bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2269: Combination of erlotinib and epigallocatechin-3-gallate induces apoptosis of squamous cell carcinoma of the head and neck through posttranslational regulation of Bim and Bcl-2
Authors: Haque Abedul , Rahman Mohammad A. , Chen Zhuo G. , Shin Dong M. , Amin A.R.M. Ruhul
Published: May 2015

Purpose: Effective chemopreventive strategies could save millions of lives. We have previously reported that the combination of the EGFR-TKI erlotinib and the green tea constituent epigallocatechin-3-gallate (EGCG) exhibited synergistic chemopreventive effects in head and neck cancers by inducing the expression of Bim, p21, p27, and inhibiting the phosphorylation of ERK and AKT and expression of Bcl-2. In the current study, we further investigated the mechanism of regulation of Bim, Bcl-2, p21 and p27, and their role in apoptosis.

Methods: Well characterized and genetically validated squamous cell carcinoma of the head and neck cell lines were used throughout the study. Annexin V staining was conducted for apoptosis assay. Expression of mRNAs and proteins were measured by RT-PCR and Western blotting, respectively. Gene overexpression and knockdown strategies were used to activate or shut down the expression of specific proteins.

Results: siRNA-mediated silencing of Bim significantly inhibited apoptosis induced by the combination of erlotinib and EGCG (p=). On the other hand, overexpression of Bcl-2 significantly protected cells from apoptosis (p=), whereas overexpression of constitutively AKT had minimal effect on apoptosis (actual values). Analysis of mRNA expression by RT-PCR revealed that neither erlotinib, EGCG nor their combination had any significant effects on the mRNA expression of Bim, p21, p27 or Bcl-2, suggesting post-transcriptional regulation of Bim, p21, p27 and Bcl-2 by the combination of erlotinib and EGCG. Furthermore, we found that erlotinib or the combination of EGCG and erlotinib inhibited the phosphorylation of Bim, and that inhibition of protein translation by cycloheximide pretreatment stabilized Bim, suggesting posttranslational regulation of Bim.

Conclusion: Our results strongly suggest that the combination of erlotinib and EGCG induces apoptosis of SCCHN cells by regulating Bim and Bcl-2 at the post-translational level. Currently, a clinical trial is underway at Winship Cancer Institute of Emory University to inhibit or reverse the progression of oral premalignant lesions using this combination. (This study is supported by R03CA159369, P50CA128613 and Robbins Scholar Award of Winship Cancer Institute).

Citation Format: Abedul Haque, Mohammad A. Rahman, Zhuo G. Chen, Dong M. Shin, A.R.M. Ruhul Amin. Combination of erlotinib and epigallocatechin-3-gallate induces apoptosis of squamous cell carcinoma of the head and neck through posttranslational regulation of Bim and Bcl-2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2269. doi:10.1158/1538-7445.AM2014-2269
Abstract 2272: Understanding resistance to Trail and Trail agonists
Authors: Chen Hong , Carrasco Rosa A. , Feng Hui , Hollingsworth Robert E. , Xiao Zhan , Tice David A.
Published: May 2015

Trail and Trail agonists induce apoptotic cell death in cancer cells while sparing normal cells making them promising drug candidates. However, Trail and Trail agonists tested so far in clinical trials have had disappointing results. We hypothesize that one reason for the lack of success could be resistance to Trail receptor agonism. In order to understand the underling mechanisms, we set out to generate a panel of Trail resistant models. Following weeks of continuous treatment with Trail, resistant cells began to emerge. Interestingly, no cross resistance to other cytotoxic agents such as doxorubicin, taxol and camptothecin was observed in the Trail receptor agonist resistant cells suggesting that the resistance mechanisms were specific to the extrinsic cell death pathway. We examined a series of factors such as ligand binding, surface receptor levels and caspase cascade activation. Here we highlight two specific mechanisms observed in a colorectal cell line DLD1 and a pancreatic cancer cell line PSN1. In both resistant models, the activation of the intrinsic and extrinsic caspase cascades were severely diminished starting at caspase-8. In the PSN1 model, TRAILR2 surface expression is significantly suppressed. Reduction of TRAILR2 by siRNA in the parental PSN1 cells also resulted in significant reduction of Trail receptor agonist activity in contrast to what was observed in the parental DLD1 cells. On the other hand, expression of caspase-8 proenzyme was severely attenuated in the DLD1 resistant cells. There was no significant change of caspase-8 mRNA expression found in resistant DLD1 suggesting post-translational control. Together, we conclude that Trail resistance is mainly due to early extrinsic pathway blockage such as down regulation of caspase-8 and TRAILR2.

Citation Format: Hong Chen, Rosa A. Carrasco, Hui Feng, Robert E. Hollingsworth, Zhan Xiao, David A. Tice. Understanding resistance to Trail and Trail agonists. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2272. doi:10.1158/1538-7445.AM2014-2272
Abstract 2274: RIP1-mediated sensitivity to SN38 induced by TNF in colon adenocarcinoma cell lines
Authors: Cabal-Hierro Lucia , O'Dwyer Peter J.
Published: May 2015

BACKGROUND: TNF is a dual role cytokine involved both in cell death and survival that is expressed in the tumor microenvironment. However, little is known about the molecular events that mark the biological outcomes of such opposite biological events. Recently, the protein kinase RIP1 has been identified as a new intermediate involved in the responses activated after DNA damage induction.

