Abstract 1775: High resolution characterization of human hepatocellular cancer (HCC) reveals a novel inactivating mutation in the TGF-β pathway that promotes alcohol induced HCC.
Authors: Shukla Vivek , Chen Jian , Chen Jiun-Sheng , Li Ying , Katz Lior , Majumdar Avijit , Li Lei , Hittleman Walter , Su Xiaoping , Chen Junjie , Wu Xifeng , Farci Patrizia , Mishra Lopa 
Published: August 2014

Background: 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. Because inactivation of the TGF-β pathway results in DNA damage, alcohol toxicity and HCC, TGF-β has been implicated as a critical promoter of genomic stability and tumor suppression; however, the framework by which this occurs is not known. We performed Whole-genome (WGS) and Exome sequencing (WES) on 6 pairs human HCC samples and identified inactivation of TGF-beta pathway members as a prominent characteristic of alcohol induced HCC, potentially through inactivation of a TGF-b adaptor, β2SP. We therefore hypothesized that the TGF-β/β2SP/Smad3 pathway is crucial for protection against aldehyde genotoxicity through enforcing genomic stability.

Materials & Methods: We performed Whole-genome (WGS) and Exome sequencing (WES) on 6 pairs human HCC samples. β2SP mutant mice were treated with alcohol to determine their susceptibility to aldehyde-induced developmental abnormalities. Primary MEFs from β2SP+/+, β2SP+/− and β2SP−/− mice were treated with colcemid for cytogenetic analysis, and they were also treated with multiple DNA damaging agents and γ-irradiation to evaluate Mdc1, NBS1 and Rad51 foci formation. ChIP assays were performed to determine the recruitment of β2SP/Smad3/Smad4 at FancD2 promoter.

Results: (1) Through our WGS and WES analyses, we discovered a novel inactivating mutation of TGF-β/β2SP pathway in alcohol-associated HCC. This β2SP mutation results in functional disruption of TGF-β signaling. (2) Alcohol treatment in the absence of β2SP mice results in teratogenicity and spontaneous fetal alcohol-like phenotype (FAS). (3) Loss of β2SP results in premature replicative senescence and marked hypersensitivity to DNA interstrand crosslinking agents, such as mitomycin C (MMC). (4) DNA damage response induces nuclear localization of β2SP in a TGF-β-dependent manner. (5) Suppression of β2SP impairs the recruitment of DNA repair proteins in the nucleus and consequently the repair of DNA double-strand breaks. (6) Loss of β2SP correlates with loss of FANCD2 expression, but not of any of the other 12 Fanconi anemia complementation factors. Moreover, our results indicate that β2SP/Smad3/Smad4 regulate expression of FANCD2 at the transcriptional level. (7) The expression of β2SP and FANCD2 is correlated in alcoholic hepatitis and cirrhotic livers.

Conclusions: Through whole genome and Exome sequencing we found a novel inactivating mutation in β2SP which results in loss of TGF-β signaling pathway. Importantly, these results could potentially lead to new therapeutics targeting toxin-induced DNA damage and tumorigenesis.

Citation Format: Vivek Shukla, Jian Chen, Jiun-Sheng Chen, Ying Li, Lior Katz, Avijit Majumdar, Lei Li, Walter Hittleman, Xiaoping Su, Junjie Chen, Xifeng Wu, Patrizia Farci, Lopa Mishra. High resolution characterization of human hepatocellular cancer (HCC) reveals a novel inactivating mutation in the TGF-β pathway that promotes alcohol induced HCC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1775. doi:10.1158/1538-7445.AM2013-1775