MSK1 promotes colorectal cancer metastasis by increasing Snail protein stability through USP5-mediated Snail deubiquitination

Abstract

Mitogen- and stress-activated protein kinase 1 (MSK1), a Ser/Thr kinase, phosphorylates nuclear proteins to increase their stability and DNA-binding affinity. Despite the role of MSK1 in promoting cancer progression in colorectal cancer (CRC), the precise molecular mechanisms remain unelucidated. Here we show that MSK1 expression induces the epithelial-mesenchymal transition (EMT) process and increases CRC cell metastasis. Furthermore, we discovered that MSK1 interacts with Snail, a key EMT regulator, and increases its stability by inhibiting ubiquitin-mediated proteasomal degradation. Importantly, MSK1 increased Snail protein stability by promoting deubiquitination rather than inhibiting its ubiquitination. Finally, we identified USP5 as an essential deubiquitinase that binds to Snail protein phosphorylated by MSK1. Based on the experimental data, in CRC, MSK1-Snail-USP5 axis can promote EMT and metastasis of CRC. Together, our findings provide potential biomarkers and novel therapeutic targets for further research in CRC.

Fig. 1. MSK1 promotes CRC cell metastasis by inducing the EMT process.

a The association of MSK1 expression with overall survival in patients with CRC. The Kaplan–Meier plot shows the reduced survival probability of patients with high levels of MSK1 mRNA (n = 90) compared with low MSK1 mRNA levels (n = 87) (GSE 17536). Statistical analysis was performed using log-rank tests. b The association of MSK1 expression with tumor stages in patients with CRC. The box plot shows increased MSK1 mRNA levels in patients with late-stage CRC (stages III and IV, n = 250) compared with patiehts with early-stage CRC (stages I and II, n = 249) (GSE 40967). ****P < 0.0001 as determined by t-test. c The association of MSK1 expression with metastasis in patients with CRC. The box plot shows increased MSK1 mRNA levels in patients with metastasis (n = 28) compared with patients without metastasis (n = 78) (GSE 28814). **P < 0.01 as determined by t-test. d The association of cancer cell types with MSK1 expression. The box plot shows increased MSK1 mRNA levels in mesenchymal cancer cells (n = 59) compared with epithelial cancer cells (n = 119) (GSE 29682). ****P < 0.0001 as determined by t-test. e Expression levels of MSK1 protein in various CRC cell lines (SW480 and Caco2, epithelial; SW620 and HCT116, mesenchymal) analyzed by immunoblotting. f Morphological changes of MSK1-expressing SW480 cells. MSK1-overexpressing SW480 and control cells are visualized by confocal microscopy after staining with tetramethylrhodamine-isothiocyanate (TRITC)-conjugated phalloidin. Scale bars, 20 µm. g Expression levels of EMT marker proteins in MSK1-expressing SW480 and control cells analyzed by immunoblotting. h A heat map showing the relative expression levels of EMT marker genes in patients with CRC with high MSK1 expression and low MSK1 expression. Patients with high or low MSK1 expression were randomly extracted from the microarray dataset (GSE 40967) and analyzed. i The indicated cells were seeded in a six-well plate at a concentration of 1 × 10⁵ cells per well. After incubation for 1–4 days, the viable cells were counted with a hemocytometer after trypan blue staining. j Left: MSK1-overexpressing SW480 and control cells were analyzed in wound-healing assays by visualizing wound closure via phase-contrast microcopy. Wound areas were measured using WimScratch software (Wimasis). Right: the data shown represent the percentage of the wound area and are expressed as the mean ± s.d. of three individual experiments. ****P < 0.0001 as determined by t-test. k MSK1-overexpressing SW480 and control cells were seeded onto Matrigel matrix-coated top chambers, and the fold changes of invading cells were measured after 30 h. The data shown are expressed as the mean ± s.d. of three individual experiments, each performed in triplicate. ****P < 0.0001 as determined by t-test. l About 2 × 10⁶ of the SW480 cells stably overexpressing MSK1 or control cells were injected into the nude mice by tail-vein injection. Left: representative pictures of H&E staining of lung sections. Scale bars, 100 μm. Right: the number of metastatic lung nodules in individual mice was quantified at 10 weeks after tail-vein injection. The data are shown as the mean ± s.d. of five mice per group. **P < 0.01 as determined by t-test.

