Chromosomal segregational defects are a common feature of oral and other cancer cells and play an important role in destabilizing the genome during carcinogenesis. One of the best characterized segregational defects is the formation of multipolar spindles. When the spindle has more than two poles, the chromosomes cannot be separated equally, consequently, there is a difference between the numbers of chromosomes inherited by the two daughter cells. Altering chromosome number results in a change in the copy numbers of genes promoting or inhibiting the cancer cell phenotype. Multipolar spindles are a consequence of changes in both the replication and organization of the spindle pole. Spindle organization depends on an interaction between the centrosomal protein NuMA and the microtubule motor cytoplasmic dynein, in oral cancer cells, as well as other types of tumor cells, dynein is missing from the spindles. In some, but not all cases, dynein is displaced by overexpression of NuMA. When NuMA is knocked down by siRNA, dynein returns to the spindle and multipolarity is corrected. Thus, displacement of dynein is shown to be a major cause of spindle multipolarity in the tested cancer cells. Loss of dynein alone is not sufficient to cause multipolar spindles. When NuMA is overexpressed, or dynein is inhibited by overexpression of an associated peptide, multipolarity is not induced unless the cells have over-replicated centrosomes. The long-term objective of this proposal is to test the hypothesis that multipolar spindles and the consequent aneuploidy seen in tumor cells result from a two-step process involving centrosome overeplication and separation.
The Specific Aims of this proposal are to test and confirm this model for spindle multipolarity, determine if NuMA can act to inhibit dynein in cells and in solution, and test the significance of multipolar spindles and NuMA overexpression on chromosomal instability and aneuploidy. Elucidation of the etiology behind multipolar spindle formation and the consequent chromosomal instability is critical to our understanding of the biology of the cancer cell phenotype. The results of this study may lead to targeted methods for prevention, early detection, therapy, and/or eradication of tumor cells harboring multipolar spindles.
