Jessica Flory and Professor David Bearss, Physiology and Developmental Biology
Introduction
Nek2 is a kinase that promotes centrosome separation during the cell cycle. Our lab has shown that Nek2 over-‐expression is the best predictor of a poor outcome in multiple myeloma therapy, but the mechanism is unknown. The standard therapy for treating multiple myeloma is use of Bortezomib, an inhibitor of the proteasome (which degrades proteins). Mitosis is often triggered by the degradation of proteins that are unnecessary for the cell to divide. The evidence that Nek2 over-‐expression can predict the outcome of multiple myeloma therapy leads us to the hypothesis that Nek2 affects proteasome activity in multiple myeloma cells. The question we addressed is whether Nek2 is involved in regulating proteasome activity and what the mechanism of this regulation is. Multiple myeloma is a deadly cancer affecting the plasma cells in the bone marrow. It is incredibly difficult to treat, and the therapies that are available rarely lead to a permanent cure, so we are looking into new and better ways to treat patients.
Materials and Methods
Our lab has transfected HeLa (cervical), ARP1 (multiple myeloma), H1929 (multiple myeloma), and KMS28PE (multiple myeloma) cancer cell lines, one group with GFP and one group with Nek2. We cultured these cells in growth medium and lysed the cells using NP-‐40 cell lysis buffer for western blot analysis. The blots were probed with antibodies for Nek2, Pp1α (a protein of the proteasome), and beta-‐tubulin as a control. The proteasome was isolated from cell lysates by ultracentrifugation and its activity tested by the Proteasome-‐Glo Trypsin-‐Like Assay (Promega). Knock down of Nek2 and the proteasome substrates of Nek2 was achieved through use of siRNA. If we recapitulate knock down of Nek2 by knocking down its substrate, then this is the mechanism. Several known Nek2 inhibitors identified in our lab were also tested with these cells and the lysates were analyzed by Flo Cytometry (to measure apoptosis) and ADP Lite (for cell viability). This allowed us to take the first steps to identifying a new drug for multiple myeloma.
Results
This project is still continuing forward, but I expect that we will be able to confirm that Nek2 does up-‐regulate the protease and that through inhibiting Nek2 we may discover a novel target for multiple myeloma drug therapies. It is the hope that some Nek2 inhibitors we test will have positive results when used on cancer cell lines. Research from there will take these inhibitors into animal testing and then clinical trials.
Discussion
From this research, I will be a co-‐author on a paper to be published in Molecular Cancer Therapy that encompasses many years of research that I have contributed to. I also submitted an abstract to present at the American Association for Cancer Research (AACR), one of the most prestigious cancer research meetings in the country. Lastly, by performing this research I have furthered my career in cancer research and strengthened my knowledge of the field