bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-199: ZEB factors link IL-1β-induced EMT and stem cell phenotypes in colon cancer
Authors: Wang Lei , Li Yijing , Beckley Amy , Pappan Loretta , Shi Jishu
Published: August 2012

Up-regulation of inflammatory cytokines such as IL-1β in tumor microenvironment has been associated with progression of human colorectal cancer. However, the exact molecular mechanisms by which inflammatory cytokines promote colon tumor growth are still unclear. Cancer stem cells (CSCs), a distinct subpopulation with self-renewal ability, drug resistance and high oncogenic potential, are thought to be responsible for tumor initiation and progression. Epithelia-mesenchymal transition (EMT), characterized by the repression of E-cardherin expression and increased cell mobility, is associated with enhanced metastasis. The objective of this study is to determine whether IL-1β may promote cancer progression by inducing EMT and colon CSC development. Here, we showed that IL-1β promoted E-cadherin repression in both human colon cancer cell line HCT-116 cells and primary colon cancer cells. Expression of E-cadherin transcription repressors ZEB1 and ZEB2 was up-regulated in IL-1β-treated HCT-116 and primary colon cancer cells, respectively. In addition, IL-1β-treated HCT-116 cells exhibited higher cell migratory ability. Moreover, IL-1β induced the expression of stem-cell marker Bmi-1 in both HCT-116 cells and primary colon cancer cells. IL-1β increased these cells’ ability of sphere formation in serum-free conditions and chemo-resistance; properties associated with colon CSCs. Taken together, these results indicate that IL-1β can induce EMT and promote generation of colon CSCs. More importantly, we found that ZEB1 and ZEB2, but not other E-cadherin transcriptional repressors (such as Snail and Twist), were consistently up-regulated in HCT-116 cells and primary colon cancer cells, respectively, during both processes of IL-1β-induced EMT and sphere formation. This suggests that ZEB factors may mediate IL-1β-induced EMT and generation of colon CSCs. Currently, this hypothesis is under evaluation using shRNAs to knock down ZEB1 in HCT-116 cells. Understanding the role of ZEB factors in IL-1β-mediated EMT and CSC development may lead to the identification of novel molecular targets for colon cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-199. doi:1538-7445.AM2012-LB-199
Abstract LB-2: Structure-based design of G-quadruplex ligands leads to enhanced potency against pancreatic cancer cells
Authors: Micco Marialuisa , Collie Gavin , Gunaratnam Mekala , Ohnmacht Stephen A. , Reszka Anthony P. , Neidle Stephen
Published: August 2012

Certain small molecules can sequester the single-stranded overhang of human telomeric DNA into higher-order quadruplex structures. Such quadruplex-ligand complexes interfere with telomerase activity and inhibit its ability to maintain telomere integrity in many cancer cell types. This approach to selectivity interfering with cancer cell viability has been demonstrated for several classes of quadruplex-binding molecules in cells and in xenograft experiments. We have previously described a series of tetra-substituted naphthalene diimide (ND) compounds with high affinity for human telomeric quadruplex DNA. These also have high potency for growth inhibition in a panel of cancer cell lines, concomitant with telomerase inhibition. Those compounds with N-methyl-piperazine (NMP) end-groups have shown exceptional potency in a pancreatic cancer cell line panel, with the lead compound having typical IC50 values of 100-200 nM. It has significant anti-tumor activity in a pancreatic cancer xenograft model, with a 50% reduction in tumor volume on ip administration, along with inhibition of telomerase activity. We report here a structure-based design study in which the four positive charges of the tetra-NMP compound have been modulated by substituting NMP groups on two of the four side-arms of the ND core with more weakly basic groups, in order to improve drug-like features. A diminution in the highly cationic nature of these ND compounds may improve cellular uptake and tumor distribution properties, while retaining quadruplex affinity. The morpholine group has been used because it is less basic than the NMP group. We hypothesized that the presence of four NMP groups is non-essential in order to retain effective quadruplex binding, since the morpholine group is of equivalent size and so could be bound in the same way. Three co-crystals structures of NMP and morpholino-containing ND compounds with a human telomeric quadruplex have been determined. These show that the morpholino group binds in quadruplex grooves in a closely-similar manner to the NMP group and has the same hydrogen-bonding. Accordingly, several hybrid morpholino-NMP ND compounds have been synthesized and shown high quadruplex affinity, exceptional potency in selectively inhibiting growth in pancreatic cancer cell lines, with IC50 values of <10 nM, together with potent telomerase inhibition in these cells. Lead compounds from this study are currently being taken into further in vivo evaluation, with a view to identifying a compound suitable for pre-clinical development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-2. doi:1538-7445.AM2012-LB-2
Abstract LB-31: USP15 stabilizes the TGF-β receptor I and promotes oncogenesis through the activation of the TGF-β signal in glioblastoma
Authors: Eichhorn Pieter J. , Rodon Laura , Gonzalez-Junca Alba , Baselga Jose , Seoane Joan
Published: August 2012

