W1756 Regulation and Function of Chd5 in Colorectal Cancer
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of β-catenin. Immunofluorescence on AGS and MKN28 cells stably expressing t-Darpp demonstrated a significant increase in the fluorescence intensity of nuclear signals of β- catenin. To investigate transcriptional regulation of β-catenin/TCF activity by t-Darpp we performed the luciferase reporter assay using pTOP flash and its mutant pFOP flash reporter constructs. AGS and MKN28 cells transfected with t-Darpp showed more than two-fold increase in β-catenin activity as compared to control cells transfected with empty vector. Further, we provide evidence to validate involvement of t-Darpp in β-catenin/TCF pathway by demonstrating enhanced mRNA expression of downstream target genes including c-Myc and cyclin D1. In accordance with this data, we also observed an increase in protein levels of c-Myc and cyclin D1 in cells overexpressing t-Darpp. Conclusions: Taken together, our findings unveil a novel finding by which t-Darpp regulates GSK3β and β-catenin/TCF activity thereby implicating its oncogenic potential in gastrointestinal cancers. W1753 Expression of Bard1 Predicts Outcome in Colon Cancer Judith C. Sporn, Barbara H. Jung BACKGROUND: The BARD1/BRCA1 heterodimer has been implicated in several different pathways crucial for maintaining genomic integrity, DNA repair and cell survival. Complex functions have been ascribed to BARD1: It acts as a chaperone that recruits BRCA1 to the nucleus to form an ubiquitin E3 ligase complex involved in DNA repair and chromosomal stability. On the other hand, BARD1 also may promote cell survival by inhibiting BRCA1- dependent apoptosis through nuclear retention of BRCA1. Mutations of the BARD1 gene, as well as aberrant expression of BARD1 splice isoforms, have been associated with poor prognosis in breast and ovarian cancer. Here, we are the first to assess the occurrence and role of BARD1 isoforms in colon cancer. METHODS: Total RNA from primary human colon cancer samples and matched normal colon tissue was extracted and reverse transcribed. Gene specific PCR primers were designed to analyze the occurrence and pattern of BARD1 splice variants in colon cancer. Splice variants were subcloned and sequenced. Protein expression of BRCA1 and BARD1 in paraffin-embedded cancer tissues was detected by immunohistochemistry and correlated with clinicopathologic parameters. RESULTS: mRNA analysis of primary colon cancers confirmed a specific pattern of BARD1 splice isoforms distinct from normal controls. Certain isoforms appear to be tumor specific. Full length BARD1 is upregulated in non-metastatic cancer samples compared to matched normal tissue, which is in line with its anti-apoptotic function. Interestingly, full length BARD1 is dramatically downregulated in metastatic cancer tissue supporting its role as a tumor sup- pressor. Immunohistochemical analysis of colon cancer samples confirmed the loss of full length BARD1 in metastatic tissues on the protein level and revealed a significant correlation (p<0.05) between expression levels of BARD1 and outcome, in which low expression BARD1 is associated with a worse prognosis. CONCLUSION: The BRCA1 chaperone BARD1 may play a complex but significant role in colon cancer. Alternative splice variants occur commonly in colon cancer tissue and full BARD1 expression is differentially regulated along the cancer- metastasis axis affecting cancer outcome. In colon cancer, BRCA1 associated functions may be targeted via BARD1 expression. W1754 Role of p73 and Mechanisms of Its Regulation in Gastrointestinal Tumors Anna Vilgelm, Seung-Mo Hong, Jinxiong Wei, Wael M. El-Rifai, Alexander Zaika Background: The p73 protein is a member of the p53 tumor suppressor family. Despite significant functional and structural similarities between p53 and p73, some p73 isoforms, termed DeltaNp73, have strong oncogenic properties. These isoforms function as dominant- negative inhibitors of p53 and p73. We have previously demonstrated that DeltaNp73 immortalizes primary murine cells and cooperates with other oncogenes in cellular trans- formation and tumorigenesis in mice. Targeted over-expression of DeltaNp73 leads to tumor development in mice. Aims: To analyze the expression of DeltaNp73, determine its