The Tgf-Beta Effector Beta 2sp Depletion Abrogates Dna Damage Repair

Objective: Exposure to genotoxins, such as ethanol-derived acetaldehyde, leads to DNA damage, liver injury, and promotes the development of cancer. Alcohol-related genotoxicity, arising from DNA damage by metabolically generated reactive aldehydes, has recently been observed in models with genetic inactivation of members of the Fanconi anemia pathway. However, sensors for genotoxicity leading to aberrant DNA repair remain elusive. Transforming growth factor β (TGF-β) is a critical protein in the regulation of several cancer phenotypes and also functions as an extracellular sensor of ionizing radiation-induced cell damage. Yet, how the TGF-β pathway contributes to toxin-induced DNA damage repair remains unclear. We utilized the TGF-β/β2SP mutant mouse model to investigate the mechanisms in relation to β2SP-mediated TGF-β modulation of the Fanconi anemia pathway for DNA damage repair, alcohol sensitivity, and liver tumorigenesis. Methods: (1) β2SP mutant mice were treated with alcohol to determine their susceptibility to aldehyde-induced developmental abnormalities. (2) Genomic instability and sensitivity to DNA damaging agents in primary β2SP+/+, β2SP-/- MEFs were determined by clonogenic survival and metaphase chromosome aberrations analysis. (3) Defective S-phase specific DNA repair in β2SP-/- MEFs were determined by DNA replication restart assays. (4) ChIP assays were performed to determine the recruitment of β2SP/Smad3 at FancD2 promoter. (5) We investigated the clinical relevance of altered β2SP and FancD2 function using immunohistochemical analyses of 20 human liver specimens from alcoholic hepatitis (n = 5), alcoholic cirrhosis (n = 5), and alcohol-associated liver cancer (n = 5), as well as normal controls (n = 5). Results: (1) Sptbn1-deficient mice exhibit a phenotype similar to human fetal alcohol syndrome and are sensitive to ethanol exposure. (2) Sptbn1-deficient cells exhibit genomic instability and hypersensitivity to DNA damage (3) Sptbn1-deficiency delays DNA damage repair. (4) Furthermore, Sptbn1-deficient cells are defective in stalled DNA replication fork resolution and homologous recombination. (5) FancD2 ectopic expression rescues the DNA repair defect in Sptbn1 null cells. (6) β2SP and FancD2 are clinically correlated in alcoholic hepatitis and HCCs. Conclusions: Our model proposes that in response to liver toxins such as alcohol, the TGF-β/β2SP/Smad3 pathway prevents liver injury and cancer through its direct effects on DNA repair and genomic stability. Thus, characterizing the role of TGF-β in alcohol-induced injury could potentially enhance our mechanistic insight into the basis for therapeutics targeting toxin-induced DNA damage and tumorigenesis. Citation Format: Jian Chen, Vivek Shukla, Jiun-Sheng Chen, Raj K. Panditab, Yun Seong Jeong, Lior H Katz, Ji-Hyun Shin, YoungJin Gi, Lawrence N. Kwongc, Clayton R. Huntb, Patrizia Farci, Xiaoping Su, Jon White, Bibhuti Mishra, Asif Rashid, Milind Javle, Lei Li, Junjie Chen, John R, Stroehlein, Marta Davila, Rehan Akbani, Keigo Machidao, Hidekazu Tsukamoto, Tej K. Pandita, Lopa Mishra. The TGF-β effector β2SP depletion abrogates DNA damage repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3594.