In advanced cancer including glioblastoma, the TGF-β pathway acts as an oncogenic factor and is considered a therapeutic target. Using a functional RNA-interference screen, we have identified the deubiquitinating enzyme USP15 as a critical component of the TGF-β signaling pathway. USP15 binds to the SMAD7-SMURF2 complex and deubiquitinates and stabilizes the TGF-β receptor I leading to an enhanced TGF-β signal. Importantly, we find that the USP15 gene is found amplified in glioblastoma, breast and ovarian cancer and that USP15 gene amplification confers poor prognosis in glioblastoma patients. Furthermore we demonstrate that downregulation or inhibition of USP15 in a patient-derived orthotopic mouse model of glioblastoma decreases TGF-β activity. Moreover, depletion of USP15 decreases the oncogenic capacity of patient-derived glioma-initiating cells due to the repression of TGF-β signaling. Our results show that USP15 regulates the TGF-β pathway and is a critical factor in glioblastoma pathogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-31. doi:1538-7445.AM2012-LB-31
Abstract LB-315: Suppression of tumor invasion and metastasis by inhibition of c-MET and VEGF signaling together
Authors: Sennino Barbara , Ishiguro-Oonuma Toshina , Williamson Casey W. , Wei Ying , Chapman Harold A. , Christensen James G. , Aftab Dana T. , McDonald Donald M.
Published: August 2012

Treatment with inhibitors that block vascular endothelial growth factor (VEGF) signaling can make tumors more aggressive in preclinical models. The mechanism underlying this form of resistance is not fully understood, but vascular pruning, intratumoral hypoxia, and increased expression of c-MET, the tyrosine kinase receptor for hepatocyte growth factor (HGF), could be contributing factors. We found that treatment of pancreatic neuroendocrine tumors in RIP-Tag2 mice with a neutralizing anti-VEGF antibody or with sunitinib, a receptor tyrosine kinase inhibitor that blocks VEGFR and related receptors, reduced tumor burden and vascularity but increased intratumoral hypoxia, HIF-1alpha, and expression and activation of c-MET. Invasion into the exocrine pancreas and metastasis to the liver also increased. Importantly, administration of either of two c-MET inhibitors, PF-04217903 or crizotinib (PF-02341066), together with anti-VEGF therapy reduced tumor growth and vascularity and also significantly decreased local invasion and liver metastasis. Similar benefits were found after treatment with cabozantinib (XL184), a multitargeted tyrosine kinase inhibitor that simultaneously blocks c-MET and VEGF receptors. Together, these results indicate that inhibition of VEGF signaling can promote vascular pruning, intratumoral hypoxia, HIF-1alpha accumulation, and activation of c-MET. Concurrent inhibition of c-MET and VEGF signaling not only can slow tumor growth but also can reduce invasion and metastasis. Inhibition of c-MET and VEGF signaling pathways together combines the favorable tumor growth-slowing effects of inhibiting angiogenesis with suppression of invasion and metastasis, despite the intratumoral hypoxia due to vascular pruning.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-315. doi:1538-7445.AM2012-LB-315
Abstract LB-33: Identification of two unrecognized acute myeloid leukemia (AML) subtypes based on cell by cell analysis of leukemic blasts
Authors: Hsu Faye , Nolan Garry P. , Chen Tiffany , Goltsev Yury
Published: August 2012

Single cell multiparametric flow cytometry studies have previously shown that G-CSF-mediated intracellular signaling responsiveness of blast cell subsets stratified clinical outcomes of AML patients. 13 AML samples were stimulated with G-CSF, fixed, and permeabilized with a technique we developed that preserves mRNA integrity in a manner suitable for microarray-based expression analysis. From the microarray profiles we identified surface markers that could be prospectively used to separately enrich G-CSF Responsive Cells (GRCs) and non-G-CSF Responsive Cells (NGRCs) from each sample. The results demonstrate that two previously unrecognized subtypes of AML could be discerned, as portrayed by the proteomic and molecular characteristics of the GRC and NGRC subsets. First, GRC cells had a higher colony formation capacity as well as being more reactive to cytokines secreted by NGRCs In one discovered AML subtype, patients present with bone marrow that had higher percentages of GRCs (20-50%), with the remainder being NGRCs. The molecular signatures of both the GRCs and NGRCs in this subtype had a hematopoietic progenitors mRNA signature (HPMS). In the second subtype of AML there were lower percentages of GRCs (0.5-10%, which had the HPMS) but in these cases the NGRC mRNA signatures were distinct resembling those of a more differentiated myeloid phenotype. Therefore, in most AML patients tested, two blast cell populations inevitably appear, and each of these blasts represented a distinct level of hematopoietic maturity, or cytokine responsiveness. The common thread between the two subtypes is that the GRCs in each patient were more capable of colony formation, enabled by a potentially more “stem-like” phenotype. Critically, each population would be clinically termed “blast” cells and would therefore be considered malignant and a potential target for chemotherapy. These results lead to two important conclusions. The first is that the blast phenotype can exist independently of the apparent cell maturation stage. This raises the questions of whether each blast subtype is equally susceptible to standard chemotherapy, whether a strategy of defining the molecular players that drive the formation of each of these blast types should be pursued, and finally whether such information can be used to better combat these aspects of the leukemic disease spectrum.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-33. doi:1538-7445.AM2012-LB-33
Abstract LB-34: Investigating novel biological functions of tankyrases in cancer
Authors: Schoumacher Marie , Feng Yun , Mishina Yuji , Shao Wenlin
Published: August 2012