clinical relevance and investigate the mechanisms of DeltaNp73 regulation in human upper gastroin- testinal tumors. Methods and Results: When analyzing a large number of primary human gastric adenocarcinomas we found strong over-expression of ΔNp73 protein in tumor tissues. This increased expression was significantly associated with poor patient survival. In order to identify factors that regulate expression of DeltaNp73 we analyzed promoters of the p73 gene in silico as well as by chromatin immunoprecipitation, site-directed mutagenesis, promoter deletion and luciferase reporter analyses. These approaches revealed a putative binding site for transcriptional repressor HIC1 (Hypermethylated In Cancer 1), a powerful tumor suppressor, which is frequently down-regulated in gastric tumors. Increased expression of HIC1 decreased the levels of endogenous DeltaNp73 mRNA and protein, while the inhibition of cellular HIC1 with specific siRNA had an opposite effect. Conclusion: Our data show that loss of expression of HIC1 leads to increased expression of DeltaNp73 oncogenic isoforms in upper gastrointestinal tumors. This accounts for a more aggressive tumor phenotype and poor patient prognosis. W1755 Expression of p53, p16, p21, P21ras, and P27 in Patients Exhibiting Gastric Adenocarcinoma With Submucosa Invasion Roberson A. Moron, Ivan Cecconello, Carlos E. Jacob, Joaquim Gama-Rodrigues, Venancio A. Alves, Bruno Zilberstein, Kleber Simões, Kiyoshi Iryia Early gastric cancer invading submucosa has lymphonodal involvement in about 20% of cases. Determining who have a greater risk of lymphonode involvement would help to choose treatment. Authors reported a greater expression of p53 and p21ras in advanced tumors, loss of p21, p27, and p16 in advanced tumors. Eighty-one patients who had undergone gastrectomy with D2 linfadenectomy were retrospectively studied. Normal, meta- plastic mucosa and the tumor were selected for tissue microarrays. Expression of p21ras, p53, p21, p27 and p16 was evaluated. In normal, metaplastic and tumor mucosa, p53 showed positivity in 53%, 87%, and 87% of the cases. P21ras in 85%, 86%, and 96%. P16 S-733 AGA Abstracts was 46%, 91%, and 86%. P27 in 60%, 94%, and 95%. P21 in 32%, 72%, and 71%. All tumors were positive for p53. Tumors with lymphonodal involvement presented hyper- expression of p53 in 47% of the cases versus 17% without. No tumor with low positivity of p53 showed lymphonodal involvement. No tumor with negative p21ras showed lymphonodal involvement. In patients with lymphonodal involvement, p21ras presented strong positivity in 88% of the cases versus 50% without. There was a strong positivity of p21 in 71% of the tumors with lymphonodal involvement versus 28% in without. It was not observed an expression loss of p21, p27 e p16 in tumors with lymphonodal involvement. In normal mucosa, p16 showed a hyper-expression in 20% of the cases with perineural invasion versus 0% of the cases without. P16 showed a hyper-expression in 50% of the cases with vascular invasion versus 0% without. Tumors with Sm2 invasion showed low positivity of p27 in normal mucosa in 89% of the cases versus 55% of Sm1 tumors. Conclusions: Higher expression of p53, p21ras, and p21 in tumor showed relationship with lymphonodal involve- ment. A higher hyper-expression of p16 in patient's normal mucosa was related to perineural and vascular invasion. W1756 Regulation and Function of Chd5 in Colorectal Cancer Mehrnaz Fatemi, Hassan Brim, Krishan Kumar, Hassan Ashktorab Background: The chromodomain helicase DNA binding protein 5 (CHD5) is a family member of chromatin remodeling factors. CHD5 functions as a tumor suppressor in neuroblastoma and ovarian cancer. The transcriptional activity of CHD5 is silenced by hypermethylation of promoter CpG Island in human cancers. We and others have established that CHD5 is a biomarker in colorectal cancer (CRC). In this study, we have investigated the transcriptional regulation and function of CHD5 in CRC. Methods: The transcriptional activity of CHD5 was tested by reporter luciferase assay. The 1kb large fragment upstream of CHD5 transcrip- tion start site (TSS) showed luciferase activity; however, the 400 bp subfragment upstream of TSS was inactive in both RKO (CHD5 endogenous non-expressing) and HCT116 (CHD5 endogenous