bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4860: Pan-HDAC inhibitor treatment sensitizes breast cancer cells to autophagy inhibitor 2-phenylethynesulfonamide: Molecular mechanism
Authors: Rao Rekha , Nalluri Srilatha , Fiskus Warren , Mudunuru Uma , Bhalla Kapil N.
Published: February 2011

The stress phenotype of transformed cells is associated with increased levels of heat shock proteins (hsp) and autophagy to avoid cell death. Autophagy is a conserved catabolic pathway in which cytoplasmic macromolecules and organelles are sequestered in autophagosomes, which fuse with lysosomes for protein degradation and amino acid recycling. The molecular cascade initiating autophagosome formation is triggered by a lipid kinase (PI3KC3) signaling complex (Vps34, Vps15, ATG6 and ATG14) that mediates phagophore nucleation. We have previously reported that treatment with panobinostat (PS), a pan-histone deacetylase (HDAC) inhibitor induced hyperacetylation of hsp70 and hsp90. In the present study we demonstrate that treatment of MB231 cells with PS increased the association of hyperacetylated hsp70 with the PI3KC3 VPS34, ATG6 (Beclin-1) and KAP1 (an E3 ligase for sumoylation). PS treatment also induced the levels of ATG7, ATG5 and LC3-II (a phosphatidylethanolamine conjugated ATG8) in MB-231 cells. Treatment with 2-phenylethynesulfonamide (PES), a small molecule inhibitor of hsp70 function resulted in the abrogation of PS-induced interaction of hsp70 with hsp40, VPS34 and (the lysosome-associated membrane protein A (LAMP-2A), a protein involved in chaperone mediated autophagy. Consequently, as compared to treatment with each agent alone, co-treatment with PS and PES resulted in significantly more cell death in MB-231 and SUM159PT cells (p < 0.05). Co-treatment with PS also significantly enhanced lethality of breast cancer MB-231 and MCF-7 cells due to autophagy inhibitor 3-methyl-adenosine (3-MA, inhibitor of Vps34), or hydroxychloroquine (inhibitor of lysosome acidification). In orthotopically implanted MB-231 xenografts in the mammary fat pads of NOD/SCID mice, tumor size increase to 100, 200, 500, 1000 and 2000 cu mm demonstrated tumor size-dependent increase in the intracellular levels of hsp90 and hsp70, hyperacetylated hsp70, ATG7, ATG5, Beclin1 as well as LC3-II. These in vivo findings suggest that as breast tumors grow they exhibit the stress-phenotype involving proteotoxic stress and autophagy. Our findings demonstrate the combination therapy with PS and autophagy inhibitor, which eliminates breast cancer cells with stress phenotype including autophagy, may be especially effective against established and enlarging breast cancers.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4860.
Abstract 4861: The role of histone deacetylases (HDACs) in XPC gene silencing and bladder cancer development
Authors: Wang Gan , Wang Lily , Xu Xiaoxin S.
Published: February 2011

The role of histone deacetylases (HDACs) in XPC gene silencing and bladder cancer development

Xiaoxin S. Xu, Le Wang, and Gan Wang

Institute of Environmental Health Sciences, Wayne State University, 259 Mack Ave., Detroit, MI 48202

Bladder cancer is one of the most common malignancies and causes hundreds thousands of death worldwide each year. Bladder cancer is strongly associated with exposure to environmental carcinogens. It is believed that DNA damage generated by environmental carcinogens and their metabolites causes development of bladder cancer. Nucleotide excision repair (NER) is the major DNA repair pathway for repairing bulk DNA damage generated by most environmental carcinogens and XPC is a DNA damage recognition protein required for initiation of the NER process. Recent studies demonstrate reduced levels of XPC protein in tumors for a majority of bladder cancer patients. In this work we investigated the role of histone deacetylases (HDACs) in XPC gene silencing and bladder cancer development. The results of our HDAC inhibitor study revealed that the treatment of HTB4 and HTB9 bladder cancer cells with HDAC inhibitor valproic acid (VPA) caused an increase in transcription of the XPC gene in these cells. The results of our chromatin immunoprecipitation (ChIP) studies indicated that the VPA treatment caused increased bindings of both CREB1 and Sp1 transcription factors at the promoter of XPC gene for both HTB4 and HTB9 cells. The results of our immunohistochemistry (IHC) staining studies further revealed a strong correlation between the presence of HDAC4 and the occurrence of bladder tumors. In addition, the results of our caspase 3 activation studies demonstrated that the prior treatment with VPA increased the anticancer drug cisplatin-induced activation of caspase 3 in both HTB4 and HTB9 cells. All these results suggest that the HDACs negatively regulate transcription of the XPC gene in bladder cancer cells and contribute to the development of bladder cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4861.
Abstract 4869: Transcriptional upregulation of histone deacetylase 2 promotes Myc-induced oncogenic effects
Authors: Marshall Glenn M. , Gherardi Samuele , Xu Ning , Neiron Zillan , Trahair Toby , Scarlett Christopher , Chang David K. , Liu Pei Y. , Jankowski Kacper , Iraci Nunzio , Haber Michelle , Norris Murray D. , Stossi Fabio , Katzenellenbogen Benita S. , Biankin Andrew V. , Perini Giovanni , Liu Tao
Published: February 2011