PURPOSE: In this study we investigated how TNF modulates the sensitivity to SN38 in colon cancer cell lines. We focused further on HT29- and HCT116-derived cell lines in which RIP1 or RIP3 proteins expression were stably downregulated.

RESULTS: We found that TNF increases cellular sensitivity to different chemotherapeutic drugs under both normoxic and hypoxic conditions over a panel of different colon adenocarcinoma cell lines. Combinations with SN38 were highly synergistic. In HT29 and HCT116 cell lines (TNF resistant and TNF sensitive respectively), we analyzed this interaction by studying P-H2-AX induction, cell proliferation (clonogenic assays and BrdU incorporation) and cell cycle distribution. To study the role of RIP1 and RIP3 on the biological responses triggered by SN38, HT29- and HCT116-derived cell lines expressing low levels of RIP1 and RIP3 were analyzed. While knockdown of RIP3 had no effect on SN38 cytotoxicity, RIP1 down regulation produced a 2.5-fold increased resistance to SN38 under hypoxic conditions. This change in SN38 sensitivity also correlated with a reduction in the levels of P-H2-AX in the presence of TNF, suggesting dependence of DNA damage induction by SN38 on RIP1.

CONCLUSIONS: These data suggest that RIP1 may be a key intermediate in determining cell death following SN38 treatment. Additional studies in tumor samples are in progress.

Citation Format: Lucia Cabal-Hierro, Peter J. O'Dwyer. RIP1-mediated sensitivity to SN38 induced by TNF in colon adenocarcinoma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2274. doi:10.1158/1538-7445.AM2014-2274
Abstract 228: Withaferin A downregulates tubulins and covalently binds β-tubulin at cysteine-303 in human breast cancer cells
Authors: Singh Shivendra V. , Antony Marie Lue , Lee Joomin , Hahm Eun-Ryeong , Kim Su-Hyeong , Romero Guillermo , Marcus Adam I. , Yang Zhen , Kumari Vandana , Ji Xinhua , Vowell Courtney L. , Wipf Peter , Uechi Guy T. , Yates Nathan A.
Published: May 2015

We have shown previously that withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from an Ayurvedic medicine plant (Withania somnifera), not only inhibits growth of human breast cancer cells in vitro and in vivo but also prevents mammary cancer development in a transgenic mouse model without any toxicity. However, the mechanisms underlying cancer preventive effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA in human breast cancer cells. WA treatment resulted in G2 and mitotic arrest in MCF-7 (estrogen-receptor positive), SK-BR-3 (estrogen-receptor negative), and SUM159 (triple-negative) cells in association with a marked decrease in protein level of β-tubulin. These effects were not observed with the naturally-occurring C6,C7-epoxy analogs of WA (withanone and withanolide A). In addition, a non-tumorigenic normal human mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest and tubulin downregulation by WA treatment compared with breast cancer cells. Downregulation of tubulin protein in WA-treated cells was due to transcriptional repression as well as proteasomal degradation. Vehicle-treated control breast cancer cells exhibited a normal bi-polar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of the normal spindle morphology in breast cancer cells. Unlike breast cancer cells, WA-treated MCF-10A cells exhibited aggregation of tubulin around nucleus. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA at cysteine-303 of β-tubulin in MCF-7 cells. Molecular docking studies indicated that the WA-binding pocket was located on the surface of the β-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of anticancer effect of WA. This work was supported by the grants RO1 CA142604 and P30 CA047904 awarded by the National Cancer Institute.

Citation Format: Shivendra V. Singh, Marie Lue Antony, Joomin Lee, Eun-Ryeong Hahm, Su-Hyeong Kim, Guillermo Romero, Adam I. Marcus, Zhen Yang, Vandana Kumari, Xinhua Ji, Courtney L. Vowell, Peter Wipf, Guy T. Uechi, Nathan A. Yates. Withaferin A downregulates tubulins and covalently binds β-tubulin at cysteine-303 in human breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 228. doi:10.1158/1538-7445.AM2014-228
Abstract 2280: Role of apoptosis signal-regulating kinase 1 in the cell apoptosis in oral leukoplakia
Authors: Tang Xiaofei , Zhang Jian-Fei , Niu Wen-wen , Zhang Min
Published: May 2015

Oral leukoplakia is a common precancerous lesion. The mechanism is not clear. The aim is to investigate the roles of apoptosis signal-regulating kinase 1(ASK1) in the cell apoptosis in oral leukoplakia. Archival tissue sections of 10 oral leukoplakia,23 oral leukoplakia with mild to moderate dysplasia, 7 normal oral mucosa were detected by real time PCR and immunohistochemical staining of markers of ASK1 and p-ASK1.The cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated nucleotide shift enzyme(TdT) mediated d-UTP end labeling(TUNEL). The results showed The mRNA and protein expression of ASK1 in oral leukoplakia with mild to moderate dysplasia was higher than the normal mucosa(p<0.05).From oral normal mucosa to oral leukoplakia to mild to moderate dysplasia, the expression of p-ASK1 was increased with the increasing grade( p<0.05).The apoptosis in oral leukoplakia and mild to moderate dysplasia increased more than normal mucose(p<0.05). It suggests that ASK1 and p-ASK1 are positive correlation with the increased apoptosis in the development of oral leukoplakia, its related signal pathways and cascade may play an important role in the cell apoptosis in oral leukoplakia.

