Her2 Breast Cancer Cells Form Super‐Enhancers which Indicate a New Mechanism of Aggression

Abstract

BackgroundHer2 positive breast cancer (BCa) occurs in 25–30% of cases and is associated with an aggressive phenotype. Multiple HER2‐stimulated pathways are known but the list of genes regulated by these events is poorly understood. We previously used RNA polymerase II (POL II) immunoprecipitation (ChIP‐chip) together with expression analysis in order to identify gene changes specifically regulated by the HER2 oncogene in cell lines and in breast cancer (BCa) tissues (1). We identified 737 genes that bind POL II in HER2 (+) breast cancer cell lines but not in HER2 (−) cell lines. Of 737 such genes “poised” for expression only in HER2(+) cell lines, 51 were differentially expressed (P < 0.05) in HER2+/− BCa cell lines. Of the remaining 686 poised genes 113 were statistically significantly differentially expressed in breast tumors in a HER2‐dependent manner using available expression arrays of 812 patients. The remaining 573 genes are not differentially expressed in cell lines nor in the 812 examined BCa cases termed “poised” genes.MethodsThe 737 genes were examined using pathway finding tools and the MIT database for Super‐enhancers. The expression of the breast cancer stem cell marker ALDH and the NOS genes in attached cell cultures and in mammosphere cultures were examined by qPCR.ResultsSuper‐enhancers (SE) are DNA structures formed by looping that associate multiple bound transcription and DNA‐modifying factors in proximity to target promoters. Many of the 113 genes products commonly occur in SEs. Examples include Mediator1 and the Bromdomain protein BRD2, and DNA binding factors such as HDAC and CREB1‐cofactors. Moreover, many of these genes have known associations with the pluripotency genes NANOG, OCT3/4, and SOX2 (NOS) which do not occur or are very low in HER2(−) cells and commonly interact with SEs as associated factors and targets. We confirmed by qPCR that the three NOS genes exhibit increased expression in mammosphere of MCF7 cells engineered to express HER2, but not in control cells.Using the MIT Super enhancer and dbSUPER (2) databases, we have identified 70 genes of the 113 group (62%) that encode factors that are located in or near SE in all available cell types in the database including HER2+ breast cancer cell line HCC1954. 33 (65%) of the 51 differentially expressed genes in HER2+/− cell lines were associated with SEs. In contrast, the class of 573 genes that are poised in HER2 (+) cells but not in HER2 (−) cells, and are not differentially expressed in breast cancer can be considered as controls for this enrichment: A much lower fraction of these genes 213 (37%)) encode potential members or targets of SE.Pathway analyses of the 113 genes indicates that major pathways enriched in these genes (p < 0.001) include EGFR and Map Kinase signaling, notch pathway signaling, hedgehog, Wnt, Integrin‐mediated cell and cytokine signaling (LI‐1, Il‐4, Interferon type 1).ConclusionThe expression of HER2 in breast cancer cells facilitates numerous gene expression changes including differential expression of 113 genes in tumor tissue and not in cell lines. Among these 113 genes 70 (62%) encode factors that are associated with super enhancers, whereas only 213 of the 573 (37%) poised genes encode factors associated with SEs. These observations suggest the hypothesis that HER2 regulates BCa via a variety of pathways including through regulation of Super Enhancers.Support or Funding Information1. NCI 1 U01 CA152738‐01 (PI: Mercola)