HDAC8 Promotes Liver Metastasis of Colorectal Cancer via Inhibition of IRF1 and Upregulation of SUCNR1

Abstract

Histone deacetylases (HDACs) are well-characterized for their involvement in tumor progression. Herein, the current study set out to unravel the association of HDAC8 with colorectal cancer (CRC). Bioinformatics analyses were carried out to retrieve the expression patterns of HDAC8 in CRC and the underlying mechanism. Following expression determination, the specific roles of HDAC8, IRF1, and SUCNR1 in CRC cell functions were analyzed following different interventions. Additionally, tumor formation and liver metastasis in nude mice were operated to verify the fore experiment. Bioinformatics analyses predicted the involvement of the HDAC8/IRF1/SUCNR1 axis in CRC. In vitro cell experiments showed that HDAC8 induced the CRC cell growth by reducing IRF1 expression. Meanwhile, IRF1 limited SUCNR1 expression by binding to its promoter. SUCNR1 triggered the growth and metastasis of CRC by inhibiting cell autophagy. HDAC8 blocked IRF1-mediated SUCNR1 inhibition and thereby inhibited autophagy, accelerating CRC cell growth. Lastly, HDAC8 facilitated the development of CRC and liver metastasis by regulating the IRF1/SUCNR1 axis in vivo. Taken together, our findings highlighted the critical role for the HDAC8/IRF1/SUCNR1 axis in the regulation of autophagy and the resultant liver metastasis in CRC.

Figure 1

Significance of the HDAC8/IRF1/SUCNR1 axis in CRC. (a) A box plot of the differential expression of HDAC8 in the colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) samples included in TCGA and GTEx (red box plots represent tumor samples, and gray box plots represent normal samples; in COAD, there are 275 tumor samples and 349 normal samples; in READ, there are 92 tumor samples and 318 normal samples). (b) Venn diagram of HDAC8 downstream regulatory genes and transcription factors (the left is the downstream genes of HDAC8 predicted by the starBase database, the right is the transcription factor annotation, and the center represents the intersection of the two sets of data). (c) Interaction analysis of the candidate transcription factors; each circle in the figure represents a gene, and the line between circles indicates interaction between two genes; the darker color of the circle where the gene is located reflects more interaction genes, higher core degree in the interaction network, and higher degree value. (d) Statistics of degree value of core genes in the gene interaction network (the abscissa represents the degree value and the ordinate represents the gene name). (e) KEGG enrichment analysis of the candidate transcription factors (the abscissa represents the gene ratio, the ordinate represents the KEGG entry identifier, and the histogram on the right is the color scale). (f) A box plot of the differential expression of SUCNR1 in the CRC included in TCGA and GTEx (red box plots represent tumor samples, and gray box plots represent normal samples; in COAD, there are 275 tumor samples and 349 normal samples; in READ, there are 92 tumor samples and 318 normal samples). ^∗p < 0.05.

Figure 2

HDAC8 suppresses the expression of IRF1 and thus facilitates the growth and metastasis of CRC cells. (a) HDAC8 and IRF1 mRNA expression in CRC and adjacent normal tissues determined by RT-qPCR (n = 58). (b) HDAC8 and IRF1 mRNA expression in SW480, SW620, HT29, HCT-116, and FHC cell lines determined by RT-qPCR. (c) Histone acetylation levels in the IRF1 promoter region in CRC and adjacent normal tissues determined by ChIP. (d) mRNA expression of HDAC8 and IRF1 in HCT-116 cells treated with sh-HDAC8 or oe-HDAC8 determined by RT-qPCR. (e) Western blot analysis of HDAC8 and IRF1 proteins in HCT-116 cells treated with sh-HDAC8 or oe-HDAC8. (f) mRNA expression of HDAC8 and IRF1 in HCT-116 cells treated with PCI-34051 determined by RT-qPCR. (g) H3K9Ac levels in the IRF1 promoter region in HCT-116 cells treated with PCI-34051 determined by ChIP. (h) HDAC8 and IRF1 mRNA expression in HCT-116 cells treated with sh-HDAC8 or combined with sh-IRF1 determined by RT-qPCR. (i) Viability of HCT-116 cells treated with sh-HDAC8 or combined with sh-IRF1 measured by CCK-8 assay. (j) Migration of HCT-116 cells treated with sh-HDAC8 or combined with sh-IRF1 measured by Transwell assay. (k) Invasion of HCT-116 cells treated with sh-HDAC8 or combined with sh-IRF1 measured by Transwell assay. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001, compared with adjacent normal tissues, FHC cells, or DMSO- or sh-NC-treated HCT-116 cells. ^#p < 0.05, ^###p < 0.001, and ^####p < 0.0001, compared with sh-HDAC8+sh-NC-treated HCT-116 cells. The experiment was conducted three times independently.