expressing) cell lines. This suggests that the transcriptionally active sites of CHD5 are located within -400 to -1000. There are two consensus TCF binding sites in the 1Kb promoter regions. In order to investigate whether Wnt pathway affects CHD5 transcrip- tion, we treated the RKO and HCT116 cell lines with 6-bromoindirubin-3'oxime (BIO), a GSK-3a inhibitor, which activates the β-Catenin. We tested the Wnt pathway activity in RKO and HCT116 cell lines by using the TCF/LEF responsive luciferase construct. The methylation status of CHD5 in 51 CRC and 6 adenoma from AAs were analyzed by MSP. Chromosomal aberrations at the CHD5 location (1p36) was analyzed using CGH array. Results: The activity of TCF/LEF responsive element increased by 17.5 folds in RKO cell line and by 7.3 folds in HCT116 cell line after BIO treatment. Interestingly, the luciferase activity of 1kb CHD5 promoter increased after treating with BIO, and no changes were observed in activity of the 400 bp fragment. This suggests that the TCF/LEF and β-Catenin might act as coactivators for CHD5 transcriptional regulation. We also analyzed the prolifera- tion of the RKO cell line after stably transfecting with CHD5 expressing plasmid (pcDNA). The results showed that the exogenous expression of CHD5 reduces the proliferation of the RKO cell line by two folds compared to the wild type and the mock controls. In AAs CRC samples, CHD5 was hypermethylated in 78% of cases while microdeletion of chromosome 1p36 (including CHD5) was 36%. Conclusion: The function of CHD5 is not well understood in CRC. Here, we suggest that the wnt/β-catenin pathway may be involved in the transcrip- tional regulation of CHD5. In addition, In Vivo CHD5 microdeletion and methylation data suggest that CHD5 act as tumor suppressor in CRC. W1757 The Role of the p53 Family of Transcription Factors in Down-Regulation of Survivin Expression by Sulindac in Gastric Cancer Cells Shiun-Kwei Chiou, Amy M. Hodges Background: Usage of NSAIDs such as sulindac is associated with reduced risk of gastrointesti- nal cancers. NSAIDs can activate the function of the p53 tumor suppressor gene, which is the most commonly mutated gene in human cancers and is known to illicit growth-arrest and/or apoptosis in gastric cancer cells. p53 and its relative, p73, belong in the same family of transcription factors that induce apoptosis and regulate cell cycle checkpoint control through transcriptional control of an overlapping set of target genes via the p53 responsive sequence in the target genes' promoter. Both p53 and p73 regulate the expression of the apoptosis inhibitor, survivin, in various cancer cells such as T cell lymphoma and MCF7 breast cancer cells. We recently showed that NSAIDs down-regulate survivin expression and induce gastric cell apoptosis. The mechanism of survivin down-regulation by NSAIDs in gastric cells is unknown. In this study, we examined whether p53 and/or p73 plays a role in down-regulation of survivin expression in gastric cancer AGS cells. Methods: AGS cells were maintained in RPMI-1640 medium supplemented with 10% FBS and antibiotics. Sulindac is a prodrug that is highly insoluble in culture medium. Therefore, we used sulindac AGA Abstracts
Transcript of W1756 Regulation and Function of Chd5 in Colorectal Cancer
of β-catenin. Immunofluorescence on AGS and MKN28 cells stably expressing t-Darppdemonstrated a significant increase in the fluorescence intensity of nuclear signals of β-catenin. To investigate transcriptional regulation of β-catenin/TCF activity by t-Darpp weperformed the luciferase reporter assay using pTOP flash and its mutant pFOP flash reporterconstructs. AGS and MKN28 cells transfected with t-Darpp showed more than two-foldincrease in β-catenin activity as compared to control cells transfected with empty vector.Further, we provide evidence to validate involvement of t-Darpp in β-catenin/TCF pathwayby demonstrating enhanced mRNA expression of downstream target genes including c-Mycand cyclin D1. In accordance with this data, we also observed an increase in protein levelsof c-Myc and cyclin D1 in cells overexpressing t-Darpp. Conclusions: Taken together, ourfindings unveil a novel finding by which t-Darpp regulates GSK3β and β-catenin/TCF activitythereby implicating its oncogenic potential in gastrointestinal cancers.
