ARID1A deficiency promotes malignant proliferation of hepatocellular carcinoma by activating HDAC7/ENO1 signaling pathway

Abstract

Background: Epigenetic dysregulation constitutes one of the mechanisms in the pathogenesis of HCC, thereby underscoring its critical scientific importance for early diagnosis and therapeutic interventions. The chromatin remodeling factor AT-rich interaction domain 1A (ARID1A) serves as an essential epigenetic regulator and is frequently subject to inactivating mutations across various cancer types. Nevertheless, the precise molecular pathways by which ARID1A deficiency facilitates the progression of HCC remain inadequately elucidated.

Methods: RNA sequencing (RNA-seq) was employed to identify genes that were significantly upregulated following the knockdown of ARID1A. Immunohistochemistry was used to assess the expression levels of ARID1A and histone deacetylase 7 (HDAC7) in HCC samples. Chromatin immunoprecipitation and luciferase reporter assays were conducted to validate PU.1 as a positive transcriptional regulator of HDAC7. Protein immunoprecipitation coupled with mass spectrometry was used to identify potential substrates of HDAC7. A xenograft assay was performed to evaluate the therapeutic potential of HDAC7 inhibitors in ARID1A-deficient HCC.

Results: Our investigation demonstrated that the loss of ARID1A in HCC cells led to an upregulation of HDAC7 expression. Mechanistic analyses revealed that ARID1A deficiency facilitated PU.1-mediated transcriptional regulation of HDAC7. Additionally, the overexpression of HDAC7 resulted in a reduced acetylation level of Enolase 1 (ENO1), thereby enhancing the malignant proliferation of HCC cells. Targeting HDAC7 effectively inhibited the growth of ARID1A-deficient tumors in tumor-bearing mice.

Conclusions: This study provides novel insights into the regulatory interplay between chromatin remodeling and acetylation modification, 2 pivotal epigenetic processes influencing tumorigenesis. It also suggests that pharmacological inhibition of HDAC7 offers a promising therapeutic strategy for treating HCC with ARID1A mutations.

Keywords: ARID1A; HCC; HDAC7; acetylation modification; chromatin remodeling.

FIGURE 1

ARID1A deficiency renders upregulation of HDAC7 expression in HCC cells. (A) GO enrichment analysis of upregulated differentially expressed genes between shARID1A or control group. (B) Heatmap depicting relative expression of the top 25 differentially expressed genes in the chromatin modification pathway between the shARID1A and control group. (C) Fold changes in mRNA expression of HDAC family genes from RNA-seq between the shARID1A versus control group. (D and E) Relative mRNA expression of ARID1A and HDACs in Huh7 or HepG2 cells±ARID1A knockdown. (F and G) Western blot of HDAC7 in HepG2 or Huh7 cells±ARID1A knockdown. (H) Relative mRNA expression of ARID1A and HDACs in SNU449 cells±ARID1A overexpression. (I) Western blot of HDAC7 in SNU449 cells±ARID1A overexpression. (J and K) Western blot of pan-acetylation level in Huh7 or HepG2 cells±ARID1A knockdown. (L) Western blot of pan-acetylation level in SNU449 cells±ARID1A overexpression. qRT-PCR data (mean±SD; n=3) were analyzed by unpaired 2-tail t test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Abbreviations: ARID1A, AT-rich interaction domain 1A; GO, Gene Ontology; HDAC7, histone deacetylase 7; WT, wild type.

FIGURE 2

HDAC7 is upregulated in HCC, and inhibition of HDAC7 restrains the proliferation of ARID1A-deficient cells. (A) Box plots showing the expression of HDAC7 mRNA in 369 HCC tumor samples and 160 corresponding normal samples from the TCGA database using the GEPIA website. (B) Kaplan-Meier survival analysis of HDAC7 expression in 364 HCC cases from the TCGA database using the GEPIA website. (C) IHC staining of ARID1A and HDAC7 protein in HCC tissues and the matched noncancerous liver tissues. Shown are representative images. Scale bar, 100 μm. (D) Correlation of H-scores for ARID1A and HDAC7 proteins in HCC samples and the paired noncancerous liver tissues, n=15. The correlation score was analyzed using the Pearson correlation test. (E and F) Proliferation was measured by EdU incorporation assay of Huh7 or HepG2 cells±ARID1A knockdown treated with or without 2 μM TMP269 for 24 hours. Nuclei were counterstained with Hoechst. Scale bar, 100 μm. G and H, Proliferation was measured by CCK8 assay of Huh7 or HepG2 cells±ARID1A knockdown treated with or without 2 μM TMP269 for 24 hours. Data (mean±SD; n=3) were analyzed by repeated-measures ANOVA. I and J, Proliferation was measured by colony formation assay of Huh7 or HepG2 cells±ARID1A knockdown treated with or without 2 μM TMP269 for 24 hours. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Abbreviations: ARID1A, AT-rich interaction domain 1A; HDAC7, histone deacetylase 7.