Figure 3

IRF1 downregulates the expression of SUCNR1 by binding to its promoter in CRC cells. (a) SUCNR1 mRNA expression in CRC and adjacent normal tissues determined by RT-qPCR (n = 58). (b) SUCNR1 mRNA expression in SW480, SW620, HT29, HCT-116, and FHC cell lines determined by RT-qPCR. (c) Prediction of IRF1 binding sites in the SUCNR1 promoter and mutation sequence generated by site mutation determined by ChIP. (d) Binding between IRF1 and SUCNR1 confirmed by dual-luciferase reporter assay. (e) Enrichment of IRF1 in the promoter of SUCNR1 determined by ChIP. (f) IRF1 and SUCNR1 mRNA expression in HCT-116 cells treated with sh-IRF1 or oe-IRF1 determined by RT-qPCR. (g) Western blot analysis of IRF1 and SUCNR1 proteins in HCT-116 cells treated with sh-IRF1 or oe-IRF1. ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001, compared with adjacent normal tissues, FHC cells, IgG group, or sh-NC-treated HCT-116 cells. ^###p < 0.001 and ^####p < 0.0001, compared with oe-NC-treated HCT-116 cells. The experiment was conducted three times independently.

Figure 4

SUCNR1 promotes the migration and invasion of CRC cells by blunting tumor cell autophagy. (a) SUCNR1 mRNA expression in HCT-116 cells treated with sh-SUCNR1 determined by RT-qPCR. (b) Viability of HCT-116 cells following SUCNR1 knockdown or combined with 3-MA measured by CCK-8 assay. (c) Migration of HCT-116 cells following SUCNR1 knockdown or combined with 3-MA measured by Transwell assay. (d) Invasion of HCT-116 cells following SUCNR1 knockdown or combined with 3-MA measured by Transwell assay. (e) Flow cytometric analysis of the HCT-116 cell apoptosis following SUCNR1 knockdown or combined with 3-MA. (f) Western blot analysis of LC3-II/LC3-I ratio in HCT-116 cells following SUCNR1 knockdown or combined with 3-MA. (g) Immunofluorescence detection of the number of GFP-LC3 spots in HCT-116 cells following SUCNR1 knockdown or combined with 3-MA (scale bar = 50 μm). (h) Number of autophagic vacuoles in HCT-116 cells following SUCNR1 knockdown or combined with 3-MA under a TEM. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001, compared with HCT-116 cells transfected with sh-NC. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001, compared with HCT-116 cells treated with sh-SUCNR1+DMSO. The experiment was conducted three times independently.

Figure 5

HDAC8 suppresses cell autophagy to boost the migration and invasion of CRC cells by regulating the IRF1/SUCNR1 axis. HCT-116 cells were transfected with sh-HDAC8 or combined with oe-SUCNR1. (a) SUCNR1 mRNA expression in HCT-116 cells determined by RT-qPCR. (b) Viability of HCT-116 cells measured by CCK-8 assay. (c) Migration of HCT-116 cells measured by Transwell assay. (d) Invasion of HCT-116 cells measured by Transwell assay. (e) Western blot analysis of LC3-II/LC3-I ratio in HCT-116 cells. (f) Immunofluorescence detection of the number of GFP-LC3 spots in HCT-116 cells (scale bar = 50 μm). (g) Number of autophagic vacuoles in HCT-116 cells under a TEM. ^∗p < 0.05 and ^∗∗p < 0.01, compared with HCT-116 cells transfected with sh-NC. ^#p < 0.05, ^##p < 0.01, and ^####p < 0.0001, compared with HCT-116 cells transfected with sh-HDAC8+oe-NC. The experiment was conducted three times independently.

Figure 6

HDAC8 promotes tumorigenesis and liver metastasis of CRC cells by regulating the IRF1/SUCNR1 axis in nude mice. (a) Tumor growth of mice treated with sh-HDAC8 or combined with oe-SUCNR1. (b) Ki67 immunohistochemical staining images of tumor tissues of nude mice treated with sh-HDAC8 or combined with oe-SUCNR1 as well as the semiquantitative analysis. (c) mRNA expression of HDAC8, IRF1, and SUCNR1 in tumor tissues of mice treated with sh-HDAC8 or combined with oe-SUCNR1 determined by RT-qPCR. (d) HE staining analysis of number of liver metastases in the liver tissues of nude mice treated with sh-HDAC8 or combined with oe-SUCNR1 (scale bar = 50 μm). n = 10 for mice upon each treatment. ^∗p < 0.05 and ^∗∗∗∗p < 0.0001, compared with mice treated with sh-NC. ^#p < 0.05 and ^####p < 0.0001, compared with mice treated with sh-HDAC8+oe-NC.

Figure 7

Molecular mechanism by which HDAC8 regulates the progression of CRC. Histone deacetylase HDAC8 upregulates SUCNR1 by downregulating IRF1 and consequently inhibits CRC cell autophagy, ultimately contributing to the CRC growth and liver metastasis.