Myc oncoproteins induce cancer cell proliferation by transcriptional modulation. Histone deacetylases (HDACs) enhance cancer cell proliferation and survival, in part, by effects on tumour suppressor gene transcription, and thus HDAC inhibitors are among the most promising new classes of anticancer drugs. Here we show that N-Myc and c-Myc up-regulated HDAC2 gene expression in neuroblastoma and pancreatic cancer cells respectively, which contributed to N-Myc- and c-Myc-induced cell proliferation. Affymetrix microarray revealed that a subset of genes, including cyclin G2 (CCNG2), was commonly repressed by N-Myc and HDAC2 in neuroblastoma cells. PCR analysis confirmed that CCNG2 gene transcription was commonly repressed by N-Myc and HDAC2 in neuroblastoma cells and by c-Myc and HDAC2 in pancreatic cancer cells, and could be reactivated by HDAC inhibitors. BrdU incorporation assays demonstrated that transcriptional repression of CCNG2 was, in part, responsible for N-Myc-, c-Myc and HDAC2-induced cell proliferation. Dual cross-linking chromatin immunoprecipitation and protein co-immunoprecipitation assays showed that N-Myc acted as a transrepressor, by recruiting the HDAC2 protein to an Sp1-binding site at the CCNG2 core promoter, in a manner distinct from its action as a transactivator. Moreover, HDAC2 was up-regulated, and CCNG2 down-regulated, in pre-cancerous and neuroblastoma tissues from MYCN (N-Myc) transgenic mice, and c-Myc over-expression correlated with up-regulation of HDAC2 and repression of CCNG2 in tumour tissues from pancreatic cancer patients. Taken together, our data indicate the critical roles of up-regulation of HDAC2 and suppression of CCNG2 in Myc-induced oncogenesis, and have significant implications for the application of HDAC inhibitors in the prevention and treatment of Myc-driven cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4869.
Abstract 487: RNAi-mediated silencing of TCF7L2 sensitizes colorectal cancer cells to radiation therapy
Authors: Grade Marian , Kendziorra Emil , Emons Georg , Ahlborn Kerstin , Spitzner Melanie , Gaedcke Jochen , Rave-Frank Margret , Pukrop Tobias , Beissbarth Tim , Becker Heinz , Ghadimi Michael , Ried Thomas
Published: February 2011

Background:

The standard treatment for locally advanced rectal carcinomas consists of preoperative chemoradiotherapy (CT/RT), followed by radical surgery. However, the clinical response to this treatment strategy is very heterogeneous. To determine the molecular basis of disparate response, we recently profiled a series of responsive and resistant rectal cancers using gene expression microarrays and identified a set of differentially expressed genes. One gene that was significantly over-expressed in the resistant tumors was TCF7L2 (also known as TCF4), the main downstream effector of the Wnt signaling pathway. The aim of this study was to understand its role in treatment response by testing the hypothesis that RNAi-mediated silencing of TCF7L2 sensitizes tumor cells to radiation therapy.

Methods:

The colorectal cancer cell line SW480, which is relatively resistant to radiation, was transfected with two different shRNA-vectors targeting TCF7L2, and a non-silencing control. Subsequently, stable single cell clones were established, and knockdown efficiency was analyzed by Western blot analysis. For each vector, individual clones were irradiated at 2, 4, 6 and 8 Gy. Colonies were counted after 10 days to calculate the respective surviving fractions.

Results:

TCF7L2 protein levels were reduced to ∼ 34% for shRNA_1, and ∼ 29% for shRNA_2. This led to a ∼ 1.6-fold growth inhibition (plating efficiency) compared to the non-silencing control. Importantly, RNAi-mediated silencing of TCF7L2 significantly reduced colony-formation after radiation (dose reduction factors of ∼ 1.5 and ∼ 2.0 at 37% survival for shRNA_1 and shRNA_2, respectively). These results were validated in a second cell line (HT29). In addition, we analyzed global gene expression profiles after RNAi to analyze the global transcriptomic changes induced by silencing TCF7L2.

