IGF2BP2-modified UBE2D1 interacts with Smad2/3 to promote the progression of breast cancer

Abstract

Recent studies have suggested that ubiquitin-conjugating enzyme E2D1 (UBE2D1) is involved in tumor progression. In this study, we found that UBE2D1 expression was upregulated in breast cancer (BC) and was related to the prognosis of BC patients. Through in vitro and in vivo experiments, we demonstrated the aberrant expression of UBE2D1 promoted the proliferation and migration of BC cells, and the IGF2BP2-mediated N6-methyladenosine (m6A) modification increased the stability of UBE2D1 mRNA. Mechanistically, UBE2D1 expression regulated the activity of TGF-β signaling through modulating the expression and the phosphorylation level of Smad2/3. Furthermore, UBE2D1 directly bound to Smad2/3 and affected the subsequent binding of Smad2 and Smad3, which is a necessary step for TGF-β signaling activation. Thus, our study reveals a pro-oncogenic role of UBE2D1 in the progression of BC and may provide novel strategies for BC treatment.

Keywords: IGF2BP2; Smad2/3; UBE2D1; breast cancer; tumor progression.

Figure 1

UBE2D1 is highly expressed in breast cancer cells and associated with poor prognosis. A. UBE2D1 expression in pancarcinoma; B, C. UBE2D1 expression in BC samples from the TCGA database; D. Prognostic model based on UBE2D1 expression in BC; E. UBE2D1 expression in BC tissues; F. UBE2D1 expression in BC cell lines as determined by western blot.

Figure 2

UBE2D1 promoted the proliferation and migration of BC cells. A. The successful knockdown or overexpression of UBE2D1 was determined by western blotting. B. The proliferation of UBE2D1-overexpressing cells was determined by CCK-8 assay. C. Colony formation assay of BC cells with UBE2D1 knockdown or overexpression. D. Transwell assays of BC cells with UBE2D1 knockdown or overexpression. E. The proliferation of cells with UBE2D1 knockdown or overexpression was measured by EdU assay. F. The growth of UBE2D1 knockdown-cell-derived tumor in nude mice.

Figure 3

m6A reader protein IGF2BP2 bound to and stabilized UBE2D1 mRNA in BC cells. A. Correlation between UBE2D1 and IGF2BP2; B, C. After IGF2BP2 was knocked down and overexpressed, the expression of Western blot analysis of UBE2D1 in cells with IGF2BP2 knockdown or overexpression; D, E. Western blot analysis of UBE2D1 in cells with METTL3/4 knocked down or overexpression; F. The distribution of m6A modification sites on UBE2D1 and the sites with high confidence on UBE2D1 mRNA; G. MeRIP assay showed the m6A binding site in UBE2D1; H. IGF2BP2 binding to UBE2D1 as determined by RIP assay; I. The stability of UBE2D1 after actinomycin D treatment.

Figure 4

IGF2BP2 promoted BC cell proliferation and migration. A. The proliferation of IGF2BP2 was measured by CCK-8 assay. B. Colony formation assay of IGF2BP2. C. Transwell assays of BC cells with IGF2BP2 knockdown or overexpression. D. Wound healing assays of BC cells with IGF2BP2 knockdown or overexpression.

Figure 5

UBE2D1 promoted the activation of TGF-β/Smad2/3 signaling pathway in BC. A. Pathway enrichment analysis of UBE2D1 target genes. B, C. Correlation between UBE2D1 and Smad2/3 levels. D. Western blot analysis of Smad2/3 and p-Smad2/3. E. qPCR analysis of Smad2/3 and p-Smad2/3. F. Western blot assay of Smad2/3 and p-Smad2/3 in CHX and MG132-treated cells. G. Western blot analysis of the nucleoplasmic distribution of target genes. H. Co-IP indicated the interaction between UBE2D1 and Smad2/3.

Figure 6

TGF-β/Smad2/3 was an important oncogenic pathway in BC. A. The effect of Smad2/3 on cell proliferation was measured by CCK-8 assay. B. The effect of Smad2/3 on colony formation. C. The effect of Smad2/3 on cell migration was determined by transwell assay.

Figure 7

UBE2D1 participated in BC cell proliferation and migration by promoting the interaction of Smad2 and Smad3. A. Co-IP of Smad2 and Smad3 in cells with UBE2D1 knockdown. B. The enrichment of Smad2 target genes. C. The enrichment of Smad3 target genes. D. The enrichment of UBE2D1 target genes. E. The intersection genes of UBE2D1 and SMAD2/3. F. Enrichment of 382 cross-target genes. G. The changes in the mRNA expression levels of target genes determined by qPCR.