Activation of phosphatidylinositol 3-kinase/AKT/snail signaling pathway contributes to epithelial-mesenchymal transition-induced multi-drug resistance to sorafenib in hepatocellular carcinoma cells

Abstract

Sorafenib, an orally available kinase inhibitor, is the standard first-line systemic drug for advanced hepatocellular carcinoma (HCC), and it exerts potent inhibitory activity against epithelial-mesenchymal transition (EMT) and multidrug resistance (MDR) by inhibiting mitogen-activated protein kinase (MAPK) signaling in HCC. However, after long-term exposure to sorafenib, HCC cells exhibit EMT and resistance to sorafenib. The activation of AKT by sorafenib is thought to be responsible for the development of these characteristics. The present study aims to examine the underlying mechanism and seek potential strategies to reverse this resistance and the progression to EMT. Sorafenib-resistant cells showed increased metastatic and invasive ability, with a higher expression of P-glycoprotein (P-gp), compared with the parental cells. This phenomenon was at least partially due to EMT and the appearance of MDR in sorafenib-resistant HCC cells. Moreover, MDR was a downstream molecular event of EMT. Silencing Snail with siRNA blocked EMT and partially reversed the MDR, thereby markedly abolishing invasion and metastasis in sorafenib-resistant HCC cells, but silencing of MDR1 had no effect on the EMT phenotype. Additionally, HCC parental cells that were stably transfected with pCDNA3.1-Snail exhibited EMT and MDR. Two sorafenib-resistant HCC cell lines, established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition but were sensitive to MK-2206, a novel allosteric AKT inhibitor. Thus, the combination of sorafenib and MK-2206 led to significant reversion of the EMT phenotype and P-gp-mediated MDR by downregulating phosphorylated AKT. These findings underscore the significance of EMT, MDR and enhanced PI3K/AKT signaling in sorafenib-resistant HCC cells.

Fig 1. EMT and MDR appears in sorafenib-resistant HCC cells.

A, The sorafenib-resistant HepG2-SR and Huh7-SR cells and parental HepG2 and Huh7 cells were incubated with increasing concentrations of sorafenib for 48 hours. Cell viability (%) was assessed and compared with the corresponding untreated cells. B, Morphological changes were evaluated in and sorafenib-resistant cells under ×400 magnification. C, Migration of HCC cells was measured by with a Transwell assay. D, Invasion of HCC cells was also measured with a Transwell assay. E, EMT- and MDR-related proteins, including E-cadherin, Vimentin, MMP-9, Snail and P-gp, were assessed by western blotting in parental and resistant cells. β-actin was used as a loading control in the western blot analysis. Data represent three independent experiments. ** indicates P<0.001.

Fig 2. Silencing of Snail blocked EMT and reversed the drug resistance in sorafenib-resistant HCC cells.

A, The sorafenib-resistant HepG2-SR and Huh7-SR cells were transfected with Snail siRNA and MDR siRNA, and cells were then incubated with increasing concentrations of sorafenib for 48 hours. Cell viability (%) was assessed and compared with the corresponding untransfected cells. B, Knockdown of snail abolished the metastatic potential of HepG2-SR cells. C, Knockdown of snail abolished the invasive ability of HepG2-SR cells. D, HepG2-SR cells were transfected with control or Snail and/or MDR1 siRNA for 24 hours, and the protein levels of EMT and MDR markers were detected by western blot. β-actin served as the loading control in the western blot analysis. Data represent three independent experiments. N.S., not significant. * indicates P<0.05, and ** indicates P<0.001.

Fig 3. HCC parental cells stably transfected with pCDNA3.1-Snail exhibited EMT and MDR.

A, Untransfected HepG2 and Huh7 cells or cells transfected with control or pCDNA3.1-Snail were incubated with increasing concentrations of sorafenib for 48 hours. Cell viability (%) was assessed and compared with the corresponding control cells. B, High expression of Snail upgraded the metastatic ability of HCC cells. C, High expression of Snail upgraded the invasion potential of HCC cells. D, The protein expression profiles of untransfected HepG2 and Huh7 cells or cells transfected with control or pCDNA3.1-Snail were detected by western blot analysis. β-actin served as the loading control in the western blot analysis. E, Cells from (D) were subjected to real-time PCR to detect the expression of Snail and the multidrug resistance protein P-gp; the results were normalized to β-actin. F, Subcutaneous tumors formed from Huh7, Huh7-SR and Huh7/Snail cells were established in mice, which received 30 mg/kg sorafenib for 15 days as described in MATERIALS AND METHODS. The sizes (mm³) of tumors were recorded. Data represent three independent experiments. N.S., not significant. * indicates P<0.05, and ** indicates P<0.001.

Fig 4. Activation of the PI3K/AKT signaling pathway is associated with the EMT phenotype and P-gp-mediated multidrug resistance in sorafenib-resistant HCC cells.

A, The sorafenib-resistant cells and parental cells were incubated with 0 or 10 μmol/L sorafenib for 48 hours. The protein expression profiles were detected by western blot analysis; β-actin served as the loading control. B, HepG2-SR cells were exposed for 48 hours to different concentrations of MK-2206 or 10 μmol/L MK-2206 for different periods of time in the presence or absence of sorafenib (10 μmol/L). Cell viability (%) was assessed and compared with the corresponding untreated cells. C, HepG2-SR cells were incubated with 10 μmol/L MK-2206 and/or 10 μmol/L sorafenib for 48 hours. The protein expression profiles were detected by western blot analysis; β-actin served as the loading control. D, HepG2-SR or HepG2-SR/Snail cells were incubated with 0 or 10 μmol/L MK-2206 for 48 hours. The protein expression profiles were detected by western blot analysis; β-actin served as the loading control. The data represent three independent experiments. N.S., not significant. * indicates P<0.05, and ** P<0.001. # indicates P<0.05 and ## indicates P<0.001 vs. untreated control; ‡ indicates P <0.001 vs. sorafenib alone.