Fig. 2. MSK1 increases Snail protein stability by suppressing ubiquitination-dependent Snail degradation.

a The interaction between exogenous MSK1 and Snail. HA–Snail and Flag–MSK1 were transfected into HEK293T cells. Cell lysates were immunoprecipitated with an anti-Flag or anti-HA and analyzed by western blot using anti-HA (left) or anti-Flag (right) antibody, respectively. b The interaction between endogenous MSK1 and Snail. SW620 and HCT116 cell lysates were immunoprecipitated with an anti-Snail and analyzed by western blot using anti-MSK1 antibody. c The subcellular localization of MSK1 and Snail. HEK293T cells co-transfected with HA–MSK1 and Flag–Snail were examined by confocal microscopy. Superimposing the three colors (Merge) resulted in pink signals, indicating colocalization of the two proteins in the nucleus. Scale bars, 20 µm. d MSK1 was transfected into SW480 or Caco2 cells. Left: cell lysates were immunoblotted with the indicated antibodies. Right: the data are representative of three independent experiments and relative Snail levels were quantified using ImageJ software. **P < 0.01, ***P < 0.001 as determined by t-test. e Left: immunoblot analysis in MSK1-depleted SW620 or HCT116 cells. Right: the data are representative of three independent experiments, and relative Snail levels were quantified using ImageJ software. **P < 0.01, ***P < 0.001 as determined by t-test. f MSK1 was transfected into SW480 or Caco2 cells in the presence of CHX (100 µg/ml) for the indicated times. Left: cell lysates were immunoblotted by antibodies as indicated. Right: the data were quantified using ImageJ software. For normalization, α-tubulin expression was used as a control. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by t-test. g MSK1-depleted SW620 or HCT116 cells were treated with CHX (100 µg/ml) for the indicated times before collection. Left: cell lysates were immunoblotted by antibodies as indicated. Right: the data were quantified using ImageJ software. For normalization, α-tubulin expression was used as a control. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by t-test. h Left: HA–Snail was co-transfected with Flag–MSK1 into HEK293T cells (top) and then treated with 10 μM MG132 for 12 h (bottom). Cell lysates were immunoblotted with the indicated antibodies. Right: the data are representative of three independent experiments, and relative Snail levels were quantified using ImageJ software. ****P < 0.0001 as determined by t-test. i MSK1 was either overexpressed (left) or inhibited (right), and Myc–Snail was co-transfected with a plasmid expressing HA–ubiquitin as indicated in HEK293T cells, and then the cells were treated with 10 μM MG132 for 6 h. Cell lysates were immunoprecipitated using an anti-Myc antibody and then analyzed by immunoblotting using an anti-HA tag antibody.

Fig. 3. MSK1 kinase activity is essential to increase the stability of Snail protein.

a In vitro kinase assays were performed by incubating recombinant active MSK1 protein with purified MBP-fused Snail in the presence of [γ-³²P]-ATP. The resulting products were subjected to SDS–polyacrylamide gel electrophoresis and autoradiography. b Myc–Snail was co-transfected with Flag–MSK1 into HEK293T cells, and then the cells were treated with 10 μM MG132 for 6 h. Cell lysates were immunoprecipitated using an anti-Myc antibody and then analyzed by immunoblotting using an anti-phospho-Ser (p-Ser) antibody. c MSK1 was transfected into HEK293T cells, and then the cells were treated with 1 μM AZD5363 for 24 h. Left: cell lysates were immunoblotted with the indicated antibodies. Right: the data are representative of three independent experiments and relative Snail levels were quantified using ImageJ software. ***P < 0.001 as determined by t-test. d A schematic diagram showing the functional domains of MSK1 and mutation sites for kinase-dead MSK1 mutant constructs (K455M and D565A). e WT-MSK1, K455M-MSK1 or D565A-MSK1 was transfected into HEK293T cells. Left: cell lysates were immunoblotted with the indicated antibodies. Histone is one of the MSK1 substrate. S.E., short exposure; L.E., long exposure. Right: the data are representative of three independent experiments, and relative Snail levels were quantified using ImageJ software. *P < 0.05, **P < 0.01, ****P < 0.0001 as determined by t-test. f WT-MSK1, K455M-MSK1 or D565A-MSK1 was transfected into HEK293T cells in the presence of CHX (100 µg/ml) for the indicated times. Left: cell lysates were immunoblotted by antibodies as indicated. Right: the data were quantified using ImageJ software. For normalization, α-tubulin expression was used as a control. ***P < 0.001, ****P < 0.0001 as determined by t-test. g Left: HA–Snail was co-transfected with WT-MSK1, K455M-MSK1 or D565A-MSK1 into HEK293T cells (top) and then treated with 10 μM MG132 for 12 h (bottom). Cell lysates were immunoblotted with the indicated antibodies. Right: the data are representative of three independent experiments, and relative Snail levels were quantified using ImageJ software. ****P < 0.0001 as determined by t-test. h WT-MSK1, K455M-MSK1 or D565A-MSK1 was co-transfected with plasmids expressing Myc–Snail and HA–ubiquitin as indicated in HEK293T cells, and then the cells were treated with 10 μM MG132 for 6 h. Cell lysates were immunoprecipitated using an anti-Myc antibody and then analyzed by immunoblotting using an anti-HA tag antibody.

