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Su1993 Somatic Mutations in the Mismatch Repair System Are Responsible for a Majority of Unexplained Lynch Syndrome Cases: Time to Revise Lynch Syndrome Screening?
AGA Abstracts
gastric tissue samples harvested from an independent population of H. pylori-infected persons in New Orleans to assess expression and methylation status of HIF-1α in vivo. Gastric tissue specimens from African-American subjects harboring an increased risk for gastric cancer exhibited marked decreases in methylation of HIF-1α with increasing disease severity. The highest levels of HIF-1α methylation were found in patients with non-atrophic gastritis and this decreased as disease progressed to atrophic gastritis (0.5-fold) and intestinal metaplasia (0.2-fold), versus gastritis alone. Consistent with decreasing HIF-1α methylation status, gastric tissue from African-American patients harbored increased levels of HIF-1α expression with increasing disease progression and this was not observed in Caucasian patients. Collectively, these data indicate that H. pylori induces HIF-1α in gastric epithelial cells and this is augmented under conditions of iron deficiency. HIF-1α expression in vivo increases in conjunction with decreased HIF-1α methylation and the development of premalignant lesions, which may provide a mechanism underpinning the link between high altitude, iron depletion, and increased gastric cancer rates within the context of H. pylori infection.
breaks, centrosome amplification, chromosome aberrations and aneuploidy. Multiple factors regulate centrosome duplication, such as p53. AIM: To determine the role of Klf4 in regulating centrosome duplication. METHODS: MEFs wild-type (WT) or null for Klf4 (Klf4-/) and colon cancer cell line HCT116 were stained for KLF4 and p53, and their presence or absence at the centrosomes was determined by co-staining for the centrosome-specific marker g-tubulin. Additionally Klf4-/- MEFs were transfected with Klf4-expressing plasmids and localization of both p53 and overexpressed KLF4 to the centrosomes was evaluated by staining, and compared the results to wild type (Klf4+/+) and untransfected or mocktransfected Klf4-/- MEFs. RESULTS: We show that both KLF4 and p53 localize to the centrosomes in WT MEFs and in HCT116. In contrast, in the absence of KLF4, p53 failed to localize to the centrosomes in Klf4-/- MEFs. Overexpression of KLF4 in Klf4-/- MEFs restored the localization of p53 to the centrosomes. CONCLUSION: Taken together these findings provide evidence that KLF4 plays a crucial role in the regulation of centrosome duplication by localizing to the centrosomes, which is in turn required for the localization of p53 to the centrosomes.
Su1993 Su1995 Somatic Mutations in the Mismatch Repair System Are Responsible for a Majority of Unexplained Lynch Syndrome Cases: Time to Revise Lynch Syndrome Screening? Rosa M. Xicola, Timothy P. Carroll, Rajyasree Emmadi, Victoria M. Alagiozian-Angelova, Jurgis Alvikas, Priti Marwaha, Maureen Regan, Joanna Gibson, Kisha A. Mitchell, Sonia S Kupfer, Nathan A Ellis, Xavier Llor
Proteomic Identification of Mutant p53 R282w Regulated Genes That Associate With Chemoresistance of Colorectal Cancer Jie Xu, Ye Hu, Jing-Yuan Fang In gastric and colorectal cancers, mutation of the tumor suppressor TP53 is frequently associated with poor patient prognosis. We have reported the p53 R282W mutation associates with early tumor onset and poor patient survival, but the mechanism is still unclear. Here we use two-dimensional electrophoresis and mass spectrometry to identify p53 R282Wregulated genes. The R282W mutant was expressed in p53-null tumor cells, followed by proteomic analysis on altered protein expression. We identified Erp29, Prdx6 and Park7 that were upregulated by mutant p53. Interestingly, by ChIP-seq we also found that p53 mutant could bind the promoters of these genes. We show by proliferation and would healing assays and these proteins contributed, at least partially, to the aggressiveness associated with p53 mutant R282W. These findings provide insight into the gain-of-function effect of p53 mutant, and suggests potential therapeutic venues for suppressing the aggressiveness of tumors bearing mutant p53.
