Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Lenka Burgetová Supervisor: PharmDr. Martina Čečková PhD. Specialized supervisor: Dr. Kathryn Taylor PhD. Title of diploma thesis: Investigating the role of zinc transporter ZIP 6 and STAT3 in mitosis It has been shown that STAT3 (signal transducer and activator of transcription 3) plays a role in the development of cancer. ZIP6 is the downstream target of this transcription factor. Previous research has focused mainly on the activation of STAT3 by tyrosine phosphorylation, while the effect of phosphorylation at a second site, serine 727, remained relatively uninvestigated. In this study, it is proposed that serine-phosphorylated STAT3 is activated throughout mitosis in tamoxifen-resistant (TamR) breast cancer cells and that zinc transporter ZIP6 and serine-phosphorylated STAT3 are involved in a zinc-mediated mitotic mechanism. After using nocodazole to induce mitotic arrest, the expression of tyrosine- phosphorylated STAT3 protein was observed to be reduced while the expression of serine- phosphorylated STAT3 was increased. Zinc supplementation after nocodazole treatment appeared to push cells through mitotic-arrest and cause proteolytic cleavage of STAT3 suggesting a novel...

In order to equally segregate their genetic material into daughter cells during mitosis, it is essential that chromosomes undergo major restructuring to facilitate compaction. However, the process of transforming diffuse, entangled interphase chromatin into discrete, highly organised chromosomal structures is extremely complex, and currently not completely understood. The complexes involved in chromatin compaction and sister chromatid decatenation in preparation for mitosis include condensins I and II. Mutations in condensin subunits have been identified in human tumours, reflecting the importance of accurate cell division in the prevention of aneuploidy and tumour formation. Most mutations described in TCGA (The Cancer Genome Atlas) and COSMIC (Catalogue of Somatic Mutations in Cancer) are missense, and therefore likely to only partially affect condensin function. Most functional genetic studies of condensin, however, have used loss of function systems, which typically cause severe chromosome segregation defects and cell death. Mice carrying global hypomorphic mutations within the kleisin subunit of the condensin II complex develop T cell lymphomas. The Caph2nes/nes mouse model is therefore a good system for understanding how condensin dysfunction can influence tumourigenesis. However, little is known about which cellular processes are affected in mutant cells before transformation. I therefore set out to use the Caph2nes/nes mouse model to study the consequences of the condensin II deficiency on cell cycle regulation in several different hematopoietic lineages. The Caph2nes/nes mice are viable and fertile, with no obvious abnormalities other than the thymus, which is drastically reduced in size. Previous studies reported greater than a hundred-fold reduction in the number of CD4+ CD8+ thymocytes. I set out to understand why the alteration of a ubiquitously expressed protein which functions in a fundamental cellular process would result in such a cell-type specific block in development. To achieve this, I investigated the possibility that condensin II is involved in interphase processes as well as in mitosis. In addition, I studied the aspects of T cell development that may make this lineage particularly vulnerable to condensin II deficiency. Finally, I carried out a preliminary investigation into the biochemical properties of the condensin complexes. During my PhD., I found strong evidence to suggest that the Caph2nes/nes T cell-specific phenotype arises due to abnormal cell division. However, I was unable to find any evidence to support the hypothesis that the phenotype is a consequence of abnormal interphase processes. Upon systematic analysis of several stages of hematopoietic differentiation, I found that at a specific stage of T cell development, the mutation results in an increased proportion of cells with abnormal ploidy, followed by a drastic reduction in cell numbers. Erythroid cells revealed a similar increase in the frequency of hyperdiploid cells, but no reduction in cell numbers. B cells and hematopoietic precursors did not reveal an increase in hyperdiploidy, or a reduction in cell numbers in wildtype relative to mutant. Subsequently, I found preliminary evidence to suggest that the T cell-specificity may be due to more rapid progression of CD4+ CD8+ T cells from S phase to M phase, relative to other hematopoietic stages. Finally, a preliminary investigation into the biochemical properties of the condensin complex revealed apparent imbalances in the expression of condensin subunits in T, B and erythroid cells. The sedimentation profile of CAP-H2 from whole-thymus extract did not exclude the possibility that condensin subunits might be forming heavier-weight complexes with non-SMC proteins. Further work must be carried out to determine whether this sedimentation pattern is unique to T cells.

