- Email: [email protected]
42 Downregulation of TGF-ß Signaling Pathway Invokes Genomic Instability in Liver Cancers
tissues (p<0.0001). Aberrant down-regulation of ATM was also detected in 8 colon cancer cell lines relative to normal colon biopsies. Expression of miR-18a and that of ATM were inversely correlated (spearman r = -0.4562, P<0.01) in CRC tissues. To confirm the direct interaction between miR-18a and ATM, a segment of the 3'UTR of ATM with or without mutations in the seed region was sub-cloned downstream of the firefly luciferase reporter. The constructs were then co-transfected with pre-miR-18a or with pre-miR control in colon cancer cell line HCT-116 for luciferase activity assays. Transfection of pre-miR-18a enabled an approximately 1000-fold increase in miR-18a expression in HCT-116 cells. Ectopic expression of miR-18a significantly reduced the relative luciferase activity of the construct with wild-type ATM 3'UTR but not that of construct with mutant ATM 3'UTR in HCT-116, indicating a direct and specific interaction of miR-18a with ATM 3'UTR. This was further confirmed by the down-regulation of ATM protein level by miR-18a over-expression in HCT-116. As ATM is a key enzyme in DNA damage repair, we thus evaluated the effect of miR-18a on DNA double strand breaks (DSBs). DSBs were induced in HCT-116 by a genotoxic agent (etoposide 2μM). Etoposide increased tail moment, a measure of DNA DSBs, from a baseline level of 7.75±4.37 units to 15.46±6.07 units (P<0.001), as evaluated by comet assay. Ectopic expression of miR-18a significantly inhibited repair of DSBs induced by etoposide (p<0.001). Moreoever, without the induction of DSBs by etoposide, miR-18a did not show significant effect on cell proliferation in HCT-116 and HT-29 cells. However, with pre-exposure to etoposide, miR-18a significantly reduced the amount of colonies formed in both HCT-116 (p<0.05) and HT-29 (p<0.05) cells, inhibited cell growing curve in HCT116 and HT-29 cells (both p<0.05), and reduced clonogenic survival in HCT-116 (p<0.05) and HT-29 (p<0.01) cells. Conclusion: MiR-18a attenuates cellular repair of DNA double strand breaks by directly suppressing ATM expression through targeting ATM 3'UTR.
hMSH3 Deficiency Causes EMAST in Human Colorectal Cancer Cells Stephanie Tseng-Rogenski, Heekyung Chung, Shuai Zhang, Maike B. Wilk, John M. Carethers Background and Aims. Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in up to 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable colorectal cancers, where hypermethylation of the major mismatch repair (MMR) gene hMLH1 drives multiple target gene mutations, the cause of EMAST is unknown but was recently associated with reduced expression of the minor MMR protein hMSH3 in CRCs. We assessed experimentally whether hMSH3 deficiency is a cause of EMAST. Methods. We constructed plasmids containing D8S321 and D20S82 tetranucleotide loci (12 and 16 repeats of AAAG respectively) that are used to define EMAST. Sequences were cloned +1 bp out of frame immediately after the start codon of the EGFP gene. A -4 bp frameshift deletion of one AAAG unit would allow in-frame expression of EGFP. Mutationresistant counterpart plasmids were constructed by changing 2 nucleotide sequences in every 3 units of AAAG, preventing frameshift mutations. First, we created MMR proficient, hMLH1-/-, hMSH6-/-, and hMSH3-/- stable cell lines carrying the plasmids. Subsequently, we reduced hMSH3 expression via RNAi in MMR proficient cells harboring the EMAST constructs. Non-fluorescent cells were sorted and cultured for flow cytometry analysis. Mutations were examined by DNA-sequencing. Results. Sequencing confirmed frameshift mutations in fluorescent cells containing D8S321 and D20S82. Such mutations included contraction and expansion of microsatellites. D8S321 mutations occurred 31-and 40-fold higher in hMLH1-/- and hMSH3-/- cells compared to hMSH6-/- cells, respectively. D20S82 mutations occurred 82-and 49-fold higher in hMLH1-/- and hMSH3-/- cells compared to hMSH6-/- cells, respectively. When hMSH3 expression levels were reduced in MMR proficient cells upon hMSH3 shRNA transfection, significantly higher mutation rates were detected in hMSH3 knockdown cells for D8S321 (18.14 x 10-4) and D20S82 (11.14 x 10-4) compared to their individual scramble control cells (0 and 0.26 x 10-4 separately). Conclusions. EMAST is dependent upon MMR background, with hMSH3-/- more prone to frameshift mutations than hMSH6-/-, opposite to frameshift mutation observed at mononucleotide repeats. hMSH3-/mimics complete MMR failure (hMLH1-/-) in inducing EMAST. Furthermore, knock down of hMSH3 expression alone was able to elicit EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, loss of hMSH3 function appears to be the cause of EMAST.
