MEK1 signaling promotes self-renewal and tumorigenicity of liver cancer stem cells via maintaining SIRT1 protein stabilization

Abstract

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death. This high mortality has been commonly attributed to the presence of residual cancer stem cells (CSCs). Meanwhile, MEK1 signaling is regarded as a key molecular in HCC maintenance and development. However, nobody has figured out the particular mechanisms that how MEK1 signaling regulates liver CSCs self-renewal. In this study, we show that inhibition or depletion of MEK1 can significantly decrease liver CSCs self-renewal and tumor growth both in vitro and vivo conditions. Furthermore, we demonstrate that MEK1 signaling promotes liver CSCs self-renewal and tumorigenicity by maintaining SIRT1 level. Mechanistically, MEK1 signaling keeps SIRT1 protein stabilization through activating SIRT1 ubiquitination, which inhibits proteasomal degradation. Clinical analysis shows that patients co-expression of MEK1 and SIRT1 are associated with poor survival. Our finding indicates that MEK1-SIRT1 can act as a novel diagnostic biomarker and inhibition of MEK1 may be a viable therapeutic option for targeting liver CSCs treatment.

Keywords: MEK1 signaling; SIRT1; cancer stem cells (CSCs); hepatocellular carcinoma (HCC); proteasome degradation.

Figure 1. MEK1 inhibitor decreases liver CSCs proliferation ability in vitro

(A) Huh7-Nanog^Pos and PLC/PRF/5-Nanog^Pos cells under different U0126 concentrations treatment as indicated (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM) were seeded (1 × 10³) and cultured for another 6 days before analyzed with CCK8. (B) Huh7-Nanog^Pos and PLC/PRF/5-Nanog^Pos cells were cultured with or without 5 μM U0126 for 48 hours. Cells were harvested for immunofluorescence (IF) analysis by anti-Ki67 antibodies. Scale bar, 10 μm. (C) Cell cycle profiles of 5 μM U0126 treated or DMSO treated (negative control) Huh7- and PLC/PRF/5-Nanog^Pos cells followed by treatment with Sodium butyrate. Percentage in each histogram indicates the portion of cells remaining in each cell cycle phase.

Figure 2. MEK1 signaling activity is required for the maintenance of liver CSC self-renewal

(A) Huh7-Nanog^Pos cells were co-cultured with various concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) in sphere-forming conditions for 7 days, counted at the same magnification. (B) Huh7-Nanog^Pos cells were treated with different concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) and grown for 14 days. Cells were stained with crystal violet and counted. (C) Western blot analysis of stemness protein expression in Huh7- and PLC/PRF/5-Nanog^Pos cells, which co-cultured with various concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) for 48 hours. (D) Huh7-Nanog^Pos cells were treated with 5 μM U0126 for 14 days, while the negative control treated with DMSO for 14 days. Then we subcutaneous injected 1 × 10², 1 × 10³, 1 × 10⁴ cells into NOD-SCID mice. After 30 days, we harvested and counted the tumors. Extreme Limiting Dilution Analysis was acquired from http://bioinf.wehi.edu.au/software/elda/.

Figure 3. MEK1 knockdown suppresses liver CSC self-renewal and tumorgenetic capacity

(A) Western blot analysis MEK1 and the substrate ERK1/2 expression in Huh7- and PLC/PRF/5-Nanog^Pos cells which depleted MEK1 with two individual lentiviruses for 48 hours. (B) Effect of MEK1 knockdown on cellular growth rates of Huh7- and PLC/PRF/5- Nanog^Pos cells. CCK8 assay was performed after transfection with indicated times. Cell lysates were obtained from cells transiently transfected with either MEK1 shRNA or negative control shRNA. (C) Huh7- and PLC/PRF/5- Nanog^Pos cells which transfected with MEK1 shRNA or negative control shRNA cultured under non-adhesive culture system for 7 days. (D) Huh7- and PLC/PRF/5-Nanog^Pos cells were transduced with lentiviruses expressing the indicated shRNA. Cells were grown for 14 days and stained with crystal violet. (E) Western blot analysis of stemness-related proteins in MEK1-depleted cells, relative to control.

Figure 4. MEK1 mainly promotes SIRT1 expression in HCC population

(A) Western blot analysis the sirtuins expression level in CSCs and non-CSCs, compared with U0126 inhibited CSCs. (B) Western blot analysis SIRT1 expression in CSCs after co-cultured with indicated concentration of U0126 or PD98059 (D) for 48 hours. (C) Western blot analysis SIRT1 expression in CSCs which cultured with 5 μM U0126 for indicated times. (E) Western blot analysis SIRT1 expression in CSCs transduced with lentiviruses expressing the indicated shRNA for 48 hours. Those experiments were repeated in two HCC cell lines (Huh7 and PLC/PRF/5).

Figure 5. MEK1 maintains liver CSC self-renewal dependent on SIRT1

Non-CSCs (Huh7-Nanog^Neg cells) were prepared with overexpression SIRT1, and grown with 5 μM U0126 or DMSO for 48 hours. Liver CSCs (Huh7-Nanog^Pos cells) co-cultured with 5 μM U0126 for 24 hours previously and overexpression SIRT1 for next 24 hours. Colony analysis of CSCs (C) and non-CSCs (A) which were cultured for 14 days and stained with crystal violet. Sphere analysis of CSCs (D) and non-CSCs (B) which were cultured for 7 days in non-adhesive culture system. All counting were performed in triplicate. (E–F) CSCs, MEK1 inhibition CSCs and SIRT1 overexpression while MEK1 inhibition CSCs were prepared for 14 days, then subcutaneous injected in NOD-SCID mice (CSCs control group 4 mice, other two groups 8 mice each). Tumor sizes were measured with calipers in three dimensions every other day. Tumor volumes were calculated using the formula: tumor volume (cm³) = 0.52 × (W) ² × (L), where L is length and W is width. We counted and weight the tumors, 30 days later.

Figure 6. MEK1 keeps SIRT1 protein stability through proteasomal degradation inhibitory

(A) We co-cultured proteasome inhibition, MEK1 inhibition or knockdown liver CSCs with CHX (10 ng/ml) for indicated times. Western blots analyzed expression of SIRT1. Grey level was measured triplicated independently. (B) Analysis of SIRT1 expression in liver CSCs by western blots. MEK1 deletion or inhibition (U0126, 5 μM) CSCs was cultured for 48 hours, then combined with or without Proteasome inhibitor (MG132, 10 μM) for 8 hours, before harvested. Those proteins were compared with CSCs of DMSO treatment with or without MG132. Grey level was measured and marked. (C) MEK1 inhibition or knockdown CSCs (Huh7- and PLC/PRF/5-Nanog^Pos cells) were treated with 10 μM MG132 for 8 hours before harvest. Total protein extracts from an equivalent number of seedlings were prepared for Co-IP in same conditions and analyzed using immunoblot with the Poly-Ub antibody.

Figure 7. Relationship of p-MEK1/SIRT1 and HCC clinical prognosis

(A) p-MEK1, SIRT1 and Nanog protein expression were detected by IHC analysis in 148 HCC patients, representative images were shown. (B) Analysis correlation between p-MEK1 and SIRT1 expression in 148 HCC patients with Person chi-square test. Correlation analysis of p-MEK1/SIRT1 and Nanog expression in same tissue samples. *P < 0.05 was considered remarkable significant. (C) Kaplan-Meier survival analysis was performed according to p-MEK1^NegSIRT1^Low and p-MEK1^PosSIRT1^High expression of HCC patients. Survival (P < 0.001) of patients who had p-MEK1^PosSIRT1^High expression was shorter.