Citation Format: Xiaofei Tang, Jian-Fei Zhang, Wen-wen Niu, Min Zhang. Role of apoptosis signal-regulating kinase 1 in the cell apoptosis in oral leukoplakia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2280. doi:10.1158/1538-7445.AM2014-2280
Abstract 2283: Potent curcumin analog FLLL-12 targets both intrinsic and extrinsic signaling pathways to induce apoptosis in lung cancers
Authors: Amin A.R.M. Ruhul , Haque Abedul , Rahman Mohammad A. , Fuchs James R. , Chen Zhuo G. , Shin Dong M.
Published: May 2015

Background: Curcumin is the major bioactive compound isolated from the rhizome of Curcuma longa (turmeric). Despite its high margin of safety and efficacy against various types of cancers, the potential utility of curcumin as a chemopreventive/chemotherapeutic drug is compromised by its low bioavailability and poor selectivity. To circumvent these problems, more potent and selective curcumin analogues have been developed. In the current study, we investigated the mechanism of apoptosis of one such synthetic analogue, FLLL-12, against lung cancers. This compound was reported to exhibit potent anti-tumor activity against prostate, colon and breast cancer cell lines. However, its mechanism of growth inhibition has yet to be elucidated.

Methods: Multiple premalignant and fully transformed lung cancer cell lines were used throughout the study. SRB assay was used to measure cell growth inhibition. Annexin V staining was conducted for apoptosis assay. Expression of mRNAs and proteins were measured by RT-PCR and Western blotting, respectively. Gene overexpression and knockdown strategies were used to activate or shut down of specific proteins.

Results: IC50 values and apoptosis assay results showed that FLLL-12 was ∼10-fold more potent than the natural parent compound, curcumin, against lung cancer cells. Further mechanistic studies revealed that FLLL-12 induced the expression of DR5, inhibited the protein expression of EGFR, p-AKT, AKT and Bcl-2 and increased the expression of BIM. Analysis of mRNA expression suggested that FLLL-12 strongly inhibited the mRNA expression of EGFR and AKT, whereas the expression of Bcl-2 and Bim mRNAs remained unchanged. FLLL-12 also induced the expression of p53 and p73. However, inactivation of these proteins with their dominant negative construct or siRNA had no significant effects on apoptosis induction.

Conclusions: Our results strongly suggest that FLLL-12 is a potent curcumin analog that induces apoptosis of lung cancer cell lines by targeting: (1) intrinsic pathways via transcriptional inhibition of EGFR and AKT and induction of BIM, and (2) extrinsic pathway via induction of DR5. Future in vivo studies using appropriate animal models are warranted for further development of this promising compound for cancer prevention and/or treatment for lung cancer.

(Supported by R03CA171663, P50CA128613 and Robbins Scholar Award of Winship Cancer Institute of Emory University).

Citation Format: A.R.M. Ruhul Amin, Abedul Haque, Mohammad A. Rahman, James R. Fuchs, Zhuo G. Chen, Dong M. Shin. Potent curcumin analog FLLL-12 targets both intrinsic and extrinsic signaling pathways to induce apoptosis in lung cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2283. doi:10.1158/1538-7445.AM2014-2283
Abstract 2288: Pharmacological inhibition of B-Raf/MEK/ERK signaling suppresses DR5 expression and impairs cancer cell response to DR5 activation-induced apoptosis
Authors: Oh You-Take , Yue Ping , Owonikoko Taofeek K. , Khuri Fadlo R. , Sun Shi-Yong
Published: May 2015