W1753
Expression of Bard1 Predicts Outcome in Colon CancerJudith C. Sporn, Barbara H. Jung
BACKGROUND: The BARD1/BRCA1 heterodimer has been implicated in several differentpathways crucial for maintaining genomic integrity, DNA repair and cell survival. Complexfunctions have been ascribed to BARD1: It acts as a chaperone that recruits BRCA1 to thenucleus to form an ubiquitin E3 ligase complex involved in DNA repair and chromosomalstability. On the other hand, BARD1 also may promote cell survival by inhibiting BRCA1-dependent apoptosis through nuclear retention of BRCA1. Mutations of the BARD1 gene,as well as aberrant expression of BARD1 splice isoforms, have been associated with poorprognosis in breast and ovarian cancer. Here, we are the first to assess the occurrence androle of BARD1 isoforms in colon cancer. METHODS: Total RNA from primary human coloncancer samples and matched normal colon tissue was extracted and reverse transcribed.Gene specific PCR primers were designed to analyze the occurrence and pattern of BARD1splice variants in colon cancer. Splice variants were subcloned and sequenced. Proteinexpression of BRCA1 and BARD1 in paraffin-embedded cancer tissues was detected byimmunohistochemistry and correlated with clinicopathologic parameters. RESULTS: mRNAanalysis of primary colon cancers confirmed a specific pattern of BARD1 splice isoformsdistinct from normal controls. Certain isoforms appear to be tumor specific. Full lengthBARD1 is upregulated in non-metastatic cancer samples compared to matched normaltissue, which is in line with its anti-apoptotic function. Interestingly, full length BARD1 isdramatically downregulated in metastatic cancer tissue supporting its role as a tumor sup-pressor. Immunohistochemical analysis of colon cancer samples confirmed the loss of fulllength BARD1 in metastatic tissues on the protein level and revealed a significant correlation(p<0.05) between expression levels of BARD1 and outcome, in which low expression BARD1is associated with a worse prognosis. CONCLUSION: The BRCA1 chaperone BARD1 mayplay a complex but significant role in colon cancer. Alternative splice variants occur commonlyin colon cancer tissue and full BARD1 expression is differentially regulated along the cancer-metastasis axis affecting cancer outcome. In colon cancer, BRCA1 associated functions maybe targeted via BARD1 expression.