FIGURE 3

ARID1A deficiency promotes PU.1-mediated transcriptional regulation of HDAC7 in HCC cells. (A) Occupancy of ARID1A with the HDAC7 promoter was determined by ChIP-PCR in Huh7 cells. (B) Chromatin accessible sites in HDAC7 promoter were determined by ATAC-seq in Huh7 cells±ARID1A knockdown. (C) Transcription factors with potential binding sites with the HDAC7 promoter were ranked by credibility score from UCSC and the JASPAR data sets. (D–F) Western blot of HDAC7 in Huh7 cells±STAT2, SPI1, or IRF1 knockdown. (G) Occupancy of PU.1 with the HDAC7 promoter was determined by ChIP-PCR in Huh7 cells. (H) Relative occupancy of PU.1 with the HDAC7 promoter was determined by ChIP-qPCR in Huh7 cells. (I) Promoter activity of HDAC7 in HEK293T cells overexpressing PU.1 was examined by luciferase reporter assay. (J and K) Western blot of HDAC7 in Huh7 or HepG2 cells±ARID1A and/or SPI1 knockdown. (L and M) co-IP analysis of the interaction between ARID1A and PU.1 in Huh7 or HepG2 cells. (N and O) Localization of ARID1A and PU.1 protein in Huh7 or HepG2 cells was examined by immunofluorescence assay. Scale bar, 10 μm. (P) Relative occupancy of PU.1 with the HDAC7 promoter was determined by ChIP-qPCR in Huh7 cells±ARID1A knockdown. ChIP-qPCR and luciferase reporter data (mean±SD; n=3) were analyzed by unpaired 2-tail t test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Abbreviations: ARID1A, AT-rich interaction domain 1A; HDAC7, histone deacetylase 7.

FIGURE 4

ARID1A deficiency promotes HCC cell proliferation through the HDAC7-ENO1 axis. (A) Coomassie brilliant blue-stained gel showed differential bands between control and HDAC7-overexpressing samples in Huh7 cells. (B) Screening of ENO1 as a potential substrate of HDAC7. (C and D) co-IP analysis of the interaction between HDAC7 and ENO1 in Huh7 or HepG2 cells. (E and F) Western blot of Ac-ENO1 in Huh7 or HepG2 cells±HDAC7 knockdown. (G and H) Western blot of Ac-ENO1, cyclin D1, and p21 in Huh7 or HepG2 cells±HDAC7 knockdown treated with or without ENO1-K89R mutant. (I and J) Proliferation was measured by EdU incorporation assay of Huh7 or HepG2 cells±ARID1A knockdown treated with or without ENO1-K89R mutant. Nuclei were counterstained by Hoechst. Scale bar, 100 μm. (K and L) Western blot of Ac-ENO1, ENO1, cyclin D1, and p21 in Huh7 or HepG2 cells±ARID1A knockdown treated with 2 μM TMP269 for 24 hours, ENO1-K89Q mutant or ENO1-K89R mutant. (M and N) Proliferation was measured by EdU incorporation assay of Huh7 or HepG2 cells±ARID1A knockdown treated with 2 μM TMP269 for 24 hours, ENO1-K89Q mutant, or ENO1-K89R mutant. Nuclei were counterstained with Hoechst. Scale bar, 100 μm. Abbreviations: ARID1A, AT-rich interaction domain 1A; EdU, 5‑ethynyl‑2′‑deoxyuridine; ENO1, enolase 1; HDAC7, histone deacetylase 7.

FIGURE 5

Targeted inhibition of HDAC7 inhibits the progression of ARID1A-deficient HCC in vivo. (A) Image of subcutaneous tumors derived from Huh7 cells±ARID1A knockdown treated with PBS, 3 mg/kg/d TMP269, and 20 mg/kg/d SAHA at the end of scheduled treatment. (B) Growth curves of subcutaneous tumors derived from Huh7 cells±ARID1A knockdown treated with PBS, 3 mg/kg/d TMP269, and 20 mg/kg/d SAHA. Data (mean±SD; n=3) were analyzed by repeated-measures ANOVA. (C) IHC staining of ARID1A and HDAC7 protein of subcutaneous tumors derived from Huh7 cells±ARID1A knockdown treated with PBS, 3 mg/kg/d TMP269, and 20 mg/kg/d SAHA. Scale bar, 100 μm. (D and E) Quantification of IHC staining of AIRD1A and HDAC7 in C. Data (mean±SD; three animals in each group) were analyzed by unpaired 2-tail t test. F, Schematic representation of the molecular mechanism of ARID1A deficiency promoting progression of HCC through upregulating HDAC7. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Abbreviations: ARID1A, AT-rich interaction domain 1A; ENO1, enolase 1; HDAC7, histone deacetylase 7.