Conclusion:

TCF7L2 is over-expressed in rectal carcinomas resistant to chemoradiotherapy. RNAi-mediated silencing of this gene caused a significant growth inhibition and, importantly, radiosensitization of colorectal cancer cells. Thus, TCF7L2 might represent a potential molecular target to sensitize a priori resistant tumor cells. Additionally, these data provide first evidence that the Wnt signaling pathway might not only be central for the development of colorectal cancers, but also for the resistance to therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 487.
Abstract 4877: Polyamine analogues modulate gene expression by inhibiting KDM1/LSD1 and altering chromatin structure in human breast cancer cells
Authors: Zhu Qingsong , Huang Yi , Marton Laurence J. , Woster Patrick M. , Davidson Nancy E. , Casero Robert A.
Published: February 2011

Gene expression can be modulated through histone methylation, which is tightly control by the balance of histone methytransferase and demethylase. Methylation (mono-, di- and tri-methylation) of lysine 4 of histone 3 is associated with active gene transcription. Lysine-specific demethylase 1 (KDM1/LSD1) can enzymatically demethylate mono- and di-methyl H3K4 that leads to suppression of gene transcription. Our previous studies demonstrated that a novel class of polyamine analogues can effectively inhibit KDM1/LSD1 and induce re-expression of candidate epigenetically silenced genes. The purpose of this study was to examine the molecular effects of polyamine analogues, 2d and PG11144, on global gene expression profile by inhibition of KDM/LSD1 in estrogen receptor-negative MDA-MB-231 breast cancer cells. The possible mechanisms of polyamine analogues mediating modulation of gene expression were also investigated. Treatment with polyamine analogous, 2d or PG11144, significantly increases global histone 3 dimethylation at lysine 4 (H3K4me2), as well as altering gene expression. There are 274 and 625 genes changed at least 2-fold after treatment with 2d and PG11144 respectively. The genes’ expression levels of twelve 2d up-regulated and fourteen PG11144 up-regulated genes were confirmed by real-time RT-PCR. Chromatin immunoprecipitation analysis demonstrated that up-regulated gene expression by polyamine analogous is associated with up-regulated active histone marks (H3K4me, H3K4me2 and ActH3K9) and down-regulated repressive histone marks (H3K9me2 and H3K27me3) in the promoter regions of the relevant target genes. Supported by NIH CA 88843, CA51085, Breast Cancer Research Foundation, and Komen for the Cure KG080923.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4877.
Abstract 4886: Knockdown of EZH2 inhibits the malignant phenotype of lung cancer cells
Authors: Liu Fang , Xi Sichuan , Zhang Mary , Kemp Clinton , Rao Mahadev , Zhang Yuwei , Schrump David
Published: February 2011

Polycomb group (PcG) proteins, which are global epigenetic gene silencers, play key roles in the maintenance of stem cell pluripotency, multicellular development, and tumorigenesis. EZH2, a core component of PcG and H3K27 methyltranferase, is highly expressed during embryogenesis and malignant transformation. Presently the mechanism by which EZH2 mediates tumorigenesis and metastasis are unclear. Our recent studies indicate that cigarette smoke condensate (CSC) increases EZH2 expression and global levels of H3K27Me3 in immortalized human bronchial epithelial cells (HBEC). In the present study, we sought to further investigate the role of EZH2 in modulating the malignant phenotype of lung cancer cells. Western blot experiments demonstrated that EZH2 was significantly over-expressed in lung cancer cell lines but not in normal human bronchial epithelial cells (NHBE) or small airway epithelial cells (SAEC). EZH2-shRNA lentiviral vectors were used to knockdown EZH2 in A549 and Calu-6 lung cancer cells as well as CSC-treated HBEC. Knockdown of EZH2 decreased H3K27Me3 levels, and diminished proliferation and clonogenicity of these cells in soft agar. These observations coincided with down-regulation of Cyclin D1. Additional western blot and flow cytometry experiments are underway to examine the impact of EZH2 knockdown on cell cycle progression and apoptosis in lung cancer cells. Illumina gene expression array experiments are also in progress to identify PcG target genes upregulated by knockdown of EZH2 that correlate with reduced proliferation and decreased clonogenicity of these cells. Collectively, these data indicate that knockdown of EZH2 inhibits the malignant phenotype of lung cancer cells, and suggest that EZH2 is a promising target for lung cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4886.
Abstract 489: Selective inhibitor of STAT1 sensitizes breast cancer cells to radiation therapy
Authors: Timofeeva Olga A. , Tarasova Nadya , Wang Honghe , Perantoni Alan , Dritschilo Anatoly
Published: February 2011

About 70% of breast cancer patients with progressive disease are resistant to conventional cancer treatments such as adjuvant chemotherapy and/or radiation therapy. Recently, the STAT1 signaling pathway has been identified as a predictive marker of poor outcome in breast cancer patients and as a factor responsible for radiation and chemotherapy resistance. STAT1 was initially considered as a tumor suppressor, since it causes growth arrest and apoptosis after stimulation with interferons and in response to DNA damage. However, constitutive activation/overexpression of STAT1 results in pro-survival cellular signaling via enhancement of expression of pro-survival proteins Mcl-1 and Hsp27 that might be responsible for observed radiation resistance of breast cancer cells. It may therefore be beneficial to sensitize patients with resistant disease to radiation or chemotherapy by inhibiting STAT1 signaling, yet there has been little effort to develop direct inhibitors of STAT1.

