When triple negative breast cancer (TNBC) are analyzed by gene expression profiling different subclasses are identified, at least one characterized by genes related to immune signaling mechanisms supporting the role of these genes in the cancers. In an earlier study we observed differences in TNBC cell lines with respect to their expression of the cytokine IL32. Our analyses showed that certain cell lines expressed higher levels of the cytokine compared to others. Because TNBC are heterogeneous and immune-related genes appear to play a pivotal role in these cancers, we chose to examine the transcriptomes of the different cell lines based on IL32 expression. We performed group analyses of TNBC cell lines demonstrating high IL32 compared to low IL32 levels and identified IL32, GATA3, MYBL1, ETS1, PTX3 and TMEM158 as differentially associated with a subpopulation of TNBC. The six candidate genes were validated experimental and in different patient datasets. The genes distinguished a subset of TNBC from other TNBC, and TNBC from normal, luminal A, luminal B, and HER2 patient samples. The current project serves as a preliminary study in which we outline the discovery and validation of our list of six candidate genes.
BackgroundTriple negative breast cancer (TNBC) and often basal-like cancers are defined as negative for estrogen receptor, progesterone receptor and Her2 gene expression. Over the past few years an incredible amount of data has been generated defining the molecular characteristics of both cancers. The aim of these studies is to better understand the cancers and identify genes and molecular pathways that might be useful as targeted therapies. In an attempt to contribute to the understanding of basal-like/TNBC, we examined the Gene Expression Omnibus (GEO) public datasets in search of genes that might define basal-like/TNBC. The Il32 gene was identified as a candidate.FindingsAnalysis of several GEO datasets showed differential expression of IL32 in patient samples previously designated as basal and/or TNBC compared to normal and luminal breast samples. As validation of the GEO results, RNA and protein expression levels were examined using MCF7 and MDA MB231 cell lines and tissue microarrays (TMAs). IL32 gene expression levels were higher in MDA MB231 compared to MCF7. Analysis of TMAs showed 42% of TNBC tissues and 25% of the non-TNBC were positive for IL32, while non-malignant patient samples and all but one hyperplastic tissue sample demonstrated lower levels of IL32 protein expression.ConclusionData obtained from several publically available GEO datasets showed overexpression of IL32 gene in basal-like/TNBC samples compared to normal and luminal samples. In support of these data, analysis of TMA clinical samples demonstrated a particular pattern of IL32 differential expression. Considered together, these data suggest IL32 is a candidate suitable for further study.
Recent data show the IL32 gene has at least nine transcript variants. The aim of this current study is to characterize the different transcript variants based on the latest sequencing data deposited in the National Center for Biotechnology Information (NCBI) and determine which variants are responsible for the differential pattern of gene expression previously observed in MCF7 compared to MDA MB231 cell lines. Analyses of the nine transcript variants showed their sequences were incredibly similar. Other than variant 9, all of the variants differed from variant 1 by deletions. PCR analyses showed that the longer transcript variants contributed more to differential gene expression observed in the MDA MB231 compared to MCF7 cell lines. Because of the similarities between the variant sequences, when determining differential expression in the breast cell lines, investigators should consider strategies that target analyses of a combination of the longer IL32 transcript variants.
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