Inhibition of B-Raf/MEK/ERK signaling is an effective therapeutic strategy against certain types of cancers such as melanoma and thyroid cancer. While demonstrated to be effective anticancer agents, B-Raf inhibitors have also been associated with increased risk of secondary neoplasms. The induction of apoptosis following ligation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with its receptor, death receptor 5 (DR5), offers a potent anticancer strategy. Importantly, this is also a natural immunosurveillance mechanism against cancer cells. We previously demonstrated that activated B-Raf/MEK/ERK signaling positively regulates DR5 expression. Hence, our current work seeks to address whether B-Raf/MEK/ERK inhibition and the consequent suppression of DR5 expression impedes cancer cell response to DR5 activation-induced apoptosis. We found that both B-Raf (e.g., PLX4032) and MEK inhibitors (e.g., AZD6244 and PD0325901) effectively inhibited ERK1/2 phosphorylation and reduced DR5 levels in both human thyroid cancer and melanoma cells. Similar to the observed effect of genetic knockdown of the B-Raf gene, pretreatment of cancer cell lines with either B-Raf or MEK inhibitors attenuated or abolished cellular apoptotic response induced by TRAIL or the DR5 agonistic antibody AMG655. Our findings clearly showed that inhibition of the B-Raf/MEK/ERK signaling suppresses DR5 expression and impairs DR5 activation-induced apoptosis. These findings warrant further investigation of potential negative impact of B-Raf or MEK inhibition on TRAIL- or DR5-targeted cancer therapy and on TRAIL/DR5-mediated cancer immunosurveillance.

(This study was supported by the Georgia Cancer Coalition Distinguished Cancer Scholar award, NIH/NCI SPORE P50 grant CA128613 and

Winship Cancer Institute Robbins Scholar award. FR Khuri and SY Sun are Georgia Cancer Coalition Distinguished Cancer Scholars. YT Oh is a recipient of Winship Cancer Institute Robbins Scholar award)

Citation Format: You-Take Oh, Ping Yue, Taofeek K. Owonikoko, Fadlo R. Khuri, Shi-Yong Sun. Pharmacological inhibition of B-Raf/MEK/ERK signaling suppresses DR5 expression and impairs cancer cell response to DR5 activation-induced apoptosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2288. doi:10.1158/1538-7445.AM2014-2288
Abstract 2290: Rational targeting of inhibitor of apoptosis proteins (IAPs) for effective therapy of colorectal cancer
Authors: Crawford Nyree T. , Dunne Philip D. , Johnston Patrick G. , Waugh David J. , Longley Daniel B.
Published: May 2015

Background

Inhibitor of Apoptosis Proteins (IAPs) are anti-apoptotic proteins which suppress the activation of caspases. IAP proteins have been shown to be overexpressed in several cancers, including colorectal cancer and correlate with poor prognosis. SMAC mimetics are a class of apoptosis inducing drugs that target IAPs by mimicking the N-terminal IAP-binding motif of SMAC. Colorectal cancer has a pro-inflammatory microenvironment with high levels of TNFα. The aims of this study were to assess the efficacy of SMAC mimetic treatment in colorectal cancer.

Methods

Colorectal cell lines were treated with SMAC mimetics and cell viability was determined using MTT assays. Apoptosis was assessed by Western blotting for caspase activation and PARP cleavage and Flow Cytometry using Annexin V/PI staining. Protein-protein interactions were assessed using co-immunoprecipitation experiments. The pro-inflammatory microenvironment was modelled by addition of exogenous TNFα and also media transfer from and direct co-cultures with macrophages (THP-1, monocytic cell line).

Results

Colorectal cell lines were resistant to SMAC mimetic treatment alone unless co-cultured with TNFα. Pretreatment of colorectal cancer cells with 5-FU and oxaliplatin sensitised the cells to SMAC mimetic treatment. THP-1 cells were found to secrete soluble TNFα and media transfer experiments and also co-culture with colorectal cell lines, sensitised the cancer cells to SMAC mimetic mediated cell death. Cell death under these conditions was found to be dependent on soluble TNFα through the utilisation of neutralising antibodies.

Conclusion

Our results to date suggest that the pro-inflammatory microenvironment of colorectal cancer could be exploited to target overexpressed IAP proteins leading to cell death.

Citation Format: Nyree T. Crawford, Philip D. Dunne, Patrick G. Johnston, David J. Waugh, Daniel B. Longley. Rational targeting of inhibitor of apoptosis proteins (IAPs) for effective therapy of colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2290. doi:10.1158/1538-7445.AM2014-2290
Abstract 2292: Epigenetic alterations in Barrett's esophagus and esophageal adenocarcinoma: identification and evaluation of potential biomarkers
Authors: Stockdale Rachele M. , Kaz Andrew M. , Morris Shelli M. , Chak Amitabh , Willis Joseph , Brenner Dean , Anandabapasathy Sharmila , Westerhoff Maria , Wong Chao-Jen , Barnholtz-Sloan Jill , Chen Yanwen , Grady William M.
Published: May 2015