41 Nuclear Ikkβ Phosphorylates ATM in Response to Genotoxic Stimuli, Which Helps Cancer Cells to Survive DNA Damage Kei Sakamoto, Yohko Hikiba, Yoku Hayakawa, Yoshihiro Hirata, Masao Akanuma, Shin Maeda Background: In many cases of gastroenterological cancers, drug resistance of cancer cells worsens outcomes of therapies. Many kinds of anticancer drugs are expected to cause irreparable DNA damage in cancer cells and lead them to cell death. Ataxia-telangiectasia mutated (ATM) is a key molecule involved in the cellular response to DNA damage. The most part of ATM stays in the nucleus to work on DNA repair. On the other hand, a portion of ATM is exported from the nucleus into the cytoplasm, where it activates the I kappa B kinase/nuclear factor kappa B (IKK/NFκ-B) signaling pathway. It has been thought that activated IKKβ, a key molecule for NF-κB activation, generally resides in the cytoplasm and phosphorylates cytoplasmic downstream molecules. In this study, we identified a new role for IKKβ during the response to DNA damage. Methods: IKKβ and ATM were knocked down in gastric cancer cells by siRNA. Genotoxic stimuli were added using alkylating agents or a topoisomerase inhibitor. ATM phosphorylation and localization of IKKβ were examined by immunoblotting. DNA damage was quantitated by comet assay. Direct phosphorylaition of ATM by IKKβ was observed by In Vitro kinase assay. RESULTS: IKKβ-knocked down cells and control cells were treated with an alkylating agent and subjected to an alkaline comet assay 24 h after stimulation. Higher levels of DNA damage were observed in IKKβknocked down cells. Since ATM is a critical factor associated with DNA repair, we analyzed phosphorylation of ATM after stimulation. ATM phosphorylation was significantly attenuated 6-9 h after stimulation in IKKβ-knocked down cells, whereas the initial ATM activation did not differ. These observations suggested that phosphorylation of ATM consists of two phases: the initial phase and the sustained phase (after 6 h) and IKKβ is involved specifically in late-phase phosphorylation. When wild type IKKβ (IKKβ-WT) and a kinase-negative IKKβ mutant (IKKβ-KN) were expressed in cells, ATM phosphorylation was attenuated in IKKβKN-expressing cells. Direct phosphorylation of ATM by IKKβ was also observed In Vitro kinase assay. Furthermore, IKKβ protein levels in the nucleus increased in response to DNA damage when nuclear extracts of the cells were immunoblotted with anti-IKKβ. To examine the function of translocation of IKKβ into nucleus, IKKβ with the nuclear exporting signal (IKKβ-NES) and IKKβ-WT were expressed in IKKβ-knockout MEFs. Late-phase ATM phosphorylation was attenuated in IKKβ-NES -expressing cells as compared with those expressing IKKβ-WT. The amount of DNA damage after stimulation was greater in IKKβ-NES -expressing cells. Conclusion: IKKβ translocates into nucleus in response to DNA damage and phosphorylates ATM. The late-phase phosphorylation of ATM promotes DNA repair, which can help cancer cells to survive under anticancer therapy.
39 Identification of Gene Loci With Microsatellite Repeats Frequently Altered in Liver Metastasis Exhibiting Moderate Levels of Microsatellite Instability From Primary CRC Melissa Garcia, Chan Choi, Hyeong-Rok Kim, Junichi Koike, Hiromichi Hemmi, Jie Li, Takeshi Nagasaka, Clement R. Boland, Minoru Koi Background & Aims: We demonstrated that moderate microsatellite instability (MSI-M) defined by NCI reference markers and elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) markers was common in primary CRC, and was an independent predictor for recurrent distant metastasis of stage II and III (II/III) primary CRC. However, how MSI-M is linked to recurrent distant metastasis is not known. To approach this question, we aimed to identify genetic changes significantly associated with MSI-M and with liver metastasis (LM) from primary CRC. Methods: We selected 142 gene loci with polymorphic microsatellites with di-, tri- and tetranucleotide repeats by genome data mining, and examined each locus for frame-shift mutations within microsatellite repeats and loss of heterozygosity (LOH) in 24 LM exhibiting MSI-M. Results: Among 142 gene loci examined, 29 loci (20.4%) exhibited microsatellite mutations in 24 cases of MSI-M-positive LM. More loci with larger repeats showed mutations than loci with smaller repeats; 53% of loci with tetranucleotide repeats, 15% of loci with trinucleotide repeats and 4% of loci with di-nucleotide repeats showed microsatellite mutations. Among loci with mutations, RBM47 (25%), WIPF (18%), D9S303 (17%), ZNF161 (17%), D8S1179 (14%), D21S11 (13%) and KANK2 (13%) exhibited higher levels of mutations in MSI-M-positive LMs. However, the mutation frequency of these loci was no greater than the average mutation frequency of 7 EMAST markers (~20%) among 24 LM cases. Compared to microsatellite mutation, LOH was found in more loci with higher frequencies. Eighty-seven out of 142 loci (61%) exhibited LOH with more than 6% of informative cases. We found 10 loci with a frequency of LOH higher than 50%, including KDM6B (75%), MNT (71%), SMARCA2 (64%), HEC1 (60%), ANKRD5 (58%), BCL2 (58%), SEMA6D (57%), D5S818 (56%), STYK1 (50%) and BCL6B (50%). LOH at chromosomal regions where KDM6B (17p13), MNT (17p13), BCL6B (17p13), HEC1 (18p11), BCL1 (18q21), ANKRD5 (20p12) and SEMA6D (15q21) reside have been reported in CRC tissues. Conclusion: SMARCA2 at 9p24 and STYK1 at 12p13 are new loci that frequently exhibit LOH in MSI-M-positive LM tissues. A possible association of LOH at the SMARCA2 or STYK1 locus with MSI-M or with LM formation is under investigation.
