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

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Summary

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.

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Abbreviations

MMEC:

Mouse Mammary Epithelial Cells

MMTV:

Mouse Mammary Tumor Virus

DMEM:

Dulbecco's Modified Eagle's Medium

GTP:

Guanosine Triphosphate

SDS-PAGE:

Sodium Dodecylsulfate-polyacrylamide Gel Electrophoresis

E2 :

17β Estradiol

2-OHE1 :

2-Hydroxyestrone

16α-OHE1 :

16α-Hydroxyestrone

[C2-3H]E2 :

Estradiol labeled at C2 position

[C16α-3H]E2 :

Estradiol labeled at C16α-position

TDLU:

Terminal Duct Lobular Units

DMBA:

7,12 Dimethylbenz(a) Anthracene

NMU:

N-nitroso-N-methylurea

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Authors and Affiliations

  1. Breast Cancer Research Laboratory, Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, 10021, New York, NY, USA

    Nitin T. Telang & Michael P. Osborne

  2. Department of Molecular Genetics, Hoffman LaRoche Inc., Nutley, New Jersey, USA

    Ramaswamy Narayanan

  3. Institute for Hormone Research, New York, USA

    H. Leon Bradlow

Authors
  1. Nitin T. Telang
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  2. Ramaswamy Narayanan
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  3. H. Leon Bradlow
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  4. Michael P. Osborne
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Telang, N.T., Narayanan, R., Leon Bradlow, H. et al. Coordinated expression of intermediate biomarkers for tumorigenic transformation in RAS-transfected mouse mammary epithelial cells. Breast Cancer Res Tr 18, 155–163 (1991). https://doi.org/10.1007/BF01990031

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