Loss of BRG1 induces CRC cell senescence by regulating p53/p21 pathway

Abstract

Brahma-related gene-1 (BRG1) is the specific ATPase of switch/sucrose nonfermentable chromatin-remodeling complex that is aberrantly expressed or mutated in various cancers. However, the exact role of BRG1 in oncogenesis remains unknown. In this study, we demonstrate that the knockdown (KD) of BRG1 promotes cellular senescence by influencing the SIRT1/p53/p21 signal axis in colorectal cancer (CRC). In particular, we reveal that the expression level of BRG1 is inversely correlated with p21, one of the classic senescence regulators, and is decreased in senescent CRC cells. KD of BRG1 promoting senescence is indicated by the increase of senescence-associated β-galactosidase (SA-β-gal) activity, inhibition of cell proliferation, induction of cell cycle arrest, and formation of senescence-associated heterochromatin foci. BRG1 binds to SIRT1 and interferes with SIRT1-mediated deacetylation of p53 at K382. Rescue experiments by co-silencing p53 or treatment with EX527, a SIRT1-specific inhibitor, abrogated the cellular senescence induced by KD of BRG1. BRG1 KD cells resulted in smaller tumor formation than that in control cells in vivo. Collectively, our study shows that BRG1 has an important role in cellular senescence and tumor growth. The BRG1/SIRT1/p53 signal axis is a novel mechanism of cell senescence in CRC and is a new potential target for cancer therapy.

Figure 1

Relationship between BRG1 and cellular senescence in CRCs. (a) Cell senescence assay by SA-β-gal staining (up panel, representative images of SA-β-gal staining; scale bar: 50 μm; down panel, percentage of SA-β-gal-positive cells from three random microscopic fields). (b) Western blot analysis for BRG1, p53, p21, and p16 expression levels in SW48 and LoVo cells treated with H₂O₂ (250 μmol/l, 2 h) or DOX (1 μmol/l, 48 h), **P<0.01

Figure 2

KD of BRG1 promotes cellular senescence in CRC cells. (a) Western blot and real-time-PCR analysis for BRG1 expression in SW48 Sh-Con and SW48 Sh-BRG1 cells. (b) Cell senescence assay by SA-β-gal staining (left panel, representative images of SA-β-gal staining; scale bar: 50 μm; right panel, average counts of SA-β-gal-positive cells from three random microscopic fields) in SW48 Sh-Con and SW48 Sh-BRG1 cells. DOX (0.25 μmol/l, 48 h). (c) Cell proliferation analysis determined by CCK8 assay (n=3, mean±S.D.) in SW48 Sh-Con and SW48 Sh-BRG1 cells (n=3, mean±S.D.; absorption at 450 nm (A450 nm) was, respectively, detected at 0, 24, 48, and 72 h after transfection). (d) Cell cycle distribution analysis measured by propodium iodide staining and flow cytometry in SW48 Sh-Con and SW48 Sh-BRG1 cells (n=3). (e) DAPI staining for SAHF formation in SW48 Sh-Con and SW48 Sh-BRG1 cells (red, BRG1; blue, DAPI. Magnification × 630). (f) DNA sequence analysis of HCT116 showing the mutation in BRG1 gene. (g) Western blot analysis for indicated protein expression levels in HCT116 Sh-Con and HCT116 Sh-BRG1 cells. (h) SA-β-gal staining analysis in HCT116 Sh-Con and HCT116 Sh-BRG1 cells. DOX (0.25 μmol/l, 48 h); *P<0.05, **P<0.01, t-test

Figure 3

KD of BRG1 increases the expression level of p53 and p21. (a and b) Western blot analysis for expression levels of BRG1, p21, and p16, and quantitative expression analyses of BRG1, p21, and p16 mRNA by quantitative real-time PCR (n=3, mean±S.D.) in SW48 and LoVo Sh-Con and HCT116 Sh-BRG1 cells. (c) Western blot detected the expression levels of BRG1 and p21 in multiple colon cancer cell lines. BRG/p21: the relative expression ratio between BRG1 and p21. *P<0.05; **P<0.01

Figure 4

BRG1 regulates the expression of p21 and cellular senescence dependent on regulating p53 protein stability. (a) Western blot detected the expression levels of BRG1, p53, and p21 in SW48 Sh-Con and SW48 Sh-BRG1 cells. (b) SA-β-gal staining (left panel, representative images of SA-β-gal staining; scale bar: 50 μm; right panel, percentage of SA-β-gal-positive cells from three random microscopic fields) in SW48 Sh-Con and SW48 Sh-BRG1 cells transfected with or without siP53. DOX (0.25 μmol/l, 48 h). (c) SA-β-gal staining in SW620 transfected Sh-Con and Sh-BRG1 cells DOX (0.25 μmol/l, 48 h). (d) Quantitative expression analysis of p53 mRNA by quantitative real-time PCR. (e) Western blot assays for BRG1 and p53 expression in SW48 Sh-Con and Sh-BRG1 cells treated with MG132(1 mmol/l). (f) Western blot assays for BRG1 and p53 expression in SW48 Sh-Con and Sh-BRG1 cells treated with CHX (1 μg/ml) (up panel: western blot bands; down panel: plot of the p53 expression levels following CHX treatment in sh-Con cells or sh-BRG1 cells, normalized for the levels of GAPDH). *P<0.05; **P<0.01

Figure 5

BRG1 binds to SIRT1 and regulates p53 acetylation. (a) The protein–protein networks view from STRING database showing the networks of BRG1 (SMARCA4, the gene name coding BRG1), p53, and SIRT1. (b) Co-immunoprecipitation analysis in SW48 cells using anti-SIRT1 antibodies and anti-mouse-IgG antibodies or anti-BRG1 antibodies and anti-mouse-IgG antibodies, respectively. (c) Immunofluorescence images scanned by confocal laser microscope detecting the sub-cellular location of BRG1 and SIRT1 expression levels in SW48 cells. (d) Western blot detected the expression levels of BRG1, p53, p53-K382, and p21 in SW48 Sh-Con and Sh-BRG1 cells treated with or without EX527 (1 μmol/l, 48 h). (e) SA-β-gal staining (left panel, representative images of SA-β-gal staining; scale bar: 50 μm; right panel, percentage of SA-β-gal-positive cells from three random microscopic fields) in SW48 Sh-Con and Sh-BRG1 cells treated with or without EX527 (1 μmol/l, 48 h) DOX (0.25 μmol/l, 48 h). **P<0.01

Figure 6

BRG1 KD promotes senescence and inhibits cell proliferation in vivo. (a) Tumor size measured as methods and material, and the tumor growth curve is shown. (b) All mice were killed 2 weeks after injection and the tumors were collected and shown. Box plot shows the distribution of tumor weight from sh-BRG1 versus sh-Con group (n=10, mean±S.D.). (c) Immunohistochemical analysis of BRG1, SIRT1, p21, and Ki-67 in tumor sections (scale bar: 50 μm). (d) HE staining and IHC staining of indicated protein in xenograft. *P<0.05