Dual inhibition of Akt and c-Met as a second-line therapy following acquired resistance to sorafenib in hepatocellular carcinoma cells

Abstract

Sorafenib displays a limited efficacy for advanced hepatocellular carcinoma (HCC). Some patients with HCC initially respond to sorafenib, but eventually succumb to the disease, indicating that the acquired resistance to sorafenib reduces its beneficial effects. No alternative drugs are available after the failure of sorafenib therapy. Therefore, investigation of the mechanisms underlying the acquired resistance and development of second-line treatments for sorafenib-resistant HCC are urgently required. In this study, sorafenib-resistant HCC cells generated from sorafenib-sensitive human HCC cells were shown to overproduce hepatocyte growth factor (HGF) and overexpress c-Met kinase and its phosphorylated form, leading to the activation of Akt and ERK (extracellular signaling-regulated kinase) pathways. Use of specific c-Met inhibitors enhanced the effects of sorafenib by inhibiting the growth of sorafenib-resistant HCC cells. Akt inhibitors, a class of second-line therapeutic drugs under investigation for treating HCC in clinical trials, enhanced the effects of sorafenib, but also activated the c-Met pathway in sorafenib-resistant cells. Dual inhibition of Akt and c-Met by their respective inhibitors, MK2206 and capmatinib, additively or synergistically suppressed sorafenib-resistant HCC cells in vitro and sorafenib-resistant HCC xenografts in mice. The anticancer activities of MK2206 mainly rely on its ability to induce cell apoptosis and autophagic death, while capmatinib treatment leads to cell cycle arrest at phase G1. These results provide strong evidence for further investigation on the clinical utility of dual inhibition of Akt and c-Met, particularly MK2206 and capmatinib, as a second-line therapy for advanced HCC that has acquired resistance to sorafenib.

Keywords: Akt; acquired resistance; c-Met; cellular signaling pathway; hepatocellular carcinoma; sorafenib.

Figure 1

Sorafenib‐resistant HCC cells express higher levels of c‐Met. Huh7, Huh7‐SR, HepG2, and HepG2‐SR cells were cultured for 48 h and harvested for analysis. (A) The hierarchical clustering analysis of differentially expressed genes was performed by using a Human liver cancer RT² Profiler™ PCR Array on Huh7 and Huh7‐SR cells. (B) The expression of c‐Met mRNA was measured by qRT‐PCR. The level of mRNA from parental cells was defined as 1. (C) PCR products (364 bp) generated from RT‐PCR for detecting c‐Met mRNA underwent a 2% agarose gel electrophoresis. (D) The above cells were immunoblotted. The density of each band was normalized to β‐actin. ‘*’ (P < 0.05) and ‘**’ (P < 0.001) indicate a significant difference.

Figure 2

Exposure to sorafenib activates the HGF/c‐Met and PTEN/Akt pathways in HCC cells. (A) Cellular expression of HGF was detected by immunofluorescence microscopy. HGF protein was stained by an anti‐HGF antibody (green), and the cell nuclei were stained blue by DAPI. (B–D) Cells were incubated in the absence or presence of sorafenib (2.5 μm) for 48 h. (B) The expression of HGF mRNA was measured by qRT‐PCR. The level of mRNA from untreated parental cells was defined as 1. (C) The levels of HGF protein in the culture media were measured by ELISA. (D) The above cells were subjected to immunoblotting. (E, F) MHCC‐7721 and MHCC‐3M cells were incubated in the absence or presence of sorafenib (2.5 μm) and harvested at indicated time points. (E) The levels of HGF protein in the culture media were measured by ELISA. (F) Cells harvested at 48 h were immunoblotted. ‘**’ (P < 0.001) indicates a significant difference. ‘ϕ’ (P < 0.05) and ‘ϕϕ’ (P < 0.001) indicate a significant difference from respective untreated cells.