Tankyrases (TNKS1 and TNKS2) are enzymes that catalyze the addition of poly(ADP-riboses) on acceptor proteins. Through poly(ADP-ribosylation), TNKS regulate the activity and/or stability of their substrates via different mechanisms. A number of TNKS substrate proteins of diverse biological functions have been reported. More recently, in silico analysis using consensus-derived TNKS binding motif predicts a wide range of proteins may be targeted by TNKS, supporting that TNKS is involved in the regulation of diverse cellular processes. In particular, TNKS have been linked to telomere elongation, Wnt signaling and spindle assembly during mitotic progression, functions that are essential for cell proliferation. Hence, TNKS could be potential molecular targets for anti-cancer therapeutics. Using highly potent and specific TNKS inhibitors or depletion of TNKS by RNAi, we have identified several cancer models which growth is dependent on TNKS activity. In one of the TNKS-dependent cancer models, RERF-LC-MS, our studies demonstrate depletion of β-catenin does not impair cell proliferation. These data suggest TNKS regulate cell proliferation in this model via Wnt-independent functions. In an effort to elucidate the mechanism underlying RERF-LC-MS growth dependence on TNKS, we tested the hypothesis whether TNKS inhibition leads to aberrant spindle formation. We observed that inhibition of TNKS increases the formation of multipolar spindles in mitotic cells. We are currently testing whether this is mediated by NuMA, a well described substrate of TNKS whose function is required for spindle integrity. In addition, we are performing proteomic studies to identify novel binding partners of TNKS that would help to unravel novel partners of TNKS in mitosis and their functions in mediating cancer cell dependence on TNKS. Overall, this work will further increase our understanding of TNKS biology in cancer, and will impact on how we could best target TNKS as a novel anti-cancer treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-34. doi:1538-7445.AM2012-LB-34
Abstract LB-341: Cysteine dioxygenase 1 is a tumor suppressor gene silenced by promoter methylation in multiple human cancers
Authors: Brait Mariana , Ling Shizhang , Nagpal Jatin , Chang Xiaofei , Park Hannah , Lee Juna , Okamura Jun , Yamashita Keishi , Sidransky David , Kim Myoung Sook
Published: August 2012

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-341. doi:1538-7445.AM2012-LB-341
Abstract LB-350: EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition by vemurafenib
Authors: Corcoran Ryan B. , Ebi Hiromichi , Turke Alexa B. , Coffee Erin M. , Nishino Michiya , Cogdill Alexandra P. , Brown Ronald D. , Dias-Santagata Dora , Pelle Patricia Della , Hung Kenneth E. , Flaherty Keith T. , Piris Adriano , Wargo Jennifer A. , Settleman Jeffrey , Mino-Kenudson Mari , Engelman Jeffrey A.
Published: August 2012

Oncogenic BRAF mutations occur in 10-15% of colorectal cancers (CRCs) and confer adverse outcome. While RAF inhibitors such as vemurafenib (PLX4032) have produced dramatic response rates in BRAF mutant melanoma (∼60-80%), they are surprisingly ineffective in BRAF mutant CRCs (∼5% response rate), and the reason for this disparity remains unclear. Compared to BRAF mutant melanoma cells, BRAF mutant CRC cells were less sensitive to vemurafenib, and P-ERK suppression was not sustained in response to treatment. Although transient inhibition of phospho-ERK by vemurafenib was observed in CRC, rapid ERK re-activation occurred through EGFR-mediated activation of RAS and CRAF. BRAF mutant CRC cell lines and patient tumor specimens expressed higher levels of phospho-EGFR than BRAF mutant melanoma cell lines and primary tumor specimens, suggesting that CRCs are specifically poised for EGFR-mediated resistance. Combined RAF and EGFR inhibition blocked reactivation of MAPK signaling in BRAF mutant CRC cells, markedly improved efficacy in vitro, and led to robust tumor regressions in vivo in BRAF mutant xenografts models. These findings support combined RAF and EGFR inhibition as a novel and promising therapeutic strategy for evaluation in clinical trials in patients with BRAF mutant CRC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-350. doi:1538-7445.AM2012-LB-350
Abstract LB-359: Extended accumulation of acetylated histone in tumor tissues obtained from the phase I study of CG200745
Authors: Ro SeongGu , Kong Gyoung-Sun , Lee Sang-Koo , Cho Joong Myung , Cha Hyun-Ju , Kwon Sool Ki , Chang Ho-Jin , Kim Kyu-pyo , Hong Yong Sang , Bae Kyun-seop , Lee Jae-Lyun , Kim Tae Won
Published: August 2012

