Cdk1 inactivation terminates mitotic checkpoint surveillance and stabilizes kinetochore attachments in anaphase

Two mechanisms safeguard the bipolar attachment of chromosomes in mitosis. A correction mechanism destabilizes erroneous attachments that do not generate tension across sister kinetochores [1]. In response to unattached kinetochores, the mitotic checkpoint delays anaphase onset by inhibiting the anaphase-promoting complex/cyclosome (APC/CCdc20) [2]. Upon satisfaction of both pathways, the APC/CCdc20 elicits the degradation of securin and cyclin B [3]. This liberates separase triggering sister chromatid disjunction and inactivates cyclin-dependent kinase 1 (Cdk1) causing mitotic exit. How eukaryotic cells avoid the engagement of attachment monitoring mechanisms when sister chromatids split and tension is lost at anaphase is poorly understood [4]. Here we show that Cdk1 inactivation disables mitotic checkpoint surveillance at anaphase onset in human cells. Preventing cyclin B1 proteolysis at the time of sister chromatid disjunction destabilizes kinetochore-microtubule attachments and triggers the engagement of the mitotic checkpoint. As a consequence, mitotic checkpoint proteins accumulate at anaphase kinetochores, the APC/CCdc20 is inhibited, and securin reaccumulates. Conversely, acute pharmacological inhibition of Cdk1 abrogates the engagement and maintenance of the mitotic checkpoint upon microtubule depolymerization. We propose that the simultaneous destruction of securin and cyclin B elicited by the APC/CCdc20 couples chromosome segregation to the dissolution of attachment monitoring mechanisms during mitotic exit

A Cathepsin L Isoform that Is Devoid of a Signal Peptide Localizes to the Nucleus in S Phase and Processes the CDP/Cux Transcription Factor

AbstractThe subclass of cysteine proteases termed lysosomal cathepsins has long been thought to be primarily involved in end-stage protein breakdown within lysosomal compartments. Furthermore, few specific protein substrates for these proteases have been identified. We show here that cathepsin L functions in the regulation of cell cycle progression through proteolytic processing of the CDP/Cux transcription factor. CDP/Cux processing in situ was increased following ectopic expression of cathepsin L but was reduced in Cat L−/− cells. Furthermore, catalytically active cathepsin L was localized to the nucleus during the G1-S transition as detected by immunofluorescence imaging and labeling using activity-based probes. Trafficking of cathepsin L to the nucleus is accomplished through a mechanism involving translation initiation at downstream AUG sites and the synthesis of proteases that are devoid of a signal peptide. Overall, these results uncover an as yet unsuspected role for cysteine proteases in the control of cell cycle progression

<i>BCL9L</i> dysfunction impairs caspase-2 expression permitting aneuploidy tolerance in colorectal cancer

Chromosomal instability (CIN) contributes to cancer evolution, intratumor heterogeneity, and drug resistance. CIN is driven by chromosome segregation errors and a tolerance phenotype that permits the propagation of aneuploid genomes. Through genomic analysis of colorectal cancers and cell lines, we find frequent loss of heterozygosity and mutations in BCL9L in aneuploid tumors. BCL9L deficiency promoted tolerance of chromosome missegregation events, propagation of aneuploidy, and genetic heterogeneity in xenograft models likely through modulation of Wnt signaling. We find that BCL9L dysfunction contributes to aneuploidy tolerance in both TP53-WT and mutant cells by reducing basal caspase-2 levels and preventing cleavage of MDM2 and BID. Efforts to exploit aneuploidy tolerance mechanisms and the BCL9L/caspase-2/BID axis may limit cancer diversity and evolution

Oncogenic PIK3CA induces centrosome amplification and tolerance to genome doubling

Mutations in PIK3CA are very frequent in cancer and lead to sustained PI3K pathway activation. The impact of acute expression of mutant PIK3CA during early stages of malignancy is unknown. Using a mouse model to activate the Pik3ca H1047R hotspot mutation in the heterozygous state from its endogenous locus, we here report that mutant Pik3ca induces centrosome amplification in cultured cells (through a pathway involving AKT, ROCK and CDK2/Cyclin E-nucleophosmin) and in mouse tissues, and increased in vitro cellular tolerance to spontaneous genome doubling. We also present evidence that the majority of PIK3CA H1047R mutations in the TCGA breast cancer cohort precede genome doubling. These previously unappreciated roles of PIK3CA mutation show that PI3K signalling can contribute to the generation of irreversible genomic changes in cancer. While this can limit the impact of PI3K-targeted therapies, these findings also open the opportunity for therapeutic approaches aimed at limiting tumour heterogeneity and evolution

