Proteomic insights into the regulatory function of ARID1A in colon cancer cells

Abstract

The AT-rich interacting domain-containing protein 1A (ARID1A) is a tumor suppressor gene that has been implicated in several cancers, including colorectal cancer (CRC). The present study used a proteomic approach to elucidate the molecular mechanisms of ARID1A in CRC carcinogenesis. Stable ARID1A-overexpressing SW48 colon cancer cells were established using lentivirus transduction and the successful overexpression of ARID1A was confirmed by western blotting. Label-free quantitative proteomic analysis using liquid chromatography-tandem mass spectrometry identified 705 differentially altered proteins in the ARID1A-overexpressing cells, with 310 proteins significantly increased and 395 significantly decreased compared with empty vector control cells. Gene Ontology enrichment analysis highlighted the involvement of the altered proteins mainly in the Wnt signaling pathway. Western blotting supported these findings, as a decreased protein expression of Wnt target genes, including c-Myc, transcription factor T cell factor-1/7 and cyclin D1, were observed in ARID1A-overexpressing cells. Among the altered proteins involved in the Wnt signaling pathway, the interaction network analysis revealed that ARID1A exhibited a direct interaction with E3 ubiquitin-protein ligase zinc and ring finger 3 (ZNRF3), a negative regulator of the Wnt signaling pathway. Further analyses using the The Cancer Genome Atlas colon adenocarcinoma public dataset revealed that ZNRF3 expression significantly impacted the overall survival of patients with CRC and was positively correlated with ARID1A expression. Finally, an increased level of ZNRF3 in ARID1A-overexpressing cells was confirmed by western blotting. In conclusion, the findings of the present study suggest that ARID1A negatively regulates the Wnt signaling pathway through ZNRF3, which may contribute to CRC carcinogenesis.

Keywords: AT-rich interacting domain-containing protein 1A; Wnt signaling pathway; colorectal cancer; proteomics; zinc and ring finger 3.

Figure 1.

ARID1A protein expression in the empty vector control and ARID1A-overexpressing cells. (A) Western blot images of ARID1A and GAPDH (loading control). (B) Band intensity of ARID1A relative to GAPDH. The bar graph represents the mean ± standard deviation (n=3 per group). *P<0.05. ARID1A, AT-rich interacting domain-containing protein 1A; O/E, overexpression; MW, molecular weight.

Figure 2.

Functional enrichment analysis of differentially altered proteins. Bubble plot representing the top 10 significantly enriched GO (A) biological processes, (B) cellular components and (C) molecular functions, and (D) KEGG pathways. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; snRNA, small nuclear RNA; RFC, replication factor C; SH3, Src-homology 3.

Figure 3.

Involvement of ARID1A and altered proteins in the Wnt signaling pathway. (A) Wnt signaling pathway information generated using Kyoto Encyclopedia of Genes and Genomes (hsa04310). The pathway is illustrated using data from the KEGG database (https://www.genome.jp/pathway/hsa04310) (53). Red boxes indicate differentially altered proteins. (B) Western blot images for c-Myc, TCF1/TCF7 and cyclin D1, with GAPDH as the loading control. (C) Band intensity of proteins relative to GAPDH. The bar graph represents the mean ± standard deviation (n=3 per group). *P<0.05. ARID1A, AT-rich interacting domain-containing protein 1A; TCF, T cell factor; O/E, overexpression; MW, molecular weight.

Figure 4.

Association between ARID1A and altered proteins involved in the Wnt signaling pathway. (A) Interaction network of ARID1A and altered proteins involved in the Wnt signaling pathway constructed using GeneMANIA. (B) Heatmap of the HR of TCF7L1 and ZNRF3 for the overall survival of patients with COAD, created using GEPIA2. The framed block indicates a significant prognostic value. (C) Kaplan-Meier curve for overall survival of patients with COAD with different levels of ZNRF3 expression, plotted using GEPIA2. (D) Correlation between the expression of ARID1A and ZNRF3 in COAD, analyzed using GEPIA2. ARID1A, AT-rich interacting domain-containing protein 1A; TCF7L1, transcription factor 7 like 1; ZNRF3, zinc and ring finger 3; COAD, colon adenocarcinoma; GEPIA2, Gene Expression Profiling Interactive Analysis 2; HR, hazard ratio; TPM, transcripts per million.

Figure 5.

ZNRF3 protein expression in the empty vector control and ARID1A-overexpressing cells. (A) Western blot images of ZNRF3 and GAPDH (loading control) expression. (B) Band intensity of ZNRF3 expression relative to GAPDH. The bar graph represents the mean ± standard deviation (n=3 per group). *P<0.05. ZNRF3, zinc and ring finger 3; ARID1A, AT-rich interacting domain-containing protein 1A; O/E, overexpression; MW, molecular weight.