Extended Accumulation of Acetylated Histone in Tumor Tissues Obtained from the Phase I Study of CG200745 Accumulation of acetylated histone is a physiological phenomenon caused by inhibition of HDACs. We have observed such accumulation in cells, mice and humans when CG200745, our cancer therapeutic candidate, was treated. In the phase I clinical trial (NCT01226407), we had a chance to compare such accumulations in both the blood and tumor tissues. Interestingly, the accumulation in the tissues was maintained much longer than that in the blood and its level was significantly high even at 24 hours after dose. Such difference in histone accumulation has been explained by the different PK profile of CG200745 between the blood and the tumor tissues. Although we have not got PK data from the human tumor tissues yet, the difference was already observed in mice PK studies. Specifically, the half-life of CG200745 in plasma of mice was extremely short like other known HDAC inhibitors (approximately 20 mins), but in tumor tissues, that of CG200745 was as long as approximately 7 hours. In the current human studies, CG200745 is showing a much better PK profile in plasma than that of mice studies. Its half-life even in plasma is observed as several hours and clearance is much lower than other HDAC inhibitors. Actually, the AUC/dose of CG200745 is the highest among all the clinical stage HDAC inhibitors. Taking the extended accumulation of acetylated histone in the tumor tissues and better human PK profile into consideration, we believe that CG200745 with excellent HDAC inhibition will be a promising anticancer intervention in the near future.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-359. doi:1538-7445.AM2012-LB-359
Abstract LB-364: The ACRBP tumor antigen normalizes mitotic spindle function to promote cancer cell proliferation
Authors: Wehn Amy K. , Xie Yang , Schorge John O. , Whitehurst Angelique W.
Published: August 2012

Tumor cells can undergo a productive mitosis despite the wholesale presence of chromosomal aberrations. While this behavior may generate plasticity with respect to the acquisition of tumorigenic traits, it also represents a vulnerability for therapeutic intervention. Using a whole genome siRNA screen for modifiers of chemoresponsiveness, we identified the tumor antigen ACRBP/OY-TES-1 as a specifier of paclitaxel resistance. ACRBP is as a cancer-testis antigen, a class of genes whose expression is selective to the testes but frequently activated in a wide range of tumors. Scant functional data exists on the contribution of CT-antigens to tumorigenic phenotypes. Here, we find that ACRBP is both necessary and sufficient for paclitaxel resistance in ovarian cancer cell lines and ovarian tumor explants. Furthermore, depletion of ACRBP significantly sensitizes ovarian tumor cells to paclitaxel in an orthotopic xenograft model system. We identified the mitotic spindle component NuMA as an ACRBP-interacting protein that may account for the effects of ACRBP on paclitaxel sensitivity. In particular, ACRBP depletion results in mitotic errors and reduced proliferative fitness that can be rescued by NuMA co-depletion. ACRBP appears to modulate NuMA protein levels, potentially by regulating its post-translational modifications. Remarkably, NuMA overexpression, a frequent event in tumors, is sufficient to induce expression of ACRBP. We propose that activation of ACRBP is positively selected for during tumor evolution to buttress mitotic spindle fidelity and chromosome segregation. Identification and elaboration of elements of the genome that are subverted to support essential cell biological processes in tumor cells present new therapeutic opportunities.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-364. doi:1538-7445.AM2012-LB-364
Abstract LB-368: Exploiting the mechanism of intron-splicing to produce viral vectors harboring toxin genes for cancer therapy
Authors: Chen Haifeng
Published: August 2012

