Role of Beta Spectrin and Smad in Alcohol Induced Liver and GI Cell Proliferation

描述（由申请人提供）：保护肝脏免受酒精、黄曲霉毒素和病毒性肝炎等药物引起的肝损伤、纤维化和癌症的关键因素是基因组稳定性的实施。然而，基因毒性导致异常DNA修复的传感器仍然难以捉摸。TGF-ß被认为是基因组稳定性和肿瘤抑制的关键启动子；然而，发生这种情况的框架尚不清楚。我们观察到TGF-ß成员ß2SP和Smad3的缺失导致自发性DNA损伤、酒精性肝损伤和肝癌（HCC）的积累。TGF-ß/ß2SP突变的细胞系和小鼠模型（人和小鼠）积累DNA损伤，DNA交联剂如丝裂霉素C加重了这种损伤，类似于P57空细胞和范可尼贫血细胞系。外显子组和基因组序列分析发现酒精相关性HCC中存在体细胞2SP突变，导致TGF-ß信号的功能破坏。此外，已观察到谱蛋白与FANC G和D、DNA链间交联和Smad3/4相关，对FANC基因进行转录调节。我们的假设是TGF-ß是基因组稳定性的关键执行因子，而ß2SP和/或Smad3/4是通过调节Fanconi通路抑制肝损伤和癌症的关键介质。最近的研究还表明，范可尼贫血DNA修复途径在对抗酒精和乙醛诱导的小鼠遗传毒性中的关键作用。重要的是，ß2SP小鼠突变体在大量病例中表现出胎儿酒精综合征，ß2SP突变体对酒精损伤非常敏感。因此，我们的第二个假设是TGF-ß/ß2SP/Smad3对醛遗传毒性的保护至关重要。TGF-ß通路失活可导致酒精毒性、肝硬化和HCC。为了进一步了解ß2SP/Smad3在DNA修复、酒精/活性醛毒性和肿瘤发生中的作用，我们提出了以下建议：确定ß2SP和Smad3在DNA损伤反应中的作用，以及ß2SP和ß2SP/Smad3缺失是否在下调Fanc/Brca通路、损害Fanc/Brca DNA损伤反应和链间交联修复中起因果作用。目标2。检测小鼠突变体ß2SP， ß2SP/Smad3和Smad3与Aldh2零突变小鼠杂交是否会导致后代显示(a)对自发肿瘤发展的易感性增强以及乙醇致畸性(b)来自这些杂交的肝细胞和mef是否会发生进行性DNA损伤。这些研究可能会为酒精引起的肝毒性建立一个可靠的模型。目标3。(a)确定TGF-ß通路和DNA修复基因Fanc/Brca是否是肝硬化进展为HCC的预后因素；(b)确定易患HCC的新基因位点。c)扩展到精细映射。值得注意的是，在此申请下提出的研究将为科学界提供对酒精性肝损伤和肝肿瘤的仪器调节因子的新认识。

DESCRIPTION (provided by applicant): A crucial factor in protection from liver injury, fibrosis and cancer by agents such as alcohol, aflatoxins and viral hepatitis is the enforcement of genomic stability. However, sensors for genotoxicity leading to aberrant DNA repair remain elusive. TGF-ß has been implicated as a critical promoter of genomic stability and tumor suppression; Yet, the framework by which this occurs is not known. We have observed that loss of TGF-ß members ß2SP and Smad3 leads to accumulation of spontaneous DNA damage, alcohol induced liver injury and cancers including those of the liver (HCC). Cell lines, and mouse models (human and mouse) mutant for TGF-ß/ß2SP accumulate DNA damage that is exacerbated by DNA cross-linking agents such as mitomycin C similar to P57 null cells and Fanconi anemia cell lines. Exome and genomic sequence analysis identified a somatic �2SP mutation in alcohol associated HCC, that results in a functional disruption of TGF-ß signaling. 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. Our hypothesis is that TGF-ß is a crucial enforcer of genomic stability, and that ß2SP and/or Smad3/4 are key mediators in suppressing liver injury and cancer through modulation of the Fanconi pathway. Recent studies have also shown the key role of the Fanconi anemia DNA repair pathway in counteracting alcohol and acetaldehyde induced genotoxicity in mice. Importantly, the ß2SP mouse mutants phenocopy a fetal alcohol syndrome in a large number of cases and the ß2SP mutants are highly susceptible to alcohol injury. Therefore our secondary hypothesis is that TGF-ß/ß2SP/Smad3 are crucial for protection against aldehyde genotoxicity. Inactivation of the TGF-ß pathway results in alcohol toxicity, cirrhosis and HCC. To obtain further insights into the role of ß2SP/Smad3 in DNA repair, alcohol/reactive aldehyde toxicity, and neoplasia, we propose the following: AIM 1. Determine the role of ß2SP and Smad3 in the DNA damage response, and whether ß2SP and ß2SP/Smad3 loss plays a causal role in downregulation of the Fanc/Brca pathway, impairment of the Fanc/Brca DNA damage response and interstrand cross link repair. AIM 2. Examine whether mouse mutants ß2SP, ß2SP/Smad3 and Smad3 crossed with Aldh2 null mutant mice result in a progeny that show (a) enhanced susceptibility to spontaneous tumor development as well as ethanol teratogenicity (b) whether the hepatocytes and MEFs derived from such crosses undergo progressive DNA damage. Potentially these studies will generate a robust model for alcohol induced liver toxicity. AIM 3. (a) Determine whether the TGF-ß pathway and DNA repair genes Fanc/Brca are prognostic factors in cirrhosis progressing to HCC, and (b) Identify novel genetic loci that predispose to HCC. c) Expand to fine mapping. Significantly, studies proposed under this application should provide the scientific community with a new understanding of the instrumental regulators in alcohol induced liver injury and neoplasia.