The objective of this study is to determine the interaction between Plk1 and B-Raf in melanocytic cells with the idea that this interaction plays a critical role in melanocytic transformation. Constitutively active oncogenic B-RafV600E mutations play an important role in melanomagenesis. However, B-Raf mutation alone are insufficient for melanocytic transformation, suggesting the involvement of other factors. Synthetic lethality, derived from the concept of `gene interaction', is gaining popularity towards developing new means and targets for certain diseases, including neoplastic conditions. Two genes are said to be synthetic lethal if mutation of either of them alone leaves the cell viable, while simultaneous modulations leads to a different fate for the cells. Thus, one gene buffers the effect of changes in the other to compensate for the effect of its deletion. This concept can help in identifying i) the buffering relationships among interacting genes, and ii) the malfunction and diseases that may occur when these relationships are altered. Exploring these relationships may lead to information how cellular processes work when the protein products expressed by two different genes have an effect together but not separately. Identification of interacting partner of B-Raf will provide a better understanding of mechanism of melanocytic transformation as well as identification of novel approaches to combat melanomagenesis. Polo-like kinase 1 (Plk1) plays pivotal roles in multiple aspects of cell division (mitosis). We have shown that compared to normal melanocytes, Plk1 is overexpressed in transformed cells and its targeted inhibition causes mitotic catastrophe and induction of apoptosis in melanoma cells, suggesting that Plk1 possibly plays an important role in melanocyctic cell proliferation. Significantly, our preliminary data show that melanocytic cells harboring B-Raf are hypersensitive to Plk1 inhibition than cells with wild type (WT) B-Raf, and that inhibition of B-RafV600E decreases cellular response to Plk1 inhibition. Based on our published and preliminary studies and available literature, in this application, we propose to challenge our central hypothesis that Plk1 acts as a synthetic lethality interaction partner of the oncogenic B- Raf. The following specific aims are proposed: 1) To determine the association between B-Raf mutation and Plk1 expression during melanocytic transformation; 2) To determine the extent to which the oncogenic B-Raf mutation enhances the sensitivity of the cellular response to mitotic stress, 3) To compare Plk1 loss-of-function phenotypes in melanoma cells with different B-Raf backgrounds; and 4) To assess the effects of Plk1 inhibition in a xenograft model with varying B-Raf. Outcome of this study is expected to enhance our understanding of mechanism of melanocytic transformation via dissecting interaction between Plk1 and B-Raf. This will be useful in designing novel strategies against proliferative melanocytic disorders, including melanomagenesis.
These studies proposed in this application will contribute to our understanding of the role of Plk1 and its interaction with B-Raf in melanocytic cells. Outcome of this study is expected to enhance our understanding of mechanism of melanocytic transformation via dissecting interaction between Plk1 and B-Raf. This knowledge will be useful in designing novel strategies against hyperproliferative melanocytic disorders, including melanomagenesis
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