Abstract 1984: Crosstalk between sirtuins and TGF-β signaling suppresses liver cancer

Abstract

Abstract Background: Cancer stem cells that escape suppression by key pathways such as TGF-β represent one of the mechanisms for poor prognosis in hepatocellular cancer (HCC). Our mouse models with disruption of TGF-β signaling (β2SP+/-/Smad3+/-) develop HCC and phenocopy an epigenetically human cancer stem cell disorder, providing clues towards the role of TGF-β in the switch in stem cell transformation to HCC. An examination of HCC genomics revealed a surprising and strong correlation between TGF-β and Sirtuins (SIRTs) expression. SIRTs are also implicated in cancer stem cell and HCC suppression. Here we hypothesized that the TGF-β pathway harnesses Sirtuin members to maintain stem cell homeostasis, which may mediate the progression of HCC. Methods: Overall Survival (OS) analysis was carried out by using early-stage HCC (TCGA, Firehose Legacy, stage N1, n=186) from cBioportal. ChIP assay was performed in HepG2 cells using anti-Smad3 and normal rabbit IgG. Biotinylated SIRT6 promoter with/without SMAD binding sites was used as probes to pull down the DNA-interacting proteins in HepG2 cells treated with/without TGF-β for 2h. Results: Analysis of TCGA HCC dataset (cBioportal, stage N1, n=186) indicated reduced OS for HCC patients with decreased SIRT6 together with loss of SPTBN1 in the early stage (Median survival months: 40.37 vs 83.18, p=0.0417). • Chip-qPCR and the DNA-protein pulldown assay revealed that Smad3 positively upregulates the SIRT6 mRNA expression (Smad3 vs IgG, SBE1: 3.27±0.53 vs 0.99±0.01, p<0.05; SBE2: 3.32±0.74 vs 1.12±0.3, p<0.05) through direct binding to the SIRT6 promoter in HepG2 cells after TGF-β treatment. • Co-IP analysis revealed directly binding of SIRT6 with β2sp and Smad3. • Overexpression of SIRT6 represses Smad3 and β2sp expression (p<0.01) and results in inhibition of the TGF-β target genes (p<0.05) such as MMP9 (68.2% inhibited), HMGA2 (53.4% inhibited), and PAI-1 (35.4% inhibited); whereas knockdown of SIRT6 increases β2sp expression in HepG2 cells (3 fold, p<0.05). • Similar to mice with disruption of SIRT6 (SIRT6-/-) that develop liver inflammation, steatosis with progression to tumorigenesis, mice with TGF-β signaling defect (Smad3+/-Sptbn1+/-) also develop fatty liver with progression to tumorigenesis under HFD. Interestingly, we found that SIRT6 was significantly downregulated in MEF cells from mice with loss of TGF-β signaling (Smad3+/-Sptbn1+/-). Conclusions: Our findings suggest that the TGF-β signaling participates in positively regulating the expression of SIRT6. In turn, SIRT6 acts as a negative regulator of TGF-β signaling through de-acetylating Smad3 and β2sp. SIRT6 and TGF-β signaling exert integrative yet complex roles in inhibiting hepatic steatosis, inflammation, and HCC that when disrupted lead to poor prognosis in HCC. The data provide new combined driving roles for the two pathways in suppressing NASH and HCC. Citation Format: Xiyan Xiang, Sobia Zaidi, Shuyun Rao, Kazufumi Ohshiro, Zhanhuai Wang, Wilma Jogunoori, Chuxia Deng, Patricia Latham, Lopa Mishra. Crosstalk between sirtuins and TGF-β signaling suppresses liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1984.