Background Lynch syndrome (LS) is the most common inherited colorectal cancer (CRC) syndrome. LS is caused by mutations in the mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2) that result in tumor microsatellite instability (MSI) and loss of expression of the protein. About 10% of sporadic CRC are also MSI due to hyper-methylation of the promoter region of MLH1. About 1/3 of these cases have somatic BRAF V600E mutations while tumors from LS patients never do. In over half of the cases with MSI and no MLH1 methylation, no germline mutations are identified and these have been called LS-like. It is unclear if this group results from: MMR mutations that are technically undetected; nonMMR inherited mutations; or sporadic mutations. Recent studies have suggested that the later could be the cause of a significant number of these cases Hypothesis and Aims Somatic mutations might constitute a significant proportion of LS-like cases. Aim: to investigate the frequency of those cases in a non-selected, multiethnic cohort of consecutive CRC patients Methods We included 654 CRC patients from the multicenter cohort of the Chicago Colorectal Cancer Consortium. To pre-screen for LS we analyzed tumors for MSI and BRAF mutations. All MSI tumors were further analyzed for promoter methylation of MLH1 and loss of MMR expression by immunohistochemistry (IHC). Un-methylated MSI tumors were exome-sequenced in order to identify pathogenic germline and somatic mutations Results Fifty-six out of 654 tumors were MSI. Thirty of those presented with MLH1 promoter methylation and loss of MLH1/PMS2 expression. The remaining 23 were classified as potential LS. None of those had mutated BRAF. Figure 1 shows the molecular screening process and specific mutations identified. We were able to sequence 15/23 cases. A case was classified as LS when a pathogenic mutation was identified in the germline. Sporadic cases had somatic mutations or one mutation and LOH in the tumor but no mutations in the germline. Seven patients were classified as LS and 6 as sporadic. We did not identify any obvious difference regarding sex, race, location or tumor features, but the sporadic group was significantly younger at diagnosis Conclusion In a group of consecutive CRC cases, a very significant number of LS-like patients do have somatic mutations different from MLH1 promoter methylation typically associated with sporadic MSI tumors. Thus, these should not be classified as Lynch syndrome. As opposed to the sporadic MLH1 methylated tumors, these other group seems to present at much younger ages suggesting a distinct carcinogenic mechanism. The usefulness of universal colon tumor testing to screen for LS, as some guidelines have recently recommended, may have to be reevaluated according to the present findings. Somatic testing should be performed in LS-like patients with no germline MMR gene mutations
Su1996 Oncogenic BRAFV600e Downregulates DNMT3B and Does Not Directly Induce the CpG Island Hypermethylator Phenotype in Human Colorectal Adenocarcinoma Douglas J. MacKenzie, Neil A. Robertson, John Cole, Peter Adams Introduction: CpG island hypermethylator phenotype (CIMP) positive colorectal carcinoma represents a distinct clinicopathological subgroup and correlates with poor prognosis. Activated oncogenic BRAFV600E is strongly associated with a CpG island hypermethylator phenotype (CIMP) in human colorectal adenocarcinoma. It has been proposed that BRAFV600E may mediate CIMP through the de novo DNA methyltransferase, Dnmt3b. We sought to determine whether activated BRAFV600E directly induces CIMP in an in vitro system and combined this with analysis of publicly available TCGA data from human colorectal carcinoma. Methods: Primary human fibroblasts (IMR-90) were transduced with the lentiviral construct HIV-CS-CG-BRAFV600E-puromycin to stably over-express BRAFV600E. Control cells were infected with the empty vector HIV-CS-CG-puromycin. Genomic DNA was isolated, and whole genome bisulfite sequencing undertaken. Methylation, expression and mutation data from 217 patients with colorectal adenocarcinoma from The Cancer Genome Atlas (TCGA) were also analyzed to explore relationships between BRAF, Dnmt3b and CIMP in human colorectal tumors. Results: In the in vitro studies, BRAFV600E induced only very modest changes in DNA methylation with small gains of methylation at promoters and gene bodies. Specifically, BRAFV600E did not induce a CIMP phenotype. Significantly, this was associated with downregulation of Dnmt3b at the protein level. In the human colorectal carcinoma TCGA data, 11.5% of patients harbored a BRAFV600E mutation. 