Reproductive history determines ErbB2 locus amplification, WNT signalling and tumor phenotype in a murine breast cancer model [RNA-seq]

Understanding the mechanisms underlying tumor heterogeneity is key to development of treatments that can target specific tumor subtypes. We have previously targeted CRE recombinase-dependent conditional deletion of the tumor suppressor genes Brca1, Brca2, p53 and/or Pten to basal or luminal ER- cells of the mouse mammary epithelium. We demonstrated that both the cell-of-origin and the tumor-initiating genetic lesions co-operate to influence mammary tumor phenotype. Here, we use a CRE-activated HER2 orthologue to specifically target HER2/ERBB2 oncogenic activity to basal or luminal ER- mammary epithelial cells and carry out a detailed analysis of the tumors which develop. We find that in contrast to our previous studies, basal epithelial cells are refractory to transformation by the activated NeuKI allele, with mammary epithelial tumor formation largely confined to luminal ER- cells. Histologically, the majority of tumors that developed were classified as either adenocarcinomas of no special type or metaplastic adenosquamous tumors. Remarkably, the former were more strongly associated with virgin animals and were typically characterised by amplification of the NeuNT/ErbB2 locus and activation of non-canonical WNT signalling. In contrast, tumors characterised by squamous metaplasia were associated with animals that had been through at least one pregnancy and typically had lower levels of NeuNT/ErbB2 locus amplification but had activated canonical WNT signalling. Squamous changes in these tumors were associated with activation of the Epidermal Differentiation Cluster. Thus, in this model of HER2 breast cancer, cell-of-origin, reproductive history, NeuNT/ErbB2 locus amplification, and the activation of specific branches of the WNT signalling pathway all interact to drive inter-tumor heterogeneity. Overall design: Mice carrying an engineered active orthologue of the HER2/ERBB2 gene knocked into the endogenous locus but with expression blocked by a FLOX-STOP-FLOX cassette were crossed with mice carrying the Beta-lactoglobulin promoter-driven CRE recombinase transgene, which targets CRE expression to the luminal cells of the mammary gland. Some mice were put through one or more rounds of pregnancy. Ten tumors which developed in the mammary glands of ten mice were snap frozen and processed for gene expression analysis by RNA-seq. One sample failed QC. Non-negative matrix factorization was used to cluster RNAseq data to determine which characterstic (reproductive history, tumor diagnosis or amplification status of the Erbb2/Neu locus) accounted for the greatest difference between samples. Amplification status of the Erbb2/Neu locus was selected as giving the greatest difference between samples, with one sample, B67_1006, proving to be an outlier. Therefore, differential expression comparing high amplification against non/low amplification samples was carried out, excluding B67_1006.