TGFbeta overrides HNF4alpha tumor suppressing activity through GSK3beta inactivation: implication for gene therapy of HCC

The loss of transcriptional factor HNF4α is a predominant mechanism through which hepatocellular carcinoma (HCC) progress to a less differentiated and more aggressive phenotype and, therefore, HNF4α has been suggested as therapeutic tool for HCC gene therapy. The rationale of the proposed therapeutic strategy has been supported by several experimental data showing that its re-expression reduced tumor growth/invasivity in vitro and tumor formation in mice. Furthermore, we recently showed HNF4α expression in low-expressing HCC cell lines can directly hamper the epithelial-to-mesenchymal transition (EMT) program, through the transcriptional repression of EMT master genes, Snail and Slug (1).The tumor progression final output, however, comes not only from cell autonomous properties, acquired through mutations, but also from microenvironmental cues influencing tumor niche. The transforming growth factor β (TGFβ) is a major microenvironmental factor for HCC influencing tumor dedifferentiation, induction of EMT and acquisition of metastatic properties. Its pathway was found constitutively activated in HCC patients and correlated with a poor prognosis. In the attempt to investigate the influence of TGFβ on the anti-EMT and tumor suppressor HNF4α activity, we demonstrated that the presence of TGFβ impairs the efficiency of HNF4α as tumor suppressor. We found that TGFβ induces HNF4α post-translational modifications (PTMs) that correlate with the early loss of HNF4α DNA binding activity on target gene promoters. Furthermore, we identified the GSK3β kinase as one of the TGFβ targets mediating HNF4α functional inactivation and PTM. The GSK3β chemical inhibition results, indeed, in HNF4α DNA binding impairment and correlates with its dephosphorylation while a constitutively active GSK3β mutant significantly impairs the TGFβ-induced inhibitory effect on HNF4α transcriptional and tumor suppressive activity. Overall, our data indicate that the presence of TGFβ represents a limit for the HNF4α-mediated gene therapy of HCC and suggest the need to develop a TGFβ-insensitive HNF4α protein in order to circumvent the influence of tumor microenvironment.