- Email: [email protected]
The Protective Role of Vitamin D3 in Colitis-Associated Colorectal Cancer in a Mouse Model
Mo1860
46,305,727), rs887946 (Chr19: 46,313,588), rs2311056 (Chr19: 46,314,265), rs4071668 (Chr19: 46,315,651), rs759300 (chr19: 46,316,995), rs3944020 (Chr19: 46,318,188) and rs4803930 (Chr19: 46,324,572). Results: The smallest overlapping LOH in LMs was mapped to a 680 bp region between rs887946 and rs2311056. This region contains DNase I hypersensitive sites and several putative FOXA1 (aka hepatocyte nuclear factor-3A or HNF3A) sites (UCSC, Genomic Browser: genome.ucsc.edu). Because our preliminary data shows lower levels of HIF3A protein expression are associated with the presence of LOH in LM, the loss of this region may negatively control HIF3A expression. To prove this hypothesis, CRC cell lines that have lost FOXA1 sites within this region are being assessed for HIF3A expression and metastatic potential. Conclusion: The novel target of HIF3A LOH found in LMs derived from primary CRCs contains FOXA1 sites embedded in a DNase hypersensitive region. This finding suggests that specific targeting of HIF3A via LOH removes transcriptionally-active sites that may modify MSI-L/EMAST CRC aggressiveness.
AGA Abstracts
THE PROTECTIVE ROLE OF VITAMIN D3 IN COLITIS-ASSOCIATED COLORECTAL CANCER IN A MOUSE MODEL Li Ma, Xin Yu, Hong Lv, Hongying Wang, Jiaming QIan Objective: Epidemiological and cytological studies have found a potential protective function of Vitamin D3 in colitis-associated colorectal cancer (CAC). In this study, we aim to decipher this effect in an azoxymethane (AOM) and dextran sulfate sodium (DSS) induced CAC mouse model and explore the underlying mechanism primarily focusing on the β-catenin signaling pathway. Method: In vitro, colon cancer cell line SW480 were treated with 1,25(OH)2D3 at 1, 10, 50 and 100nmol/L for 24, 48 and 72h and then quantified. β-catenin transcriptional activity assay was performed in SW480 cells both nontreated (control) and treated with 1,25(OH)2D3 (100nmol/L) for 48h, while the expression levels of β-catenin were determined. The same assay was applied to SW480 cells with FOXM1 and nVDR siRNA knockdowns separately. In animal experiment, C57BL/6 mice were divided into 6 groups. Group 1 served as the naïve control, which was given normal saline. Group 2 to 6 were given AOM (12.5mg/kg) by intraperitoneal injection and 2.5% DSS by gastric lavage sequentially to establish CAC model. Group 2 served as the model control; Group 3 to 5 received Vitamin D3 at different doses before AOM/DSS treatment, while Group 6 (postdose group) received 60IU/g/w VitD3 5 days after DSS intake. Mice were sacrificed after 14 weeks. Macroscopic and pathologic evaluations were carried out to assess the severity of CAC. The mRNA and protein levels of β-catenin were also detected. Results: The proliferation of SW480 cells was significantly inhibited by 1,25(OH)2D3 in a both dose-dependent and time-dependent manner (p<0.05). 1,25(OH)2D3 significantly decreased the transcriptional activity of β-catenin in SW480 cells by promoting the nuclear export of β-catenin (p<0.05) without affecting the mRNA and protein levels of β-catenin, which could be recovered by knocking down either FOXM1 or nVDR. In mice, Vitamin D intervention groups (Group 4,5,6) showed a lower tumor number (TN) and smaller tumor load (TL) than the model control group (Group 2) (p<0.05). However, there were no significant difference between the high dose group (Group 5) and post-dose group (Group 6). In addition, Vitamin D intervention groups presented lower β-catenin mRNA levels than the model control group (p<0.05). The amount of nuclear β-catenin decreased as the VitD3 dose increased. Finally, the total protein level of β-catenin was significantly lower in high dose group (Group 5) than the model control group (Group 2) (p<0.05). Conclusion: 1,25(OH)2D3 downregulates the transcriptional activity of β-catenin by expelling β-catenin out of nucleus and therefore severely dampens the proliferation of SW480 human colon cancer cells. In mice model, VitD3 treatment successfully inhibits the progression from AOM/DSS-induced colitis to colorectal cancer potentially by downregulating the activity of β-catenin signaling pathway.
