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Functional Genomic Analysis of Centrosome Organization: a New Approach for Breast Cancer Therapeutics
Background: Effective cancer therapeutics must exploit biological differences between tumor cells and the normal cells from which they arose. One striking feature of solid tumors, specifically breast cancer, is the presence of extra centrosomes, the major microtubule organizing centers in cells. Cells containing multiple centrosomes have a propensity to undergo a catastrophic multipolar mitosis that leads to chromosome gain and loss, and cell death. To survive, tumors containing extra centrosomes often achieve bipolar mitosis by clustering additional centrosomes into two groups. This centrosome clustering is a remarkable phenomenon and is poorly understood at a mechanistic level. Objective/Hypothesis: The defining feature of many breast tumors is the presence of extra centrosomes. Thus, drugs that induce multipolar mitoses might selectively kill breast cancer cells. A first step towards creating such a drug would be to identify genes that promote bipolar mitosis in the presence of extra centrosomes. Specific aims/Study design :(1) Which genes prevent multipolar mitosis in cells with extra centrosomes? I will perform a genome-wide RNAi screen in Drosophila S2 cells at the Drosophila RNAi Screening Center at Harvard Medical School to identify all genes that if inhibited result in multipolar mitoses. (2) I will determine whether the genes identified in (1) are required for centrosome clustering in p53-/- tetraploid mouse mammary epithelial cells (MMECs) or in human cancer cell lines containing extra centrosomes using shRNA knock down. Special emphasis will be given to genes that are essential for viability in tetraploid MMECs containing extra centrosomes but not in diploid MMECs. (3) I will determine if the genes identified in (2) are required for breast cancer development using a recently developed mouse model where tumorigenesis is initiated by blocking cytokinesis in primary p53-/-MMECs, generating tetraploid cells that contain extra centrosomes. siRNAs that inhibit centrosome clustering will be introduced into p53-/-diploid and tetraploid MMECs by lentivirus vectors to test the effects on tumor development . Potential outcomes: Understanding the mechanisms of centrosome clustering may open new avenues for therapeutics because cancer cells depend on these mechanisms to proliferate in the presence of extra centrosomes while normal cells do not. I have outlined a genome-wide strategy to identify novel targets for the development of new breast cancer therapies.
Breast cancer is a leading cause of cancer deaths in women worldwide. There have been important advances in the early diagnosis of breast cancer and in the development of some targeted treatments. However, more effective therapies, especially for advanced stage breast cancers remains a major medical challenge. The basis for any effective breast cancer therapy must be a biological difference between the tumor cells and the normal breast cells from which they arise. One obvious difference between tumor and normal cells is that tumor cells often contain increased numbers of a cellular structure called the centrosome, the major microtubule organizing center in cells. The centrosome organizes the machinery that distributed the chromosomes in a dividing cell. Normal dividing cells have two centrosomes that help distribute chromosomes to the two daughter cells after division (a bipolar mitosis). Many breast cancer cells have more than two centrosomes. This creates the potential that during cell division, the chromosomes will be pulled in multiple directions and that the dividing cell will therefore die (a multipolar mitosis). Surprisingly, cancer cells usually divide effectively even with extra centrosomes. Only rarely they undergo a multipolar mitosis that would lead to genetic changes in daughter cells. They accomplish this by "clustering" their extra centrosomes into two groups (a bipolar distribution), thus dividing cells in two. How centrosome clustering is achieved is not understood at a mechanistic level. The goal of this proposal is to identify all of the genes required to cluster extra centrosomes in a model cell system. We will then determine if these genes are required to cluster centrosomes in breast epithelial cells and in breast cancer cells. Finally, using a new breast cancer mouse model recently developed by this laboratory, I will determine if these genes are required for the development of breast cancer. The goal of this work is to identify potential targets for the development of novel breast cancer therapeutics. Because centrosome clustering is a unique phenomenon observed in tumor but not normal cells, proteins required for clustering centrosomes could be highly selective targets for chemotherapeutic attack.