W1754
Role of p73 and Mechanisms of Its Regulation in Gastrointestinal TumorsAnna Vilgelm, Seung-Mo Hong, Jinxiong Wei, Wael M. El-Rifai, Alexander Zaika
Background: The p73 protein is a member of the p53 tumor suppressor family. Despitesignificant functional and structural similarities between p53 and p73, some p73 isoforms,termed DeltaNp73, have strong oncogenic properties. These isoforms function as dominant-negative inhibitors of p53 and p73. We have previously demonstrated that DeltaNp73immortalizes primary murine cells and cooperates with other oncogenes in cellular trans-formation and tumorigenesis in mice. Targeted over-expression of DeltaNp73 leads to tumordevelopment in mice. Aims: To analyze the expression of DeltaNp73, determine its clinicalrelevance and investigate the mechanisms of DeltaNp73 regulation in human upper gastroin-testinal tumors. Methods and Results: When analyzing a large number of primary humangastric adenocarcinomas we found strong over-expression of ΔNp73 protein in tumor tissues.This increased expression was significantly associated with poor patient survival. In orderto identify factors that regulate expression of DeltaNp73 we analyzed promoters of the p73gene in silico as well as by chromatin immunoprecipitation, site-directed mutagenesis,promoter deletion and luciferase reporter analyses. These approaches revealed a putativebinding site for transcriptional repressor HIC1 (Hypermethylated In Cancer 1), a powerfultumor suppressor, which is frequently down-regulated in gastric tumors. Increased expressionof HIC1 decreased the levels of endogenous DeltaNp73 mRNA and protein, while theinhibition of cellular HIC1 with specific siRNA had an opposite effect. Conclusion: Ourdata show that loss of expression of HIC1 leads to increased expression of DeltaNp73oncogenic isoforms in upper gastrointestinal tumors. This accounts for a more aggressivetumor phenotype and poor patient prognosis.
W1755
Expression of p53, p16, p21, P21ras, and P27 in Patients Exhibiting GastricAdenocarcinoma With Submucosa InvasionRoberson A. Moron, Ivan Cecconello, Carlos E. Jacob, Joaquim Gama-Rodrigues,Venancio A. Alves, Bruno Zilberstein, Kleber Simões, Kiyoshi Iryia
Early gastric cancer invading submucosa has lymphonodal involvement in about 20% ofcases. Determining who have a greater risk of lymphonode involvement would help tochoose treatment. Authors reported a greater expression of p53 and p21ras in advancedtumors, loss of p21, p27, and p16 in advanced tumors. Eighty-one patients who hadundergone gastrectomy with D2 linfadenectomy were retrospectively studied. Normal, meta-plastic mucosa and the tumor were selected for tissue microarrays. Expression of p21ras,p53, p21, p27 and p16 was evaluated. In normal, metaplastic and tumor mucosa, p53showed positivity in 53%, 87%, and 87% of the cases. P21ras in 85%, 86%, and 96%. P16
S-733 AGA Abstracts
was 46%, 91%, and 86%. P27 in 60%, 94%, and 95%. P21 in 32%, 72%, and 71%. Alltumors were positive for p53. Tumors with lymphonodal involvement presented hyper-expression of p53 in 47% of the cases versus 17% without. No tumor with low positivity ofp53 showed lymphonodal involvement. No tumor with negative p21ras showed lymphonodalinvolvement. In patients with lymphonodal involvement, p21ras presented strong positivityin 88% of the cases versus 50% without. There was a strong positivity of p21 in 71% ofthe tumors with lymphonodal involvement versus 28% in without. It was not observed anexpression loss of p21, p27 e p16 in tumors with lymphonodal involvement. In normalmucosa, p16 showed a hyper-expression in 20% of the cases with perineural invasion versus0% of the cases without. P16 showed a hyper-expression in 50% of the cases with vascularinvasion versus 0% without. Tumors with Sm2 invasion showed low positivity of p27 innormal mucosa in 89% of the cases versus 55% of Sm1 tumors. Conclusions: Higherexpression of p53, p21ras, and p21 in tumor showed relationship with lymphonodal involve-ment. A higher hyper-expression of p16 in patient's normal mucosa was related to perineuraland vascular invasion.
