Changes in chromosome number in cancer cells can have drastic effects on proliferation, cell death, and drug resistance. The vast majority of tumors are aneuploid, yet the mechanisms responsible are not completely known. The mitotic checkpoint ensures equal chromosome segregation to daughter cells, but still functions in many human tumor-derived cell lines. More likely, subtle defects in the strength or fidelity of the checkpoint may underlie aneuploidy. Cancer cells are defined by mis-regulation of growth factor pathways and associated signal transduction machinery. We have detected a requirement for the signal transducing kinase Gsk3 in the mitotic checkpoint. Gsk3 is required for the recruitment of checkpoint proteins Mad2 and BubR1 to kinetochores and for formation of the mitotic checkpoint complex (MCC) that keeps cells in mitosis until chromosomes properly attach to the spindle. We also observed that Gsk3 did not affect the mitotic checkpoint in cells lacking the deacetylase SIRT2 indicating a possible target. Gsk3 activity is silenced by a number of growth factor pathways, with the WNT pathway being one of the best studied examples. WNT activation enhances aneuploidy in some experimental systems. Given that WNT signaling is elevated in many cancer cells, Gsk3 may provide a link between this pathway, the strength of the mitotic checkpoint and aneuploidy. We propose three specific aims to determine the mechanism by which Gsk3 regulates the mitotic checkpoint and the linkage between WNT signaling, growth factor signaling, Gsk3 activity and mitotic regulation.
AIM 1. REGULATION OF MCC BY GSK3. Inhibiting Gsk3 disrupts the MCC composed of Mad2, Cdc20, BubR1 and Bub3 and reduces Mad2 and BubR1 at kinetochores. We plan to analyze the kinetochore localization of additional key checkpoint proteins and to determine whether Gsk3 regulates assembly or disassembly of the MCC. These experiments will help to determine the mechanism by which Gsk3 regulates the mitotic checkpoint apparatus.
AIM 2. PROXIMAL TARGETS OF GSK3 IN THE MITOTIC CHECKPOINT. The kinetochore effects of Gsk3 may be several steps away from direct Gsk3 substrates. SIRT2 is one potential target of Gsk3 in the checkpoint response. We will analyze effects of specific mutants of SIRT2 and analyze SIRT2 deacetylase activity to determine whether this protein acts downstream of Gsk3. Axin2 and Adenomatous polyposis coli also regulate the mitotic checkpoint and interact with Gsk3. Additional experiments will determine whether these proteins are responsible for the effects of Gsk3 on mitosis.
AIM 3. MODULATION OF MITOSIS BY UPSTREAM REGULATORS OF GSK3. Regulation of the mitotic checkpoint by Gsk3 potentially links many signal transduction pathways upregulated in cancer with a core process controlling chromosome stability. To investigate this linkage, WNT signaling will be disrupted followed by analysis of the mitotic checkpoint. We will also determine whether Akt, another signal transducer that regulates Gsk3 can modify the strength of the mitotic checkpoint.
More than half a million people in the US die every year due to cancer, a disease characterized by uncontrolled cell division, and defects in the segregation of chromosomes. Losses and gains of whole chromosomes in cancer cells is a common characteristic that is not understood at the molecular level, yet contributes to cancer progression and malignancy. Our research has uncovered a new connection between a large number of growth regulator pathways and the fidelity of mitotic chromosome segregation, and will help to understand the processes responsible for cancer formation and drug resistance.