We have recently developed a selective inhibitor ST1-Hel2A-2 that directly binds to the N-terminal domain of STAT1, but not other STAT family members, and inhibits STAT1 transcriptional activity (ACS Chem Biol, 2007. 2(12): 799). The inhibitor kills breast cancer cells at nanomolar concentrations but does not effect survival of normal mammary epithelial cells. We have now found that inhibition of STAT1 N-domain can further sensitize breast cancer cells to ionizing radiation. The mechanism for observed sensitization at least partially depends on down-regulation of STAT1 target genes, including pro-survival Hsp27. In addition, ST1-Hel2A-2 is capable of disrupting the STAT1 N-domain protein-protein interactions. Yeast two-hybrid screening has revealed that the STAT1 N-domain associates with double-stranded RNA-activated protein kinase (PKR) and DNA glycosylase NEIL1. Both PKR and NEIL1 facilitate DNA repair after ionizing radiation and protect mammalian cells from radiation-mediated cell death. ST1-Hel2A-2 disrupts STAT1 interactions with both proteins resulting in a reduced DNA repair capacity.

Our data suggest that a novel inhibitor of STAT1 decreases radiation resistance of breast cancer cells through downregulation of pro-survival genes and inhibition of DNA repair. Inhibition of STAT1 signaling appears to be a promising strategy in overcoming radiation resistance and can be used as an effective treatment of metastatic breast cancer when combined with conventional therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 489.
Abstract 4890: ING2, a p53- and chromatin-interacting protein, is essential to mammalian spermatogenesis: Implications in male infertility in humans
Authors: Saito Motonobu , Kumamoto Kensuke , Horikawa Izumi , Robles Ana I. , Furusato Bungo , Okamura Shu , Goto Akiteru , Yamashita Taro , Nagashima Makoto , Lee Tin-Lap , Baxendale Vanessa , Rennert Owen M. , Takenoshita Seiichi , Yokota Jun , Sesterhenn Isabell A. , Trivers Glenwood E. , Hussain Perwez , Harris Curtis C.
Published: February 2011

ING2 (inhibitor of growth family, member 2) plays pivotal roles in the regulation of cellular senescence, apoptosis, DNA damage repair, gene transcription and chromatin modification. Our previous in vitro studies on cellular senescence suggested that ING2 functionally interplays with the p53 tumor suppressor protein in two different manners: endogenous ING2 inhibits senescence and the transcriptional repression of ING2 by p53 abrogates this inhibition; and overexpressed ING2 enhances p53 acetylation and stability to induce senescence. ING2, as a subunit of the mSin3A-HDAC1 (histone deacetylase 1) complex, specifically binds to tri-methylated lysine 4 of histone H3 (H3K4me3) via its plant homeodomain (PHD) finger and regulates gene expression through chromatin modifications in response to DNA damage. This study investigates the in vivo developmental and physiological functions of ING2. Abundant expression of ING2 in mouse and human testes and its decreased expression associated with male infertility and defective spermatogenesis in humans suggested an essential role of ING2 in spermatogenesis, which is a process tightly regulated by chromatin modifications. Consistently, male mice deficient for Ing2 were infertile. Their testes had degeneration of seminiferous tubules, which became more severe with age, and showed enhanced p53-dependent and -independent apoptosis. Spermatogenesis arrest at meiotic phase in Ing2−/− testes was due to impaired HDAC1 accumulation and deregulated chromatin acetylation. This study establishes ING2 as a novel mammalian regulator of spermatogenesis, suggests that an HDAC1/ING2/H3K4me3-regulated, stage-specific coordination of chromatin modifications is essential to normal spermatogenesis, and provides a model system to study idiopathic and iatrogenic infertility in men, including those who had cancer chemotherapy and radiotherapy. Spontaneous tumor incidence and spectrum were similar in wild-type and Ing2-deficient mice.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4890.
Abstract 4898: Polycomb group molecule PHC3 regulates polycomb complex composition and prognosis of osteosarcoma
Authors: Iwata Shintaro
Published: February 2011

PHC3 is a member of the human polycomb complex and has been regarded as a candidate tumor suppressor of osteosarcoma. We performed a mutation survey and PHC3 expression analysis by quantitative real-time PCR using ten osteosarcoma cell lines and 42 primary osteosarcoma samples. Relative PHC3 expression values of clinical samples were analyzed with clinical outcomes, and it was suggested that lower PHC3-expressing patients had significantly worse overall survival. Relative PHC3 values of clinical samples were less than those of normal bone tissues, whereas greater than those of cell lines. By denaturing high performance liquid chromatography analysis and direct sequencing, we found a PHC3 missense mutation in U2OS cells, which resulted in arginine56 to proline substitution. The same point mutation existed in four of 42 primary osteosarcoma samples. Regarding functional analysis, PHC3 expression significantly suppressed colony formation of tumor cells. Intriguingly, polycomb repressive complex 1 members, Bmi1 and Ring1b proteins, were reduced in PHC3-expressing cells. Deletion mutant PHC3 expression suggested that the carboxyl terminus of PHC3 has a role in suppression; the above-mentioned point mutation of PHC3 also lost inhibitory activities. Conversely, Bmi1 expression reduced PHC3 at the mRNA level and induced the proliferation of osteosarcoma cells. Taken together, we confirmed the role of PHC3 as a tumor suppressor in osteosarcoma cells and found that PHC3-dependent tumor suppression may be caused by modification of the composition of polycomb repressive complex 1 in cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4898.
Abstract 4901: Polycomb group proteins in myelodysplastic syndromes and its progression to acute myeloid leukemia
Authors: Chen Zi-Xing , Shen Hong-jie , Cen Jian-nong , Wang yuan-yuan , He Jun , Qi Xiao-fei
Published: February 2011