42 Downregulation of TGF-β Signaling Pathway Invokes Genomic Instability in Liver Cancers Vivek Shukla, Ying Li, Lopa Mishra Background: Loss of TGF-β adaptor protein β2SP leads to delayed liver regeneration and extensive DNA damage in mice. The Smad3/4 adaptor protein, β2SP, is emerging as potent regulator of tumorigeneisis by its ability to affect TGF-β tumor suppressor function. Deletion of β2SP results in a dramatic and spontaneous formation of liver cancer and gastrointestinal cancers. Moreover, β2SP+/- and β2SP+/- /Smad3+/- mutant mice phenocopy human Beckwith-Wiedemann syndrome (BWS), a hereditary human cancer stem cell syndrome imprinting disorder with loss of p57, increased IGF2, associated with an 800 fold increase of cancers that include those of the liver. Delayed liver regeneration and extensive DNA damage was observed in β2SP heterozygote mice. In addition, spectrins have been observed to associate with FANC G and D, DNA interstrand cross links, and Smad3/4 to transcriptionally regulate FANC genes. Hypothesis: We therefore hypothesised that TGF-β is a crucial enforcer of genomic stability, and that β2SP and/or Smad3/4 are key mediators in suppressing liver injury and cancer through modulation of the DNA repair pathway. Materials & Methods: To determine the role of β2SP and Smad3 in the DNA damage response, and whether β2SP and β2SP/Smad3 loss plays a causal role in the downregulation of the Fanc/Brca pathway, we used our mouse models as well as the mouse embryonic fibroblasts. Oxidative DNA damage was induced by H2O2 treatment and assayed tail length analysis. Irradiation was
40 MicroRNA-18a Attenuates DNA Damage Repair by Directly Controlling the Expression of ATM in Colon Cancer Chung-Wah Wu, Yu Juan Dong, Xin Qi He, Simon Ng, Francis K. L. Chan, Joseph J. Sung, Jun Yu Background and Aims: MiR-18a is one of the most up-regulated miRNAs in colorectal cancers (CRC) compared with the adjacent normal tissues based on miRNA profiling. However, the function of miR-18a in cancer development remains unclear. Methods and Results: The up-regulation of miR-18a was validated and confirmed in 46 CRC tumors compared with their adjacent normal tissues (p<0.0001). Up-regulation of miR-18a was also demonstrated in colon cancer cell lines relative to normal colon biopsies. Potential target genes of miR-18a were predicted in silico by algorithms TargetScan and MiRanda. Ataxia Telangiectasia Mutated (ATM) was identified as one of the potential targets of miR18a. Downregulation of ATM was detected in the CRC tumors compared to adjacent normal
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induced by 8Gy IR and DNA was studied by immunofluorescence of DNA damage sensors γH2AX 532BP1, pChk2. DNA repair was assayed by Mdc1, NBS1 and Rad51 focus formation at different time points after irradiation. Results: β2SP+/− and β2SP+/−/Smad3+/− mice exhibited an increased prevalence of HCC and GI cancer and many phenotypic characteristics observed in BWS patients. Absence of β2SP results in impaired liver regeneration and dramatic spontaneous DNA damage in β2SP+/- mouse livers after PHx in p53 independent manner. Loss of β2SP results in premature replicative senescence with spontaneous DNA damage. β2SP deficiency results in increased sensitivity to exogenous DNA damage along with insufficient DNA repair and impairs the loading of repair proteins and repair of DNA double-strand breaks. Loss of FancD2 expression in HCC and GI cancer cell lines correlates with loss of β2SP. Moreover, β2SP-/- and β2SP+/−/Smad3+/− cell lines accumulate DNA damage that is further exacerbated by DNA cross-linking agents such as mitomycin C similar to P57 null cells and Fanconi anemia cell lines. Conclusions: Downregulation of TGF-β signaling pathway impels genomic instability through regulation of DNA impairment of the Fanc/Brca DNA damage response and interstrand link repair and inactivation of TGFpathway results in alcohol toxicity, cirrhosis and liver cancers.
45 NOD2 Deficiency is Associated With Increased Mucosal Regulatory Response to Commensal Microorganisms Antonello Amendola, Alessia Butera, Monica Boirivant Background: It has been reported that Peyer's patches (PPs) of Nod2 KO mice show, as a consequence of the presence of ileal microbiota, increased tissue content of IFN-γ and TNFα that are responsible for an increased permeability of the epithelium covering PP (Gut 2010;59:207), yet the mice do not show intestinal inflammation. Moreover, Nod2 variants in healthy Crohn's Disease relatives associate with increased intestinal permeability (Gut. 2006; 55:342). Therefore, Nod2 mutation is not sufficient “per se” to establish inflammatory lesions both in humans and in animal models. We observed, after the induction of a transient increase of intestinal permeability that follows an induced breach of epithelial barrier associated or not with epithelial damage, a microflora-dependent mucosal regulatory response characterized by the expansion of T regulatory cells expressing surface TGF-β latency-associated- peptide (LAP+ T cells)(Gastroenterology 2008;135:1612). Aim: To evaluate the influence of NOD2 deficiency on mucosal regulatory cells and cytokines. Methods: In separated groups of untreated and ethanol-treated Nod2KO and WT mice, we evaluated: intestinal permeability by serum quantification of fluorescent particles after intrarectal administration of FITC-dextran; IL-12p70 and TGF-β colonic tissue content by ELISA on tissue protein extracts and the % of regulatory cells by immunofluorescence on isolated lamina propria mononuclear cells. Results: we found that untreated Nod2 KO mice, when compared to WT mice, showed an increased intestinal permeability. This feature was associated with a significant increase of IL12-p70 (5.6±1.3 vs. 2.8±0.7 pg/mg protein, mean ± SE, Nod2 KO vs. WT mice, *P =.03) and TGF-β (345.6±23.2 vs. 261.0±37.0 pg/mg protein, mean ± SE, *P =.03 Nod2 KO vs. WT mice) colonic tissue content. In addition, we observed an increased % of CD4+LAP+ T cells in lamina propria mononuclear cells isolated from colons of Nod2 KO mice, when compared to WT mice (6.9%±0.9 vs. 3.9%±0.8, mean ± SE, *P = .01). No difference was observed in the % of CD4+Foxp3+ cells. The induction of a transient increase of intestinal permeability by intrarectal ethanol administration was associated with an increased TGF-β content in both Nod2 KO and WT mice. However, % of CD4+LAP+ T cells in Nod2 KO mice were significant increased when compared to WT mice 1 day after ethanol administration(10.5%±1.6 vs. 4.9%±0.9, mean ± SE, *P =.007). The data suggest that Nod2 deficiency is associated with increased regulatory response to commensal microorganisms. Conclusions: The increased regulatory response to microflora associated with NOD2 mutation might explain why NOD2 deficiency is not sufficient to establish inflammatory lesions.