Patients diagnosed with esophageal adenocarcinoma (EAC) have poor long-term survival rates. Currently, patients identified with Barrett's esophagus (BE), the strongest risk factor for EAC, are placed in costly and invasive surveillance programs. The impact of these surveillance programs on decreasing EAC related mortality is debatable, presumably because many patients with BE are not diagnosed until they develop cancer and because of suboptimal BE risk markers for EAC. Thus, there is a need for more accurate biomarkers. Epigenetic alterations in BE have the potential to be detection and risk markers for BE and EAC. In order to identify individual gene methylation patterns of normal squamous (SQ) epithelium, BE, low-grade dysplasia, high-grade dysplasia and EAC tissues, we have conducted genome-wide methylation assessments of these tissue types using HumanMethylation450 arrays (Illumina). Unique methylation signatures for each histological subtype have been identified by unsupervised clustering. Additionally, we determined the mean methylation state (‘beta value’) for more than 480,000 CpGs for each sample group. After normalization and filtering, differentially methylated loci (FDR p < 0.05) with mean beta values with differences greater than 0.20 between the different tissue types were used to identify potential biomarkers. These biomarkers were then subjected to technical and clinical validation studies. We have currently identified and validated four potential biomarkers that may be useful in detection assays for BE and EAC. We have found two CpGs that distinguish EAC from BE, and two other CpGs that distinguish BE from SQ epithelium. The best performing biomarker has 100% sensitivity and 87.5% specificity in its ability to distinguish BE from EAC in the validation set of tissue samples. We presently have a number of additional candidates in various stages of validation. In summary, we have shown that differential methylation patterns exist between the various stages of progression from normal SQ epithelium to BE and ultimately EAC. Furthermore, some of these methylated CpGs have potential to be used as detection markers for BE or EAC.

Citation Format: Rachele M. Stockdale, Andrew M. Kaz, Shelli M. Morris, Amitabh Chak, Joseph Willis, Dean Brenner, Sharmila Anandabapasathy, Maria Westerhoff, Chao-Jen Wong, Jill Barnholtz-Sloan, Yanwen Chen, William M. Grady. Epigenetic alterations in Barrett's esophagus and esophageal adenocarcinoma: identification and evaluation of potential biomarkers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2292. doi:10.1158/1538-7445.AM2014-2292
Abstract 2295: Epigenetic regulation of KLHL34 may be predictive of pathologic response to preoperative chemoradiotherapy in rectal cancer patients
Authors: Ha Yejin
Published: May 2015

Aims: Radiation therapy is used to treat half of all cancer patients, but response to radiation therapy varies widely among patients. Studies aimed at predicting individual responsiveness to preoperative radiation therapy (CRT) are urgently needed, especially considering the risks associated with poorly responsive patients.

Methods: This study adopted a three-step process to select radiosensitive candidates of methylation genes. In the first step, a genome-wide creening of methylation genes was performed using Infinium humanMethylation450K Beadchip according to the correlation with istopathologic tumor regression grade (TRG) of 45 patients with locally advanced rectal cancer (LARC). In the second step, methylation status of selected sites was analyzed with pyrosequencing in the other 98 LARC patients. Finally, candidate gene identified by second screening was verified by biological utility assessment using cell viability assays of transfected colorectal cancer (CRC) cells. Results: Genome-wide creening identified that hypermethylation of 7 CpG sites (DZIP cg24107021, DZIP cg26886381, ZEB1 cg04430381, DKK3 cg041006961, STL cg00991794, KLHL34 cg01828474, ARHGAP6 cg07828380) were associated with preoperative CRT responses. Patients carrying hypermethylated KLHL34 cg14232291 were confirmed to show radiosensitivity by pyrosequencing in additional patients. Knockdown of KLHL34 significantly reduced colony formation and increased cytotoxicity in LoVo cells. It increased radiation induced caspase-3 activity and sub G1 population.Conclusions: In this study, we showed that methylation status of KLHL34 cg14232291 may be a predictive marker of radiosensitivity to preoperative CRT, although further validation is needed in large cohorts.

Citation Format: Yejin Ha. Epigenetic regulation of KLHL34 may be predictive of pathologic response to preoperative chemoradiotherapy in rectal cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2295. doi:10.1158/1538-7445.AM2014-2295
Abstract 2296: IDH2 mutation induced histone and DNA hypermethylation is progressively reversed by small molecule inhibition
Authors: Kernytsky Andrew , Wang Fang , Hansen Erica , Schalm Stefanie , Straley Kimberly , Gliser Camelia , Yang Hua , Travins Jeremy , Murray Stuart , Dorsch Marion , Agresta Sam , Schenkein David P. , Biller Scott A. , Su Shinsan M. , Liu Wei , Yen Katharine E.
Published: May 2015

Mutations of IDH1 and IDH2 which produce the oncometabolite 2-hydroxyglutarate (2HG), have been identified in several tumors including acute myeloid leukemia (AML). Recent studies have shown that expression of the IDH mutant enzymes results in high levels of 2HG and a block in cellular differentiation that can be reversed with IDH-mutant specific small molecule inhibitors. To further understand the role of IDH mutations in cancer, we conducted mechanistic studies in the TF-1 IDH2 R140Q erythroleukemia model system and found that IDH2 mutant expression caused both histone and genomic DNA methylation changes that can be reversed when IDH2 mutant activity is inhibited. Specifically, histone hypermethylation is rapidly reversed within days whereas reversal of DNA hypermethylation proceeds in a progressive manner over the course of weeks. Pathway enrichment analysis revealed several pathways involved in tumorigenesis of leukemia and lymphoma, indicating a selective modulation of relevant cancer genes by IDH mutations. As methylation of DNA and histones is closely linked to mRNA expression and differentiation, these results indicate that IDH2 mutant inhibition may function as a cancer therapy via histone and DNA demethylation at genes involved in differentiation and tumorigenesis.