Proper attachment of chromosomes to microtubules is important for the accurate segregation of chromosomes and genome stability. The initial engagement of chromosomes happens along the lateral wall of microtubules through a highly specialised protein structure assembled on the centromeric DNA, the kinetochore. Ultimately, kinetochores must be attached to the ends of microtubules (a geometry called end- on attachment). A series of highly dynamic steps called the end-on conversion process, converts the initial immature lateral attachments into mature end-on attachments. How this process is finely tuned by phosphorylation and dephosphorylation to achieve stable attachments is still unclear. Furthermore, what is the role of microtubule-associated proteins in the stabilisation of kinetochore-microtubule attachments is unknown. This project aimed to study the role of phosphatases in the regulation of the end-on conversion process. First, I investigated the different contribution of the two outer-kinetochore phosphatases - BubR1- recruited PP2A-B56 and KNL1-recruited PP1 - in counteracting Aurora B kinase during the end-on conversion process. I found that BubR1-recruited PP2A-B56 plays an essential role in the process, but KNL1-recruited PP1 does not. I also investigated whether the HEC1/Ndc80 N-tail is a critical substrate of Aurora B phosphorylation for the stabilisation of the end-on attachments. Using a phospho-dead mutant of the HEC1/Ndc80 N-tail, I discovered that cells are still susceptible to Aurora B activity, indicating downstream pathways independent of HEC1/Ndc80. Then, I studied the biological role of the Astrin C-terminus, where an evolutionarily conserved RVMF motif, a putative PP1 binding site, is located. My findings show C-terminal Astrin mutants fail to localise at kinetochores of both monopolar and bipolar spindles; induce defects in the end-on conversion process in monopolar spindles and prolong mitosis time with increased Mad2 levels at the outer-kinetochore. A kinase inhibitor assay showed that kinetochore-microtubule attachment defects in Astrin mutant expressing cells could be rescued when both Aurora B and Cdk1 kinases are inhibited, suggesting a role for Astrin’s C-terminus in counteracting Aurora B and Cdk1 activity. Finally, I probed the putative interaction of the Astrin C-terminus and PP1 using biochemistry, cell biology and fluorescence microscopy techniques. I discovered that artificially targeting PP1 onto the Astrin C-terminus but not on the N-terminus rescues mutants localisation defects at the kinetochore. In summary, my results indicate that Astrin and PP1 interact at the kinetochore of living cells. In conclusion, my work shows that mitotic phosphatases have distinctive contributions in the regulation of the dynamic steps of the end-on conversion process and that Astrin is a potential PP1 phosphatase recruiter at the outer-kinetochore, where is necessary for the stabilisation of end-on attachments.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide. Current treatments are subpar, with late stage diagnosis and poor prognosis contributing to limited treatment options. The evolutionarily conserved, ubiquitously expressed transcription factor LSF is overexpressed in HCC, and its expression is positively correlated with disease severity. Certain small molecules, known as Factor Quinolinone Inhibitors (FQIs), specifically inhibit LSF DNA-binding activity, inhibit HCC cell proliferation in vitro and prevent tumor growth in an endogenous mouse liver cancer model without apparent toxicity. The targeting of transcription factors by small molecule inhibitors has been historically difficult (Dunker and Uversky, 2010), warranting further molecular investigation into the requirement for LSF in HCC to confirm that the anti-tumor effects of FQIs are the consequence of LSF inhibition. This body of work investigates a dual approach for inhibiting LSF function in order to determine the molecular consequences for HCC cells. To identify the specific point of the cell cycle where LSF is required for HCC proliferation, synchronous HCC cells were treated with FQI or with short interfering RNA to reduce levels of LSF. The results indicate that LSF is required for proper mitotic progression in HCC cells. Specifically, these data show a reduction of key mitotic regulators Aurora Kinase B and Cdc20, at the level of mRNA and protein expression. Time-lapse microscopy also demonstrated an increase in the time for progression through mitosis, with a prometaphase/metaphase delay. Immunofluorescence analysis revealed a prometaphase delay plus aberrant cell division and generation of multi-nucleated cells. These findings were consistent with both FQI1 treatment and RNA interference. Additionally, shorter incubation with FQI1 surprisingly revealed a distinct, non-transcriptional regulation of mitosis in HCC cells, suggesting that mitotic regulation by LSF is multi-faceted. As a targeted therapy for use in the clinic, the in vivo toxicity of FQIs is critical to investigate. Whole blood provides populations of rapidly dividing normal cells that can test susceptibility to anti-mitotic compounds. When mice were treated with FQI1, the blood analysis showed no toxicity. Taken together, these findings indicate that LSF is a mitotic regulator in HCC, further supporting the therapeutic promise of molecular therapies targeting LSF. / 2019-03-04T00:00:00Z