43 Dietary Fat-Induced Taurocholic Acid Production Promotes Pathobiont and Colitis in IL-10-/- Mice Suzanne Devkota, Yunwei Wang, Mark W. Musch, Vanessa Leone, Hannah Fehlner-Peach, Anuradha Nadimpalli, Dionysios Antonopoulos, Bana Jabri, Eugene B. Chang The composite human microbiome of Western populations has changed over the past century, brought on by new environmental triggers that often have a negative impact on human health. Here we show that consumption of a diet high in saturated (milk derived)fat (MF) but not polyunsaturated (safflower oil)-fat (PUFA) changes the conditions for microbial assemblage and promotes expansion of a rare, immunogenic, sulfite-reducing pathobiont, Bilophila wadsworthia. This was associated with a proinflammatory TH1 immune response and the development of colitis in genetically susceptible IL-10-/- mice. Taurineconjugated hepatic bile acids provide a sulfur source for the bile-loving B. wadsworthia, therefore we examined the possibility that the MF effects are mediated by changes in bile composition. Mass spectrometry analysis of gall bladder aspirates from mice consuming MF revealed 85% of total bile was taurocholic acid, compared to 64% in PUFA and 43% in low-fat (LF). When these aspirates were added to pure cultures of B. wadsworthia, only bile obtained from MF-fed mice promoted robust B. wadsworthia growth In Vitro. In addition, a bloom of B. wadsworthia and development of colitis was observed when mice were fed a LF diet supplemented with taurocholic, but not with glycocholic acid. Furthermore, when germ-free IL-10-/- mice were monoassociated with B. wadsworthia, the bacteria survived only when provided with the MF diet or taurocholic acid, and resulted in colitis, which was not observed when MF or taurocholic acid were fed alone. The marked colitis in these mice was accompanied by increased IFNγ and IL12p70, but not IL-17 or IL-23 in the colonic mucosa and mesenteric lymph nodes, which indicates B. wadsworthia polarizes naive T cells toward a pro-inflammatory TH1 mediated colitis. Together these data show that dietary fats differ in their effects on the host microbiota and immune system. The data also provide a plausible mechanistic basis by which Western type diets high in saturated fats may increase the prevalence of complex immune-mediated diseases like inflammatory bowel diseases in genetically susceptible hosts.
46 Pouch Inflammation is Associated With Crohn's Disease-Like Dysbiosis and May Be Predicted by Microbiota Analysis and Follow up Amir Kovacs, Elhanan Meirovithz, Amos Ofer, Tanir Zion, Lior Yahav, Hagit Tulchinsky, Uri Gophna, Iris Dotan Background: Total proctocolectomy and Ileal pouch-anal anastomosis, ("pouch surgery") is the operation of choice for complicated ulcerative colitis (UC) patients. Inflammation of the pouch, termed pouchitis, is the most common complication, occurring in up to 60% of the patients, but is rare in patients undergoing surgery due to familial adenomatous polyposis (FAP). The exact etiology of pouchitis is unknown; however it is believed that similarly to inflammatory bowel diseases (IBD), pouchitis is driven by an aberrant immune response towards the commensal microbiota in genetically susceptible individuals. Aim: To longitudinally investigate the role of the gut microbiota in pouch inflammation. Methods: Patients were prospectively followed up at a comprehensive pouch clinic. Clinical, demographic, endoscopic and histological data were recorded and pouch status (normal, acute, recurrent acute and chronic pouchitis) was defined. Stool samples were collected longitudinally, and their microbial composition was assessed using 16SrRNA gene pyrosequencing. Results: Eighty seven patients were recruited, 30 with normal pouches, 25 with chronic pouchitis, 27 with recurrent acute pouchitis and 5 FAP pouch patients with normal pouches. In 26 patients 2 or more samples were collected longitudinally in an interval of 4.8±0.4 months. The bacterial composition of most normal pouches (83%) was characterized by dominance of the phyla Firmicutes and Bacteroidetes (70%-100% and 0%-30% of all bacteria identified, respectively). All FAP patients' pouches were dominated by Firmicutes as well (over 90%). The level of Proteobacteria was significantly associated with pouch inflammation: Chronic pouchitis patients had the highest abundance (25.1±4.4%), followed by recurrent acute pouchitis (12.0±2%), normal pouches (10.3±1.9%) and FAP patients having the lowest abundance (3.2±1.1%) (ANOVA, p-value=4.67E10-4). Longitudinally, clinical stability or changes in disease activity were reflected by microbiota stability or volatility, respectively, in 73% of cases. In chronic pouchitis, patients treated with immunomodulatory and biological therapies had bacterial composition similar to that of normal pouch patients, while chronic treatment with antibiotics was associated with severe dysbiosis and with continuous disease activity. Conclusions: Normal pouch microbiota is characterized by dominance of Firmicutes and Bacteroidetes. An increase in Proteobacteria is significantly associated with pouch inflammation. Thus, dysbiosis in pouch patients resembles that observed in Crohn's disease. Detection of microbiota composition enables follow up of pouch disease activity. The association of severe dysbiosis and disease activity with chronic antibiotic treatment may challenge the concept of antibiotic treatment in pouch patients.
44 Mucosal Surface Sampling and Metaproteomic Analysis Identify DiseaseRelated Biologic Neighborhoods XiaoXiao Li, James F. LeBlanc, David Elashoff, Ian McHardy, Maomeng Tong, Bennett Roth, Andrew Ippoliti, Thomas Graeber, Lee Goodglick, Jonathan Braun Background: Aberrant interactions between the host and the intestinal bacteria at the mucosal surface are important in the pathogenesis and disease activity of inflammatory bowel diseases. Previously, we have established a novel metaproteomic method to collect colon lavage samples during colonoscopy, and protein analysis of the mucosal functional state by highthroughput matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) associated with microbial mucosa-associated microbial composition (L Presley, 2011 Inflamm Bowel Dis; X Li, 2011 PLoS One). Methods: 272 samples from 54 subjects (20 normal, 13 ulcerative colitis, 21 Crohn's disease) underwent endoscopic saline lavage sampling at 6 sites (cecum, ascending, transverse, descending, sigmoid, rectum). A simple clinic procedure for rectal mucosal sampling using a sponge swab was also assessed. Each sample was analyzed by MALDI-TOF MS. High-resolution spectra were preprocessed, and permutation test and linear mixed-effect model were used to examine regional and disease-related features. Protein modules were calculated and visualized using weighted correlation network analysis (WGCNA). Neighborhood members were further identified by MALDI-immunoprecipitation or MALDI-LIFT-TOF/TOF MS. Results: Significant differences in the mucosal metaproteome were observed between the distal (rectum, sigmoid) and proximal colon as well as the three disease groups. Module analysis showed 9 distinct but reproducible modules, which are defined by clusters of proteins that are coordinately displayed at the mucosal surface, and were also detectable by rectal sponge sampling. Certain modules have a significant disease association. To further characterize those disease-related modules, we identified 10 hub proteins from 4 modules, and validated the disease association and cellular origins of each protein using immunoblot and immunohistochemistry. Those hub proteins include human neutrophil defensins1-3, alpha-defensin 5, beta-defensin 1 and 2, hepcidin, transferrin, elastase-2, and peptidase M20. Direct visualization of these hub proteins in whole-mount human mucosa will be presented to document the size and frequency of protein modules. Conclusions: These findings reveal that the human colonic mucosal surface is organized into biologic neighborhoods, which may be identified and characterized by surface protein analysis, including sponge swab sampling as a clinic procedure. Such neighborhoods offer an integrative target for therapeutic intervention to restore and sustain disease-free mucosal state, and a mucosal surface sampling method to monitor such interventions.
