39 identification of gene loci with microsatellite repeats frequently altered in liver metastasis...
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hMSH3 Deficiency Causes EMAST in Human Colorectal Cancer CellsStephanie 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 majormismatch repair (MMR) gene hMLH1 drives multiple target gene mutations, the cause ofEMAST is unknown but was recently associated with reduced expression of the minor MMRprotein hMSH3 in CRCs. We assessed experimentally whether hMSH3 deficiency is a causeof EMAST.Methods. We constructed plasmids containing D8S321 and D20S82 tetranucleot-ide loci (12 and 16 repeats of AAAG respectively) that are used to define EMAST. Sequenceswere cloned +1 bp out of frame immediately after the start codon of the EGFP gene. A -4bp frameshift deletion of one AAAG unit would allow in-frame expression of EGFP. Mutation-resistant counterpart plasmids were constructed by changing 2 nucleotide sequences inevery 3 units of AAAG, preventing frameshift mutations. First, we created MMR proficient,hMLH1-/-, hMSH6-/-, and hMSH3-/- stable cell lines carrying the plasmids. Subsequently, wereduced hMSH3 expression via RNAi in MMR proficient cells harboring the EMAST con-structs. Non-fluorescent cells were sorted and cultured for flow cytometry analysis. Mutationswere examined by DNA-sequencing. Results. Sequencing confirmed frameshift mutationsin fluorescent cells containing D8S321 and D20S82. Such mutations included contractionand expansion of microsatellites. D8S321 mutations occurred 31-and 40-fold higher inhMLH1-/- and hMSH3-/- cells compared to hMSH6-/- cells, respectively. D20S82 mutationsoccurred 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 uponhMSH3 shRNA transfection, significantly higher mutation rates were detected in hMSH3knockdown cells for D8S321 (18.14 x 10-4) and D20S82 (11.14 x 10-4) compared to theirindividual scramble control cells (0 and 0.26 x 10-4 separately). Conclusions. EMAST isdependent upon MMR background, with hMSH3-/- more prone to frameshift mutations thanhMSH6-/-, opposite to frameshift mutation observed at mononucleotide repeats. hMSH3-/-
mimics complete MMR failure (hMLH1-/-) in inducing EMAST. Furthermore, knock downof hMSH3 expression alone was able to elicit EMAST. Given the observed heterogeneousexpression of hMSH3 in CRCs with EMAST, loss of hMSH3 function appears to be thecause of EMAST.
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Identification of Gene Loci With Microsatellite Repeats Frequently Altered inLiver Metastasis Exhibiting Moderate Levels of Microsatellite Instability FromPrimary CRCMelissa 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 referencemarkers and elevatedmicrosatellite alterations at selected tetranucle-otide repeats (EMAST) markers was common in primary CRC, and was an independentpredictor 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 livermetastasis (LM) from primary CRC. Methods: We selected 142 gene loci with polymorphicmicrosatellites with di-, tri- and tetranucleotide repeats by genome data mining, and examinedeach 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 largerrepeats showed mutations than loci with smaller repeats; 53% of loci with tetranucleotiderepeats, 15% of loci with trinucleotide repeats and 4% of loci with di-nucleotide repeatsshowed microsatellite mutations. Among loci with mutations, RBM47 (25%), WIPF (18%),D9S303 (17%), ZNF161 (17%), D8S1179 (14%), D21S11 (13%) and KANK2 (13%) exhib-ited higher levels of mutations in MSI-M-positive LMs. However, the mutation frequencyof 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 moreloci with higher frequencies. Eighty-seven out of 142 loci (61%) exhibited LOH with morethan 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 atchromosomal regions where KDM6B (17p13), MNT (17p13), BCL6B (17p13), HEC1(18p11), BCL1 (18q21), ANKRD5 (20p12) and SEMA6D (15q21) reside have been reportedin CRC tissues. Conclusion: SMARCA2 at 9p24 and STYK1 at 12p13 are new loci thatfrequently exhibit LOH in MSI-M-positive LM tissues. A possible association of LOH at theSMARCA2 or STYK1 locus with MSI-M or with LM formation is under investigation.
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MicroRNA-18a Attenuates DNA Damage Repair by Directly Controlling theExpression of ATM in Colon CancerChung-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 colorectalcancers (CRC) compared with the adjacent normal tissues based on miRNA profiling.However, the function of miR-18a in cancer development remains unclear. Methods andResults: The up-regulation of miR-18a was validated and confirmed in 46 CRC tumorscompared with their adjacent normal tissues (p<0.0001). Up-regulation of miR-18a wasalso demonstrated in colon cancer cell lines relative to normal colon biopsies. Potentialtarget 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 miR-18a. Downregulation of ATM was detected in the CRC tumors compared to adjacent normal
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tissues (p<0.0001). Aberrant down-regulation of ATM was also detected in 8 colon cancercell lines relative to normal colon biopsies. Expression of miR-18a and that of ATM wereinversely correlated (spearman r = -0.4562, P<0.01) in CRC tissues. To confirm the directinteraction between miR-18a and ATM, a segment of the 3'UTR of ATM with or withoutmutations 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 coloncancer cell line HCT-116 for luciferase activity assays. Transfection of pre-miR-18a enabledan approximately 1000-fold increase in miR-18a expression in HCT-116 cells. Ectopicexpression of miR-18a significantly reduced the relative luciferase activity of the constructwith 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 furtherconfirmed by the down-regulation of ATM protein level by miR-18a over-expression inHCT-116. As ATM is a key enzyme in DNA damage repair, we thus evaluated the effect ofmiR-18a on DNA double strand breaks (DSBs). DSBs were induced in HCT-116 by agenotoxic 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 bycomet assay. Ectopic expression of miR-18a significantly inhibited repair of DSBs inducedby etoposide (p<0.001). Moreoever, without the induction of DSBs by etoposide, miR-18adid 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 formedin both HCT-116 (p<0.05) and HT-29 (p<0.05) cells, inhibited cell growing curve in HCT-116 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 doublestrand breaks by directly suppressing ATM expression through targeting ATM 3'UTR.
