Over-expression of cell cycle kinases, cyclin D-Cdk4, cyclin E-Cdk2, and Aurora A kinase in estrogen-induced pre-neoplastic mammary lesions and tumors in the female ACI rat.

摘要

Breast cancer (BC) is the leading cause of cancer in women in developed countries. Common features of early human BC are cell cycle deregulation, centrosome amplification, chromosome instability, and aneuploidy. Chromosome instability, an incipient event in pre-malignant breast cells, may result from over-expression of mitotic kinases like Aurora A (Aur-A) and subsequent centrosome amplification. Since chromosome segregation and cell cycle progression are intimately linked, it is not surprising that over-expression of cell-cycle proteins, such as cyclins, may promote chromosome instability and aneuploidy. Aur-A and cyclins D1, D3, and E1 were examined in female ACI rats during estradiol-17beta (E2)-induced mammary oncogenesis. Low serum E2 levels (∼60-120 pg/ml) were sufficient to induce pre-malignant mammary gland lesions and tumors (MGTs) that remarkably resemble human ductal BC at the histopathologic and molecular levels. Small focal dysplasias were commonly seen at 3.0-3.6 months, large focal dysplasias, including atypical ductal hyperplasia at 3.6-4.3 months, ductal carcinoma in-situ (DCISs) at 4.3-5.0 months, and invasive ductal carcinomas at 5.0-6.0 months after E2-treatment. Western blot analysis of the E2-induced MGTs revealed a marked rise in the expression of centrosome proteins Aur-A, centrin, and gamma-tubulin, and cyclins D1, D3, and E1 compared to age-matched untreated controls. A significant elevation in cyclins D1, D3, and E1 kinase activity was detected by in-vitro kinase assays. Pre-malignant lesions of focal dysplasia and DCIS, and invasive ductal carcinomas exhibited over-expression of cyclins D1, D3, and E1, and centrosome amplification. However, cyclins D3 and E1 over-expression and centrosome amplification were confined essentially to early pre-malignant lesions and primary MGTs, with less than 1-5% of resting and normal hyperplasic breast cells staining positive. Semi-quantitative RT-PCR analysis of E2-induced MGTs revealed increased mRNA expression of Aur-A, cyclins D1, D3, and E1. However, using quantitative real-time Q-PCR, a significant amplification of cyclin E1 gene was detected. Thus, we conclude that low constant E2-treatment is causally linked to Aur-A over-expression, centrosome amplification, chromosome instability, and aneuploidy leading to BC in susceptible mammary gland cells. E2-induced pre-malignant lesions differentially and selectively express cyclins D3 and E1, contributing to the distinct growth advantage of these pre-neoplasias relative to E 2-elicited normal hyperplasia.