Fig. 4. MSK1 kinase activity is essential to promote CRC cell metastasis.

a Morphological changes of WT-MSK1-, K455M-MSK1- or D565A-MSK1-expressing SW480 cells. MSK1-overexpressing SW480 and control cells visualized by confocal microscopy after staining with TRITC-conjugated phalloidin. Scale bars, 20 µm. b Expression levels of EMT marker proteins in WT-MSK1-, K455M-MSK1- or D565A-MSK1-expressing SW480 and control cells were analyzed by immunoblotting. c The indicated cells were seeded in a six-well plate at a concentration of 1 × 10⁵ cells per well. After incubation for 1–4 days, the viable cells were counted with a hemocytometer after trypan blue staining. d Left: WT-MSK1-, K455M-MSK1- or D565A-MSK1-expressing SW480 and control cells were analyzed in wound-healing assays by visualizing wound closure via phase-contrast microcopy. Wound areas were measured using WimScratch software (Wimasis). Right: the data shown represent the percentage of the wound area and are expressed as the mean ± s.d. of three individual experiments. *P < 0.05, ****P < 0.0001 as determined by t-test. e WT-MSK1-, K455M-MSK1- or D565A-MSK1-expressing SW480 and control cells were seeded onto Matrigel matrix-coated top chambers, and the fold changes of invading cells were measured after 30 h. The data shown are expressed as the mean ± s.d. of three individual experiments, each performed in triplicate. *P < 0.05, **P < 0.01, ****P < 0.0001 as determined by t-test. f About 2 × 10⁶ of the SW480 cells stably overexpressing WT-MSK1, K455M-MSK1, D565A-MSK1 or control cells were injected into the nude mice by tail-vein injection. Left: representative pictures of HE staining of lung sections. Scale bars, 100 μm. Right: the number of metastatic lung nodules in individual mice was quantified at 10 weeks after tail-vein injection. The data are shown as the mean ± s.d. of five mice per group. **P < 0.01 as determined by t-test.

Fig. 5. Snail is essential for MSK1-induced EMT and an increase in CRC cell metastatic ability.

a Morphological changes of Snail-depleted MSK1-overexpressing SW480 cells. Snail-depleted MSK1-overexpressing SW480 and control cells visualized by confocal microscopy after staining with TRITC-conjugated phalloidin. Scale bars, 20 µm. b Expression levels of EMT marker proteins in Snail-depleted MSK1-overexpressing SW480 and control cells were analyzed by immunoblotting. c The indicated cells were seeded in a six-well plate at a concentration of 1 × 10⁵ cells per well. After incubation for 1–4 days, the viable cells were counted with a hemocytometer after trypan blue staining. d Left: Snail-depleted MSK1-overexpressing SW480 and control cells were analyzed in wound-healing assays by visualizing wound closure via phase-contrast microcopy. Wound areas were measured using WimScratch software (Wimasis). Right: the data shown represent the percentage of the wound area and are expressed as the mean ¡± s.d. of three individual experiments. ****P < 0.0001 as determined by t-test. e Snail-depleted MSK1-overexpressing SW480 and control cells were seeded onto Matrigel matrix-coated top chambers, and the fold changes of invading cells were measured after 30 h. The data shown are expressed as the mean ± s.d. of three individual experiments, each performed in triplicate. ****P < 0.0001 as determined by t-test. f About 2 × 10⁶ of the Snail-depleted MSK1-overexpressing SW480 or control cells were injected into the nude mice by tail-vein injection. Left: representative pictures of H&E staining of lung sections. Scale bars, 100 μm. Right: the number of metastatic lung nodules in individual mice was quantified at 10 weeks after tail-vein injection. The data are shown as the mean ± s.d. of five mice per group. *P < 0.05 as determined by t-test. g Left: representative IHC images of phospho-MSK1 and Snail in colorectal tumors. Scale bars, 50 μm. Left: correlation of the Snail expression levels with the phospho-MSK1 levels, as determined using a colorectal tumor TMA. Statistical significances were determined by Pearson’s χ² test and Fisher’s exact test.