Suicide gene therapy has been widely investigated for the treatment of human immunodeficiency virus (HIV) infection, for controlling graft-versus-host disease, and also for the treatment of cancer. While the production of viral vectors carrying suicide genes such as the herpes simplex virus thymidine kinase (HSV-TK) or cytosine deaminase are easily achievable, the generation of viral vectors containing toxin genes such as diphtheria toxin A fragment (DT-A), Pseudomonas exotoxin (PE), and barnase has always been a challenge very difficult to fulfill due to the extreme toxicity of these toxins. We developed a novel way to produce in insect cells baculoviral and adeno-associated viral (AAV) vectors harboring toxin genes such as DT-A or Barnase by exploiting the different intron-splicing mechanisms between insect and mammalian cells. Since most of the mammalian introns are not functional in insect cells, we cloned two mammalian introns to interrupt the DT-A or Barnase coding sequences and produced recombinant baculoviral and AAV vectors at the normal level as compared with the baculoviral or AAV vectors carrying just the green fluorescent protein (GFP) gene. After transduction of the toxin gene-harboring baculoviral or AAV vectors into mammalian HEK293 cells, the mammalian introns were spliced out and toxins expressed and killed the HEK293 cells. This is the first report so far as we know that the differences in intron-splicing between insect and mammalian cells can be utilized to manufacture viral vectors harboring toxin genes. Our finding indicates that this novel method provides a valuable new tool for the development of toxin-based suicide gene cancer therapy. Bac, baculovirus; AAV, adenoassociated virus; inDTA (hGH), DT-A gene interrupted by human growth hormone intron; inBar (SV40)-GFP, Barnase gene interrupted by SV40 LT antigen intron and fused in-frame with GFP; vg, vector genome copy number; pfu, plaque-forming unit; ND, not done.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-368. doi:1538-7445.AM2012-LB-368
Abstract LB-378: The outlier approach discriminates drivers from passengers among genes methylated in cancers
Authors: Kikuyama Mizuho , Takeshima Hideyuki , Kinoshita Takayuki , Okochi-Takada Eriko , Wakabayashi Mika , Akashi-Tanaka Sadako , Ogawa Toshihisa , Seto Yasuyuki , Ushijima Toshikazu
Published: August 2012

A large number of genes are methylated in cancers, and most of them are methylated as passengers of carcinogenesis, and this fact hampers identification of drivers, tumor-suppressor genes (TSGs), silenced by aberrant methylation of promoter CpG islands (CGIs). To overcome this issue, we focused on the rule that the vast majority of genes methylated in cancers lack, in normal cells, RNA polymerase II (Pol II) and have trimethylation of histone H3 lysine 27 (H3K27me3) in their promoter CGIs. However, approximately 5% of the genes methylated in cancers are against this general rule, constituting a group of “outliers”. It is expected that TSGs belong to this group of outliers since they are expressed or ready to be expressed in normal cells. Here, we aimed to demonstrate that TSGs belong to the group of outliers, and that searching for outliers enables us to identify TSGs. First, we demonstrated that some of known TSGs in breast and colon cancers had Pol II and lacked H3K27me3 in normal cells, being outliers of the rule. We then made a genome-wide search for outlier genes in breast cancers. Based on Pol II binding and H3K27me3 statuses in normal cells and DNA methylation statuses in five breast cancer cell lines, 14 outlier genes were identified from 280 methylated genes. Among these genes, four genes were confirmed to be methylated in primary breast cancer samples, and two (HOXA5 and FBN2) of these were known TSGs. Among the remaining two genes, DZIP1 was shown to suppress growth of breast cancer cells, suggesting it to be a novel TSG. These results showed that some of known TSGs are the outlier genes and that TSGs can be efficiently identified by searching for outliers against the rule, the outlier approach.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-378. doi:1538-7445.AM2012-LB-378
Abstract LB-38: Kure It cancer research
Authors: Jones Karen
Published: August 2012

Kure It funds researchers conducting innovative projects in the fields of kidney cancer and other underfunded malignancies. Kiure It raised funds in several ways: through annual special events; contributions from family and community foundations; and partnerships with the self storage industry, through which we've created tailored programs and events aimed at raising funding and awareness. Since its inception in 2007, Kure It has raised over one milliion for research. The goal is to raise one million annually going forward. Kure It supports the work of researchers at City of Hope and Cedars-Sinai.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-38. doi:1538-7445.AM2012-LB-38
Abstract LB-381: Biochemical characterization of recombinant β-glucosyltransferase and analysis of global 5-hydroxymethylcytosine in unique genomes
Authors: Terragni Jolyon
Published: August 2012

5-hydroxymethylcytosine (5-hmC) is an enzymatic oxidative product of 5-methylcytosine (5-mC). Ten Eleven Translocation (TET) family of enzymes catalyze the conversion of 5-mC to 5-hmC. Phage encoded glucosyltransferases are known to glucosylate 5-hmC, which can be utilized to detect and analyze the 5-hmC an epigenetic mark in the mammalian epigenome. Here we have performed a detailed biochemical characterization and steady-state kinetic parameter analysis of T4 phage β-glucosyltransferase (β-GT). Recombinant β-GT glucosylates 5-hmC DNA in a non-processive manner and binding to either 5-hmC DNA or uridine diphosphoglucose (UDP-glucose) substrates is random, with both binary complexes being catalytically competent. Product inhibition studies with β-GT demonstrated that UDP is a competitive inhibitor to UDP-glucose and a mixed inhibitor to 5-hmC DNA. Similarly, the glucosylated-5-hmC (5-ghmC) DNA is a competitive inhibitor to 5-hmC DNA and mixed inhibitor to UDP-glucose. 5-hmC DNA binds ∼10 fold stronger to the β-GT enzyme when compared to its glucosylated product. The numbers of 5-hmC on target sequences influenced the turnover numbers for recombinant β-GT. Furthermore, we have utilized recombinant β-GT to estimate global 5-hmC content in a variety of genomic DNAs. Most of the genomic DNAs derived from vertebrate tissue/cell-lines contained 5-hmC. DNA from mouse, human and bovine brains displayed 0.5 to 0.9% of the total nucleotides as 5-hmC, which was higher compared to other tissues. A comparison between cancer and healthy tissue genomes suggested a lower percentage of 5-hmC in cancer, which may reflect the global hypomethylation of 5-mC observed during oncogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-381. doi:1538-7445.AM2012-LB-381
Abstract LB-384: BARX1, CYGB, and other epigenetic targets of gastric cancer
Authors: Agarwal Rachana , Mori Yuriko , Cheng Yulan , Abraham John M. , Montgomery Elizabeth , Meltzer Stephen J.
Published: August 2012