Polycomb group BMI-1 and EZH2 in addition to SALL4 and P16 were detected in 89 MDS bone marrow including 49 newly diagnosed MDS and 40 treated MDS by using qRT-PCR. We found the expression level of BMI-1, EZH2, SALL4 and P16 were remarkably higher than that in non-MDS cytopenias, such as iron-deficiency anemia (P<0.05). BMI1 expression level has an increasing tendency from MDS-RA to MDS-RAEB without statistically significant. On the contrary, the EZH2 expression level of MDS-RAEB is lower than that of MDS-RA (P<0.05). Although BMI1 expression level was down-regulated when MDS patients were in morphological remission, EZH2, SALL4 and P16 expression levels still remain abnormally high level (P<0.05). We also found that BMI-1 expression level in MDS CD34+ cells was correlated with International Prognostic Scoring System (IPSS) score (R=0.712, P=0.012). BMI-1 had the highest positive rate in MDS-AML. To explore the function of BMI-1 and its possible role in the pathogenesis of MDS transformation into acute leukemia. We have cloned cDNA of BMI-1 and transfected into U937, K562 leukemic cells mediated by MSCV retroviral vector. The viability In vitro of BMI1 transfected cell was higher than control and BMI1 transfected cell also inhibited As2O3-induced apoptosis. Furthermore, BMI1 repressed the expression of EZH2, PTEN and P16 in U937, and repressed the expression of EZH2 and PTEN in K562. Our results provide evidence suggesting that the clonal disorder of MDS patients remained in molecular aberration even after achieving clinical remission. The In vitro results suggest that BMI-1 may play a key role in MDS progression via P16 pathway or PTEN pathway.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4901.
Abstract 4902: Methylation profiles in head and neck cancer
Authors: Demokan Semra , Yalniz Zubeyde , Suoglu Yusufhan , Ulusan Murat , Dalay Nejat
Published: February 2011

Purpose: Among human malignancies, head and neck cancer (HNSCC) is the sixth most common cancer type in the world. Multiple genetic and epigenetic changes may contribute to the development of HNSCC. One of the epigenetic alterations, aberrant methylation in the promoter regions of multiple genes that lead to transcriptional silencing, has been shown in various cancer types including HNSCC. In this study we aimed to evaluate the epigenetic changes specific to HNSCC by investigating aberrant promoter hypermethylation of a panel of 24 tumor suppressor genes.

Experimental Design: We investigated methylation of the promoter regions by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). The methylation status of the selected genes were analyzed in 126 patients with HNSCC. Tumor and corresponding normal tissue samples were obtained during surgical resection. Probes added to the samples hybridized with their target sequence and were amplified by PCR depending on the methylation status of the target. The amplified products were detected by sequence type capillary electrophoresis. Peak sizes and areas were normalized and the relative signal peaks were compared to determine the methylation status.

Results: 18 of 24 (75 %) genes displayed methylation in the tumor samples while none of the corresponding normal tissue samples were methylated. In 67.5 % (85/126) of the tumors the promoter regions were hypermethylated in at least one of the genes. The most frequently methylated genes were RARB (retinoic acid receptor beta), CHFR (checkpoint with forkhead and ring-finger domains) and CDH13 (Cadherin 13) genes. In 50 (39.7 %) patients, methylation was observed in more than one gene.

Conclusions: Methylation of the CHFR, RARB and CDH13 gene promoters is a frequent event in HNSCC, demonstrating potential for these genes as biomarkers in detection strategies.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4902.
Abstract 491: The roles of homologous recombination repair and cell cycle checkpoints in radiosensitization by the Chk1/2 inhibitor, AZD7762
Authors: Morgan Meredith Ann , Parsels Leslie , Zhao Lili , Parsels Joshua , Davis Mary , Arumugarajah Sankari , Canman Christine , Zabludoff Sonya , Maybaum Jonathan , Lawrence Theodore
Published: February 2011