Fig. 6. USP5 is essential for the MSK1-induced increase in the stability of Snail protein.

a HA-tagged WT- or L73P-ubiquitin was co-transfected with plasmids expressing Myc–Snail and Flag–MSK1 as indicated in HEK293T cells, and then the cells were treated with 10 μM MG132 for 6 h. Cell lysates were immunoprecipitated using an anti-Myc antibody and then analyzed by immunoblotting using an anti-HA tag antibody. b Left: each of the five DUB-specific siRNAs was co-transfected with MSK1 in HEK293T cells. Cell lysates were immunoblotted with the indicated antibodies. S.E., short exposure; L.E., long exposure. Right: the data are representative of three independent experiments, and relative Snail levels were quantified using ImageJ software. **P < 0.01 as determined by t-test. c Left: USP5-specific siRNA was co-transfected with MSK1 in HEK293T cells. Cell lysates were immunoblotted with the indicated antibodies. Right: the data are representative of three independent experiments, and relative Snail levels were quantified using ImageJ software. ****P < 0.0001 as determined by t-test. d USP5-specific siRNA was co-transfected with MSK1 in SW480 or Caco2 cells. Left: cell lysates were immunoblotted with the indicated antibodies. Right: the data are representative of three independent experiments, and relative Snail levels were quantified using ImageJ software. ****P < 0.0001 as determined by t-test. e USP5-specific siRNA was co-transfected with MSK1 in SW480 or Caco2 cells in the presence of CHX (100 µg/ml) for the indicated times. Left: cell lysates were immunoblotted by antibodies as indicated. Right: the data were quantified using ImageJ software. For normalization, α-tubulin expression was used as a control. ****P < 0.0001 as determined by t-test. f USP5-specific siRNA was co-transfected with plasmids expressing Myc–Snail, Flag–MSK1 and HA–ubiquitin as indicated in HEK293T cells, and then the cells were treated with 10 μM MG132 for 6 h. Cell lysates were immunoprecipitated using an anti-Myc antibody and then analyzed by immunoblotting using an anti-HA tag antibody. g The interaction between exogenous USP5 and Snail. Myc–Snail and HA–USP5 were transfected into HEK293T cells. Cell lysates were immunoprecipitated with an anti-HA or anti-Myc and analyzed by western blot using anti-Myc (top) or anti-HA (bottom) antibody, respectively. h The interaction between exogenous USP5 and Snail in the presence of WT-MSK1, K455M-MSK1 or D565A-MSK1. Myc–Snail and HA–USP5 were co-transfected with WT-MSK1, K455M-MSK1 or D565A-MSK1 into HEK293T cells and then treated with 10 μM MG132 for 6 h. Cell lysates were immunoprecipitated with an anti-Myc and analyzed by western blot using anti-HA or anti-Flag antibody. i The direct interaction between USP5 and Snail in vitro. The kinase reactions were performed by incubating recombinant active MSK1 protein, recombinant Snail protein and recombinant USP5 protein in the kinase buffer with or without the presence of ATP for 1 h at 30 °C. After IP of each sample with anti-Snail antibody, western blot analysis was conducted using anti-USP5 antibody. j The interaction between exogenous USP5 and Snail in the presence of λ-phosphatase. Myc–Snail and HA–USP5 were transfected into HEK293T cells. Cell lysates were treated with λ-phosphatase for 30 min at 30 °C, and immunoprecipitated with an anti-Myc and analyzed by western blot using anti-HA antibody.

Fig. 7. MSK1 regulates Snail protein expression and its interaction with USP5 under physiological conditions.

a HCT116 cells were incubated for the indicated times in serum-free medium and then stimulated with DMEM medium including 10% FBS. Cell lysates were immunoblotted with the indicated antibodies. b MSK1-depleted HCT116 cells were incubated for 24 h in serum-free medium and then stimulated with DMEM medium including 10% FBS for 1 h. Cell lysates were immunoblotted with the indicated antibodies. c Serum-induced interaction between Snail and USP5. MSK1-depleted HCT116 cells were incubated for 24 h in serum-free medium and then stimulated with DMEM medium including 10% FBS for 1 h. The cells were treated with 10 μM MG132 for 6 h before collection. Cell lysates were immunoprecipitated with an anti-Snail and analyzed by western blot using anti-USP5 or anti-MSK1 antibody. The data are representative of three independent experiments. d A schematic diagram showing how stabilization of Snail by MSK1 leads to EMT in CRC. In normal or early-stage CRC cells, MSK1 is inactivated (or the expression of MSK1 is low) and Snail is readily degraded through UPS, thereby suppressing EMT. In malignant CRC cells, MSK1 is highly activated, and activated MSK1 can increase Snail protein stability by enhancing USP5-mediated Snail deubiquitination, with the resulting high Snail expression being able to promote EMT and tumor metastasis.