The basic helix-loop-helix (bHLH) proteins in breast cancer progression

Abstract

The basic helix-loop-helix (bHLH) proteins are the major transcription factors acting as transcriptional enhancers or inhibitors of various genes through binding to the canonical E-box sequence. The bHLH proteins are highly conserved in both vertebrates and invertebrates. The bHLH domain is approximately 60 amino acids in length consisting of a DNA-binding basic region followed by two alpha-helices separated by a variable loop. The DNA-binding basic region associates with the hexanucleotide E-box sequence. The human genome contains 121 bHLH genes which regulate various cellular processes including embryonic development [1, 2]. Many of them are involved in cell proliferation, differentiation and oncogenesis, as well as apoptosis. For example, MyoD and NeuroD family bHLH proteins contribute to myogenesis, neurogenic differentiation and pancreatic development, while Myc family bHLH proteins are directly involved in cell differentiation, proliferation and oncogenesis [3]. The bHLH proteins are mainly subdivided in six major groups (A–F). This classification is based on the target E-box sequence. While A–C group members target 6 different hexanucleotide sequences, group D members, ID (inhibitor of differentiation/DNA-binding) family proteins lack the basic domain and are thus incapable of interacting with DNA. The ID family proteins form protein–protein dimers and function as dominant negative regulator of other bHLH proteins. Expression of ID proteins and their target bHLH proteins differs with the cell type and stage of differentiation, growth or development, and the balance of bHLH and ID activity is critical to maintain normal cellular processes [3]. Thus, imbalance in these bHLH proteins contributes to abnormal gene regulation, which is very often observed in cancer [3]. In this report, we studied expression of bHLH proteins in breast cancer. Micro-array data available for breast cancer patients were downloaded from NCBI gene expression omnibus (GEO). Data were thoroughly checked for platforms, and only Affymetrix Human Genome U133A Array (GPL96) was selected for this study. The methods used for data processing were described previously [4–9]. Recent studies suggest that several bHLH proteins are involved in breast cancer control by inducing apoptosis or controlling antiapoptotic genes [10–12]. To understand which genes are deregulated in breast cancer, we analyzed mRNA expression data from more than 2,500 patient samples. The complete set of 121 bHLH genes was used for this analysis [1]. We used classical multidimensional scaling (MDS) and principal component analysis (PCA) to identify deregulated bHLH genes. The distance matrix was created using ‘‘dist’’ command in R package. Then, ‘‘cmdscale’’ command was used to reduce dimensions. This analysis resulted in a list of 14 deregulated genes (Fig. 1a). Then, we applied PCA analysis using ‘‘prcomp’’ command on the same dataset. PCA analysis also resulted in a list of 13 deregulated genes (Fig. 1a). We furthermore analyzed dataset with SAM which generated a list of 30 genes (Fig. 1a). Eight genes BHLHE40, CDK5RAP3, HES2, HIF1A, ID2, MAX, MLXIP and USF2 were common in all Electronic supplementary material The online version of this article (doi:10.1007/s12032-013-0666-9) contains supplementary material, which is available to authorized users.