The combination of gemcitabine and radiation is superior to gemcitabine alone for locally advanced pancreatic cancer however the median survival is only approximately one year. We have previously demonstrated that inhibition of Chk1 (checkpoint kinase 1) sensitizes pancreatic cancer cell lines, as well as patient-derived tumor xenografts, to both radiation and gemcitabine-radiation. The exact mechanism(s) for this sensitization, however, remains unclear. Based on the abilities of Chk1 to induce cell cycle arrest and promote HRR (homologous recombination repair) in response to DNA damage, we set out to determine the relevance of these mechanisms to radiosensitization by AZD7762, an AstraZenca Chk1/2 inhibitor currently in Phase I clinical trials. We tested the effects of AZD7762 on survival, cell cycle checkpoints, Rad51 focus formation and HRR activity in response to radiation and gemcitabine-radiation in two pancreatic cancer cell lines, Panc-1 and MiaPaCa-2. We began this study with the observation that AZD7762 sensitized MiaPaCa-2 cells to radiation and gemcitabine-radiation with enhancement ratios of 1.5 ± 0.08 (AZD7762), 1.2 ± 0.07 (50 nM gemcitabine), and 1.9 ± 0.16 (AZD7762 + 50 nM gemcitabine) but did not sensitize Panc-1 cells where enhancement ratios were 1.0 ± 0.03 (AZD7762), 1.5 ± 0.11 (100 nM gemcitabine), and 1.4 ± 0.05 (AZD7762 + 100 nM gemcitabine). AZD7762 inhibited Chk1 in both cell lines as evidenced by accumulation of Cdc25A and abrogation of the radiation-induced G2 arrest. To begin to determine the contribution of HRR to radiosensitization by AZD7762, we utilized a DR-GFP reporter assay which measures homology directed repair of an I-SceI endonuclease-induced DNA double strand break in an integrated GFP reporter gene in MiaPaCa-2 and Panc-1 cells. Treatment of cells expressing the DR-GFP reporter with 100 nM AZD7762 produced a 2- to 3-fold (P<0.05) inhibition of HRR activity in MiaPaCa-2 cells but minimal to no inhibition in Panc-1 cells. Inhibition of HRR by AZD7762 was accompanied by a reduction in the number of MiaPaCa-2 cells which stained positive for Rad51 foci in response to radiation (32 ± 3% versus 9 ± 3%; P<0.05). Taken together these results suggest that inhibition of HRR may play a role in the mechanism(s) of radiosensitization by Chk1 inhibition. It will be important in future studies to further decipher the contributions of cell cycle checkpoint abrogation and HRR inhibition to the mechanisms of radiosensitization by Chk1 inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 491.
Abstract 4915: Global epigenomic modification platform
Authors: Lin Yi-Shiuan , Leu Yu-Wei , Hou Pei-Chi Peggy , Teng I-Wen , Wang Shi-Gang , Shaw Yeng-Jeng , Lee Kuan-Der , Hsu Chia-Chen , Hung Joyce Yu-Hung , Lin Shih-Cheih , Sun H. Sunny , Chang Yu-Sun , Huang Tim H.-M. , Tsai Shaw-Jenq , Hsiao Shu-Huei
Published: February 2011

DNA methylation is an epigenetic mechanism that governs gene silencing and this silencing could be reversed. Pre-deposited global DNA methylation during differentiation is associated with cell fate determination and abnormal DNA methylation could lead to tumorigenesis. A global DNA methylation manipulation system is thus hypothesized to control the differentiation and tumorigenesis more efficiently. Two platforms were established to loss or gain of DNA methylation genome-wide. For the loss of DNA methylation, an in vivo methylation monitoring system was employed to screen 169 procainamide derivatives and identified three distinct classes of these compounds based on their cytotoxicity and demethylation potency. For the gain of DNA methylation, a global DNA methylation subtraction method was developed to identify and methylate target loci genome-wide. After targeted methylation globally, the drug resistance of cancer cells was reverted to become drug sensitive. The same method was used to identify and methylate the hypomethylated loci within somatic stem cells when compared with the differentiated neuronal cells. The targeted methylation accelerated the differentiation of the somatic stem cell to become neuron-like. With both platforms, we wish to evaluate if global DNA methylation changes is sufficient to steer the cell fate. (Supported in part by NSC 97-2320-B-194-003-MY3, NSC 98-3112-B-194-001 and NSC 97-2627-B-006-003)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4915.
Abstract 4921: Selective promoter methylation of the cell differentiation marker, intestinal alkaline phosphatase (iALP), in microsatellite unstable colon cancer
Authors: Shin Joongho , Azarbayejani Azadeh , Lai Victoria , Ahmed Naseem , Hodin Richard , Malo Madhu , Arango Diego , Weber Thomas , Mariadason John M.
Published: February 2011

