New mechanism of transforming growth factor-β signaling in hepatoma: Dramatic up-regulation of tumor initiating cells and epidermal growth factor receptor expression

Transforming growth factor-beta (TGF-beta) has dual activity in tumor cells. We studied the effect of TGF-beta on tumor-initiating cells (TICs), which are similar in self-renewal and differentiation features to normal adult stem cells. We used side population (SP) cells that exclude DNA binding dye Hoechst 33342 to obtain TICs, studied the differences in the kinetics of the SP cell response to TGF-beta treatment between hepatic tumor cell lines, and performed gene analysis. SP cells from all cell lines have higher proliferative ability compared to non-SP cells and they are drug resistant. TGF-beta treatment increased the percentage of SP cells (%SP) and the survival rate; chemotherapeutic drug resistance developed only in K-251 SP cells. Gene analysis showed that TGF-beta up-regulated epidermal growth factor receptor (EGFR) only in K-251 cells. There were no EGFR mutations in K-251, which had been reported in lung cancer. Knockdown of Smad4 using the small-interfering RNA technique in K-251 cells inhibited EGFR overexpression and significantly decreased the %SP. In contrast, the JNK inhibitor had little effect on EGFR expression or the %SP. TGF-beta treatment of K-251 cells causes tumor progression and the anti-cancer drug resistant phenotype by increasing SP.