Systematic analysis of TGF-β/Smad signaling and (loss of) cytostatic outcome in human hepatocellular carcinoma cell lines

Abstract

Primary liver cancer, of which hepatocellular carcinomas (HCCs) accounts for 90 %, is the fifth most common and third deadliest cancer worldwide. As HCCs are strongly heterogenic and with symptoms occurring late, the development of efficient treatment strategies is until now challenging. Transforming growth factor beta (TGF-β) is a multifactorial cytokine and a driver of chronic liver disease progression, which eventually ends in HCC. TGF-β, which acts as a tumor suppressor during cancer development, may switch to tumor-supporting characteristics during cancer progression. The ambiguous nature of TGF-β, together with the strong heterogeneity of HCC, leads to a highly complex situation in regard to TGF-β signaling during hepatocarcinogenesis. As TGF-β signaling is frequently altered in HCC, it is an interesting target to develop new therapeutic strategies. However, this requires deep knowledge about the time point and underlying mechanisms of the switch in TGF-β signaling. This question was addressed by a systematic in-depth analysis of cytostatic effects of TGF-β and mechanisms of resistance in ten commonly used liver cancer cell lines. TGF-β induced cell death or growth arrest in one group of cell lines (PLC/PRF/5, Hep3B, HuH7 and HepG2) in a Smad3 dependent manner. These cells commonly expressed relatively low basal levels of TGF-β1 and its inhibitor Smad7, but elevated levels of TβRII (TGF-β receptor II). Furthermore, they exhibited a high inducibility of Smad3 and TβRI expression, Smad3 transcriptional activity and target gene expression, as exemplified for Bim, PAI-1 and Smad7. Interestingly, all cell lines have previously been described to express an early TGF-β-response gene signature, which was also identified in HCC patients and correlates with a better prognosis. In contrast, the second group (HLE, HLF, FLC-4 and to some extent HuH6 cells) was resistant against cytostatic effects of TGF β and further characterized by high Smad7 and TGF-β1, low TβRII expression and low TGF-β induced Smad3 transcriptional activity. In addition, all but HuH6 cells had a highly motile phenotype. HCC-M and HCC-T cells shared characteristics of both groups, showing, e.g., resistance against TGF-β induced cytostasis but low TGF-β1, Smad7 and TβRII levels. Finally, analysis of patient samples showed an overexpression of Smad7 in 59 % of HCCs with a significant correlation to tumor size in a HBV related subgroup from China. In conclusion, HCC cell lines can be allocated into two main groups regarding the cytostatic TGF-β response, which is accompanied by distinct differences in basal and TGF-β induced expression patterns of TGF-β signaling components and target genes. Smad7 upregulation seems to be an important mechanism in transformed hepatocytes to establish insensitivity against TGF-β induced cytostasis, and once this is accomplished, a more malignant phenotype may develop.