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hMSH3 Deficiency Causes EMAST in Human Colorectal Cancer Cells Stephanie Tseng-Rogenski, Heekyung Chung, Shuai Zhang, Maike B. Wilk, John M. Carethers Background and Aims. Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in up to 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable colorectal cancers, where hypermethylation of the major mismatch repair (MMR) gene hMLH1 drives multiple target gene mutations, the cause of EMAST is unknown but was recently associated with reduced expression of the minor MMR protein hMSH3 in CRCs. We assessed experimentally whether hMSH3 deficiency is a cause of EMAST. Methods. We constructed plasmids containing D8S321 and D20S82 tetranucleotide loci (12 and 16 repeats of AAAG respectively) that are used to define EMAST. Sequences were cloned +1 bp out of frame immediately after the start codon of the EGFP gene. A -4 bp frameshift deletion of one AAAG unit would allow in-frame expression of EGFP. Mutationresistant counterpart plasmids were constructed by changing 2 nucleotide sequences in every 3 units of AAAG, preventing frameshift mutations. First, we created MMR proficient, hMLH1-/-, hMSH6-/-, and hMSH3-/- stable cell lines carrying the plasmids. Subsequently, we reduced hMSH3 expression via RNAi in MMR proficient cells harboring the EMAST constructs. Non-fluorescent cells were sorted and cultured for flow cytometry analysis. Mutations were examined by DNA-sequencing. Results. Sequencing confirmed frameshift mutations in fluorescent cells containing D8S321 and D20S82. Such mutations included contraction and expansion of microsatellites. D8S321 mutations occurred 31-and 40-fold higher in hMLH1-/- and hMSH3-/- cells compared to hMSH6-/- cells, respectively. D20S82 mutations occurred 82-and 49-fold higher in hMLH1-/- and hMSH3-/- cells compared to hMSH6-/- cells, respectively. When hMSH3 expression levels were reduced in MMR proficient cells upon hMSH3 shRNA transfection, significantly higher mutation rates were detected in hMSH3 knockdown cells for D8S321 (18.14 x 10-4) and D20S82 (11.14 x 10-4) compared to their individual scramble control cells (0 and 0.26 x 10-4 separately). Conclusions. EMAST is dependent upon MMR background, with hMSH3-/- more prone to frameshift mutations than hMSH6-/-, opposite to frameshift mutation observed at mononucleotide repeats. hMSH3-/mimics complete MMR failure (hMLH1-/-) in inducing EMAST. Furthermore, knock down of hMSH3 expression alone was able to elicit EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, loss of hMSH3 function appears to be the cause of EMAST.
41 Nuclear Ikkβ Phosphorylates ATM in Response to Genotoxic Stimuli, Which Helps Cancer Cells to Survive DNA Damage Kei Sakamoto, Yohko Hikiba, Yoku Hayakawa, Yoshihiro Hirata, Masao Akanuma, Shin Maeda Background: In many cases of gastroenterological cancers, drug resistance of cancer cells worsens outcomes of therapies. Many kinds of anticancer drugs are expected to cause irreparable DNA damage in cancer cells and lead them to cell death. Ataxia-telangiectasia mutated (ATM) is a key molecule involved in the cellular response to DNA damage. The most part of ATM stays in the nucleus to work on DNA repair. On the other hand, a portion of ATM is exported from the nucleus into the cytoplasm, where it activates the I kappa B kinase/nuclear factor kappa B (IKK/NFκ-B) signaling pathway. It has been thought that activated IKKβ, a key molecule for NF-κB activation, generally resides in the cytoplasm and phosphorylates cytoplasmic downstream molecules. In this study, we identified a new role for IKKβ during the response to DNA damage. Methods: IKKβ and ATM were knocked down in gastric cancer cells by siRNA. Genotoxic stimuli were added using alkylating agents or a topoisomerase inhibitor. ATM phosphorylation and localization of IKKβ were examined by immunoblotting. DNA damage was quantitated by comet assay. Direct phosphorylaition of ATM by IKKβ was observed by In Vitro kinase assay. RESULTS: IKKβ-knocked down cells and control cells were treated with an alkylating agent and subjected to an alkaline comet assay 24 h after stimulation. Higher levels of DNA damage were observed in IKKβknocked down cells. Since ATM is a critical factor associated with DNA repair, we analyzed phosphorylation of ATM after stimulation. ATM phosphorylation was significantly attenuated 6-9 h after stimulation in IKKβ-knocked down cells, whereas the initial ATM activation did not differ. These observations suggested that phosphorylation of ATM consists of two phases: the initial phase and the sustained phase (after 6 h) and IKKβ is involved specifically in late-phase phosphorylation. When wild type IKKβ (IKKβ-WT) and a kinase-negative IKKβ mutant (IKKβ-KN) were expressed in cells, ATM phosphorylation was attenuated in IKKβKN-expressing cells. Direct phosphorylation of ATM by IKKβ was also observed In Vitro kinase assay. Furthermore, IKKβ protein levels in the nucleus increased in response to DNA damage when nuclear extracts of the cells were immunoblotted with anti-IKKβ. To examine the function of translocation of IKKβ into nucleus, IKKβ with the nuclear exporting signal (IKKβ-NES) and IKKβ-WT were expressed in IKKβ-knockout MEFs. Late-phase ATM phosphorylation was attenuated in IKKβ-NES -expressing cells as compared with those expressing IKKβ-WT. The amount of DNA damage after stimulation was greater in IKKβ-NES -expressing cells. Conclusion: IKKβ translocates into nucleus in response to DNA damage and phosphorylates ATM. The late-phase phosphorylation of ATM promotes DNA repair, which can help cancer cells to survive under anticancer therapy.