Gastric cancer (GC), a disease with a very poor prognosis, is the second leading cause of cancer-related deaths worldwide. Although few studies have identified genetic and epigenetic alterations to be involved in gastric carcinogenesis, the molecular pathogenesis of this disease remains incompletely understood, and largely unexplored. With an aim to understand, and identify the molecular pathways important in the pathogenesis of GC, and to identify targets for its detection and therapy, we conducted a pilot microarray-based genome-wide search for identification of epigenetic targets in GC. This study identified 11 prioritized targets of aberrant epigenetic inactivation in gastric neoplasia (GN), including the tight junction protein claudin-11 (CLDN11), the transcription factor BarH-like homeobox (BARX1), basonuclin1 (BNC1), Coagulation factor C homolog (COCH), filamin C gamma (FLNC), cytoglobin B (CYGB), glutamine-fructose-6-phospahe-transaminase-2 (GFPT2), heat-shock-70 kDa protein-6 (HSPA6), snail homolog 1(SNAL1), skin calmodulin-related factor (SCARF), and tumor protein p53 binding protein 2 (TP53BP2). Our initial validations of these targets, particularly, CLDN11, BARX1 and CYGB confirmed these genes to be hypermethylated in their promoter region in gastric cancer cell lines and patient specimens, and their mRNA expression levels to be reduced in gastric cancer tissues and cell lines. More interestingly, our recent preliminary studies demonstrate that overexpression of the BARX1, in GC cells which lack its endogenous expression led to dramatic phenotypic changes in these transfected cells. Moreover, viable cell count was reduced in BARX1 transfected cells. BARX1 is a transcription factor which is known to be involved in the differentiation of embryonic endoderm into stomach. This study for the first time, identifies a potential role of BARX1 in gastric carcinogenesis. Further, large-scale validation studies are underway to confirm the role of BARX1 and other genes as potential targets for GC detection and therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-384. doi:1538-7445.AM2012-LB-384
Abstract LB-386: The NuRD complex cooperates with DNMTs to maintain silencing of colorectal tumor suppressor genes.
Authors: Cai Yi , Geutjes Ernst-Jan , Roepman Paul , Yu Li-Rong , Blijswijk Janneke V. , Wang Wei , Mohammad Helai , de Rink Iris , Baylin Stephen , Bernards Rene
Published: August 2012

Many Tumor Suppressor Genes (TSGs) are silenced through synergistic layers of epigenetic regulation including abnormal DNA hypermethylation of promoter CpG islands, repressive chromatin modifications and enhanced nucleosome deposition over transcription start sites. Many of the protein complexes responsible for silencing of such TSGs remain to be identified. A subset of silenced TSGs controlling key regulatory signaling pathways in colorectal cancer cells can be partially reactivated by in cells disrupted for the DNA methyltransferase 1 and 3B (DNMT1 and 3B) or by DNMT inhibitors (DNMTi). Herein, we used proteomic and functional genomic approaches to identify additional proteins that cooperate with DNMTs in silencing these key silenced TSGs in colon cancer cells. We discovered that DNMTs and the core components of the NuRD nucleosome remodeling complex, chromo domain helicase DNA-binding protein 4 (CHD4), histone deacetylases 1 and 2 (HDAC1 and 2), occupy the promoters of several of these key hypermethylated TSGs and physically and functionally interact to maintain their silencing. Consistent with this, we find an inverse relationship between expression of HDAC1 and 2 and these TSGs in a large panel of primary colorectal tumors. We demonstrate that this DNMT-NuRD complex maintains the silencing of several negative regulators of the WNT signaling pathway. We find that depletion of CHD4 is synthetic lethal with DNMT inhibition in correlation with reactivation of TSGs, suggesting that their combined inhibition may be beneficial for the treatment of colon cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-386. doi:1538-7445.AM2012-LB-386
Abstract LB-387: EZH2 knockdown suppresses the growth of human inflammatory breast cancer cells both in vitro and in vivo in xenograft models
Authors: Mu Zhaomei , Li Hua , Zhang Rugang , Cristofanilli Massimo
Published: August 2012

