Terminally differentiated cells are largely post-mitotic, but can become dedifferentiated under certain conditions, which can lead to the development of cancer. Much emphasis has been placed on understanding the mutations enabling dedifferentiation, but less is known about the mechanisms of preventing dedifferentiation. The centrosome presents an attractive target for investigation the prevention of dedifferentiation due to its role in cell cycle regulation and its functional absence in skeletal muscle fibers, which are highly resistant to dedifferentiation. Despite studies demonstrating the loss of some centrosomal proteins during myogenic differentiation, the mechanism mediating this loss and the ultimate fate of the centrosome as a physical structure remains unclear. However, preliminary studies suggest the potential for the presence of centrioles during terminal differentiation. Therefore, we hypothesize that functional, rather than physical, loss of the centrosome is a key step in preventing the dedifferentiation of differentiated cells.
Two specific aims are addressed in this proposal: (1) Determine the role of the centrosome in dedifferentiation; (2) Determine the mechanism of centrosome loss during differentiation with respect to the (a) necessity and (b) sufficiency of the candidate proteins for mediating functional centrosome loss, HDAC6, Aurora A kinase, and MST1/STK4. Through the use of microscopy, pharmacological manipulation, and genome engineering, we will investigate the status and role of the centrosome during differentiation and dedifferentiation in immortalized and primary murine myoblasts and in newt myoblasts, which are able to dedifferentiate and proliferate in response to muscle damage. To address the necessity and sufficiency of the candidate proteins, we will use genome engineering to generate conditionally stabilized and inducible variants of the proteins to manipulate functional centrosome loss during differentiation and to examine their effects on purified, functional centrosomes in an in vitro microtubule-organizing assay. The results of these investigations will identify novel pathways for the treatment of cancer.
As part of the progression of cancer, cancerous cells may acquire multiple functional centrosomes and can differentiate to a more plastic state. Mature skeletal muscle, however, is very resistant to dedifferentiation and may not possess functional centrosomes. Characterization of the role of the centrosome in dedifferentiation of skeletal muscle will identify novel targets for the treatment of cancer.