39 Identification of Gene Loci With Microsatellite Repeats Frequently Altered in Liver Metastasis Exhibiting Moderate Levels of Microsatellite Instability From Primary CRC Melissa Garcia, Chan Choi, Hyeong-Rok Kim, Junichi Koike, Hiromichi Hemmi, Jie Li, Takeshi Nagasaka, Clement R. Boland, Minoru Koi Background & Aims: We demonstrated that moderate microsatellite instability (MSI-M) defined by NCI reference markers and elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) markers was common in primary CRC, and was an independent predictor for recurrent distant metastasis of stage II and III (II/III) primary CRC. However, how MSI-M is linked to recurrent distant metastasis is not known. To approach this question, we aimed to identify genetic changes significantly associated with MSI-M and with liver metastasis (LM) from primary CRC. Methods: We selected 142 gene loci with polymorphic microsatellites with di-, tri- and tetranucleotide repeats by genome data mining, and examined each locus for frame-shift mutations within microsatellite repeats and loss of heterozygosity (LOH) in 24 LM exhibiting MSI-M. Results: Among 142 gene loci examined, 29 loci (20.4%) exhibited microsatellite mutations in 24 cases of MSI-M-positive LM. More loci with larger repeats showed mutations than loci with smaller repeats; 53% of loci with tetranucleotide repeats, 15% of loci with trinucleotide repeats and 4% of loci with di-nucleotide repeats showed microsatellite mutations. Among loci with mutations, RBM47 (25%), WIPF (18%), D9S303 (17%), ZNF161 (17%), D8S1179 (14%), D21S11 (13%) and KANK2 (13%) exhibited higher levels of mutations in MSI-M-positive LMs. However, the mutation frequency of these loci was no greater than the average mutation frequency of 7 EMAST markers (~20%) among 24 LM cases. Compared to microsatellite mutation, LOH was found in more loci with higher frequencies. Eighty-seven out of 142 loci (61%) exhibited LOH with more than 6% of informative cases. We found 10 loci with a frequency of LOH higher than 50%, including KDM6B (75%), MNT (71%), SMARCA2 (64%), HEC1 (60%), ANKRD5 (58%), BCL2 (58%), SEMA6D (57%), D5S818 (56%), STYK1 (50%) and BCL6B (50%). LOH at chromosomal regions where KDM6B (17p13), MNT (17p13), BCL6B (17p13), HEC1 (18p11), BCL1 (18q21), ANKRD5 (20p12) and SEMA6D (15q21) reside have been reported in CRC tissues. Conclusion: SMARCA2 at 9p24 and STYK1 at 12p13 are new loci that frequently exhibit LOH in MSI-M-positive LM tissues. A possible association of LOH at the SMARCA2 or STYK1 locus with MSI-M or with LM formation is under investigation.
42 Downregulation of TGF-β Signaling Pathway Invokes Genomic Instability in Liver Cancers Vivek Shukla, Ying Li, Lopa Mishra Background: Loss of TGF-β adaptor protein β2SP leads to delayed liver regeneration and extensive DNA damage in mice. The Smad3/4 adaptor protein, β2SP, is emerging as potent regulator of tumorigeneisis by its ability to affect TGF-β tumor suppressor function. Deletion of β2SP results in a dramatic and spontaneous formation of liver cancer and gastrointestinal cancers. Moreover, β2SP+/- and β2SP+/- /Smad3+/- mutant mice phenocopy human Beckwith-Wiedemann syndrome (BWS), a hereditary human cancer stem cell syndrome imprinting disorder with loss of p57, increased IGF2, associated with an 800 fold increase of cancers that include those of the liver. Delayed liver regeneration and extensive DNA damage was observed in β2SP heterozygote mice. In addition, spectrins have been observed to associate with FANC G and D, DNA interstrand cross links, and Smad3/4 to transcriptionally regulate FANC genes. Hypothesis: We therefore hypothesised that TGF-β is a crucial enforcer of genomic stability, and that β2SP and/or Smad3/4 are key mediators in suppressing liver injury and cancer through modulation of the DNA repair pathway. Materials & Methods: To determine the role of β2SP and Smad3 in the DNA damage response, and whether β2SP and β2SP/Smad3 loss plays a causal role in the downregulation of the Fanc/Brca pathway, we used our mouse models as well as the mouse embryonic fibroblasts. Oxidative DNA damage was induced by H2O2 treatment and assayed tail length analysis. Irradiation was
40 MicroRNA-18a Attenuates DNA Damage Repair by Directly Controlling the Expression of ATM in Colon Cancer Chung-Wah Wu, Yu Juan Dong, Xin Qi He, Simon Ng, Francis K. L. Chan, Joseph J. Sung, Jun Yu Background and Aims: MiR-18a is one of the most up-regulated miRNAs in colorectal cancers (CRC) compared with the adjacent normal tissues based on miRNA profiling. However, the function of miR-18a in cancer development remains unclear. Methods and Results: The up-regulation of miR-18a was validated and confirmed in 46 CRC tumors compared with their adjacent normal tissues (p<0.0001). Up-regulation of miR-18a was also demonstrated in colon cancer cell lines relative to normal colon biopsies. Potential target genes of miR-18a were predicted in silico by algorithms TargetScan and MiRanda. Ataxia Telangiectasia Mutated (ATM) was identified as one of the potential targets of miR18a. Downregulation of ATM was detected in the CRC tumors compared to adjacent normal
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induced by 8Gy IR and DNA was studied by immunofluorescence of DNA damage sensors γH2AX 532BP1, pChk2. DNA repair was assayed by Mdc1, NBS1 and Rad51 focus formation at different time points after irradiation. Results: β2SP+/− and β2SP+/−/Smad3+/− mice exhibited an increased prevalence of HCC and GI cancer and many phenotypic characteristics observed in BWS patients. Absence of β2SP results in impaired liver regeneration and dramatic spontaneous DNA damage in β2SP+/- mouse livers after PHx in p53 independent manner. Loss of β2SP results in premature replicative senescence with spontaneous DNA damage. β2SP deficiency results in increased sensitivity to exogenous DNA damage along with insufficient DNA repair and impairs the loading of repair proteins and repair of DNA double-strand breaks. Loss of FancD2 expression in HCC and GI cancer cell lines correlates with loss of β2SP. Moreover, β2SP-/- and β2SP+/−/Smad3+/− cell lines accumulate DNA damage that is further exacerbated by DNA cross-linking agents such as mitomycin C similar to P57 null cells and Fanconi anemia cell lines. Conclusions: Downregulation of TGF-β signaling pathway impels genomic instability through regulation of DNA impairment of the Fanc/Brca DNA damage response and interstrand link repair and inactivation of TGFpathway results in alcohol toxicity, cirrhosis and liver cancers.
