Abstract 1982: The HER2 Regulon:Identification of 113 genes that are directly controlled by HER2 and define four nodes of cancer stem cell networks

Abstract

Abstract We recently developed a combination method using RNA polymerase II immunoprecipitation together with expression analysis in order to identify genes regulated by the HER2 oncogene: the HER2 Regulon. We identified 737 genes which are poised for transcription. 113 of these genes are differentially expressed (p < 0.05) in HER2+/- as determined by analysis of available expression arrays of 812 breast cancer tissues. Thus for these genes, expression may depend upon features of the in situ organization and/or features of the microenvironment in addition to the action of high HER2. We further noted that 58/113 genes are members of pathways related by four “nodes”: NANOG, SOX2, OCT3/4, and CREB1.We hypothesize that high HER2 expression and factors of breast cancer cells in situ, possibly including microenvironment factors, determine the concerted expression of the four node proteins and the 58 associated genes. These genes may participate in the formation of CSC cells or cells with enhanced “stemness” in high HER2-expressing breast cancer. Methods The HER2+/- breast cancer cell lines were grown in medium supplied with FBS, the sub-cultures were depleted from FBS and cultured on ultra-low adherent plate supplemented with EGF/FGF/B27. The formed spheres were collected and used for expression analysis of ALDH1, SOX2, NANOG and OCT3/4 and 58 microenvironment dependent genes employing quantitative real time PCR. Immunofluorescence staining was used to measure protein expression levels of ALDH1, NANOG, SOX2 and OCT3/4. Results We used PCR to compare the amount of transcripts formed in high- and low-HER2 expression cells both as attached cultures and as “mammospheres”. We observed all four node transcript levels were strikingly higher in mammospheres of HER2-expressing cells compared to attached cultures or compared to HER2-null control cells. We used immunofluorescence to confirm that the corresponding protein levels were also increased. Thus, in the context of mammospheres, node genes exhibit HER2-dependent and greatly increased expression analogous to the conditions required for the 113 genes. This observation suggests that the anchorage-independent growth mode of mammospheres growing in factor-supplemented medium may replicate key features of cancer cells in situ not observed in attached cultures. If so, mammospheres may provide an in vitro model for the dissection of the key in situ and microenvironment features influencing HER2-dependent expression in breast cancer. Conclusion A “poised” class of genes in HER2+ cell lines has POLII binding and low RNA expression and is differentially expressed in primary tumors in a HER2-dependent manner. These genes include key genes with a role in stem cell proliferation. These genes become expressed when cell lines are grown as three dimensional mammospheres. These observations strongly suggest a role of 3D structure. Note: This abstract was not presented at the meeting. Citation Format: Farah Rahmatpanah, Yuanjie Hu, Xin Chen, Michael McClelland, Frank Jones, Dan Mercola. The HER2 Regulon:Identification of 113 genes that are directly controlled by HER2 and define four nodes of cancer stem cell networks. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1982. doi:10.1158/1538-7445.AM2015-1982