29.5% of patients were CIMP positive. Whilst there was a significant overlap (68%) between BRAFV600E mutation and CIMP positive, 32% of BRAFV600E mutant patients were CIMP negative. Furthermore, 73.4% of CIMP positive patients were BRAF wild-type. Dnmt3b mRNA levels were upregulated in 19.3% of patients, but in none of the patients with BRAFV600E. Dnmt3b mRNA levels were upregulated in only 12.5% of CIMP positive patients, and down-regulated in 31.25% of CIMP positive patients. Discussion: Together these data argue against BRAF being the primary mediator of CIMP in human colorectal carcinoma and suggest that BRAFassociated CIMP is not driven by Dnmt3b. Su1997 Colorectal Cancer With Methylator Phenotype. Clinical Characterisitics, Prognosis and Response to Adjuvant Chemotherapy Oscar Murcia, Maria Rodriguez-Soler, Miriam Juarez-Quesada, Carla Guarinos, Eva Hernandez-Illan, Montserrat Andreu, Antoni Castells, Xavier Llor, Angel Carracedo, Rodrigo Jover
Su1994
INTRODUCTION There is some controversy about prognostic and response to adjuvant Chemotherapy (CT) of colorectal cancer (CRC) following the carcinogenetic pathway due to methylation of CpG islands (CIMP). AIM The aim of this study is to determine differential characteristics of CIMP CRC and to evaluate its prognostic and response to CT. MATERIAL AND METHODS There were been included 701 CRC patients consecutively from the national and multicentre Epicolon II project. Hypermethylation of CpG islands was studied by MS-MLPA method using RUN-X3, CACNA1G, IGF2, MLH1, NEUROG1, CRABP1, SOCS1 and CDKN2A markers in 614 patients, taking into account for CIMP+ the methylation of 5 or more of these markers. Adjuvant chemotherapy was based in 5-FU in all the patients, a 64.6% of them had 5-FU associated to oxaliplatin. RESULTS The median of age was 72 years old [34-93], being men 58,9%. The median of follow-up was 57,7 months. Tumour
Krüppel-Like Factor 4 (KLF4) Is Required for the Localization of p53 to the Centrosomes Enas Gad El-karim, Amr Ghaleb, Vincent W. Yang BACKGROUND: Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and regulates proliferation, differentiation, apoptosis and somatic cell reprogramming. Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer. We previously showed that KLF4 inhibits cell cycle progression following DNA damage and that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by increased rates of cell proliferation, and the presence of DNA double strand
AGA Abstracts
S-570
gastric tissue samples harvested from an independent population of H. pylori-infected persons in New Orleans to assess expression and methylation status of HIF-1α in vivo. Gastric tissue specimens from African-American subjects harboring an increased risk for gastric cancer exhibited marked decreases in methylation of HIF-1α with increasing disease severity. The highest levels of HIF-1α methylation were found in patients with non-atrophic gastritis and this decreased as disease progressed to atrophic gastritis (0.5-fold) and intestinal metaplasia (0.2-fold), versus gastritis alone. Consistent with decreasing HIF-1α methylation status, gastric tissue from African-American patients harbored increased levels of HIF-1α expression with increasing disease progression and this was not observed in Caucasian patients. Collectively, these data indicate that H. pylori induces HIF-1α in gastric epithelial cells and this is augmented under conditions of iron deficiency. HIF-1α expression in vivo increases in conjunction with decreased HIF-1α methylation and the development of premalignant lesions, which may provide a mechanism underpinning the link between high altitude, iron depletion, and increased gastric cancer rates within the context of H. pylori infection.
breaks, centrosome amplification, chromosome aberrations and aneuploidy. Multiple factors regulate centrosome duplication, such as p53. AIM: To determine the role of Klf4 in regulating centrosome duplication. METHODS: MEFs wild-type (WT) or null for Klf4 (Klf4-/) and colon cancer cell line HCT116 were stained for KLF4 and p53, and their presence or absence at the centrosomes was determined by co-staining for the centrosome-specific marker g-tubulin. Additionally Klf4-/- MEFs were transfected with Klf4-expressing plasmids and localization of both p53 and overexpressed KLF4 to the centrosomes was evaluated by staining, and compared the results to wild type (Klf4+/+) and untransfected or mocktransfected Klf4-/- MEFs. RESULTS: We show that both KLF4 and p53 localize to the centrosomes in WT MEFs and in HCT116. In contrast, in the absence of KLF4, p53 failed to localize to the centrosomes in Klf4-/- MEFs. Overexpression of KLF4 in Klf4-/- MEFs restored the localization of p53 to the centrosomes. CONCLUSION: Taken together these findings provide evidence that KLF4 plays a crucial role in the regulation of centrosome duplication by localizing to the centrosomes, which is in turn required for the localization of p53 to the centrosomes.
