Wang Liyi , Sena Johnny , Hu Cheng-Jun
Published: July 2012

Hypoxia inducible factor HIF1α, and in particular HIF2α have been shown to drive malignant progression by activation of angiogenesis, proliferation, anaerobic metabolism, and other processes that enable tumor cells to survive or to escape their O2-deficient microenvironments. The dominant role of HIF2α in tumor biology is likely due to its selective activation of genes such as cyclin D1, TGFa, PDGFb, and OCT4 that are not regulated by HIF1α. The mechanism through which the highly conserved HIF1 and HIF2 regulate distinct target genes has not been established. We have previously shown that the transcriptional domain, but not the DNA binding domain of HIF determines its target gene specificity, suggesting that HIFs achieve their target gene specificity by interacting with other transcriptional factors or cofactors via their transactivation domains. Here we reported that HIF2α achieves its target gene specificity and enhanced transcriptional activity by interacting with the b/HLH/LZ transcription factor USF2 (upstream stimulatory factor 2) on promoters of HIF2α specific genes. We observed that siRNA-mediated USF2 knockdown significantly inhibited hypoxic induction of HIF2α (but not HIF1α) target genes in Hep3B and RCC4T cells. Additionally, overexpression of a USF2 dominant negative construct in Hep3B cells significantly reduced the levels of HIF2α (but not HIF1α) target genes under hypoxia. These data determined that USF2 is required for HIF2α to activate its target gene under hypoxia. By HIF2 target gene promoter functional analysis, gel-shift, and Chromatin-IP, we found that USF2 enhances HIF2α transcriptional activity by binding to HIF2α specific gene promoters and recruiting co-activator CBP/P300. In summary, we have uncovered a novel function of USF2 in HIF2α target gene regulation and demonstrated that USF2 is required for HIF2, but not HIF1 to activate its target genes. This finding has laid a foundation for the development of specific HIF2α inhibitors for use in solid tumor treatment.

NIH T32-GM08730; RO1 CA134687 NIH/NCI (Hu); Cancer League of Colorado (Hu)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2048. doi:10.1158/1538-7445.AM2011-2048
Abstract 2052: Inhibition of aldose reductase prevents hypoxic stress signaling in human colon cancer cells
Authors: Srivastava Satish K. , Tammali Ravinder , Ramana Kota V.
Published: July 2012

Hypoxia, a hallmark of invasive solid tumors and increased angiogenesis, is strongly associated with the progression of malignant phenotypes, poor prognosis, and resistance to chemo and radiation therapies. In a variety of cancers, hypoxic stress-induces inflammatory signaling which is mediated by specific cytokines, chemokines and growth factors. We have recently shown that inhibition of aldose reductase (AR), an enzyme that catalyzes the reduction of lipid aldehydes and their glutathione conjugates, prevents human colon cancer cell growth in culture as well as in nude mice xenografts by inhibiting the NF-kB-dependent activation of oxidative stress-mediated inflammatory and carcinogenic markers. However, the role of AR in mediating hypoxic stress signals is not known. We therefore investigated the molecular mechanisms by which AR inhibition prevents the hypoxia-induced human colon cancer cells growth and invasion. Our results indicate that AR inhibition by pharmacological inhibitor fidarestat or ablation by AR specific siRNA prevents hypoxia-induced proliferation of HT29 colon cancer cells. Further, hypoxia- induced increase in the level of HIF1a in HT29 cells was significantly decreased by AR inhibition. During hypoxic conditions treatment of HT29 cells with AR inhibitor, fidarestat significantly decreased the expression of VEGF, a down target of HIF1a, at both mRNA and protein levels and also prevented the activation of PI3K/AKT and GSK3b. Further, AR inhibition also prevented hypoxia-induced inflammatory molecules such as Cox-2 and PGE2 and expression of extracellular matrix proteins such as MMP2, vimentin, uPAR and LOX2. In addition, AR inhibition also prevented the hypoxia -induced tube formation (angiogenesis) in vascular endothelial cells. In conclusion, our results indicate that AR mediates hypoxia -induced tumor progression, invasion and angiogenesis which can be prevented by AR inhibition or ablation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2052. doi:10.1158/1538-7445.AM2011-2052
Abstract 2053: Differential regulation of hypoxia-inducible factors 1α and 2α by hypoxia
Authors: Lin Qun , Cong Xiangyu , Yun Zhong
Published: July 2012

The hypoxia-inducible transcription factors (HIF)-1α and -2α are the key transcription factors regulating the expression of hypoxia-induced genes critical for a wide range of tumor cell functions from survival to clonal selection to metastasis. Elevated HIF-α expression correlates with poor patient survival in a large number of cancers. Recent evidence suggests that HIF-2α appears to be preferentially expressed in neuronal tumor cells that exhibit cancer stem cell characteristics. Furthermore, elevated HIF-2α expression is co-localized in vivo with expression of neural crest progenitor markers, suggesting a preferential association of HIF-2α expression with the immature stem cell-like neuroblastoma cells. These observations suggest that expression of HIF-1α and -2α is differentially regulated in the hypoxic tumor microenvironment. However, the underlying mechanisms remain to be fully investigated. In this study, we investigated the transcriptional regulation HIF-1α and -2α under different hypoxic conditions. In addition to conventional hypoxia treatment, we developed an adaptive chronic hypoxia approach by preconditioning tumor cells at 5% O2 before reducing pO2 to hypoxia levels (below 2% O2) to mimic in vivo tumor hypoxia. We found that transcription of HIF-2α was consistently increased by hypoxia in a panel of neuroblastoma cell lines, whereas transcription of HIF-1α showed variable levels of repression. Mechanistically, differential regulation of HIF-α transcription involved hypoxia-induced changes in actetylation of core histones H3 and H4 associated with the proximal promoters of the HIF-1α or HIF-2α gene. We also found that, although highly stable under acute hypoxia, HIF-1α and HIF-2α proteins become destabilized under chronic hypoxia. Our results have thus provided new mechanistic insights into the differential expression and localization of HIF-1α and -2α proteins within the hypoxic tumor microenvironment. These findings further underscore the importance of HIF-2α in the regulation of tumor progression, especially in the regulation of the stem cell-like tumor cell population as observed in neuronal tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2053. doi:10.1158/1538-7445.AM2011-2053