Mo1864 HIGH FREQUENCY OF RNF43 R117H MISSENSE MUTATION IN SSA/PS PREDISPOSES TO TRUNCATING R117FS IN MICROSATELLITE UNSTABLE COLORECTAL CANCER Sophia Ang, Matthew Cook, Stephanie J. Austin, Nikolajs Zeps, Naomi Fletcher, Paul Whiting, Mitali Fadia, Doug R. Taupin Background Serrated lesions of the colon give rise to perhaps 25-30% of all colorectal cancers (CRCs) and are associated with MLH1 silencing, microsatellite instability (MSI) and BRAF oncogenic mutation. Truncating mutations of RNF43, a transmembrane E3 ubiquitin ligase which functions to suppress frizzled-dependent Wnt/βcatenin signaling, are pathogenic in familial serrated polyposis syndrome (SPS) and arise somatically in up to 20% of CRCs. Somatic frameshift (fs) mutations arise in two homo-polymeric tracts of 6Cs and 7Gs mononucleotide repeats, which include R117 and G659, respectively. We sought evidence of somatic alterations of RNF43 in serrated precursor lesions. Methods Two independent, series of sessile serrated polyps (SSA/Ps) at Canberra (series 1, n=61 samples from 34 patients) and Perth (series 2, n=93) were reviewed by a pathologist. We performed analysis for mutations of the hotspots R117 and G659, as well as R113 and R132 regions mutated in SPS. A curated set of 93 MLH1- CRC was then analyzed. Sanger sequencing of RNF43 was performed on DNA extracted from FFPE samples. RNF43 PCR products were cloned and resequenced. Results Somatic missense single nucleotide variation at R117 (R117H, G-A transition) occurred in 63% of SSA/Ps (Series 1, 29/61; Series 2, 68/93) (73%) in the two independent series. There was no association between the presence of BRAF V600E mutation and R117H. Germ line calls of R117H were also observed, but at rates below the reported allelic frequency. No G659 or R113 fs mutations were identified in SSA/Ps although 5% harbored mutations at R132. In MSI+ CRC, 75% (70/93) carried R117H (n=67, 72%) and truncating R117fs (n=3, 3%) while G659fs was found in 27% (26/95). R117H and R117fs mutations were confirmed by cloning and sequencing of tumor-derived PCR products. To further investigate the three occurrences of R117fs, metachronous and synchronous SSA/Ps from these cases were retrieved; all carried R117H mutation with one previous metachronous lesion harboring R117fs. Conclusion In this large series of 247 cases we found somatic R117H variants of RNF43 in 63% of SSA/Ps and 75% MLH1- CRC. While germ line R117H allele of RNF43 has a reported frequency of 0.22, the variant is predicted to be damaging using in silico tools. We observed concordance of R117H mutation in SSA/P with R117fs+ CRC and one instance of R117fs in a precursor SSA/P. We propose mechanistically that the G-A transition at R117 predisposes to deletion in the homopolymeric tract at this locus in the setting of MSI that results in truncating R117fs. This molecular transition from missense to truncating mutation emerges as a plausible important step in progression to cancers in SSA/Ps.