Tumorigenesis is characterized by excessive cell growth, resistance to apoptosis and aberrant cellular differentiation. In particular, MSI colorectal cancer is frequently characterized by poorly differentiated histology. Histone Deacetylase inhibitors (HDACi) are a novel class of cancer therapeutics that induce growth arrest, apoptosis and differentiation of a variety of tumour types, including colon cancer cells. Differentiation inducing agents have been used effectively to treat hematopoietic malignancies, prompting us to examine the effects of HDACi treatment on differentiation of colon cancer cells, and to compare the effects induced in MSS and MSI colon cancers. Remarkably, while HDACi treatment robustly and consistently induced differentiation induction as measured by alkaline phosphatise activity and iALP gene expression in multiple MSS colon cancer cell lines, approximately 70% of MSI colon cancer cell lines were highly resistant to HDACi-induced ALP induction. Examination of the coding sequence of the iALP gene failed to identify any significant repeat elements suggesting mutation is unlikely to be responsible for lack of iALP induction. Furthermore, transient transfection of an iALP promoter reporter construct demonstrated robust induction in response to butyrate treatment in both MSS and MSI lines, suggesting loss of a key regulatory factor in MSI cell lines is not likely to be responsible. Conversely, examination of iALP promoter methylation by bisulphite conversion and direct sequencing demonstrated multiple CpG sites, including a Sp1/Sp3 binding site, to be methylated in 4 of 5 MSI lines examined, while no methylation was observed in MSS lines. These findings were confirmed using the sequenom assay. ALP promoter methylation was independent of CIMP status. The importance of Sp1/Sp3 in butyrate-induced iALP induction was demonstrated by the ability of the Sp1/Sp3 inhibitor, mithramycin, to attenuate butyrate-induced ALP activity. Finally, consistent with methylation being a key determinant of iALP expression in MSI lines, azacytidine treatment was able to re-induce iALP expression, and butyrate-induction of ALP expression was markedly enhanced in isogenic HCT116 cells lacking the DNA methyltransferases Dnmt1 and Dnmt3b (DKO), compared to parental cells. These findings demonstrate selective promoter methylation of iALP in MSI colon cancer cell lines.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4921.
Abstract 4929: Hypermethylation of tumor suppressor gene promoters in basal cell carcinoma
Authors: Brinkhuizen Tjinta , van Engeland Manon , van Marion Ariënne , van Steensel Maurice
Published: February 2011

Background

Basal-cell carcinoma (BCC) is the most frequent malignancy in Caucasians, with increasing incidence. Overactivity of the Sonic Hedgehog pathway is implicated in BCC etiology, however recent literature suggests that BCC carcinogenesis may be more complex.

Silencing of tumor suppressor genes by promoter hypermethylation, influenced by hypoxia, results in deregulation of cancer-related pathways. Since BCC arises in a hypoxic micro-environment, we assumed that epigenetic modifications may play a major part in BCC tumorigenesis. In this pilot we investigated promoter hypermethylation of tumor suppressor genes selected for their possible involvement in BCC. Eventually, knowledge of epigenetic modifications in BCC may impact diagnosis, classification, and treatment.

Methods

DNA was isolated from 33 formalin fixed paraffin-embedded BCC of 3 different histological subtypes. Marked tumor tissue was macroscopically scraped from slides and collected. CpG island methylation status of 10 promoter regions was analysed by methylation-specific PCR on sodium bisulfite-treated DNA. Data were compared with 36 normal skin tissue samples. All data were analysed with SPSS version 17.0. We used the Pearson Chi-Square test. Statistical significance was defined as a p-value ≤0.05.

Results

DNA methylation levels of the examined candidate gene promoters in tumor tissue compared to normal skin tissue were significantly higher for the following genes: APC 100% vs. 47,1% (p<0,001); sFRP5 93,9% vs. 42,4% (p<0,001); SHH 90,9% vs. 50,0% (p< 0,001) and 81,8% vs. 56,3% (p= 0,029) for CylD. In addition, the PTCH1 promoter showed a tendency to hypermethylation. The remaining promoters of sFRP1, sFRP2, sFRP4, TSC1 and RASSF1A showed no hypermethylation.

Conclusions

From our preliminary data we conclude that in BCC, promoters of several tumor suppressor genes, which may normally inhibit BCC growth, show aberrant methylation resulting in silencing of these genes. As a consequence, signalling pathways implicated in cancerous growth such as the SHH-, WNT- and NFκB- routes may be affected. Our data provide evidence that epigenetic modifications may play a role in BCC tumorigenesis. Genome-wide analyses will be our next step to screen for more relevant genes and to chart the full extent of genomic methylation changes in BCC. This entirely new approach may lead to new insights into development and treatment of BCC.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4929.
Abstract 4931: Methylation of the hTERT promoter: a potential cancer biomarker for leptomeningeal metastasis detection in cerebrospinal fluids
Authors: Benhattar Jean , Bougel Stéphanie , Gallagher Gabrielle , de Flaugergues Jeanne-Chantal , Janzer Robert
Published: February 2011

Background: The diagnosis of leptomeningeal metastases is usually based on the finding of malignant cells by cytological examination in the cerebrospinal fluid (CSF). However, cytology is only moderately sensitive, and sensitive and specific cancer biomarkers may improve the detection of tumor cells in the CSF. Expression of telomerase hTERT subunit might be such a marker but its use is hampered by proliferating normal lymphocyte cells. hTERT methylation, characterizing most cancer cells, might be an alternative as proliferating lymphocytes have unmethylated hTERT promoter. The aim of this study was to develop a specific, sensitive, quantitative, and fast method for detection of hTERT methylation and to explore its use as a cancer biomarker in the diagnosis of metastatic tumors in CSF.