PURPOSE: Inflammatory breast cancer (IBC) is a rare but very aggressive type of advanced breast cancer with a poor prognosis. Thus, there is an urgent need for novel IBC therapeutics. Enhancer of zeste homolog 2 (EZH2) is of the catalytic subunit of the polycomb repressive complex 2, which silences the expression of its target genes by generating trimethylation of lysine 27 on histone H3 (H3K27Me3). EZH2 is overexpressed in several types of cancer including IBC and its overexpression correlates with a poor prognosis in IBC patients. Here, we investigated the molecular basis by which EZH2 promotes IBC. METHODS: IBC cell lines SUM149 and FC-IBC-02 which were developed from pleural effusion of an IBC patient were used in this study. Specific knockdown of EZH2 was performed using short hairpin RNA (shRNA) specific to the human EZH2 gene. MTS and soft-agar colony formation assays were used for anchorage-dependent and -independent cell growth in vitro, respectively. xCELLigence system was used to determine the cell migration and invasion. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice. RESULTS: EZH2 was expressed at higher levels in all tested human IBC cell lines when compared with non-transformed human mammary epithelial cells. Knockdown of EZH2 caused a significant reduction in the levels of H3K27Me3 in human IBC cell lines. Notably, knockdown of EZH2 expression significantly suppressed both anchorage-dependent and -independent growth of human IBC cells in vitro due to induction of apoptosis. In addition, EZH2 knockdown inhibited the migration and invasion of IBC cells. Significantly, EZH2 knockdown suppressed the growth IBC cells in vivo in an orthotopic IBC model. CONCLUSIONS: EZH2 knockdown suppresses the growth of human IBC cells in vitro and in vivo in an orthotopic IBC model in immunodeficient mice. In addition, EZH2 knockdown inhibits the invasion of human IBC cells. Together, we conclude that EZH2 contributes to IBC by promoting proliferation and invasion of human IBC cells. These results suggest that targeting EZH2 may provide a novel therapeutic strategy for the treatment of patients with IBC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-387. doi:1538-7445.AM2012-LB-387
Abstract LB-388: Regulation of histone deacethylase 8 expression by inhibitory GTP binding proteins in H1299 non-small-cell lung cancer cells
Authors: Hahn Hwa-jeong , juhnn Yong-sung
Published: August 2012

Histone deacetylases (HDACs) have been reported to regulate the cell growth, cell proliferation, cell cycle, and apoptosis, and increased expression of HDAC (classI) was observed in many kinds of cancer. HDAC8, most recently discovered subtype of HDAC classI, seems to be involved in tumor growth and progression because it is highly expressed in several cell lines and human cancer tissues. Inhibitory heterotrimeric GTP-binding proteins (Gi) are activated by seven transmembrane receptors bound with ligands, and regulate cellular metabolism and proliferation by regulating the activity of adenylate cyclase, activate phospholipase C beta, and ion channels. Thus, we made a hypothesis that Gi signaling system may regulate the expression of HDAC8, and this study investigated the effects of Gi signaling system on the expression of HDAC8 and the underlying mechanism in lung cancer cells. When Gi signaling system was activated by expressing Gαi1Q204L, a constitutively active mutant Gαi1, the expression of both HDAC8 protein and mRNA was increased in H1299 non-small-cell lung cancer (NSCLC) cells. However, the HDAC8 promoter activity did not increased significantly, and treatment with Actinomycin D did not block the increase in mRNA. The degradation of HDAC8 mRNA was facilitated in Gαi1Q204L-expressing cells, indicating Gi signaling system increases HDAC8 expression by stabilizing mRNA. Furthermore, treatment with adenosine or CCPA (2-chloro-N(6)cyclopentyladenosine), which bind to adenosine receptors leading to Gαi1 activation, also increased both HDAC8 mRNA and protein levels in H1299 cells. The effect of adenosine was blocked by treatment with DPCPX (8-Cyclopentyl-1,3-dipropylxanthine), an adenosine A1 receptor antagonist. These results indicate that Gi signaling system increases the HDAC8 expression by stabilizing its mRNA in H1299 NSCLC cells

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-388. doi:1538-7445.AM2012-LB-388
Abstract LB-390: High throughput, quantitative ligand binding assays for human bromodomains
Authors: Quinn Elizabeth , Hunt Jeremy , Wodicka Lisa , Ciceri Pietro , Treiber Daniel K.
Published: August 2012

