ID4 an oncogene in TNBC with properties to repress luminal genes

REAL, SEBASTIAN; NASIF DANIELA; GOMEZ DIAZ ALDANA ; LAURITO SERGIO; ROQUÉ, MARÍA; BRANHAM MARIA T

Mendoza

Congreso; Reunión anual Edición LVIII de la Sociedad Argentina de Bioquímica y Biología Molecular; 2022

Background: Inhibitor of differentiation protein 4 (ID4) is a dominant negative regulator of the basic helix-loop-helix (bHLH) family of transcription factors. In different tumor types ID proteins are associated with loss of differentiation, stemness, and neoangiogenesis. In breast cancer, ID4 is highly expressed in the triple-negative breast cancer (TNBC) subtype and high ID4 expression is associated with stem-like phenotype and poor prognosis. It has been proposed that ID4 regulates the expression of genes that participate in luminal differentiation such as estrogen receptor (ER), FOXC1, and NOTCH1. Since ID4 regulates the expression genes involved in luminal specialization and since ID4 is overexpressed and meets an oncogenic function in TN tumors, we hypothesize that silencing ID4 in TN tumors will unlock the expression of genes required for luminal differentiation and will revert the aggressive behavior of this tumor type. Methods: In vitro ID4 transient and stable silencing by siRNA or by shRNA in MDA-MB231 TN cell line, gene and protein expression were analyzed by RT-qPCR and western blot. To assess the impact of ID4 on development of tumors in vivo we inoculated MDA-MB231 cells lines (silenced and control) in the fourth mammary gland of NSG mice. In-silico analyses involved the evaluation of differentially expressed genes according to ID4 expression on human breast tumors of public datasets. Results: We first evaluated differential gene expression patterns among breast cancer samples with high vs low ID4 expression. To do so we performed in silico differential gene expression analysis from the TCGA database from 680 samples. Our analysis revealed a total of 922 differentially expressed genes (DEGs) (290 down-regulated and 632 up-regulated) (logFC > 1.5 and logFC < -1.5, p-value < 0.05). Amongst downregulates genes we found that ER, GATA3, and FOXA1 were the ones with the highest fold change values and highest significance. To determine the biological processes in which the downregulated genes are involved, we did pathway analysis which revealed that the most significant downregulated genes (logFC