45 NOD2 Deficiency is Associated With Increased Mucosal Regulatory Response to Commensal Microorganisms Antonello Amendola, Alessia Butera, Monica Boirivant Background: It has been reported that Peyer's patches (PPs) of Nod2 KO mice show, as a consequence of the presence of ileal microbiota, increased tissue content of IFN-γ and TNFα that are responsible for an increased permeability of the epithelium covering PP (Gut 2010;59:207), yet the mice do not show intestinal inflammation. Moreover, Nod2 variants in healthy Crohn's Disease relatives associate with increased intestinal permeability (Gut. 2006; 55:342). Therefore, Nod2 mutation is not sufficient “per se” to establish inflammatory lesions both in humans and in animal models. We observed, after the induction of a transient increase of intestinal permeability that follows an induced breach of epithelial barrier associated or not with epithelial damage, a microflora-dependent mucosal regulatory response characterized by the expansion of T regulatory cells expressing surface TGF-β latency-associated- peptide (LAP+ T cells)(Gastroenterology 2008;135:1612). Aim: To evaluate the influence of NOD2 deficiency on mucosal regulatory cells and cytokines. Methods: In separated groups of untreated and ethanol-treated Nod2KO and WT mice, we evaluated: intestinal permeability by serum quantification of fluorescent particles after intrarectal administration of FITC-dextran; IL-12p70 and TGF-β colonic tissue content by ELISA on tissue protein extracts and the % of regulatory cells by immunofluorescence on isolated lamina propria mononuclear cells. Results: we found that untreated Nod2 KO mice, when compared to WT mice, showed an increased intestinal permeability. This feature was associated with a significant increase of IL12-p70 (5.6±1.3 vs. 2.8±0.7 pg/mg protein, mean ± SE, Nod2 KO vs. WT mice, *P =.03) and TGF-β (345.6±23.2 vs. 261.0±37.0 pg/mg protein, mean ± SE, *P =.03 Nod2 KO vs. WT mice) colonic tissue content. In addition, we observed an increased % of CD4+LAP+ T cells in lamina propria mononuclear cells isolated from colons of Nod2 KO mice, when compared to WT mice (6.9%±0.9 vs. 3.9%±0.8, mean ± SE, *P = .01). No difference was observed in the % of CD4+Foxp3+ cells. The induction of a transient increase of intestinal permeability by intrarectal ethanol administration was associated with an increased TGF-β content in both Nod2 KO and WT mice. However, % of CD4+LAP+ T cells in Nod2 KO mice were significant increased when compared to WT mice 1 day after ethanol administration(10.5%±1.6 vs. 4.9%±0.9, mean ± SE, *P =.007). The data suggest that Nod2 deficiency is associated with increased regulatory response to commensal microorganisms. Conclusions: The increased regulatory response to microflora associated with NOD2 mutation might explain why NOD2 deficiency is not sufficient to establish inflammatory lesions.
43 Dietary Fat-Induced Taurocholic Acid Production Promotes Pathobiont and Colitis in IL-10-/- Mice Suzanne Devkota, Yunwei Wang, Mark W. Musch, Vanessa Leone, Hannah Fehlner-Peach, Anuradha Nadimpalli, Dionysios Antonopoulos, Bana Jabri, Eugene B. Chang The composite human microbiome of Western populations has changed over the past century, brought on by new environmental triggers that often have a negative impact on human health. Here we show that consumption of a diet high in saturated (milk derived)fat (MF) but not polyunsaturated (safflower oil)-fat (PUFA) changes the conditions for microbial assemblage and promotes expansion of a rare, immunogenic, sulfite-reducing pathobiont, Bilophila wadsworthia. This was associated with a proinflammatory TH1 immune response and the development of colitis in genetically susceptible IL-10-/- mice. Taurineconjugated hepatic bile acids provide a sulfur source for the bile-loving B. wadsworthia, therefore we examined the possibility that the MF effects are mediated by changes in bile composition. Mass spectrometry analysis of gall bladder aspirates from mice consuming MF revealed 85% of total bile was taurocholic acid, compared to 64% in PUFA and 43% in low-fat (LF). When these aspirates were added to pure cultures of B. wadsworthia, only bile obtained from MF-fed mice promoted robust B. wadsworthia growth In Vitro. In addition, a bloom of B. wadsworthia and development of colitis was observed when mice were fed a LF diet supplemented with taurocholic, but not with glycocholic acid. Furthermore, when germ-free IL-10-/- mice were monoassociated with B. wadsworthia, the bacteria survived only when provided with the MF diet or taurocholic acid, and resulted in colitis, which was not observed when MF or taurocholic acid were fed alone. The marked colitis in these mice was accompanied by increased IFNγ and IL12p70, but not IL-17 or IL-23 in the colonic mucosa and mesenteric lymph nodes, which indicates B. wadsworthia polarizes naive T cells toward a pro-inflammatory TH1 mediated colitis. Together these data show that dietary fats differ in their effects on the host microbiota and immune system. The data also provide a plausible mechanistic basis by which Western type diets high in saturated fats may increase the prevalence of complex immune-mediated diseases like inflammatory bowel diseases in genetically susceptible hosts.
