Human breast cancer cells generated by oncogenic transformation of primarymammary epithelial cells

A number of genetic mutations have been identified in human breast cancers, yet the specific combinations of mutations required in concert to form bre ast carcinoma cells remain unknown. One approach to identifying the genetic and biochemical alterations required for this process involves the transfo rmation of primary human mammary epithelial cells (HMECs) to carcinoma cell s through the introduction of specific genes. Here we show that introductio n of three genes encoding the SV40 large-T antigen, the telomerase catalyti c subunit, and an H-Ras oncoprotein into primary HMECs results in cells tha t form tumors when transplanted subcutaneously or into the mammary glands o f immunocompromised mice. The tumorigenicity of these transformed cells was dependent on the level of ras oncogene expression. Interestingly, transfor mation of HMECs but not two other human cell types was associated with ampl ifications of the c-myc oncogene, which occurred during the in vitro growth of the cells. Tumors derived from the transformed HMECs were poorly differ entiated carcinomas that infiltrated through adjacent tissue. When these ce lls were injected subcutaneously, tumors formed in only half of the injecti ons and with an average latency of 7.5 weeks. Mixing the epithelial tumor c ells with Matrigel or primary human mammary fibroblasts substantially incre ased the efficiency of tumor formation and decreased the latency of tumor f ormation, demonstrating a significant influence of the stromal microenviron ment on tumorigenicity. Thus, these observations establish an experimental system for elucidating both the genetic and cell biological requirements fo r the development of breast cancer.