Song-Tao Liu - US grants

Intellectual merit: The research objective of the project is to investigate the assembly, disassembly and function of a fundamentally important protein complex that prevents erroneous chromosome segregation. The genome of a eukaryotic cell is packaged into individual chromosomes. Faithful transmission of a full complement of chromosomes into daughter cells requires delicate control mechanisms during cell division (mitosis). Of these critical mechanisms, the mitotic checkpoint (or spindle assembly checkpoint) ensures that duplicated sister chromatids are not separated until they are properly attached by microtubules emanating from opposite spindle poles. At the molecular level, the checkpoint is executed by inhibiting an enzyme termed the Anaphase Promoting Complex/Cyclosome (APC/C) whose activation underlies anaphase onset and exit from mitosis. How the APC/C is inhibited during early mitosis is not yet resolved. A four-subunit Mitotic Checkpoint Complex (MCC), comprised of BUBR1 (MAD3 in yeast), BUB3, CDC20 and MAD2, was found to bind and inhibit the APC/C efficiently under physiologically relevant protein concentrations in cells. However, there have been continuous debates in the past decade concerning whether MAD2 is an integral component of the MCC. Partly due to this identity crisis', how MCC activity is regulated remains elusive. The PI's group has recently demonstrated that a specific conformation of MAD2 (C-MAD2) and a previously unknown direct interaction between BUBR1 and C-MAD2 are essential for the assembly and function of the MCC. The discoveries, made by graduate and undergraduate researchers, help resolve earlier inconsistencies and open the door to further characterization of the MCC. Three specific aims will be addressed in the proposal to investigate how MCC is assembled; how MCC inhibits APC/C; and how MCC disassembly is regulated. A combination of molecular, cellular, and biochemical approaches will be used in the project. Cell division is one of the most fundamental processes for life. If successful, the project will significantly advance current understandings of the control mechanisms for the timing of chromosome segregation. A coherent model of the MCC:APC/C interaction will further accelerate discoveries of additional factors that impact the fidelity of chromosome segregation through modulating MCC and APC/C activities.
Broader impact: The primary educational objective of the project is to retain aspiring young researchers in science careers through research-based engagement. The natural beauty of mitosis is appealing to graduate, undergraduate and high school students, but how to help young people to convert transient interest in science into lasting passion for research is still challenging. Level-appropriate career guidance and research activities will be bundled to engage students, particularly those from underrepresented groups, in scientific exploration. Different programs managed by the University of Toledo Office of Undergraduate Research (OUR-UT) and the UT High School Outreach Initiatives Office will help the PI to identify and recruit enthusiastic young researchers. The PI is also interested in exploring efficient channels to communicate scientific progress with the public. An interactive lab website and a potential museum exhibit are proposed as means to introduce their research to a broader audience. The results of proposed educational activities will not only support realization of specific research goals, but will also aid in design of effective curricula and activities to stimulate the interest in science careers among young people, and promote public understanding of science.

DESCRIPTION (provided by applicant): Aneuploidy and chromosomal instability (CIN) are remarkably common in solid tumors including breast cancers. Identifying and characterizing recurring genetic and epigenetic changes in aneuploid cancers will have tremendous impact on cancer prevention, diagnosis and treatment. TRIP13 was ranked as one of the top genes associated with CIN (#12 in the CIN70 signature), and its overexpression was also observed in breast cancers with poor prognosis in multiple microarray studies. However, the working mechanism of TRIP13 and the pathological significance of TRIP13 overexpression in breast cancer development remain unclear. Our preliminary work found that TRIP13, a member of the AAA-ATPases, is a novel kinetochore protein and directly interacts with mitotic checkpoint silencing protein p31comet. TRIP13 knockdown delayed metaphase-to- anaphase transition and significantly reduced breast cancer cell proliferation in 2D culture and soft agar plates. The objective of current proposal is to elucidate mitotic functions of TRIP13 and examine the relationship between TRIP13 overexpression and breast cancer development. The central hypothesis is that TRIP13 overexpression promotes untimely mitotic checkpoint silencing, driving CIN and cancer development in mammary epithelial cells. Three specific aims will test the central hypothesis. Aim 1 will assess biochemical functions of TRIP13 in the mitotic checkpoint. Specifically, we will test that TRIP13, through its interaction with p31comet and its ATPase activity disrupts protein complexes required for mitotic checkpoint signaling. In Aim 2, we will use inducible cell lines to modulate TRIP13 expression level and determine the impact on chromosomal stability and cell proliferation in 2D and 3D cell culture models. Aim 3 will examine the effects of TRIP13 overexpression or knockdown on breast cancer cell growth in xenografted mouse models. Together, the proposed work will test potential oncogenic functions of TRIP13 ATPase and assess the possibility to target TRIP13 as a novel means to control breast cancers. The work also addresses some critical knowledge gaps in mitotic checkpoint signaling studies. Dissecting TRIP13 functioning mechanisms represents an important step in our continuous efforts to understand and exploit aneuploidy and CIN in breast cancers.

PROJECT SUMMARY Maternal Embryonic Leucine Zipper Kinase (MELK) is listed among clinically used Mammaprint and Prosigna (PAM50) breast cancer signature genes. MELK overexpression has also been reported in other cancers and cancer stem cells. These studies suggested that MELK overexpression predicts poor survival of cancer patients. A small molecule MELK inhibitor OTS167 is currently tested in Phase I clinical trials. However, recent results questioned the specificity of MELK inhibitors, and CRISPR/Cas9 mediated MELK knockout suggested MELK is not an essential kinase for cell proliferation. This has posed a paradox: why then is MELK overexpressed in cancer cells? There is clearly knowledge gap and clinical urgency to better delineate MELK functions. Particularly, we still lack the understanding about MELK action at the individual cell level. Based on literature survey and preliminary data, we propose the central hypothesis that MELK regulates cell cortex stability during late mitosis and controls cell division symmetry. The hypothesis leads to the conceptual innovation that MELK overexpression might not affect proliferation of the mass of cancer cells, but could still exert functional significance through amplifying cancer stem cells by engaging symmetric cell division. The protein level, phosphorylation level and kinase activity of MELK all peak during mitosis. Previously we have identified MELK as a gene co-expressing with core centromere/kinetochore proteins. MELK is also a top-ranking chromosomal instability (CIN) signature gene. We reason that MELK does have some functions during mitosis, and in proper contexts its mitotic activities may impact cell fates. Stem cells including cancer stem cells normally go through asymmetric cell division. However, in certain situations stem cells also divide symmetrically to increase the pool of stem cells. The central hypothesis will be tested in three specific aims. Aim 1. To elucidate the spatio- temporal control of MELK activity during mitosis; Aim 2. To test whether MELK amplifies cancer stem cells by regulating cell division symmetry; Aim 3. To assess surrogate markers for MELK inhibition. The project is expected to bridge mechanistic insights into MELK cell biology and its known overexpression in breast cancers. The project is expected to identify at least one urgently needed surrogate marker to evaluate the efficacy of MELK inhibition in cells. It will be collaborated with several experts and will become a perfect training platform for undergraduate and graduate students in the Department of Biological Sciences at the University of Toledo.