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Nuclear Ikkβ Phosphorylates ATM in Response to Genotoxic Stimuli, WhichHelps Cancer Cells to Survive DNA DamageKei Sakamoto, Yohko Hikiba, Yoku Hayakawa, Yoshihiro Hirata, Masao Akanuma, ShinMaeda
Background: In many cases of gastroenterological cancers, drug resistance of cancer cellsworsens outcomes of therapies. Many kinds of anticancer drugs are expected to causeirreparable DNA damage in cancer cells and lead them to cell death. Ataxia-telangiectasiamutated (ATM) is a key molecule involved in the cellular response to DNA damage. Themost part of ATM stays in the nucleus to work on DNA repair. On the other hand, a portionof ATM is exported from the nucleus into the cytoplasm, where it activates the I kappa Bkinase/nuclear factor kappa B (IKK/NFκ-B) signaling pathway. It has been thought thatactivated IKKβ, a key molecule for NF-κB activation, generally resides in the cytoplasm andphosphorylates cytoplasmic downstream molecules. In this study, we identified a new rolefor IKKβ during the response to DNA damage. Methods: IKKβ and ATM were knockeddown in gastric cancer cells by siRNA. Genotoxic stimuli were added using alkylating agentsor a topoisomerase inhibitor. ATM phosphorylation and localization of IKKβ were examinedby immunoblotting. DNA damage was quantitated by comet assay. Direct phosphorylaitionof ATM by IKKβ was observed by In Vitro kinase assay. RESULTS: IKKβ-knocked downcells and control cells were treated with an alkylating agent and subjected to an alkalinecomet assay 24 h after stimulation. Higher levels of DNA damage were observed in IKKβ-knocked down cells. Since ATM is a critical factor associated with DNA repair, we analyzedphosphorylation of ATM after stimulation. ATM phosphorylation was significantly attenuated6-9 h after stimulation in IKKβ-knocked down cells, whereas the initial ATM activation didnot 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 inlate-phase phosphorylation. When wild type IKKβ (IKKβ-WT) and a kinase-negative IKKβmutant (IKKβ-KN) were expressed in cells, ATM phosphorylation was attenuated in IKKβ-KN-expressing cells. Direct phosphorylation of ATM by IKKβ was also observed In Vitrokinase assay. Furthermore, IKKβ protein levels in the nucleus increased in response to DNAdamage when nuclear extracts of the cells were immunoblotted with anti-IKKβ. To examinethe 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 phos-phorylation was attenuated in IKKβ-NES -expressing cells as compared with those expressingIKKβ-WT. The amount of DNAdamage after stimulationwas greater in IKKβ-NES -expressingcells. Conclusion: IKKβ translocates into nucleus in response to DNA damage and phos-phorylates ATM. The late-phase phosphorylation of ATM promotes DNA repair, which canhelp cancer cells to survive under anticancer therapy.
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Downregulation of TGF-β Signaling Pathway Invokes Genomic Instability inLiver CancersVivek Shukla, Ying Li, Lopa Mishra
Background: Loss of TGF-β adaptor protein β2SP leads to delayed liver regeneration andextensive DNA damage in mice. The Smad3/4 adaptor protein, β2SP, is emerging as potentregulator of tumorigeneisis by its ability to affect TGF-β tumor suppressor function. Deletionof β2SP results in a dramatic and spontaneous formation of liver cancer and gastrointestinalcancers. Moreover, β2SP+/- and β2SP+/- /Smad3+/- mutant mice phenocopy humanBeckwith-Wiedemann syndrome (BWS), a hereditary human cancer stem cell syndromeimprinting disorder with loss of p57, increased IGF2, associated with an 800 fold increaseof cancers that include those of the liver. Delayed liver regeneration and extensive DNAdamage was observed in β2SP heterozygote mice. In addition, spectrins have been observed toassociate with FANC G and D, DNA interstrand cross links, and Smad3/4 to transcriptionallyregulate FANC genes. Hypothesis: We therefore hypothesised that TGF-β is a crucial enforcerof genomic stability, and that β2SP and/or Smad3/4 are key mediators in suppressing liverinjury 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 β2SPand 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 DNAdamage was induced by H2O2 treatment and assayed tail length analysis. Irradiation was
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