Centrosome Amplification: A Potential Marker of Breast Cancer Agressiveness

Maintenance of normal breast epithelial cells depends on the balance between mitogen and tumor suppressor signaling pathways. One of the mechanisms responsible for the origin of phenotypic heterogeneity commonly observed in breast tumors is the development of centrosome amplification leading to mitotic spindle abnormalities and consequent chromosomal instability. However, how mitogen and tumor suppressor pathways are mechanistically coupled to the centrosome cycle in breast cancer cells has not been established. In this study we show that in MCF-7 cells, hormone withdrawal led to arrest of DNA replication and centriole duplication, inhibition of G1/S cyclins activity and retinoblastoma (Rb) hypo-phosphorylation. Stimulation of arrested cells with 17-β estradiol, EGF and IGF-I, led to their orderly progression through the cell cycle characterized by the sequential expression of G1/S cyclins, progressive Rb phosphorylation and the timing of centriole duplication corresponding to G1/S transition. In contrast, MCF-7 cells with abrogated p53 function (vMCF-7) showed uncoupling of DNA replication and centriole duplication following hormone withdrawal. After mitogen stimulation, these cells showed shortened G1/S cell cycle progression compared to the parental cell line. Importantly, while normal timing of cyclinD1 expression was maintained in these cells, their accelerated cell cycle progression was linked to premature expression of cyclin E and A, Rb hyper-phosphorylation and centrosome amplification. Furthermore, these cells also showed a high frequency of pseudobipolar and multipolar mitotic spindles. In conclusion, this study demonstrates that mitogen-signaling pathways coordinate centrosome duplication and cell cycle progression through the integrity of p53 function in hormone dependent breast cancer cells. Introduction The progression of human breast cancer from an estrogen dependent to an estrogen independent phenotype represents a major clinical problem that limits the long term usefulness of endocrine therapeutic strategies (1, 2). Among estrogens, 17-β estradiol is the major promoter of cell proliferation in both normal and neoplastic breast epithelium through its binding to high-affinity estrogen receptor (ERα) (3, 4). ERα functions as an estrogen activated transcription factor and mediates the stimulation of estrogen target genes involved in the regulation of cell proliferation and inhibition of apoptosis of the breast epithelium (5-7). Excessive stimulation of the ERα pathway due to increased hormonal secretion, prolonged exposure to estrogens or increased levels of the receptor may lead to deregulation of cell proliferation and thus increase the risk to develop breast cancer (8, 9). In addition to estrogens, growth factors, such as EGF and IGF-I act in an autocrine and/or paracrine fashion to induce the proliferation of breast epithelial cells (10). It has been demonstrated that aberrant cross-talk between estrogens and growth factors signaling pathways play an important role in the development and progression of breast cancer (11-13). Chromosomal instability represents a hallmark of breast cancer and is responsible for the evolution of cancer cells with more aggressive behavior (14, 15). Recent studies show that development of centrosome amplification drives chromosomal instability and the consequent phenotypic heterogeneity of breast tumors, highlighting the centrosome as a key organelle in the control of chromosomal stability (15-17). The correct timing of centrosome duplication during cell cycle progression plays a critical role in the maintenance of a diploid karyotype (18, 19). In order to ensure the formation of a bipolar mitotic spindle leading to equal chromosome segregation during cytokinesis, centrosome duplication must be strictly coordinated with DNA replication during the G1/S progression of the cell cycle generating no more than two centrosomes at the end of the G2 phase (20-22). Breast tumors as well as breast cancer cell lines often show multiple centrosomes (centrosome amplification) linked to the formation of aberrant mitotic figures (23, 24). Aberrant mitoses resulting in unbalanced chromosome segregation, promote the generation of aneuploid cancer cells (25, 26). Inhibition of centrosome reduplication depends on the balance between oncogene and tumor suppressor activities (27). The tumor suppressors p53 and retinoblastoma inhibit centriole over-duplication (28, 29), while over-expression of cyclin/cdk2 complex and the centrosome associated kinases Aurora-A and PLK1 promote the development of amplified centrosomes (30-34). Recent studies also demonstrate that anticancer genotoxic agents induce centrosome amplification and chromosomal instability due to the uncoupling of DNA replication and centriole duplication in cancer cells lacking p53 function (35, 36). These findings suggest that p53 controls centrosome homeostasis through activation of the G1/S cell cycle checkpoint that monitors genomic integrity. However, whether p53 also plays a role in monitoring centrosome duplication following mitogen stimulation has not been established. In this study, we investigated the role of 17-β estradiol, EGF and IGF-I in the regulation of the timing of centrosome duplication in the MCF-7 breast cancer cells with endogenous wild-type p53, or in these cells engineered to over-express a mutant p53 construct (vMCF-7). Our findings demonstrate that in hormone stimulated MCF-7 cells with wild-type p53, centriole duplication is well coordinated with DNA replication. On the contrary, hormone stimulated MCF-7 cells with abrogated p53 function showed a deregulated G1/S cell cycle progression characterized by premature expression of cyclin E and A and Rb hyper-phosphorylation. Interestingly, these cells also developed centrosome amplification leading to an increased number of pseudobipolar and multipolar mitotic spindles. In conclusion, the results presented here highlight an important role for p53 in maintaining centrosome homeostasis and mitotic fidelity in hormone stimulated breast cancer