TGF-β1/SMAD signaling downregulates KLF4 expression via direct transcriptional suppression and FAT10-mediated proteasomal degradation in hepatocellular carcinoma.

backgroundLoss of tumor suppressor Krüpple-like factor 4 (KLF4) expression in hepatocellular carcinoma (HCC) is crucial for aggressive tumor progression and poor patient prognosis. However, the mechanisms underlying KLF4 loss in human HCC are poorly characterized. Here, we explore the mechanisms by which canonical TGF-β1 signaling suppresses KLF4 in HCC.

methodsThe expression and correlation of KLF4, FAT10 and phospho-SMAD2/3 (pSMAD2/3) were evaluated by using tissue microarray and immunohistochemistry. Cellular and molecular impacts of TGF-β1 signaling on FAT10 and KLF4 expression as well as the interaction between FAT10 and KLF4 were determined using western blot, quantitative real-time PCR, chromatin immunoprecipitation, reporter promoter assay, co-immunoprecipitation and immunofluorescence.

resultsWe found that elevated p-SMAD2 and p-SMAD3 expression correlated with increased FAT10 but decreased KLF4 expression in primary HCC. High p-SMAD2 and p-SMAD3 expression also associated with reduced overall and recurrence-free survival after surgery in HCC. Activation of TGF-β1 signaling in HCC cells transcriptionally upregulated FAT10, but downregulated KLF4 expression. Mechanistically, activation of TGF-β1 signaling enhanced the direct binding of SMAD2/3 to FAT10 and KLF4 promoter. Furthermore, FAT10 directly interacted with KLF4 protein, and deletion of the C-terminal diglycine motif of FAT10 or the Zinc-finger domain of KLF4 abrogated the interaction. Moreover, FAT10 promoted KLF4 proteasomal degradation in a ubiquitin-independent manner in HCC cells.

conclusionsOur data suggest that active TGF-β1/SMAD signaling contributes to the loss of KLF4 expression in human HCC through FAT10-mediated ubiquitin-independent proteasomal degradation and direct transcriptional suppression.