Citation Format: Andrew Kernytsky, Fang Wang, Erica Hansen, Stefanie Schalm, Kimberly Straley, Camelia Gliser, Hua Yang, Jeremy Travins, Stuart Murray, Marion Dorsch, Sam Agresta, David P. Schenkein, Scott A. Biller, Shinsan M. Su, Wei Liu, Katharine E. Yen. IDH2 mutation induced histone and DNA hypermethylation is progressively reversed by small molecule inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2296. doi:10.1158/1538-7445.AM2014-2296
Abstract 2310: Epigenetic silencing of a potential tumor suppressor NR4A3 by aberrant JAK/STAT signaling predicts prognosis in gastric cancer
Authors: Chan Michael W. Y. , Zeng Li-Han , Chang Liang-Yu , Dittner Claudia , Chou Jian-Liang , Huang Yao-Ting , Cheng Alfred S.L. , Lin Jiayuh , Yeh Kun-Tu
Published: May 2015

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

Citation Format: Michael W. Y. Chan, Li-Han Zeng, Liang-Yu Chang, Claudia Dittner, Jian-Liang Chou, Yao-Ting Huang, Alfred S.L. Cheng, Jiayuh Lin, Kun-Tu Yeh. Epigenetic silencing of a potential tumor suppressor NR4A3 by aberrant JAK/STAT signaling predicts prognosis in gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2310. doi:10.1158/1538-7445.AM2014-2310
Abstract 2319: Dynamics of TET methylcytosine dioxygenases in 5-methylcytosine and 5-hydroxymethylcytosine patterning in human cancer cells
Authors: Putiri Emily L. , Tiedemann Rochelle L. , Choi Jeong-Hyeon , Robertson Keith D.
Published: May 2015

The Ten-eleven translocation (TET) family of dioxygenases hydroxylate 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. 5mC provides important epigenetic instructions during development, and its aberrant control is a major contributor to cellular transformation; however TET functions in regulating the epigenome, particularly in cancer, remain largely unknown. We targeted TET1, TET2, and TET3 for siRNA-mediated depletion in pluripotent human embryonic carcinoma cells and examined the impact on 5mC and 5hmC genome-wide localization. TET1, TET2, and TET3 co-regulate 5hmC at many sites, and depletion of only one of the TETs is sufficient to reduce 5hmC at these co-regulated sites, suggesting a functional co-dependence for TETs. Depletion of TET1 and TET2 had the greatest impact on 5hmC levels at high and low CpG density promoters, respectively, indicating that TETs exhibit DNA sequence-based functional specificity. All TETs prevent hypermethylation throughout the genome, especially in CpG island shores, where TET depletion resulted in prolific hypermethylation. Promoter hypermethylation resulting from TET depletion was associated with histone H2AK119 monoubiquitination, DNMT1, and DNMT3B occupancy. Surprisingly, TETs also promote cytosine methylation, as many loci became hypomethylated following TET depletion. Induction of differentiation generally caused 5hmC reduction, except at transcriptionally activated genes, which become enriched for 5hmC. Importantly, genes prone to promoter hypermethylation in cancer become depleted of intragenic 5hmC and 5mC with TET deficiency. This study highlights the multi-dimensional functions of TETs in mediating DNA methylation, hydroxymethylation, and gene expression patterns, and the results reveal that chromatin landscape and DNA sequence composition are regulators of TET function.

Citation Format: Emily L. Putiri, Rochelle L. Tiedemann, Jeong-Hyeon Choi, Keith D. Robertson. Dynamics of TET methylcytosine dioxygenases in 5-methylcytosine and 5-hydroxymethylcytosine patterning in human cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2319. doi:10.1158/1538-7445.AM2014-2319
Abstract 2326: Understand the telomerase-dependent transcriptional regulation in cancer cells
Authors: Yan TingDong , Liu Chang-Ching , Poon Lai-Fong , Chai Xiaoran , Fullwood Melissa Jane , Ghosh Sujoy , Li Shang
Published: May 2015

We and others have previously demonstrated that modulation of telomerase activity induces rapid and defined changes in genome-wide gene expression profiles in telomerase-positive cancer cells in culture, which point to the possibility of telomerase's involvement in gene regulation. Previous results also indicated the direct involvement of telomerase catalytic subunit-hTERT in transcriptional regulation through its interaction with Brg1 as well as NFkB. Such transcriptional function of hTERT is not dependent on its reverse transcriptase activity, suggesting a novel telomere-independent function of telomerase that promote stem cell proliferation and survival via activation of key genes regulating cell proliferation and survival. To further explore the potential role of hTERT in direct regulation of genome-wide gene expression in cancer cells, we have engineered cancer cells expressing 3xFlag-tagged hTERT from endogenous loci using AAV-mediated recombination. These genetically engineered cells allow the reliable detection and immunopurification of endogenous and functional hTERT in cancer cells. Such unique tools provide the key to identify potential transcriptional targets regulated by hTERT directly in vivo. Using ChIP-seq, we have mapped the genome-wide targets of potential hTERT binding sites in both HeLa S3 cells and HCT116 cells.

