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Characterization of a Novel Protein Implicated in Breast Cancer Progression
Centrosomes play critical role in processes that ensure proper segregation of chromosomes and maintain the genetic stability of human cells. We have recently identified a novel protein that locates to the centrosome, which hereafter will be referred to as CMAP (centrosome- and midbody-associated protein). Characterisation of cells depleted of CMAP by siRNA reveals that CMAP participates in cytokinesis. The EST corresponding to CMAP, FLJ10540, has recently been shown to be consistently upregulated in invasive ductal carcinoma relative to ductal carcinoma in situ using micro-array technology. We propose to further characterise the functions of CMAP and define its mechanisms of action in breast cancer progression.
Hypothesis: CMAP plays a role in cellular processes that allow normal division and prevent aneuploidy.
Aim 1: To determine CMAP mitosis-specific phosphorylation sites and to determine the effect of this modification on its function. We have shown that CMAP is localised at the centrosomes and moves to midbody during the telophase. CMAP is also phosphorylated during entry of cells into mitosis, which we think might be important for microtubule nucleation. We will map the mitosis specific site (s) of phosphorylation in CMAP and identify the mitotic kinase that phosphorylates CMAP.
Aim 2: To characterise CMAP functions in cell. The major function of centrosomes in the cell is to nucleate microtubules to set up the mitotic spindle. To determine if CMAP is required for microtubule nucleation we will compare the centrosomes of GFP siRNA and CMAP siRNA expressing U2OS cells for their ability to nucleate microtubule arrays by performing in vivo microtubule nucleation assays. We will also investigate the role of CMAP in completion of cytokinesis.
Aim3: To determine the role of CMAP in breast cancer progression. We will examine CMAP expression in collection of breast cancer specimens that have associated detailed pathology, patient outcome and are already characterized for other prognostic markers. This will establish to what degree CMAP expression is linked to breast cancer progression.
Relevance: Proteins associated with centrosomes have been shown to play a major role in the cell division and centrosome abnormalities have long been recognised as distinguishing feature of cancer cells. This proposal will determine whether upregulated expression of a centrosome protein can be used as marker for development of more aggressive phenotype during tumor progression. Ultimately, this will have important implications for breast cancer prognosis, and treatment.
Background: Cell division is a highly regulated process involving many components to produce two daughter cells, which contain an equal amount of DNA. The incorrect expression and localization of proteins that regulate this process cause cell division errors, which underlie various human diseases in particular cancer. We have recently identified a novel protein called CMAP that is involved in the final stages of cell division, which involves the cleavage of cell memberane to produce two daughter cells. There is some evidence that CMAP is over-expressed in high grade breast cancers.
Hypothesis: CMAP plays an important role in controlling normal cell division and prevents cancer.
Aims and Study Design: In this proposal, experiments are planned to characterize the normal functions of CMAP in cells using a series of well established as well as novel methodologies. Experiments are also planned to study the level of expression of CMAP in breast tumor samples at various stages of disease
to understand its role in breast cancer progression.
Relevance: Defects in cell division underlie a number of major diseases like cancer and understanding this process has enormous implications for breast cancer.