Figure 3

Inhibition of c‐Met by capmatinib enhances the sensitivity of sorafenib‐resistant HCC cells to sorafenib. (A, B) Huh7‐SR and HepG2‐SR cells were incubated for 48 h with various concentrations of capmatinib and subjected to immunoblotting (A) or cell viability assays (B). (C) Cells were incubated for 48 h with various concentrations of capmatinib in the presence or absence of sorafenib (5 μm). (D) Huh7‐SR cells were incubated with sorafenib (5 μm), capmatinib (2 nm), or the combination, and harvested at indicated time points. (B–D) Cell viability (%) was normalized to the respective untreated cells. (E–G) Huh7‐SR cells were incubated for 48 h with sorafenib (5 μm), capmatinib (2 nm), or the combination, and subjected to cytometry for measuring cell apoptosis (%) (E, F), or to immunoblotting analysis (G). The density of each band was normalized to β‐actin. ‘*’ (P < 0.05) and ‘**’ (P < 0.001) indicate a significant difference. ‘†’ (P < 0.05) and ‘††’ (P < 0.001) vs. sorafenib alone; ‘ϕ’ (P < 0.05) and ‘ϕϕ’ (P < 0.001) vs. capmatinib alone. ‘#’ (P < 0.05) and ‘##’ (P < 0.001) indicate a significant increase, while ‘∇’ (P < 0.05) and ‘∇∇’ (P < 0.001), a significant reduction, versus controls.

Figure 4

Inhibition of Akt suppresses sorafenib‐resistant HCC cells and activates the c‐Met pathway. (A) Huh7‐SR and HepG2‐SR cells were incubated for 48 h with various concentrations of MK2206 in the presence or absence of sorafenib (5 μm). Cell viability (%) was normalized to the respective untreated cells. (B) Cells were incubated for 48 h with sorafenib (5 μm), MK2206 (1 μm), or the combination. Untreated cells served as controls. Cell apoptosis (%) was measured by cytometry. (C) Huh7‐SR cells were incubated in media containing MK2206 (0.5 μm) or GDC0068 (5 μm), which were refreshed every 24 h. Cell viability (%) was measured at indicated time points and normalized to untreated cells. (D) Cells treated with MK2206 in (C) were immunoblotted. (E) Huh7‐SR cells were transfected with scrambled siRNA or Akt‐siRNA for 48 h and then subjected to immunoblotting. ‘*’ (P < 0.05) and ‘**’ (P < 0.001) indicate a significant difference. ‘††’ (P < 0.001) vs. sorafenib alone; ‘ϕϕ’ (P < 0.001) vs. MK2206 alone. ‘##’ (P < 0.001) indicates a significant increase, while ‘∇’ (P < 0.05) and ‘∇∇’ (P < 0.001), a significant reduction, versus controls.

Figure 5

Dual inhibition of Akt and c‐Met induces apoptosis and autophagy of sorafenib‐resistant HCC cells. Huh7‐SR and HepG2‐SR cells were incubated for 48 h with capmatinib (2 nm), or MK2206 (1 μm), or the combination. (A, B) Cell apoptosis (%) was measured by cytometry. (C) Representative images were taken from cells stained with annexin V/propidium iodide (magnification ×100). (D) Representative images were taken from acridine orange‐stained cells (magnification ×400). (E) The fold change of acridine orange fluorescence intensity (FL3) versus untreated controls was calculated. The FL3 in untreated controls was defined as 1. (F) Lysates of the above Huh7‐SR cells were immunoblotted. ‘#’ (P < 0.05) and ‘##’ (P < 0.001) indicate a significant increase from controls. ‘ϕϕ’ (P < 0.001) vs. capmatinib alone; ‘†’ (P < 0.05) and ‘††’ (P < 0.001) vs. MK2206 alone.

Figure 6

Dual inhibition of Akt and c‐Met as a second‐line therapy suppresses sorafenib‐resistant HCC tumors. (A) Animal experimental schedule was described in Materials and Methods. (B) The size (mm³) of tumors was recorded. Tumors harvested at the end of experiments were weighed (C) and photographed (D). (E) The bodyweights of mice were monitored ‘**’ (P < 0.001) indicates a significant difference.

Figure 7

Proposed molecular mechanisms by which dual inhibition suppresses sorafenib‐resistant HCC cells by targeting the Akt and c‐Met pathways. ‘→’ indicates positive regulation or activation; ‘⊥’, negative regulation or blockade. Solid lines ‘____’ indicate mechanisms examined in the present study, while dotted lines ‘‐‐‐‐‐‐’, mechanisms in previous studies (He et al., 2015; Llovet et al., 2008; Zhai et al., 2014). Abbreviations and explanations: c‐Kit, also called stem cell growth factor receptor or CD117; c‐Met, also called hepatocyte growth factor receptor [HGFR]; ERK, extracellular signaling‐regulated kinase; GSK‐3β, glycogen synthase kinase‐3β; HGF, hepatocyte growth factor; LC3, microtubule‐associated protein 1 light chain 3; mTOR, mammalian target of rapamycin; PDGF, platelet‐derived growth factor; PDGFR, platelet‐derived growth factor receptor; PTEN, phosphatase and tensin homolog; SCF, stem cell factor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.