Coordinated expression of intermediate biomarkers for tumorigenic transformation in RAS-transfected mouse mammary epithelial cells

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Abstract
Deregulated expression of the RAS oncogene is associated with tumorigenic transformation of mammary cells. Because of the complex, multiphasic nature of cancer progression, it is important to systematically identify the biomarkers specific for initiation, promotion, and progression of breast cancer. Mouse mammary epithelial cells (MMEC) were transfected with normal c-Ha-RAS proto oncogene (pH06N) and with mutant c-Ha-RAS oncogene (pH06T). The parental MMEC and the cloned transfectants pH06N1, pH06N2, pH06T1, and pH06T12 were evaluated for the acquisition of transformed characteristics by determining altered cellular metabolism of estradiol, increased ability for anchorage-independent growth, and ability to form tumors at the transplant site in athymic ‘nude’ mice. Persistent, functional integration of c-Ha-RAS was evidenced by the presence of a 1.2 kb c-Ha-RAS transcript in the four transfectants but not in MMEC. All the transfectants also exhibited a substantial increase in the binding of c-Ha-RAS p21 to [α-32P] GTP relative to MMEC (P<0.003). The relative extent of estradiol metabolism leading to the formation of 16α-hydroxyestrone was increased (P<0.004) in all the four transfectants. These four transfectants also showed a 100–400 fold increase in colony forming efficiency in 0.33% agar, relative to MMEC (P<0.0009), and formed rapidly growing tumors within 3–5 weeks of transplantation. Our results demonstrate that i) persistent expression of normal and mutant c-Ha-RAS can bring about tumorigenic transformation of mouse mammary epithelial cells; and ii) alteration in estradiol metabolism and acquisition of anchorage-independent growth precede the emergence of a tumorigenic phenotype. These endpoints therefore may constitute useful intermediate biomarkers to examine oncogene-induced tumorigenic transformation of mammary epithelial cells.