Su1993 Su1995 Somatic Mutations in the Mismatch Repair System Are Responsible for a Majority of Unexplained Lynch Syndrome Cases: Time to Revise Lynch Syndrome Screening? Rosa M. Xicola, Timothy P. Carroll, Rajyasree Emmadi, Victoria M. Alagiozian-Angelova, Jurgis Alvikas, Priti Marwaha, Maureen Regan, Joanna Gibson, Kisha A. Mitchell, Sonia S Kupfer, Nathan A Ellis, Xavier Llor
Proteomic Identification of Mutant p53 R282w Regulated Genes That Associate With Chemoresistance of Colorectal Cancer Jie Xu, Ye Hu, Jing-Yuan Fang In gastric and colorectal cancers, mutation of the tumor suppressor TP53 is frequently associated with poor patient prognosis. We have reported the p53 R282W mutation associates with early tumor onset and poor patient survival, but the mechanism is still unclear. Here we use two-dimensional electrophoresis and mass spectrometry to identify p53 R282Wregulated genes. The R282W mutant was expressed in p53-null tumor cells, followed by proteomic analysis on altered protein expression. We identified Erp29, Prdx6 and Park7 that were upregulated by mutant p53. Interestingly, by ChIP-seq we also found that p53 mutant could bind the promoters of these genes. We show by proliferation and would healing assays and these proteins contributed, at least partially, to the aggressiveness associated with p53 mutant R282W. These findings provide insight into the gain-of-function effect of p53 mutant, and suggests potential therapeutic venues for suppressing the aggressiveness of tumors bearing mutant p53.
Background Lynch syndrome (LS) is the most common inherited colorectal cancer (CRC) syndrome. LS is caused by mutations in the mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2) that result in tumor microsatellite instability (MSI) and loss of expression of the protein. About 10% of sporadic CRC are also MSI due to hyper-methylation of the promoter region of MLH1. About 1/3 of these cases have somatic BRAF V600E mutations while tumors from LS patients never do. In over half of the cases with MSI and no MLH1 methylation, no germline mutations are identified and these have been called LS-like. It is unclear if this group results from: MMR mutations that are technically undetected; nonMMR inherited mutations; or sporadic mutations. Recent studies have suggested that the later could be the cause of a significant number of these cases Hypothesis and Aims Somatic mutations might constitute a significant proportion of LS-like cases. Aim: to investigate the frequency of those cases in a non-selected, multiethnic cohort of consecutive CRC patients Methods We included 654 CRC patients from the multicenter cohort of the Chicago Colorectal Cancer Consortium. To pre-screen for LS we analyzed tumors for MSI and BRAF mutations. All MSI tumors were further analyzed for promoter methylation of MLH1 and loss of MMR expression by immunohistochemistry (IHC). Un-methylated MSI tumors were exome-sequenced in order to identify pathogenic germline and somatic mutations Results Fifty-six out of 654 tumors were MSI. Thirty of those presented with MLH1 promoter methylation and loss of MLH1/PMS2 expression. The remaining 23 were classified as potential LS. None of those had mutated BRAF. Figure 1 shows the molecular screening process and specific mutations identified. We were able to sequence 15/23 cases. A case was classified as LS when a pathogenic mutation was identified in the germline. Sporadic cases had somatic mutations or one mutation and LOH in the tumor but no mutations in the germline. Seven patients were classified as LS and 6 as sporadic. We did not identify any obvious difference regarding sex, race, location or tumor features, but the sporadic group was significantly younger at diagnosis Conclusion In a group of consecutive CRC cases, a very significant number of LS-like patients do have somatic mutations different from MLH1 promoter methylation typically associated with sporadic MSI tumors. Thus, these should not be classified as Lynch syndrome. As opposed to the sporadic MLH1 methylated tumors, these other group seems to present at much younger ages suggesting a distinct carcinogenic mechanism. The usefulness of universal colon tumor testing to screen for LS, as some guidelines have recently recommended, may have to be reevaluated according to the present findings. Somatic testing should be performed in LS-like patients with no germline MMR gene mutations