Post translationally appended acetyl-lysine marks on histone tails are key regulatory features of the epigenetic code. Bromodomains are “readers” of this specific lysine acetylation code, playing an important role in chromatin remodeling and regulation of gene expression. Bromodomains have emerged as an important new druggable target class in small-molecule inhibitor drug discovery, and several bromodomain-containing proteins have been associated with disease. There are 57 bromodomains contained in 41 different proteins, however, few small molecule inhibitors of bromodomains have been identified to date. The primary factor limiting the discovery of new inhibitors is the absence of a comprehensive, biochemical screening platform for bromodomains. Here we describe the application of DiscoveRx Corporation's novel competitive binding assay technology (used to build the KINOMEscan panel of >450 kinase assays) to the development of quantitative ligand binding assays for human bromodomains. By applying our established methodologies, we have rapidly developed an assay panel that covers >15% of the bromodomain family, with the ultimate goal of developing a comprehensive panel, comparable to the KINOMEscan kinase assay panel. A robust bromodomain assay panel suitable for high throughput screening that delivers quantitative ligand binding data will facilitate the identification and optimization of potent and selective small molecule bromodomain inhibitors suitable for both pharmaceutical use and as tool compounds to further elucidate the roles of bromodomains in human disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-390. doi:1538-7445.AM2012-LB-390
Abstract LB-391: HDAC6 is a dual enzyme with deacetylase and E3 ligase activities for MSH2 degradation
Authors: Zhang Mu
Published: August 2012

MSH2 is a key DNA mismatch repair (MMR) protein that functions as an MSH2/MSH6 heterodimer (MutSa) in repairing errors after DNA replication as well as sensing certain types of DNA damage. It is believed that reduction of MutSα attenuates recognition of DNA adducts which renders a declined cell cycle arrest and apoptosis upon DNA damage and subsequently leads to chemotolerance. However, the mechanisms of down-regulation of MutSa are largely unknown. Here we show that histone deacetylase 6 (HDAC6) is co-purified with MutSa in HeLa nuclear extracts. Interestingly, we have found that HDAC6 itself serves as a ubiquitin E3 ligase to promote MSH2 polyubiquitination which renders MSH2 degradation by proteasome pathway. Intriguingly, HDAC6 prefers to target MSH2 monomer over MutSa for ubiquitination and degradation. Mechanistically, HDAC6 E3 ligase activity primarily resides in the N-terminal region encompassing the previously identified deacetylase domain 1 (DAC1) whereas its deacetylase activity resides in the deacetylase domain 2 (DAC2). We also showed that HDAC6 E3 ligase activity towards acetylated MSH2 is facilitated by its deacetylase activity in vivo. MSH2 lysine 249 and 892 can be either acetylated or ubiqutinated in vivo. These data suggest that HDAC6 sequentially deacetylates and ubiquitinates MSH2 via its DAC2 and DAC1 domains to target MSH2 degradation in vivo. Moreover, cells lacking or depleted of HDAC6 exhibit elevated levels of MSH2 due to impaired ubiquitin-proteasome-mediated degradation and subsequently prolonged half-life, compared with control cells. In agreement with this, HDAC6 knockout cells display higher MMR activity compared with wild type cells. Importantly, absence or reduction of HDAC6 leads to an increased cellular sensitivity to 6-TG and MNNG, whose DNA lesions were recognized by MutSa, suggesting that HDAC6 governs MSH2-dependent cytotoxicity induced by 6-TG or MNNG. Overall, our results identified a novel ubiquitin E3 ligase activity of HDAC6 and revealed that HDAC6 plays an important role in response to genotoxic stress.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-391. doi:1538-7445.AM2012-LB-391
Abstract LB-393: Genotoxicity of arsenic in primary human bronchial cells
Authors: Xie Hong , Martin Sara , Wise John , Huang Shouping
Published: August 2012

Arsenic originates from both geochemical and numerous anthropogenic activities including mining, combustion of fossil fuels, wood preservation, agriculture and metallurgy. Exposure of the general public to significant levels of arsenic is widespread. Arsenic is a well-documented human carcinogen. Long-term exposure to low levels of arsenic in drinking water have been linked to bladder, lung, kidney, liver, prostate, and skin cancer. Among them, lung cancer is of great public concern. However, little is known about how arsenic causes lung cancer. The purpose of this study was to determine the cytotoxicity and genotoxicity in human primary bronchial fibroblasts cells (NHBF). Our data show that arsenic induces a concentration-dependent increase in cell death after acute (24 h) or chronic (120 h) exposure. 0.1, 1 and 10 uM arsenic for 24 h induced 85, 64 and 35 percent relative survival, respectively, and induced 73, 57 and 11 percent of relative survival, respectively after 120 h treatment. 24 h exposure of 5 and 10 uM arsenic induced 15 and 32 chromosome damage compared to the control. Comet assay was used to measure the formation of DNA double strand breaks. We found that 120 h exposure induced a concentration-dependent increase in double strand breaks. Future study will focus on arsenic cytotoxicity and genotoxicity in primary bronchial epithelial cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-393. doi:1538-7445.AM2012-LB-393