46 Pouch Inflammation is Associated With Crohn's Disease-Like Dysbiosis and May Be Predicted by Microbiota Analysis and Follow up Amir Kovacs, Elhanan Meirovithz, Amos Ofer, Tanir Zion, Lior Yahav, Hagit Tulchinsky, Uri Gophna, Iris Dotan Background: Total proctocolectomy and Ileal pouch-anal anastomosis, ("pouch surgery") is the operation of choice for complicated ulcerative colitis (UC) patients. Inflammation of the pouch, termed pouchitis, is the most common complication, occurring in up to 60% of the patients, but is rare in patients undergoing surgery due to familial adenomatous polyposis (FAP). The exact etiology of pouchitis is unknown; however it is believed that similarly to inflammatory bowel diseases (IBD), pouchitis is driven by an aberrant immune response towards the commensal microbiota in genetically susceptible individuals. Aim: To longitudinally investigate the role of the gut microbiota in pouch inflammation. Methods: Patients were prospectively followed up at a comprehensive pouch clinic. Clinical, demographic, endoscopic and histological data were recorded and pouch status (normal, acute, recurrent acute and chronic pouchitis) was defined. Stool samples were collected longitudinally, and their microbial composition was assessed using 16SrRNA gene pyrosequencing. Results: Eighty seven patients were recruited, 30 with normal pouches, 25 with chronic pouchitis, 27 with recurrent acute pouchitis and 5 FAP pouch patients with normal pouches. In 26 patients 2 or more samples were collected longitudinally in an interval of 4.8±0.4 months. The bacterial composition of most normal pouches (83%) was characterized by dominance of the phyla Firmicutes and Bacteroidetes (70%-100% and 0%-30% of all bacteria identified, respectively). All FAP patients' pouches were dominated by Firmicutes as well (over 90%). The level of Proteobacteria was significantly associated with pouch inflammation: Chronic pouchitis patients had the highest abundance (25.1±4.4%), followed by recurrent acute pouchitis (12.0±2%), normal pouches (10.3±1.9%) and FAP patients having the lowest abundance (3.2±1.1%) (ANOVA, p-value=4.67E10-4). Longitudinally, clinical stability or changes in disease activity were reflected by microbiota stability or volatility, respectively, in 73% of cases. In chronic pouchitis, patients treated with immunomodulatory and biological therapies had bacterial composition similar to that of normal pouch patients, while chronic treatment with antibiotics was associated with severe dysbiosis and with continuous disease activity. Conclusions: Normal pouch microbiota is characterized by dominance of Firmicutes and Bacteroidetes. An increase in Proteobacteria is significantly associated with pouch inflammation. Thus, dysbiosis in pouch patients resembles that observed in Crohn's disease. Detection of microbiota composition enables follow up of pouch disease activity. The association of severe dysbiosis and disease activity with chronic antibiotic treatment may challenge the concept of antibiotic treatment in pouch patients.
44 Mucosal Surface Sampling and Metaproteomic Analysis Identify DiseaseRelated Biologic Neighborhoods XiaoXiao Li, James F. LeBlanc, David Elashoff, Ian McHardy, Maomeng Tong, Bennett Roth, Andrew Ippoliti, Thomas Graeber, Lee Goodglick, Jonathan Braun Background: Aberrant interactions between the host and the intestinal bacteria at the mucosal surface are important in the pathogenesis and disease activity of inflammatory bowel diseases. Previously, we have established a novel metaproteomic method to collect colon lavage samples during colonoscopy, and protein analysis of the mucosal functional state by highthroughput matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) associated with microbial mucosa-associated microbial composition (L Presley, 2011 Inflamm Bowel Dis; X Li, 2011 PLoS One). Methods: 272 samples from 54 subjects (20 normal, 13 ulcerative colitis, 21 Crohn's disease) underwent endoscopic saline lavage sampling at 6 sites (cecum, ascending, transverse, descending, sigmoid, rectum). A simple clinic procedure for rectal mucosal sampling using a sponge swab was also assessed. Each sample was analyzed by MALDI-TOF MS. High-resolution spectra were preprocessed, and permutation test and linear mixed-effect model were used to examine regional and disease-related features. Protein modules were calculated and visualized using weighted correlation network analysis (WGCNA). Neighborhood members were further identified by MALDI-immunoprecipitation or MALDI-LIFT-TOF/TOF MS. Results: Significant differences in the mucosal metaproteome were observed between the distal (rectum, sigmoid) and proximal colon as well as the three disease groups. Module analysis showed 9 distinct but reproducible modules, which are defined by clusters of proteins that are coordinately displayed at the mucosal surface, and were also detectable by rectal sponge sampling. Certain modules have a significant disease association. To further characterize those disease-related modules, we identified 10 hub proteins from 4 modules, and validated the disease association and cellular origins of each protein using immunoblot and immunohistochemistry. Those hub proteins include human neutrophil defensins1-3, alpha-defensin 5, beta-defensin 1 and 2, hepcidin, transferrin, elastase-2, and peptidase M20. Direct visualization of these hub proteins in whole-mount human mucosa will be presented to document the size and frequency of protein modules. Conclusions: These findings reveal that the human colonic mucosal surface is organized into biologic neighborhoods, which may be identified and characterized by surface protein analysis, including sponge swab sampling as a clinic procedure. Such neighborhoods offer an integrative target for therapeutic intervention to restore and sustain disease-free mucosal state, and a mucosal surface sampling method to monitor such interventions.
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