Materials and methods: hTERT methylation levels were assayed by several quantitative techniques including methylation-sensitive dot blot assay (MS-DBA), methylation-sensitive high resolution melting (MS-HRM), and a newly developed real-time MS-HRM assay. We used MS-HRM assays for the analysis of 59 CSF specimens from 50 patients including 31 CSFs from 23 patients with a known malignancy suspected for leptomeningeal metastasis.

Results: MS-HRM assays both allowed hTERT methylation quantification in CSF samples. The MS-HRM detected samples containing more than 10% of hTERT methylated alleles whereas real-time MS-HRM detected methylation below 10%. PCR products were obtained from 54 CSF samples (92%). hTERT methylation was only detected in the CSF from patients with a known malignancy.

Conclusion: The real-time MS-HRM analysis is a fast, sensitive, and specific technique for methylation assessment in many diagnostic and research applications. As an adjunct to the traditional examination of CSF, the hTERT MS-HRM approach could be perform as routine diagnosis of leptomeningeal metastases and could become a valuable tool for further management and planning of treatment.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4931.
Abstract 4932: Genistein Can Induce Demethylation Of Wnt Negative Regulatory Genes In Prostate Cancer Cells
Authors: Phillip Cornel J. , Moreno Carlos S.
Published: February 2011

Among American men, prostate cancer is a prevalent malignancy that accounts for an estimated 192,280 new cases and 27,360 deaths in 2009. Previous work has determined that the adenomatous polyposis coli (APC) gene that negatively regulates the Wnt pathway is hypermethylated in a high proportion of prostate cancers. We hypothesized that methylation of additional Wnt negative regulatory genes may also play a role in prostate cancer progression such as SRY box 7 (SOX7), Wnt inhibitory factor 1 (WIF1), secreted frizzled-related protein 1 (SFRP1) and dickkopf-related protein 3 (DKK3). To test this hypothesis, the basal level methylation status was tested in several prostate cancer patient tumor and adjacent normal samples by methylation specific PCR. When the methylation status of some of these genes was explored, there was variation in the methylation of some of these genes, particularly DKK3 among different patients. This preliminary finding may suggest that the methylation status of some of these genes may play a role in the progression of prostate cancer. In this study, we propose that demethylation of these Wnt negative regulatory genes may allow for a decrease activity of the Wnt signaling pathway. Genistein is a soy isoflavone that has many potentially therapeutic activities, including demethylation of DNA. We hypothesized that treatment of prostate cancer cells could lead to demethylation and re-expression of these genes. To test our hypothesis, prostate cancer cell lines LNCAP and DU145 were treated with for 6 days with concentrations of 5, 10, and 20 µM genistein. We observed a dose-dependent reduction in methylation of SOX7, DKK3, WIF1, and SFRP1. Taken together, these data indicate that genistein may have therapeutic value for patients with prostate cancer. As a therapeutic agent, genistein treatment has potential solely or in combination with other compounds to demethylate Wnt negative regulatory genes and possibly impact patient outcomes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4932.
Abstract 4936: Promoter methylation in prostate cancer: A pilot study
Authors: Delgado-Cruzata Lissette , Gonzalez Karina , Santella Regina M. , Shen Jing
Published: February 2011

Prostate cancer is a leading cause of cancer death among men in the United States. The current method used for the screening for prostate cancer, the prostate-specific antigen (PSA) test, has poor specificity and sensitivity. This has lead to the search for new biomarkers that can improve the prostate cancer screening, either by themselves or in combination with established markers. Epigenetic modifications, believed to occur early during tumorgenesis are one of the new measurements that could aid in improving prostate cancer screening. We conducted a case-control study to determine the association of promoter methylation of p16 INK4a and GSTP1 and prostate cancer risk. Twenty seven histologically confirmed prostate cancer patients were matched by age and ethnicity to twenty four controls. DNA was extracted from plasma, and percentage of DNA methylation was determined by pyrosequencing. We found that methylation levels were consistently higher in cases than in controls, but this difference was not statistically significant (p16 INK4a OR (95%CI)=2.38 (0.75-7.50); GSTP1 OR (95%CI)=2.57 (0.79-8.40). Methylation levels in these regions were also investigated in tumor and normal adjacent tissue samples from cases. Promoter regions of these genes are hypermethylated in prostatic tumors (7.2% for p16INK4a and 95.8% for GSTP1). Hypermethylation of both regions is statistically significantly associated with tumor status with OR p16INK4a =5.0, 95%CI: 1.3-19.1 and ORGSTP1=22.9, 95%CI: 2.7-198.6, respectively. The correlations of GSTP1 and p16INK4a methylation levels in matched tissue and plasma samples were also determined. Pearson's correlation coefficient was 0.46 for GSTP1 methylation when comparing tumor tissue and plasma (p=0.037). However, the tissue-plasma correlation for p16INK4a methylation levels was not statistically significant. Our samples size is small, but our results are encouraging suggesting that plasma DNA methylation levels at the promoter regions of p16INK4a and GSTP1 are relevant in the etiology of prostate cancer and may be useful as markers of the onset of prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4936.