ID (Inhibitor Of DNA Binding) Proteins Regulate Differentiation Of Breast Epithelial Cells And Influence Breast Cancer Prognosis

Objective:Inhibitors of DNA binding (ID) proteins are known as important modulators in regulating cell proliferation and differentiation. This study sought to investigate the expression profile and prognostic value of IDs in breast cancer tissues, and furthermore figured out the concrete biological processes.Methods:The prognostic role of ID proteins in human breast cancer was investigated in 250 breast cancers via tissue microarrays (TMA). We applied three dimensional cultivation of control MCF10A cells and MCF10A cells overexpressing ID proteins to investigate regulation of ID family on differentiation of normal breast epithelial cells. Then we used RNAseq technique and IPA analysis to try to find the probable biological variations. At last, we carried out immunofluorescence, point mutations and dual-luciferase reporter assay to find out the mechanism.Results:1D1, ID2 and ID3 were all examined by immunohistochemistry to have high expression levels, especially ID2 whose expression level was found to be much higher in breast cancer tissues than normal breast tissues. We found that ID2 and ID3 have higher expression levels in ER positive breast cancer cells, and by Chi square test ID2 and ID3 were proved to be statistically correlated with ER status, P value was 0.016 and 0.006 respectively. The survival analysis by Kaplan-Meier and Cox regression showed ID2 expression level served as an independent prognostic factor for disease-free survival (P=0.013; HR=2.358, 95%CI 1.065-5.218). The prognostic value of ID2 was most significant in triple-negative patients (DFS; P=0.009). During three dimensional cultivation, we observed that overexpression of ID proteins in normal breast epithelial cells inhibited their differentiation, promoted proliferation and caused transformed phenotype. Then by RNAseq and the following bioinformatics analysis, we figured out that a number of signaling pathways were activated by overexpression of IDs. We chose the remodeling of epithelial adherens junctions pathway to validate and found prominent reduction of its key factor E-cadherin. We constructed dual-luciferase reporter plasmid of E-cadherin promoter region and observed prominent inhibition of the promoter activity when ID proteins were added in the dual-luciferase reporter assay. By employing point mutations of the E-box elements in the wild-type plasmid above, we found that ID proteins transcriptionally inhibited E-cadherin by indirectly interacting with the E-box -1286 to -1281bps upstream TSS. Re-evaluated by tissue array, we also found that ID2 was negatively correlated with E-cadherin expression by (P=0.020, Pearson’s R=-0.155). Then using a combination of ID2 and E-cadherin expression, the patients were classified into four subgroups with different DFS (P=0.023).Conclusions:ID proteins had high expression levels in breast cancers, in which ID2 predicted worse clinical outcome. The exact biological rationale was recovered that ID proteins inhibited differentiation of breast epithelial cells and promoted proliferation and transformation of epithelial cells. A number of signaling pathways were found to change including the remodeling of epithelial adherens junctions. ID proteins were found to transcriptionally inhibit E-cadherin expression indirectly. And a combination of ID2 and E-cadherin could better predict prognosis of breast cancer.