ERK Dephosphorylation through MKP1 Deacetylation by SIRT1 Attenuates RAS-Driven Tumorigenesis

Abstract

The role of Situin 1 (SIRT1) in tumorigenesis is still controversial due to its wide range of substrates, including both oncoproteins and tumor suppressors. A recent study has demonstrated that SIRT1 interferes in the Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven activation of the Raf-mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway, thereby inhibiting tumorigenesis. However, the molecular mechanism of SIRT1 as a tumor suppressor in RAS-driven tumorigenesis has been less clearly determined. This study presents evidence that the ectopic expression of SIRT1 attenuates RAS- or MEK-driven ERK activation and reduces cellular proliferation and transformation in vitro. The attenuation of ERK activation by SIRT1 results from prompt dephosphorylation of ERK, while MEK activity remains unchanged. We identified that MKP1, a dual specific phosphatase for MAPK, was deacetylated by SIRT1. Deacetylation of MKP1 by direct interaction with SIRT1 increased the binding affinity to ERK which in turn facilitated inactivation of ERK. Taken together, these results suggest that SIRT1 would act as a tumor suppressor by modulating RAS-driven ERK activity through MKP1 deacetylation.

Keywords: ERK; MKP1; RAS; SIRT1; deacetylation; tumorigenesis.

Figure 1

Protective role of SIRT1 in human cancer. (A–C) The Kaplan–Meier curves showing overall survival (OS) of cancer patients stratified by SIRT1-high and -low groups. Patients were divided into SIRT1-high and -low expression groups, using the median SIRT1 expression as a cutoff. OS stratified by SIRT1-high and -low groups in patients in The Cancer Genome Atlas (TCGA) pan-cancer cohort (A), lung (B) and breast (C) cancers in the Gene Expression Omnibus (GEO) datasets. (D) A bar graph showing the prevalence of Kirsten rat sarcoma viral oncogene homolog (KRAS) alterations across TCGA tumor samples grouped by tumor types. The information on KRAS alterations was obtained from cBioPortal. For visualization, ten cancer types including at least five patients with KRAS mutation at glycine 12 (G12) were shown. The number of patients with KRAS mutations is indicated on the bar graph. (E,F) The Kaplan–Meier curves showing OS stratified by SIRT1-high and -low groups in TCGA pancreatic cancer patients (E) and those carrying KRAS G12 (F).

Figure 2

SIRT1 suppresses tumorigenesis. (A) Gene set enrichment analysis (GSEA) plots showing the enrichment of tumorigenesis-related genes in a ranked list of genes differentially expressed between SIRT1-high and -low groups in TCGA pan-cancer cohort (n = 9435). Tumorigenesis-related gene sets were obtained from MSigDB: ‘CROMER TUMORIGENESIS UP’ (left) and ‘IWANAGA CARCINOGENESIS BY KRAS PTEN DN’ (right). The normalized enrichment score (NES) and nominal p-value were calculated by the GSEA tool. (B) Comparison of growth rates between Ctrl-iRas and Sirt1-iRas cells. The cell proliferation rate was assessed by the JuLI stage at each time point. Representative images (left, original magnification, 40×) and growth curves (right) of Ctrl-iRas and Sirt1-iRas NIH3T3 cells. (C) The soft-agar assays were performed in Ctrl-iRas or Sirt1-iRas NIH3T3 cells with doxycycline (Dox) treatment. Representative images of soft-agar assay (left, original magnification, 40×) and a dot plot represents the number of colonies (right).

Figure 3

SIRT1 impairs RAS-mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK signaling. (A) Immunoblotting (IB) analysis shows phospho-ERK levels in Ctrl-iRas and Sirt1-iRas NIH3T3 cells. The cells were treated with Dox for 24 h at the indicated concentration. α-tubulin, a protein loading control for IB. (B) IB analysis of phospho-ERK in Sirt1-iRas NIH3T3 cells treated with or without nicotinamide (NAM) and Dox for 24 h. (C,D) IB reveals phospho-ERK levels after transfection with HRAS in the 293T cells depending on Sirt1 expression (C) or NAM treatment (D). β-actin and proliferating cell nuclear antigen (PCNA), protein loading controls for IB. (E,F) IB shows phospho-ERK levels after transfection with dominant negative (DN) or constitutively active (CA) MEK1 in the 293T cells depending on Sirt1 expression (E) or NAM treatment (F). ERK2 and PCNA, protein loading controls for IB. The whole western blot images please find in Figure S7.

Figure 4

MAPK phosphatase 1 (MKP1) is responsible for SIRT1-dependent ERK dephosphorylation. (A) IB analysis of MKP1 expression and acetylation in 293T cells. Cells were transiently transfected with indicated vectors and then immunoprecipitation (IP) was performed with an anti-pan-acetyl lysine (acK) antibody, followed by IB with an anti-Myc antibody. IgG, a negative control for IP. (B) IB analysis of acetylation status of MKP1 upon overexpression of p300 and/or SIRT1 in 293T cells. IP was performed with an anti-Myc antibody, followed by IB with an anti-pan acK antibody. (C) IB analysis of phospho-ERK levels depending on SIRT1 expression in the presence or absence of a MKP1/6 inhibitor (E/Z)-BCl Hcl in Dox-exposed Ctrl-iRas (Lane# 1, 3, 5, and 7) and Sirt1-iRas (Lane# 2, 4, 6, and 8) cells. MKP1/6 inhibitor (E/Z)-BCl Hcl and Dox was treated for 3h. ERK2 and α-tubulin, protein loading controls for IB. The whole western blot images please find in Figure S7.

Figure 5

SIRT1 enhances interaction between MKP1 and ERK. (A) CA-RAS-overexpressing 293T cells were transfected with vectors containing Myc-tagged MKP1-CS plus or minus of SIRT1. IP was performed with an anti-Myc antibody, followed by IB with an anti-ERK2 antibody. IgG, a negative control for IP. (B) Co-IP was performed with an endogenous MKP1 in Ctrl-iRas and Sirt1-iRas cells treated with or without Dox for 3 h. (C) CA-MEK1-overexpressing 293T cells were transfected with Myc-tagged MKP1-CS plus or minus SIRT1, then treated with or without NAM. IP was performed with an anti-Myc antibody, followed by IB with an anti-ERK2 antibody. IgG NC, a negative control for IP. The whole western blot images please find in Figure S7.

Figure 6

Prognostic significance of MKP1 with SIRT1 expression. (A–C) The Kaplan–Meier plots showing the OS of cancer patients in TCGA data. The OS stratified by MKP1-high and -low patient groups in a pan-cancer cohort (A); the OS stratified by MKP1-high and -low groups in patients of kidney renal clear cell carcinoma (TCGA KIRC) (B); the OS stratified by the combination of SIRT1-high/-low and MKP1-high/-low groups in patients of TCGA KIRC (C). HR and p-values derived from comparisons among individual groups are placed in Figure S6.