Mo1861 LONG NONCODING RNA NEAT1 MEDIATES GASTRIC CARCINOGENESIS AND CISPLATIN RESISTANCE BY FUNCTIONING AS A COMPETING ENDOGENOUS RNA Jin Yan Long noncoding RNA NEAT1 has been considered as a pro-oncogene in multiple cancers. However, its role in gastric cancer (GC) and cisplatin resistance, and the precise mechanism remain largely elusive. Here, we investigated the function and mechanism of NEAT1 in GC patients, GC cell models, and a xenograft mouse model. The regulatory network between miR-433 and NEAT1 was elucidated by RNA immunoprecipitation and luciferase reporter assays. Moreover, relationship between miR-433 and its predicted target methylation associated gene MBD2 was validated with luciferase reporter assay, real time- PCR, and western blot. In this study, we found that NEAT1 was up-regulated in GC tissues and correlated with tumor stage. We also reported that NEAT1 promoted GC cell proliferation, migration, and cisplatin resistance in vivo. Besides, NEAT1 promoted GC in mouse model. Moreover, negatively regulated miR-433 expression in GC, which might be mediated through an NEAT1-Ago2-miR-433 way. Further investigation revealed that MBD2 was demonstrated to be a functional target of miR-433 and the methylation were activated in NEAT1-upregulated tumorigenesis. In human GC specimens, NEAT1 and MBD2 were concordantly upregulated whereas miR-433 was downregulated in these tissues. In conclusion, NEAT1 activates MBD2 by sponging miR-433, resulting in the tumorigenesis and cisplatin resistance in GC.
Mo1865 WELL-BEING IN A DOUBLE-BLIND RANDOMIZED CLINICAL TRIAL COMPARING RACECADOTRIL 100 MG CAPSULES AND DEXECADOTRIL 75 MG TABLETS WITH PLACEBO IN ADULT OUTPATIENTS SUFFERING FROM ACUTE DIARRHEA Marion Eberlin, Erika Richter, Tobias Mueck, Harald Weigmann Background Racecadotril is a prodrug of the potent enkephalinase inhibitor Thiorphan. Thiorphan has an anti-diarrheic efficacy without any effect on motility of the gut. It also reduces diarrhea associated symptoms like abdominal pain and distension. However, little is known about the effect of symptomatic treatment of diarrhea with Racecadotril on patient's quality of life. Dexecadotril is the [R]-isomer of the racemic Racecadotril [R,S] which is also metabolized into Thiorphan. Methods A multi-center, randomized, double-blind, doubledummy, comparative study comparing Racecadotril 100 mg capsules and Dexecadotril 75 mg tablets with placebo included adult patients suffering from acute diarrhea of presumed infectious origin. At day one patients were randomized to either taking Racecadotril 100 mg (n=86) 3 times a day or Dexecadotril 75 mg (n=86) or placebo (n=87) 4 times a day until recovery (defined as 12 hours without stools or 2 consecutive normal stools) at most for 5 days. As one of the secondary objectives the impairment of daily activities, diet, sleep and feeling of discomfort were assessed by the patients daily until recovery (or day 5 = end of treatment) as follows: 0 = none, 1 = mild, 2 = moderate, 3 = severe. The Well-being Index (WBI) was calculated as the sum of the 4 items, summed up over the treatment days until recovery (or day 5) and analyzed by an analysis of covariance (ANCOVA) model, adjusting for baseline values, with the intent-to-treat (ITT) population. Results The adjusted means of WBI in patients treated with Racecadotril or Dexecadotril were lower in comparison to the placebo group indicating better development of well-being within the Racecadotril and Dexecadotril group: The mean (SE) and 95% confidence interval for the treatment difference of Racecadotril versus Placebo were −6.42 (1.695); [-9.746,-3.102]; p = 0.0002. The mean (SE) and 95% confidence interval for the treatment difference of Dexecadotril versus Placebo were −8.86 (1.708); [−12.21, −5.517]; p <.0001. Conclusions Patients being treated with Racecadotril or Dexecadotril showed better well-being compared to placebo treated patients. In conclusion, treatment of acute diarrhea with Racecadotril or Dexecadotril improves patient's disease-related quality of life.