Citation Format: TingDong Yan, Chang-Ching Liu, Lai-Fong Poon, Xiaoran Chai, Melissa Jane Fullwood, Sujoy Ghosh, Shang Li. Understand the telomerase-dependent transcriptional regulation in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2326. doi:10.1158/1538-7445.AM2014-2326
Abstract 2328: COX-2 induce mediated multidrug resistance by activating JNK signal transduction pathway in colorectal cancer
Authors: Li Qi , Sui Hua
Published: May 2015

Chemotherapy is an important therapeutic option for most cancer patients; however, drug resistance causes the failure of chemotherapy and becomes a major obstacle for successful chemotherapy. The overexpression of P-gp has been associated with the resistance to a wide range of anticancer drugs. In the present study, drug-sensitive and multidrug resistant MDR colon carcinoma cell lines were used to examine the role of JNK signaling pathway in P-gp-mediated MDR associated with COX-2. We have found that sp600125, a JNK inhibitor or NS-398, a COX-2 inhibitor, reduced the degree of MDR in MDR cells. This phenomenon was accompanied by a significant decrease in gene level of ABCB1 and protein level of P-gp in MDR cells. In contrast, addition of a JNK inhibitor had no significant effect on the expression of COX-2. Interestingly, inhibition of COX-2 activity led to a decreased level of p-c-Jun at Ser63/73 due to JNK activation. In addition, there is an overexpression of p-c-Jun in MDR cells transfected with recombinant vector pGL3-Basic-COX-2 promoter. Moreover, the intracellular vincristine accumulation significantly increased with treatment of sp600125, or NS-398. In conclusion, our study has provided the first direct evidence that COX-2 induced up-regulation of P-gp in MDR cells, which was associated with an enhanced activity of the c-Jun phosphorylation at Ser63/73. Our findings may facilitate the development of novel antitumor drugs that are more effective against both chemosensitive and MDR cells.

Note: This abstract was not presented at the meeting.

Citation Format: Qi Li, Hua Sui. COX-2 induce mediated multidrug resistance by activating JNK signal transduction pathway in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2328. doi:10.1158/1538-7445.AM2014-2328
Abstract 2332: Oncogenic mutant KRAS modulates EZH2 expression through MEK-ERK signaling by remodeling gene expression in NSCLC
Authors: Riquelme Erick M. , Shen Li , Wang Jing , Behrens Carmen , Minna John D. , Wistuba Ignacio I.
Published: May 2015

Abstract:

Background. EZH2 overexpression occurs in lung cancer and is associated with a poor outcome. The mechanisms driving EZH2 expression in lung cancer are not fully understood. In this study, we investigated whether pharmacological disruption of signaling MEK-ERK pathway would affect EZH2 expression in a panel of NSCLC cell lines with and without KRAS mutation. Moreover, we analyzed the transcriptome expression following knockdown of EZH2 expression in NSCLC cell lines with different types of KRAS mutations.

Methods. NSCLC cell lines were treated with different doses of MEK inhibitor AZD6244 (0, 0.5 and 1μM) and the expressions of EZH2, MEK and MAPK were determined by Western-blots. Cell lines were transfected with gene-specific EZH2 siRNA and control siRNA. Gene expression profiling was performed using the Illumina Human HT-12 v 4.0 platform. Functional pathway analysis was conducted using the software Ingenuity Pathways Analysis.

Result. We found that pharmacological disruption of signaling MEK-ERK pathways with AZD6244 decreases the expression of MAPK p44/42 and phospho-MAPK p44/42 in all NSCLC cell lines analyzed wild-type and of KRAS mutant status. However, after AZD6244 treatment, EZH2 expression in NSCLC cell lines varied according to the KRAS mutation status: strong reduction only in NSCLC cell lines harboring GtoC mutation, a partial reduction in cells with GtoS and GtoR mutation, and no change in cell lines with GtoD or GtoV mutation and with wild-type KRAS. The gene expression analysis revealed that 3,235 genes were similarly regulated for mutant KRAS GtoC, GtoD and GtoV in NSCLC following EZH2 knockdown compared with KRAS wild-type (P < 0.05). Functional pathways analysis showed that EZH2 knockdown modulated differentially pathways commonly activated in cancer as growth factor signaling, molecular mechanism of cancer and cellular growth, proliferation and development, between cell lines that harbor mutant KRAS GtoC, GtoD or GtoV compared with KRAS wild-type.

