Aneuploidy produced by chromosome missegregation or errors in cytokinesis is a common characteristic of cancer cells. Here we propose three directions for identifying how aneuploidy is prevented in the normal situation and to test the consequences of single chromosome missegregation or spindle pole amplification in driving tumorigenesis. The mitotic checkpoint (or spindle assembly checkpoint) serves as the primary guard against chromosome missegregation in mammals. This cell cycle control mechanism delays anaphase onset until all chromosomes have attached to spindle microtubules. Unattached kinetochores are the central elements that produce a ?wait anaphase? signal which blocks premature destruction of cyclin B and securin by selectively inhibiting Cdc20 stimulated recognition of them by a multisubunit E3 ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC/C). Checkpoint silencing releases APC/CCdc20 for ubiquitination of securin and cyclin B1. Very recent studies have hypothesized that the joint action of the AAA+ ATPase TRIP13 and p31comet can mediate catalytic disassembly of the mitotic checkpoint inhibitor(s) and for initial mitotic checkpoint activation. Biochemical evidence has supported ATPase activity of TRIP13 as important for triggering conformation change of closed Mad2 to its inactive open form as a means to inactivate checkpoint signaling. Here we propose to use gene replacement with CRISPR-Cas9 genome editing to replace both endogenous TRIP13 alleles with ones encoding auxin-inducible degron tags so that TRIP13 can be inducibly eliminated at any cell cycle point. With these, we will determine the contribution of TRIP13 (and its ATPase activity), along with any contribution of p31comet to activating or silencing the mitotic checkpoint and in cell cycle advance. By replacing TRIP13 with a variant defective in ATP hydrolysis ? and thereby serving as a ?substrate trap? ? we will identify the substrates of TRIP13 throughout the cell cycle. Aneuploidy is frequent in human cancers. The linkage of aneuploidy to tumorigenesis has long been recognized. The great German cytologist Theodor Boveri initially proposed related hypotheses that aneuploidy drives tumorigenesis from missegregation of individual chromosomes or an aberrant mitosis caused by centrosome amplification. Using mice that missegregate chromosomes at high frequency from reduced levels of the centromere motor protein CENP-E, we showed in the previous grant period that whole chromosomal aneuploidy can facilitate tumorigenesis in some genetic contexts, but does not affect tumorigenesis caused by mutations in DNA repair, and delays tumorigenesis when combined with genetic lesions that also increase aneuploidy. We now will test how centrosome amplification affects tumorigenesis. Using a conditional mouse model in which extra centrosomes can be transiently induced, we will now determine whether centrosome amplification promotes 1) cellular transformation or 2) the formation of spontaneous tumors, 3) is capable of facilitating the development of carcinogen-induced tumors, and 4) is able to accelerate the development (or increase the aggressiveness or metastatic potential) of tumors driven by the loss of a tumor suppressor gene. Lastly, sequencing efforts from human tumors have uncovered a striking feature shared among a broad range of cancers in which whole or localized regions of chromosomes undergo gross rearrangements. These rearrangements, called chromothripsis or chromoanagenesis, are typically confined to one (or two) chromosomes, which appear to have been shattered into tens to hundreds of small genomic fragments and religated back together in random order. Recent sequencing experiments from human cells and genetic plant models suggest that chromothripsis is consequence of chromosomes initially missegregated into micronuclei. We propose now to test mechanisms of chromothripsis using an approach to generate missegregation of a specific chromosome (the Y) into micronuclei at high efficiency. By exploiting a unique feature of the human Y centromere, we have produced cells in which we can produce selective, transient inactivation of the Y centromere, with the Y chromosome missegregated into micronuclei at high frequency. We will use this approach to determine whether sustained and/or transient centromere inactivation can produce stably heritable chromothripsis from chromosomes fragmented within micronuclei and to determine the repair mechanisms underlying reassembly of fragment micronuclear chromosomes to generate chromothripsis.
The essential function of mitosis is the delivery of a complete set of chromosomes to each daughter cell. An abnormal chromosome number, aneuploidy, has long been linked to human tumors. The effort here will identify key steps through which the major cell cycle mechanism in mitosis, the mitotic checkpoint, acts to prevent chromosome missegregation. Additionally, amplification of centrosomes, the microtubule organizing centers of mitotic spindles, has also long been associated with tumorigenesis. Using mice in which extra centrosome replication can be transiently induced, it will be determined whether centrosome amplification promotes cellular transformation, the formation of spontaneous tumors and accelerates the development of tumors that develop from loss of a tumor suppressor gene. Finally, sequencing efforts from human tumors have uncovered that a frequent event in a broad range of cancers is chromothripsis, a phenomenon in which a chromosome appears to have been shattered into tens to hundreds of small genomic fragments and religated back together in random order. We propose now to test mechanisms of chromothripsis using an approach to generate missegregation of a specific chromosome (the Y) into micronuclei at high efficiency.
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