Transcriptional changes associated with overexpression of TBX3 isoforms (TBX3iso1 and TBX3iso2) in the DCIS-like 21NT cell line (ChIP)

Purpose: The acquisition of cellular invasiveness of breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Very little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like 21NT cells results in increased survival, growth, and invasiveness. In this study we explore the role of TBX3 in breast cancer progression pathways, focusing specifically on its involvement in the induction of EMT and the transition from DCIS to invasive mammary carcinoma. Methods: Total mRNA was isolated from 21NT transfected cell lines. Sequencing was conducted using an Illumina HiSeq 2500 sequencer, comparing the resultant transcriptional profiles with overexpression of either TBX3 isoform (TBX3iso1 or TBX3iso2) as fold changes relative to the empty vector control. RNA-Seq data was integrated with ChIP-array data downstream to assess direct targets of TBX3 isoforms and relate it to the process of EMT. TBX3, SLUG and TWIST1 expression were assessed by immunohistochemistry in two independent early-stage (Stage 0 and Stage 1) breast cancer cohorts. Results: An EMT-related transcriptional profile was observed with overexpression of TBX3iso1 and TBX3iso2. Using genome-wide bioinformatic approaches in conjunction with more conventional in vitro studies, we have identified SLUG and TWIST1 as downstream targets of TBX3 and have assessed their expression by immunohistochemistry in two different patient cohorts. Our findings suggest that TBX3 facilitates the process of early invasion in DCIS lesions by promoting the induction of EMT and tumor progression through the low-grade pathway. Finally, we propose a progression model in which SLUG is an important and necessary effector downstream of TBX3, leading to increased motility, invasiveness, and induction of key invasiveness-associated genes. Conclusions: This is the only existing study which has conducted assessment of transcriptional changes associated with TBX3 isoforms (TBX3iso1 and TBX3iso2). Our published study has identified the transcriptional regulator TBX3 as an enabler of EMT in non-high grade, pre-invasive lesions of the breast, inducing SLUG and promoting the transition from in situ (DCIS) to invasive breast cancer. Assessed direct transcriptional targets of TBX3 isoforms; chromatin immunoprecipitation (ChIP) was conducted in duplicate using either a rabbit anti-TBX3 antibody or rabbit IgG antibody in 3 cell lines (empty vector, TBX3iso1 overexpression, or TBX3iso2 overexpression) Peaks from the non-specific IgG ChIPs were subtracted from specific TBX3 ChIPs for each cell line. Differential probe intensities between TBX3iso1 or TBX3iso2 vs empty vector were determined by ANOVA and enriched regions of significance were determined using the MAT algorithm.