W1756
Regulation and Function of Chd5 in Colorectal CancerMehrnaz Fatemi, Hassan Brim, Krishan Kumar, Hassan Ashktorab
Background: The chromodomain helicase DNA binding protein 5 (CHD5) is a family memberof chromatin remodeling factors. CHD5 functions as a tumor suppressor in neuroblastomaand ovarian cancer. The transcriptional activity of CHD5 is silenced by hypermethylationof promoter CpG Island in human cancers. We and others have established that CHD5 isa biomarker in colorectal cancer (CRC). In this study, we have investigated the transcriptionalregulation and function of CHD5 in CRC. Methods: The transcriptional activity of CHD5was tested by reporter luciferase assay. The 1kb large fragment upstream of CHD5 transcrip-tion start site (TSS) showed luciferase activity; however, the 400 bp subfragment upstreamof TSS was inactive in both RKO (CHD5 endogenous non-expressing) and HCT116 (CHD5endogenous expressing) cell lines. This suggests that the transcriptionally active sites ofCHD5 are located within -400 to -1000. There are two consensus TCF binding sites in the1Kb promoter regions. In order to investigate whether Wnt pathway affects CHD5 transcrip-tion, we treated the RKO and HCT116 cell lines with 6-bromoindirubin-3'oxime (BIO), aGSK-3a inhibitor, which activates the β-Catenin. We tested the Wnt pathway activity inRKO and HCT116 cell lines by using the TCF/LEF responsive luciferase construct. Themethylation status of CHD5 in 51 CRC and 6 adenoma from AAs were analyzed by MSP.Chromosomal aberrations at the CHD5 location (1p36) was analyzed using CGH array.Results: The activity of TCF/LEF responsive element increased by 17.5 folds in RKO cellline and by 7.3 folds in HCT116 cell line after BIO treatment. Interestingly, the luciferaseactivity of 1kb CHD5 promoter increased after treating with BIO, and no changes wereobserved in activity of the 400 bp fragment. This suggests that the TCF/LEF and β-Cateninmight act as coactivators for CHD5 transcriptional regulation. We also analyzed the prolifera-tion of the RKO cell line after stably transfecting with CHD5 expressing plasmid (pcDNA).The results showed that the exogenous expression of CHD5 reduces the proliferation of theRKO cell line by two folds compared to the wild type and the mock controls. In AAs CRCsamples, CHD5 was hypermethylated in 78% of cases while microdeletion of chromosome1p36 (including CHD5) was 36%. Conclusion: The function of CHD5 is not well understoodin CRC. Here, we suggest that the wnt/β-catenin pathway may be involved in the transcrip-tional regulation of CHD5. In addition, In Vivo CHD5 microdeletion and methylation datasuggest that CHD5 act as tumor suppressor in CRC.
W1757
The Role of the p53 Family of Transcription Factors in Down-Regulation ofSurvivin Expression by Sulindac in Gastric Cancer CellsShiun-Kwei Chiou, Amy M. Hodges
Background: Usage of NSAIDs such as sulindac is associated with reduced risk of gastrointesti-nal cancers. NSAIDs can activate the function of the p53 tumor suppressor gene, which isthe most commonly mutated gene in human cancers and is known to illicit growth-arrestand/or apoptosis in gastric cancer cells. p53 and its relative, p73, belong in the same familyof transcription factors that induce apoptosis and regulate cell cycle checkpoint controlthrough transcriptional control of an overlapping set of target genes via the p53 responsivesequence in the target genes' promoter. Both p53 and p73 regulate the expression of theapoptosis inhibitor, survivin, in various cancer cells such as T cell lymphoma and MCF7breast cancer cells. We recently showed that NSAIDs down-regulate survivin expression andinduce gastric cell apoptosis. The mechanism of survivin down-regulation by NSAIDs ingastric cells is unknown. In this study, we examined whether p53 and/or p73 plays a rolein down-regulation of survivin expression in gastric cancer AGS cells. Methods: AGS cellswere maintained in RPMI-1640 medium supplemented with 10% FBS and antibiotics.Sulindac is a prodrug that is highly insoluble in culture medium. Therefore, we used sulindac
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