CHARACTERIZING THE CELLULAR CONSEQUENCES OF CENTROSOME LOSS: DISCOVERY OF A MITOTIC SURVEILLANCE PATHWAY

Centrosomes are the major microtubule-organizing centers of the cell, and play a key role in organizing the bipolar mitotic spindle. Precise regulation of centrosome number is therefore critical for accurate chromosome segregation and the maintenance of genomic integrity. The consequences of centrosome loss, however, have been difficult to study due to a lack of specific tools that allow persistent and reversible centrosome depletion. In the first chapter of this thesis, we combined gene targeting with an auxin-inducible degradation system to achieve rapid, titratable, and reversible control of Polo-like kinase 4 (Plk4), a master regulator of centrosome biogenesis. Depletion of Plk4 led to a failure of centrosome duplication that, unexpectedly, produced an irreversible cell cycle arrest within a few divisions. This arrest was not a result of a prolonged mitosis, chromosome segregation errors or cytokinesis failure. Depleting p53 allowed cells that failed centrosome duplication to proliferate indefinitely, indicating the presence of a p53-dependent surveillance mechanism that protects against genome instability by preventing cell growth following centrosome duplication failure. However, the mechanism by which p53 is activated in response to centrosome loss was unknown. In the second chapter, we describe how we performed a genome-wide CRISPR/Cas9 knockout screens to identify a USP28-53BP1-p53-p21 signaling axis at the core of the centrosome surveillance pathway. We show that USP28 and 53BP1 act to stabilize p53 following centrosome loss and demonstrate this function to be independent of their previously characterized role in the DNA damage response. Surprisingly, the USP28-53BP1-p53-p21 signaling pathway is also required to arrest cell growth following a prolonged prometaphase. We therefore propose that centrosome loss or a prolonged mitosis activate a common signaling pathway that acts to prevent the growth of cells with an increased propensity for mitotic errors. Notably, most cancer cells have a disrupted p53 or p21 pathway, which allows them to proliferate indefinitely in the absence of centrosomes. In the last chapter of this thesis, we illustrate an exception to this trend, identifying a subset of breast cancers that exhibit profound sensitivity to centrosome loss that is independent of the mitotic surveillance pathway. We find this sensitivity to be dependent on the overexpression of TRIM37, an E3 ubiquitin ligase encoded within the 17q23 amplicon found in this subset of breast cancers. Timelapse studies show that centrosome depletion in these cells leads to dramatically prolonged mitoses that largely end in mitotic slippage. Subsequent analyses show that pericentriolar material (PCM) is depleted when TRIM37 is overexpressed, and that this suppresses the ability of these cells to organize a bipolar mitotic spindle through compensating, non-centrosomal pathways. Together, these results reveal a synthetic lethal vulnerability in breast cancers harboring the 17q23 amplicon that could be exploited therapeutically. In summary, this thesis describes the development of precise tools with which to study centrosome depletion, and reports the cellular consequences of centrosome loss in distinct cellular contexts

p53 protects against genome instability following centriole duplication failure

Centriole function has been difficult to study because of a lack of specific tools that allow persistent and reversible centriole depletion. Here we combined gene targeting with an auxin-inducible degradation system to achieve rapid, titratable, and reversible control of Polo-like kinase 4 (Plk4), a master regulator of centriole biogenesis. Depletion of Plk4 led to a failure of centriole duplication that produced an irreversible cell cycle arrest within a few divisions. This arrest was not a result of a prolonged mitosis, chromosome segregation errors, or cytokinesis failure. Depleting p53 allowed cells that fail centriole duplication to proliferate indefinitely. Washout of auxin and restoration of endogenous Plk4 levels in cells that lack centrioles led to the penetrant formation of de novo centrioles that gained the ability to organize microtubules and duplicate. In summary, we uncover a p53-dependent surveillance mechanism that protects against genome instability by preventing cell growth after centriole duplication failure

A USP28-53BP1-p53-p21 signaling axis arrests growth after centrosome loss or prolonged mitosis

Precise regulation of centrosome number is critical for accurate chromosome segregation and the maintenance of genomic integrity. In nontransformed cells, centrosome loss triggers a p53-dependent surveillance pathway that protects against genome instability by blocking cell growth. However, the mechanism by which p53 is activated in response to centrosome loss remains unknown. Here, we have used genome-wide CRISPR/Cas9 knockout screens to identify a USP28-53BP1-p53-p21 signaling axis at the core of the centrosome surveillance pathway. We show that USP28 and 53BP1 act to stabilize p53 after centrosome loss and demonstrate this function to be independent of their previously characterized role in the DNA damage response. Surprisingly, the USP28-53BP1-p53-p21 signaling pathway is also required to arrest cell growth after a prolonged prometaphase. We therefore propose that centrosome loss or a prolonged mitosis activate a common signaling pathway that acts to prevent the growth of cells that have an increased propensity for mitotic errors

Gorab is a Golgi protein required for structure and duplication of Drosophila centrioles.

We demonstrate that a Drosophila Golgi protein, Gorab, is present not only in the trans-Golgi but also in the centriole cartwheel where, complexed to Sas6, it is required for centriole duplication. In addition to centriole defects, flies lacking Gorab are uncoordinated due to defects in sensory cilia, which lose their nine-fold symmetry. We demonstrate the separation of centriole and Golgi functions of Drosophila Gorab in two ways: first, we have created Gorab variants that are unable to localize to trans-Golgi but can still rescue the centriole and cilia defects of gorab null flies; second, we show that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype overcome by mutations preventing Golgi targeting. Our findings suggest that during animal evolution, a Golgi protein has arisen with a second, apparently independent, role in centriole duplication.D.M.G. is grateful for a Wellcome Investigator Award, which supported this work. The study was initiated with support from Cancer Research UK

Centrosome amplification arises before neoplasia and increases upon p53 loss in tumorigenesis

The uploaded article version is the Epub Ahead of Print version of the article, posted online 8 May 2018. It has been submitted to peer-review.The deposited article version contains attached the supplementary materials within the pdf.Centrosome abnormalities are a typical hallmark of human cancers. However, the origin and dynamics of such abnormalities in human cancer are not known. In this study, we examined centrosomes in Barrett's esophagus tumorigenesis, a well-characterized multistep pathway of progression, from the premalignant condition to the metastatic disease. This human cancer model allows the study of sequential steps of progression within the same patient and has representative cell lines from all stages of disease. Remarkably, centrosome amplification was detected as early as the premalignant condition and was significantly expanded in dysplasia. It was then present throughout malignant transformation both in adenocarcinoma and metastasis. The early expansion of centrosome amplification correlated with and was dependent on loss of function of the tumor suppressor p53 both through loss of wild-type expression and hotspot mutations. Our work shows that centrosome amplification in human tumorigenesis can occur before transformation, being repressed by p53. These findings suggest centrosome amplification in humans can contribute to tumor initiation and progression.Fundação para a Ciência e a Tecnologia–Harvard Medical School Program Portugal grant: (HMSP-CT/SAU-ICT/0075/2009); Liga Portuguesa Contra o Cancro; European Molecular Biology Organization Installation; Sociedade Portuguesa de Gastroenterologia.N/

Genome-Wide Analyses Reveal a Role for Peptide Hormones in Planarian Germline Development

Genomic/peptidomic analyses of the planarian Schmidtea mediterranea identifies >200 neuropeptides and uncovers a conserved neuropeptide required for proper maturation and maintenance of the reproductive system

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field