Molecular mechanisms of senescence response to transforming growth factor-beta in liver cancer

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. HCC is associated with several etiological factors including infections with hepatitis B and C viruses, heavy alcohol consumption and chronic aflatoxin B1 exposure. Due to its multi-step disease hallmark characterized with genetic heterogeneity, liver cancer has very limited therapeutic options. In light of many previous findings, cellular senescence acts as a barrier against immortalization and prohibits the proliferation of premalignant cells in various tumors including HCCs. However, implications of this anti-tumor mechanism in hepatic tissues are not wellknown.

TGF-β is a multifunctional cytokine implicated in diverse cellular processes including senescence arrest as well as liver physiology and pathophysiology. Although TGF-β-induced senescence has been described in different cell types, this issue has never been addressed for hepatic cells. According to our recent data, TGF- β1 expression pattern in various HCC malignancies closely correlated with reported frequencies of SABG activities in these corresponding disease stages. Therefore, we hypothesized that TGF-β signaling might play key role in hepatocellular senescence. Well-differentiated (WD) five cell lines characterized with epithelial-like morphology displayed TGF-β-induced growth inhibition associated with SABG activity, with lack of evidence of apoptosis induction. Even a brief exposure to TGF- β was sufficient to trigger a massive senescence response. Senescence arrest in WD cell lines was linked to c-myc down-regulation and a reciprocal increase in p21Cip1 and p15Ink4b protein levels. In addition, TGF-β-induced senescence was correlated with Nox4 induction, intracellular accumulation of reactive oxygen species (ROS) and sustained 53BP1 foci formation as a mark of DNA-damage response. Moreover, intratumoral injection of TGF-β in human HCC tumors, generated subcutaneously in immunodeficient mice, induced expanded SABG that was associated with a strong anti-tumor response activity. On the other hand, poorly differentiated (PD) HCC cell lines with mesenchymal-like characteristics appeared to be resistant to TGF−β-induced senescence. However, PD cell lines had intact TGF-β signaling from cell membrane to nucleus. Resistance of PD cell lines was partially due to zeb2 overexpression, homozygous p15Ink4b deletion and lack of pRb expression. Besides, PD cells did not display Nox4 upregulation and also lacked ROS accumulation upon TGF-β stimulation. In addition, we demonstrated that sustained exposure to TGF-β established resistant Huh7 subclone. The resistance was partially attributed to deregulated Smad signaling, permanent epithelial-mesenchymal transition-like transformation. Surprisingly enough, removal of TGF-β from culture medium of continuously treated Huh7 subclone did not resolve the resistance phenotype in the rescued subclone. Epigenetic regulations mainly histone modifications are considered as candidate mechanisms responsible for irreversible TGF-β-resistance and maintenance of mesenchymal-like phenotype. Taken together, our results establish a close link between senescence arrest and anti-tumor activity of TGF-β signaling pathway in WD cell lines by delineating the mechanisms underlying TGF-β-induced growth arrest. Moreover, we propose partial explanation for the resistance to TGF-β- mediated growth arrest in PD cell lines and thoroughly signify the potential mechanisms of acquired resistance to TGF-β in continuously treated cultures. Further studies to enlighten our knowledge about implications of TGF-β signaling in less differentiated HCCs are necessary. As a conclusion, we identify TGF-β signaling as a potent therapeutic option for well-differentiated early HCCs.