Conclusion. Our findings suggest that oncogenic mutant KRAS GtoC, GtoS and GtoR modulate EZH2 expression through MEK-ERK signaling contributing to changes of the expression of genes frequently altered in cancer (Grant support: 5 R01 CA155196 and P50CA70907)

Citation Format: Erick M. Riquelme, Li Shen, Jing Wang, Carmen Behrens, John D. Minna, Ignacio I. Wistuba. Oncogenic mutant KRAS modulates EZH2 expression through MEK-ERK signaling by remodeling gene expression in NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2332. doi:10.1158/1538-7445.AM2014-2332
Abstract 2333: Nuclear EPS8 as a potential cooperative factor for FOXM1 in cancer cell proliferation
Authors: Ngan Wan-Ling , Yao Kwok-Ming
Published: May 2015

Tumorigenesis arises very often from dysregulated cell proliferation mechanisms. The Forkhead box transcription factor FOXM1 is a critical regulator of cell cycle progression as it affects the expression of genes that control the cell cycle, such as cyclin B1, cdc25B, etc. It is upregulated in various cancers, including cervical cancer. In a yeast two-hybrid screen for partnering factors that interact with FOXM1, we identified Epidermal Growth Factor Receptor (EGFR) Pathway Substrate 8 (EPS8).

The involvement of EPS8 in the EGF signal transduction and actin cytoskeleton organization has been well documented. Despite the lack of an SH2 domain, EPS8 associates with the EGFR and is tyrosine-phosphorylated upon EGF signaling. With its level and phosphorylation strongly induced by serum stimulation, EPS8 is believed to partake in the regulation of cell proliferation. Based on recent observations that EPS8 and FOXM1 expression are both elevated in human cervical cancer cells, and expression levels of EPS8 and FOXM1 are correlated in human head and neck squamous cell carcinoma, we hypothesize that there is crosstalk between these two proteins in vivo to mediate cell cycle progression.

In this study, interaction between FOXM1 and EPS8 was confirmed by co-immunoprecipitation analysis and the domains important for the interaction were defined using yeast two-hybrid assay. The presence of putative nuclear localization and export signals suggests regulation of EPS8 function by nucleocytoplasmic shuttling, consistent with EPS8 being a partnering factor of FOXM1 in the nucleus. Indeed, immunostaining of Hela cells revealed nuclear localization of EPS8 at S and G2/M phases of the cell cycle. Treatment with nuclear export inhibitor leptomycin B led to nuclear retention of EPS8, indicating that EPS8 localization is regulated by Crm-1-dependent nuclear export. Further site-directed mutagenesis defined nuclear export signals on EPS8 which affect the nuclear export/import of EPS8. Moreover, silencing EPS8 using shRNAs in various cell lines led to decreases in FOXM1 and cyclin B expression. Taken together, these data support that EPS8 may function together with FOXM1 in the nucleus and contribute to carcinogenesis via co-regulation of downstream target genes.

Citation Format: Wan-Ling Ngan, Kwok-Ming Yao. Nuclear EPS8 as a potential cooperative factor for FOXM1 in cancer cell proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2333. doi:10.1158/1538-7445.AM2014-2333
Abstract 2334: Identification of differentially expressed genes and somatic mutations in esophageal adenocarinoma cancer patients
Authors: Oster Bodil , Joecker Anika , Hein Anne-Mette K. , Dekker Patrick , O'Neill Robert , Krejci Adam , Arens Anne , Thomson Naomi , Boysen Cecilie , Mønsted Søren , Forsberg Roald , Knudsen Bjarne , Knudsen Thomas , Lussier Richard , Hupp Ted R.
Published: May 2015

High throughput sequencing technologies are currently revolutionizing the cancer research area with rapid improvements in sequencing capacity and time consumption. As a result the most time consuming step has moved from being the sequencing process itself to being the bioinformatic data analysis.

RNA sequencing (RNA-Seq) is used in an increasing number of transcriptomic studies. The great advantage of using RNA-Seq is its ability to precisely quantify transcript levels and identify novel transcripts, isoforms, and splice junctions, while further providing information of the mutational landscape down to single base resolution. To ease the hurdles associated with RNA-Seq data analysis there is an increasing demand for tools that are specifically tailored to RNA-Seq data.

Here we describe how the newly developed CLC Cancer Research Workbench can be used to analyze and visualize RNA-Seq data with ready-to-use workflows that automatically map, quantify, and annotate transcriptomes. We identify differentially expressed genes and transcripts in Illumina HiSeq transcriptomic data from matched tumor and normal samples from four esophageal adenocarinoma cancer patients, and compare the mutational patterns in the samples with the expression values of the corresponding genes.

Results are visualized in a track based genome browser view, which provides the means for quick and easy navigation as well as allowing the user to simultaneously view and annotate multiple samples and different data types (e.g. genes, transcript expression levels, and detected variants).

Citation Format: Bodil Oster, Anika Joecker, Anne-Mette K. Hein, Patrick Dekker, Robert O'Neill, Adam Krejci, Anne Arens, Naomi Thomson, Cecilie Boysen, Søren Mønsted, Roald Forsberg, Bjarne Knudsen, Thomas Knudsen, Richard Lussier, Ted R. Hupp. Identification of differentially expressed genes and somatic mutations in esophageal adenocarinoma cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2334. doi:10.1158/1538-7445.AM2014-2334