The cellular roles of CUX1

The goal of my project was to develop cell-based assays to investigate the functions of the p110 CUX1 homeodomain transcription factor during cell cycle progression. I observed that constitutive expression of p110 CUX1 stimulates cell proliferation. Using fluorescence-activated cell sorting (FACS) analysis and BrdU incorporation, I demonstrated that p110 CUX1 can accelerate entry into S phase whether cells were coming out of quiescence, were allowed to resume progression through the cell cycle after synchronization at the G1/S or were simply enriched in G2/M using counter-flow centrifugal elutriation. Consistent with these observations, mouse embryo fibroblasts (MEFs) derived from homozygous cux1-/- mice proliferated more slowly and spent more time in G1 than wild type MEFs. I identified the cyclin E2 and cyclin A2 genes as some of the downstream targets of p110 CUX1 that mediate its stimulatory effect on the G1/S transition. Constitutive expression of p110 CUX1 in some, but not all, cell lines led to the emergence of cells with a tetraploid DNA content. Whereas the induction of tetraploidy in normal cells causes cell cycle arrest or cell death, cells expressing p110 CUX1 were able to undergo bipolar division in spite of the presence of more than two centrosomes. Using various experimental approaches, I demonstrated that p110 CUX1 contributes to the establishment of a transcriptional program that allows cells to sustain a robust spindle assembly checkpoint. Tetraploid cells eventually evolved to become aneuploid and tumorigenic. Similarly, over 80% of mammary tumor cells from CUX1 transgenic mice exhibit a near-tetraploid DNA content. I therefore propose that one mechanism by which elevated p110 CUX1 expression contributes to tumor development is through its effect on the spindle assembly checkpoint. In this manner, p110 CUX1 enables the survival of cells that harbor supernumerary chromosome and/or centrosome numbers and have the capacity to generate a large numL'objectif initial de mon projet consistait à mettre au point divers tests permettant d'étudier le rôle du facteur de transcription à homéodomaine p110 CUX1 au cours du cycle cellulaire. J'ai d'abord observé que p110 CUX1 stimule la prolifération cellulaire lorsque exprimé de façon soutenue. À partir d'analyses par cytométrie en flux et de marquages au BrdU, j'ai démontré que l'expression de p110 CUX1 suscite une accélération de l'entrée en phase S. Cet effet s'est manifesté dans trois situations : à partir de la phase de quiescence (G0), à partir d'une synchronisation en G1/S et à partir de la phase G2/M lors d'un enrichissement par centrifugation à contre-courant (élutriation). En accord avec ces résultats, les fibroblastes murins isolé à partir d'embryons Cux-/- proliférèrent plus lentement par rapport aux fibroblastes Cux+/+ à cause d'une prolongation de la phase G1. J'ai identifié les gènes cycline E2 et cycline A2 comme deux cibles transcriptionnelles de p110 CUX1 pouvant expliquer son effet sur la transition G1/S. J'ai observé qu'il se développait une sous-population tétraploïde dans certaines lignées cellulaires exprimant p110 CUX1. Alors que la tétraploïdie cause généralement un arrêt de la prolifération ou la mort cellulaire dans les cellules normales, j'ai noté que l'expression de p110 CUX1 permettait aux cellules tétraploïdes d'avoir une division bipolaire normale malgré la présence de plusieurs centrosomes. J'ai démontré que cet effet était dû à la capacité de p110 CUX1 d'induire la transcription de plusieurs gènes permettant une activation efficace et prolongée du point de contrôle mitotique. J'ai établi un lien entre l'instabilité génomique des cellules tetraploïdes exprimant p110 CUX1 et l'acquisition de caractéristiques tumorales. De plus, j'ai noté que plus de 80% des cellules de tumeurs mammaires provenant de souris transgéniques CUX1 sont quasi tétraploïdes. Je propose donc un no

The p110 Isoform of the CDP/Cux Transcription Factor Accelerates Entry into S Phase

The CDP/Cux transcription factor was previously found to acquire distinct DNA binding and transcriptional properties following a proteolytic processing event that takes place at the G(1)/S transition of the cell cycle. In the present study, we have investigated the role of the CDP/Cux processed isoform, p110, in cell cycle progression. Populations of cells stably expressing p110 CDP/Cux displayed a faster division rate and reached higher saturation density than control cells carrying the empty vector. p110 CDP/Cux cells reached the next S phase faster than control cells under various experimental conditions: following cell synchronization in G(0) by growth factor deprivation, synchronization in S phase by double thymidine block treatment, or enrichment in G(2) by centrifugal elutriation. In each case, duration of the G(1) phase was shortened by 2 to 4 h. Gene inactivation confirmed the role of CDP/Cux as an accelerator of cell cycle progression, since mouse embryo fibroblasts obtained from Cutl1(z/z) mutant mice displayed a longer G(1) phase and proliferated more slowly than their wild-type counterparts. The delay to enter S phase persisted following immortalization by the 3T3 protocol and transformation with H-Ras(V12). Moreover, CDP/Cux inactivation hindered both the formation of foci on a monolayer and tumor growth in mice. At the molecular level, expression of both cyclin E2 and A2 was increased in the presence of p110 CDP/Cux and decreased in its absence. Overall, these results establish that p110 CDP/Cux functions as a cell cycle regulator that accelerates entry into S phase

Cut homeobox 1 causes chromosomal instability by promoting bipolar division after cytokinesis failure

Cell populations able to generate a large repertoire of genetic variants have increased potential to generate tumor cells that survive through the multiple selection steps involved in tumor progression. A mechanism for the generation of aneuploid cancer cells involves passage through a tetraploid stage. Supernumerary centrosomes, however, can lead to multipolar mitosis and cell death. Using tissue culture and transgenic mouse models of breast cancer, we report that Cut homeobox 1 (CUX1) causes chromosomal instability by activating a transcriptional program that prevents multipolar divisions and enables the survival of tetraploid cells that evolve to become genetically unstable and tumorigenic. Transcriptional targets of CUX1 involved in DNA replication and bipolar mitosis defined a gene expression signature that, across 12 breast cancer gene expression datasets, was associated with poor clinical outcome. The signature not only was higher in breast tumor subtypes of worse prognosis, like the basal-like and HER2+ subtypes, but also identified poor outcome among estrogen receptor-positive/node-negative tumors, a subgroup considered to be at lower risk. The CUX1 signature therefore represents a unique criterion to stratify patients and provides insight into the molecular determinants of poor clinical outcome