Mo1862 TARGETED LOSS OF FORKHEAD BOX A1 (FOXA1) TRANSCRIPTION FACTOR SITES WITHIN THE HYPOXIA INDUCIBLE FACTOR 3A (HIF3A) LOCUS FROM LIVER METASTASIS (LM) DERIVED FROM MSI-L/EMAST PRIMARY COLORECTAL CANCER (CRC) Alexander W. Worix, Minoru Koi, Takahito Kitajima, Chan Choi, Hyeong-Rok Kim, Yuji Toiyama, Masato Kusunoki, John M. Carethers Background and Aims: We have previously demonstrated that low levels of microsatellite instability and/or elevated microsatellite alterations at selected tetra-nucleotide repeats (MSIL/EMAST) in CRC are induced by MSH3-deficiency, a defect that can trigger dinucleotide or larger microsatellite frameshifts as well as DNA double strand breaks. Tumor hypoxia or inflammation causes down-regulation of MSH3 expression or shuttling of MSH3 from the nucleus to the cytoplasm in tumor cells, leading to MSI-L/EMAST. Because MSI-L/EMAST is associated with recurrence/metastasis in stage II/III CRC, we hypothesized that genetic and/or epigenetic alterations in putative metastasis genes might be associated with MSI-L/ EMAST and identified from LMs. After surveying the cancer genome of LMs derived from MSI-L/EMAST CRCs, we focused utilizing 6 microsatellite markers present near or within the HIF3A locus on chromosome 19, and identified that loss of heterozygosity (LOH) at the HIF3A locus is frequent in LMs (59%) and associated with MSI-L/EMAST. Here, we utilized SNP markers present within the HIF3A locus to further refine the common LOH region among LMs. Methods: Genomic DNA from 75 paired normal and LMs from primary CRC was isolated. The genomic region containing each of the following 7 SNPs was amplified, sequenced and compared between normal and LM. These SNPS include rs2311054 (Chr19:
AGA Abstracts
S-804
46,305,727), rs887946 (Chr19: 46,313,588), rs2311056 (Chr19: 46,314,265), rs4071668 (Chr19: 46,315,651), rs759300 (chr19: 46,316,995), rs3944020 (Chr19: 46,318,188) and rs4803930 (Chr19: 46,324,572). Results: The smallest overlapping LOH in LMs was mapped to a 680 bp region between rs887946 and rs2311056. This region contains DNase I hypersensitive sites and several putative FOXA1 (aka hepatocyte nuclear factor-3A or HNF3A) sites (UCSC, Genomic Browser: genome.ucsc.edu). Because our preliminary data shows lower levels of HIF3A protein expression are associated with the presence of LOH in LM, the loss of this region may negatively control HIF3A expression. To prove this hypothesis, CRC cell lines that have lost FOXA1 sites within this region are being assessed for HIF3A expression and metastatic potential. Conclusion: The novel target of HIF3A LOH found in LMs derived from primary CRCs contains FOXA1 sites embedded in a DNase hypersensitive region. This finding suggests that specific targeting of HIF3A via LOH removes transcriptionally-active sites that may modify MSI-L/EMAST CRC aggressiveness.
