ID4 ACTS AS A TUMOR SUPPRESSOR IN ER+ BREAST TUMORS

NASIF DANIELA; LAURITO S; CAMPOY E; MARÍA ROQUÉ MORENO; BRANHAM MT

Congreso; SAIC 2016; 2016

SAIC

Introduction: Inhibitor of differentiation proteins 1, 2, 3 and 4 (ID1?4), are dominant negative regulators of the basic helix-loop helix (bHLH) family of transcription factors. In human tumors, an increased expression of ID proteins has been associated with reversion to an embryonic-like state, with loss of differentiation, high rates of proliferation, migration and neo-angiogenesis[1]. In breast cancer there are apparently controversial findings regarding the role of ID4 in tumorigenesis. For instance, ID4 silencing by promoter hypermethylation is a frequent event in ER+ (estrogen receptor) breast tumors and is associated with an increased risk of lymph node metastasis [2]. On the contrary, in ER- breast tumors ID4 increased expression has been associated with the ability of breast cancer cells to exhibit anchorage-independent growth. Our group has previously shown that ID4 promoter unmethylation is associated with the aggressive Triple Negative Breast cancer subtype. We also demonstrated that ID4 unmethylation and expression is associated with BRCAness phenotype and downregulation of BRCA1 gene [3, 4]. Based on our observations and on literature findings it seems that ID4 has a dual role in breast cancer. Here, we hypothesize that ID4 acts as a tumor suppressor in ER+ breast tumors.Methodologies: To test our hypothesis we used two approaches: data mining analyses from the Cancer Genome Atlas (TCGA) database and experimental cell culture experiments in MCF-7 and T47D cell lines. Data mining analyses were performed by retrieving information from Mexpress web page for methylation analyses and Xena (UCSA) platforms for expression analyses. Cell culture experiments were performed on MCF-7 and T47D cell lines. Both cell lines are ER+ and have been shown by us and others to have methylated the promoter of ID4 and do not express the gene [4]. Briefly, 3ug of the pCMV-ID4 vector and 3ug of control vectors (pCMV alone or GFP) were transfected in sub-confluent (80%) cells grown in six well plates using Lipofectamine 2000. Forty-eight hours after transfection, the cells with incorporated pCMV-ID4 or control plasmids were treated as previously described, according to the experiments to be performed: proliferation, apoptosis, cell cycle or migration [5].Results: Data mining of published microarray databases was used to determine the relative expression levels of ID4 in breast tumors. As shown in figure 1A, the expression of ID4 is significantly decreased in ER+ breast tumors with respect to ER- tumors (p