Su1996 Oncogenic BRAFV600e Downregulates DNMT3B and Does Not Directly Induce the CpG Island Hypermethylator Phenotype in Human Colorectal Adenocarcinoma Douglas J. MacKenzie, Neil A. Robertson, John Cole, Peter Adams Introduction: CpG island hypermethylator phenotype (CIMP) positive colorectal carcinoma represents a distinct clinicopathological subgroup and correlates with poor prognosis. Activated oncogenic BRAFV600E is strongly associated with a CpG island hypermethylator phenotype (CIMP) in human colorectal adenocarcinoma. It has been proposed that BRAFV600E may mediate CIMP through the de novo DNA methyltransferase, Dnmt3b. We sought to determine whether activated BRAFV600E directly induces CIMP in an in vitro system and combined this with analysis of publicly available TCGA data from human colorectal carcinoma. Methods: Primary human fibroblasts (IMR-90) were transduced with the lentiviral construct HIV-CS-CG-BRAFV600E-puromycin to stably over-express BRAFV600E. Control cells were infected with the empty vector HIV-CS-CG-puromycin. Genomic DNA was isolated, and whole genome bisulfite sequencing undertaken. Methylation, expression and mutation data from 217 patients with colorectal adenocarcinoma from The Cancer Genome Atlas (TCGA) were also analyzed to explore relationships between BRAF, Dnmt3b and CIMP in human colorectal tumors. Results: In the in vitro studies, BRAFV600E induced only very modest changes in DNA methylation with small gains of methylation at promoters and gene bodies. Specifically, BRAFV600E did not induce a CIMP phenotype. Significantly, this was associated with downregulation of Dnmt3b at the protein level. In the human colorectal carcinoma TCGA data, 11.5% of patients harbored a BRAFV600E mutation. 29.5% of patients were CIMP positive. Whilst there was a significant overlap (68%) between BRAFV600E mutation and CIMP positive, 32% of BRAFV600E mutant patients were CIMP negative. Furthermore, 73.4% of CIMP positive patients were BRAF wild-type. Dnmt3b mRNA levels were upregulated in 19.3% of patients, but in none of the patients with BRAFV600E. Dnmt3b mRNA levels were upregulated in only 12.5% of CIMP positive patients, and down-regulated in 31.25% of CIMP positive patients. Discussion: Together these data argue against BRAF being the primary mediator of CIMP in human colorectal carcinoma and suggest that BRAFassociated CIMP is not driven by Dnmt3b. Su1997 Colorectal Cancer With Methylator Phenotype. Clinical Characterisitics, Prognosis and Response to Adjuvant Chemotherapy Oscar Murcia, Maria Rodriguez-Soler, Miriam Juarez-Quesada, Carla Guarinos, Eva Hernandez-Illan, Montserrat Andreu, Antoni Castells, Xavier Llor, Angel Carracedo, Rodrigo Jover
Su1994
INTRODUCTION There is some controversy about prognostic and response to adjuvant Chemotherapy (CT) of colorectal cancer (CRC) following the carcinogenetic pathway due to methylation of CpG islands (CIMP). AIM The aim of this study is to determine differential characteristics of CIMP CRC and to evaluate its prognostic and response to CT. MATERIAL AND METHODS There were been included 701 CRC patients consecutively from the national and multicentre Epicolon II project. Hypermethylation of CpG islands was studied by MS-MLPA method using RUN-X3, CACNA1G, IGF2, MLH1, NEUROG1, CRABP1, SOCS1 and CDKN2A markers in 614 patients, taking into account for CIMP+ the methylation of 5 or more of these markers. Adjuvant chemotherapy was based in 5-FU in all the patients, a 64.6% of them had 5-FU associated to oxaliplatin. RESULTS The median of age was 72 years old [34-93], being men 58,9%. The median of follow-up was 57,7 months. Tumour
Krüppel-Like Factor 4 (KLF4) Is Required for the Localization of p53 to the Centrosomes Enas Gad El-karim, Amr Ghaleb, Vincent W. Yang BACKGROUND: Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and regulates proliferation, differentiation, apoptosis and somatic cell reprogramming. Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer. We previously showed that KLF4 inhibits cell cycle progression following DNA damage and that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by increased rates of cell proliferation, and the presence of DNA double strand
AGA Abstracts
S-570