AGA Abstracts
THE PROTECTIVE ROLE OF VITAMIN D3 IN COLITIS-ASSOCIATED COLORECTAL CANCER IN A MOUSE MODEL Li Ma, Xin Yu, Hong Lv, Hongying Wang, Jiaming QIan Objective: Epidemiological and cytological studies have found a potential protective function of Vitamin D3 in colitis-associated colorectal cancer (CAC). In this study, we aim to decipher this effect in an azoxymethane (AOM) and dextran sulfate sodium (DSS) induced CAC mouse model and explore the underlying mechanism primarily focusing on the β-catenin signaling pathway. Method: In vitro, colon cancer cell line SW480 were treated with 1,25(OH)2D3 at 1, 10, 50 and 100nmol/L for 24, 48 and 72h and then quantified. β-catenin transcriptional activity assay was performed in SW480 cells both nontreated (control) and treated with 1,25(OH)2D3 (100nmol/L) for 48h, while the expression levels of β-catenin were determined. The same assay was applied to SW480 cells with FOXM1 and nVDR siRNA knockdowns separately. In animal experiment, C57BL/6 mice were divided into 6 groups. Group 1 served as the naïve control, which was given normal saline. Group 2 to 6 were given AOM (12.5mg/kg) by intraperitoneal injection and 2.5% DSS by gastric lavage sequentially to establish CAC model. Group 2 served as the model control; Group 3 to 5 received Vitamin D3 at different doses before AOM/DSS treatment, while Group 6 (postdose group) received 60IU/g/w VitD3 5 days after DSS intake. Mice were sacrificed after 14 weeks. Macroscopic and pathologic evaluations were carried out to assess the severity of CAC. The mRNA and protein levels of β-catenin were also detected. Results: The proliferation of SW480 cells was significantly inhibited by 1,25(OH)2D3 in a both dose-dependent and time-dependent manner (p<0.05). 1,25(OH)2D3 significantly decreased the transcriptional activity of β-catenin in SW480 cells by promoting the nuclear export of β-catenin (p<0.05) without affecting the mRNA and protein levels of β-catenin, which could be recovered by knocking down either FOXM1 or nVDR. In mice, Vitamin D intervention groups (Group 4,5,6) showed a lower tumor number (TN) and smaller tumor load (TL) than the model control group (Group 2) (p<0.05). However, there were no significant difference between the high dose group (Group 5) and post-dose group (Group 6). In addition, Vitamin D intervention groups presented lower β-catenin mRNA levels than the model control group (p<0.05). The amount of nuclear β-catenin decreased as the VitD3 dose increased. Finally, the total protein level of β-catenin was significantly lower in high dose group (Group 5) than the model control group (Group 2) (p<0.05). Conclusion: 1,25(OH)2D3 downregulates the transcriptional activity of β-catenin by expelling β-catenin out of nucleus and therefore severely dampens the proliferation of SW480 human colon cancer cells. In mice model, VitD3 treatment successfully inhibits the progression from AOM/DSS-induced colitis to colorectal cancer potentially by downregulating the activity of β-catenin signaling pathway.
Mo1864 HIGH FREQUENCY OF RNF43 R117H MISSENSE MUTATION IN SSA/PS PREDISPOSES TO TRUNCATING R117FS IN MICROSATELLITE UNSTABLE COLORECTAL CANCER Sophia Ang, Matthew Cook, Stephanie J. Austin, Nikolajs Zeps, Naomi Fletcher, Paul Whiting, Mitali Fadia, Doug R. Taupin Background Serrated lesions of the colon give rise to perhaps 25-30% of all colorectal cancers (CRCs) and are associated with MLH1 silencing, microsatellite instability (MSI) and BRAF oncogenic mutation. Truncating mutations of RNF43, a transmembrane E3 ubiquitin ligase which functions to suppress frizzled-dependent Wnt/βcatenin signaling, are pathogenic in familial serrated polyposis syndrome (SPS) and arise somatically in up to 20% of CRCs. Somatic frameshift (fs) mutations arise in two homo-polymeric tracts of 6Cs and 7Gs mononucleotide repeats, which include R117 and G659, respectively. We sought evidence of somatic alterations of RNF43 in serrated precursor lesions. Methods Two independent, series of sessile serrated polyps (SSA/Ps) at Canberra (series 1, n=61 samples from 34 patients) and Perth (series 2, n=93) were reviewed by a pathologist. We performed analysis for mutations of the hotspots R117 and G659, as well as R113 and R132 regions mutated in SPS. A curated set of 93 MLH1- CRC was then analyzed. Sanger sequencing of RNF43 was performed on DNA extracted from FFPE samples. RNF43 PCR products were cloned and resequenced. Results Somatic missense single nucleotide variation at R117 (R117H, G-A transition) occurred in 63% of SSA/Ps (Series 1, 29/61; Series 2, 68/93) (73%) in the two independent series. There was no association between the presence of BRAF V600E mutation and R117H. Germ line calls of R117H were also observed, but at rates below the reported allelic frequency. No G659 or R113 fs mutations were identified in SSA/Ps although 5% harbored mutations at R132. In MSI+ CRC, 75% (70/93) carried R117H (n=67, 72%) and truncating R117fs (n=3, 3%) while G659fs was found in 27% (26/95). R117H and R117fs mutations were confirmed by cloning and sequencing of tumor-derived PCR products. To further investigate the three occurrences of R117fs, metachronous and synchronous SSA/Ps from these cases were retrieved; all carried R117H mutation with one previous metachronous lesion harboring R117fs. Conclusion In this large series of 247 cases we found somatic R117H variants of RNF43 in 63% of SSA/Ps and 75% MLH1- CRC. While germ line R117H allele of RNF43 has a reported frequency of 0.22, the variant is predicted to be damaging using in silico tools. We observed concordance of R117H mutation in SSA/P with R117fs+ CRC and one instance of R117fs in a precursor SSA/P. We propose mechanistically that the G-A transition at R117 predisposes to deletion in the homopolymeric tract at this locus in the setting of MSI that results in truncating R117fs. This molecular transition from missense to truncating mutation emerges as a plausible important step in progression to cancers in SSA/Ps.
Mo1861 LONG NONCODING RNA NEAT1 MEDIATES GASTRIC CARCINOGENESIS AND CISPLATIN RESISTANCE BY FUNCTIONING AS A COMPETING ENDOGENOUS RNA Jin Yan Long noncoding RNA NEAT1 has been considered as a pro-oncogene in multiple cancers. However, its role in gastric cancer (GC) and cisplatin resistance, and the precise mechanism remain largely elusive. Here, we investigated the function and mechanism of NEAT1 in GC patients, GC cell models, and a xenograft mouse model. The regulatory network between miR-433 and NEAT1 was elucidated by RNA immunoprecipitation and luciferase reporter assays. Moreover, relationship between miR-433 and its predicted target methylation associated gene MBD2 was validated with luciferase reporter assay, real time- PCR, and western blot. In this study, we found that NEAT1 was up-regulated in GC tissues and correlated with tumor stage. We also reported that NEAT1 promoted GC cell proliferation, migration, and cisplatin resistance in vivo. Besides, NEAT1 promoted GC in mouse model. Moreover, negatively regulated miR-433 expression in GC, which might be mediated through an NEAT1-Ago2-miR-433 way. Further investigation revealed that MBD2 was demonstrated to be a functional target of miR-433 and the methylation were activated in NEAT1-upregulated tumorigenesis. In human GC specimens, NEAT1 and MBD2 were concordantly upregulated whereas miR-433 was downregulated in these tissues. In conclusion, NEAT1 activates MBD2 by sponging miR-433, resulting in the tumorigenesis and cisplatin resistance in GC.
