An oestrogen-dependent model of breast cancer created by transformation of normal human mammary epithelial cells

Introduction About 70% of breast cancers express oestrogen receptor a ( ESR1/ ER alpha) and are oestrogen-dependent for growth. In contrast with the highly proliferative nature of ER alpha-positive tumour cells, ER alpha-positive cells in normal breast tissue rarely proliferate. Because ER alpha expression is rapidly lost when normal human mammary epithelial cells ( HMECs) are grown in vitro, breast cancer models derived from HMECs are ER alpha-negative. Currently only tumour cell lines are available to model ER alpha-positive disease. To create an ER alpha-positive breast cancer model, we have forced normal HMECs derived from reduction mammoplasty tissue to express in combination with other relevant breast cancer genes.

Methods Candidate genes were selected based on breast cancer microarray data and cloned into lentiviral vectors. Primary HMECs prepared from reduction mammoplasty tissue were infected with lentiviral particles. Infected HMECs were characterised by Western blotting, immunofluorescence microscopy, microarray analysis, growth curves, karyotyping and SNP chip analysis. The tumorigenicity of the modified HMECs was tested after orthotopic injection into the inguinal mammary glands of NOD/ SCID mice. Cells were marked with a fluorescent protein to allow visualisation in the fat pad. The growth of the graft was analysed by fluorescence microscopy of the mammary glands and pathological analysis of stained tissue sections. Oestrogen dependence of tumour growth was assessed by treatment with the oestrogen antagonist fulvestrant.

Results Microarray analysis of ER alpha-positive tumours reveals that they commonly overexpress the Polycomb-group gene BMI1. Lentiviral transduction with ER a, BMI1, TERT and MYC allows primary HMECs to be expanded in vitro in an oestrogen-dependent manner. Orthotopic xenografting of these cells into the mammary glands of NOD/ SCID mice results in the formation of ER alpha-positive tumours that metastasise to multiple organs. The cells remain wild type for TP53, diploid and genetically stable. In vivo tumour growth and in vitro proliferation of cells explanted from tumours are dependent on oestrogen.

Conclusion We have created a genetically defined model of ERa-positive human breast cancer based on normal HMECs that has the potential to model human oestrogen-dependent breast cancer in a mouse and enables the study of mechanisms involved in tumorigenesis and metastasis.