MTOR inhibition reduces growth and adhesion of hepatocellular carcinoma cells invitro.

Abstract

Mechanistic target of rapamycin (mTOR) signaling is typically increased in hepatocellular carcinoma (HCC). A panel of HCC cell lines (HepG2, Hep3B and HuH6) was exposed to various concentrations of the mTOR inhibitors, everolimus and temsirolimus, in order to investigate their effects on cell growth, clonal formation, cell cycle progression, and adhesion and chemotactic migration using MTT and clonal cell growth assays, fluorometric detection of cell cycle phases and a Boyden chamber assay. In addition, integrinαandβ adhesion receptors were analyzed by flow cytometry and blocking studies using function blocking monoclonal antibodies were conducted to explore functional relevance. The results demonstrated that everolimus and temsirolimus significantly suppressed HCC cell growth and clonal formation, at 0.1 or 1nM (depending on the cell line). In addition, the number of cells in G0/G1 phase was increased in response to drug treatment, whereas the number of G2/M phase cells was decreased. Drug treatment also considerably suppressed HCC cell adhesion to immobilized collagen. Integrin profiling revealed strong expression of integrin α1, α2, α6 and β1 subtypes; and integrin α1 was upregulated in response to mTOR inhibition. Suppression of integrin α1 did not affect cell growth; however, it did significantly decrease adhesion and chemotaxis, with the influence on adhesion being greater than that on motility. Due to a positive association between integrin α1 expression and the extent of adhesion, whereby reduced receptor expression was correlated to decreased cell adhesion, it may be hypothesized that the adhesion‑blocking effects of mTOR inhibitors are not associated with mechanical contact inhibition of the α1 receptor but with integrin α1‑dependent suppression of oncogenic signaling, thus preventing tumor cell‑matrix interaction.