Mo1865 WELL-BEING IN A DOUBLE-BLIND RANDOMIZED CLINICAL TRIAL COMPARING RACECADOTRIL 100 MG CAPSULES AND DEXECADOTRIL 75 MG TABLETS WITH PLACEBO IN ADULT OUTPATIENTS SUFFERING FROM ACUTE DIARRHEA Marion Eberlin, Erika Richter, Tobias Mueck, Harald Weigmann Background Racecadotril is a prodrug of the potent enkephalinase inhibitor Thiorphan. Thiorphan has an anti-diarrheic efficacy without any effect on motility of the gut. It also reduces diarrhea associated symptoms like abdominal pain and distension. However, little is known about the effect of symptomatic treatment of diarrhea with Racecadotril on patient's quality of life. Dexecadotril is the [R]-isomer of the racemic Racecadotril [R,S] which is also metabolized into Thiorphan. Methods A multi-center, randomized, double-blind, doubledummy, comparative study comparing Racecadotril 100 mg capsules and Dexecadotril 75 mg tablets with placebo included adult patients suffering from acute diarrhea of presumed infectious origin. At day one patients were randomized to either taking Racecadotril 100 mg (n=86) 3 times a day or Dexecadotril 75 mg (n=86) or placebo (n=87) 4 times a day until recovery (defined as 12 hours without stools or 2 consecutive normal stools) at most for 5 days. As one of the secondary objectives the impairment of daily activities, diet, sleep and feeling of discomfort were assessed by the patients daily until recovery (or day 5 = end of treatment) as follows: 0 = none, 1 = mild, 2 = moderate, 3 = severe. The Well-being Index (WBI) was calculated as the sum of the 4 items, summed up over the treatment days until recovery (or day 5) and analyzed by an analysis of covariance (ANCOVA) model, adjusting for baseline values, with the intent-to-treat (ITT) population. Results The adjusted means of WBI in patients treated with Racecadotril or Dexecadotril were lower in comparison to the placebo group indicating better development of well-being within the Racecadotril and Dexecadotril group: The mean (SE) and 95% confidence interval for the treatment difference of Racecadotril versus Placebo were −6.42 (1.695); [-9.746,-3.102]; p = 0.0002. The mean (SE) and 95% confidence interval for the treatment difference of Dexecadotril versus Placebo were −8.86 (1.708); [−12.21, −5.517]; p <.0001. Conclusions Patients being treated with Racecadotril or Dexecadotril showed better well-being compared to placebo treated patients. In conclusion, treatment of acute diarrhea with Racecadotril or Dexecadotril improves patient's disease-related quality of life.
Mo1862 TARGETED LOSS OF FORKHEAD BOX A1 (FOXA1) TRANSCRIPTION FACTOR SITES WITHIN THE HYPOXIA INDUCIBLE FACTOR 3A (HIF3A) LOCUS FROM LIVER METASTASIS (LM) DERIVED FROM MSI-L/EMAST PRIMARY COLORECTAL CANCER (CRC) Alexander W. Worix, Minoru Koi, Takahito Kitajima, Chan Choi, Hyeong-Rok Kim, Yuji Toiyama, Masato Kusunoki, John M. Carethers Background and Aims: We have previously demonstrated that low levels of microsatellite instability and/or elevated microsatellite alterations at selected tetra-nucleotide repeats (MSIL/EMAST) in CRC are induced by MSH3-deficiency, a defect that can trigger dinucleotide or larger microsatellite frameshifts as well as DNA double strand breaks. Tumor hypoxia or inflammation causes down-regulation of MSH3 expression or shuttling of MSH3 from the nucleus to the cytoplasm in tumor cells, leading to MSI-L/EMAST. Because MSI-L/EMAST is associated with recurrence/metastasis in stage II/III CRC, we hypothesized that genetic and/or epigenetic alterations in putative metastasis genes might be associated with MSI-L/ EMAST and identified from LMs. After surveying the cancer genome of LMs derived from MSI-L/EMAST CRCs, we focused utilizing 6 microsatellite markers present near or within the HIF3A locus on chromosome 19, and identified that loss of heterozygosity (LOH) at the HIF3A locus is frequent in LMs (59%) and associated with MSI-L/EMAST. Here, we utilized SNP markers present within the HIF3A locus to further refine the common LOH region among LMs. Methods: Genomic DNA from 75 paired normal and LMs from primary CRC was isolated. The genomic region containing each of the following 7 SNPs was amplified, sequenced and compared between normal and LM. These SNPS include rs2311054 (Chr19:
AGA Abstracts
S-804