Le variant d'histone CENP-A est le facteur responsable de la détermination épigéné- tique du centromère. Il permet le recrutement de nombreuses protéines centromériques, et constitue ainsi la brique fondatrice du kinétochore. Il possède un domaine amino-terminal non structuré dont la fonction précise reste encore à élucider, bien qu'il soit déjà établi chez certaines espèces que ce domaine est requis pour le bon fonctionnement du cen- tromère et conséquemment le bon déroulement de la mitose. Nous avons construit des lignées cellulaires humaines exprimant stablement diverses formes mutantes de CENP-A, qui nous ont permis de réaliser des expériences de pseudogénétique en supprimant l'ex- pression de la protéine CENP-A endogène. Nous observons une augmentation drastique du taux de défauts de ségrégation des chromosomes et de cellules plurinucléées dans des cellules exprimant uniquement le domaine globulaire de CENP-A, ce qui est en accord avec les données de la littérature et confirme l'importance du domaine amino-terminal. Un phénotype similaire est observé dans des cellules exprimant une protéine CENP-A entière mais dont le domaine amino-terminal n'est pas phosphorylable. Nos résultats montrent l'implication de la phosphorylation de la sérine de CENP-A dans le bon déroulement de la mitose, et suggèrent que la fonction mitotique du domaine amino-terminal est centrée sur cette seule phosphorylation. / The histone variant CENP-A is the epigenetic factor responsible for centromere deter- mination. It allows the recruitment of a handful of centromeric proteins, and thus acts as the primary foundation for the kinetochore. It comprises an unstructured amino-terminal domain to which no precise function has yet been assigned, although it is established in some species that the mere presence of that domain is required for proper centromere func- tion and thus successful completion of mitosis. We have established several human cell lines stably expressing GFP-tagged CENP-A constructs, allowing us to perform pseudoge- netic experiments by siRNA-mediated silencing of the endogenous CENP-A. Our results show a dramatic increase of mitotic defects and plurinuclear cells when cells express only the globular domain of CENP-A; this is in accordance with the litterature and confirms the importance of the amino-terminal tail. More importantly, a similar increase of mitotic defects is observed when cells express a full-length, but non-phosphatable, CENP-A. Our results show the involvement of the phosphatable serine 7 of CENP-A in the successful completion of mitosis, and may suggest that the role of the whole amino-terminal tail of CENP-A could be reduced to this single phosphorylation event.

The Chromosomal Passenger Complex (CPC) is a major regulator of mitosis composed of the catalytic subunit Aurora B, the inner centromere protein INCENP, Survivin and Borealin/Dasra B. The CPC controls aspects of mitosis, ranging from chromosome and spindle structure to the correction of kinetochore-microtubule attachment errors, regulation of mitotic progression and completion of cytokinesis (Carmena et al., 2012). Knocking down any one CPC component induces delocalization of the others and disrupts mitotic progression (Adams et al., 2001 ; Carvalho et al., 2003; Lens et al., 2003 ; Gassmann et al., 2004; Vader et al., 2006). Telophase Disc (TD-60), also known as RCC2, is a putative Guanine Exchange Factor (GEF) that has been suggested to be involved in completion of cytokinesis through GTPase activation (Mollinari et al., 2003). However, its mechanism of action is still unclear. Interestingly, TD-60 has a typical Chromosomal Passenger Complex (CPC) localization (Andreassen et al., 1991) and its down-regulation alters CPC localisation during early mitosis. However, it is not a member of the CPC immunoprecipitated from mitotic cells (Gassmann et al., 2004). Here, I improved human TD-60 recombinant protein production by expressing a synthetic cDNA in the baculovirus expression system. This allowed me to characterize TD-60-associated GEF activity in vitro and study its possible influence on core CPC activity in vivo. I tested purified human TD-60 against a broad selection of GTPase targets, representing each GTPase family, in an established GEF assay. My data demonstrated that TD-60 has consistent high GEF activity in vitro towards the Ras-like protein A, RalA. To understand if TD-60 links RalA GTPase function to the CPC in vivo, I performed TD-60 and RalA RNAi experiments in HeLa cells. Interestingly, both TD-60 and RalA-depleted cells exhibit destabilized kinetochore fibers, a similar defective prometaphase-like bipolar spindle structure, and an abnormal centromeric accumulation of the CPC in early mitosis. In order to confirm that phenotypes seen after TD-60 depletion were due to lack of RalA activation in vivo, I generated a constitutively active RalA mutant that I transfected into TD-60- deficient cells. Strikingly, the RalA Q72L active mutant (mimicking the GTP-bound form) rescued the abnormal bipolar spindle structure, corrected the defective kinetochore-microtubules attachments, and rescued the atypical CPC distributions observed at centromeres after TD-60 depletion. These results suggest that TD-60-associated RalA GEF activity stabilizes kinetochore-microtubule attachments in early mitosis and that, TD-60 links RalA GTPase function to the CPC during mitosis.

Kindlin-1 (Kin1) is an epithelial focal adhesion protein that plays a key role in integrin-mediated anchorage of cells to the extracellular matrix. Congenital loss of Kin1 leads to Kindler Syndrome (KS), whose symptoms include progressive epidermal atrophy, reduced keratinocyte proliferation, skin blistering and increased incidence of aggressive Squamous Cell Carcinoma (SCC). Objectives of this study were to examine the role of Kin1 in skin homeostasis and in the development of aggressive SCC in KS, as the molecular aetiologies for these pathologies are yet to be clearly understood. We first examined whether the recently discovered role of Kin1 in mitosis contributes to reduced keratinocyte proliferation observed in KS epidermis. We discovered that short-term loss of Kin1 in adult mouse epidermis reduced keratinocyte proliferation. We also found that Kin1 loss increased mitotic spindle misorientation that, according to the model of cell division in skin homeostasis, decreases cell proliferative potential, and, thus, may account for the reduced proliferation in our model. As spindle misorientation can stem from microtubule instability, we believe that the reduction in acetylated α-tubulin (ac-tub), a known marker of stable microtubules, that we also observed in mouse epidermis following Kin1 loss could account for the defective spindle orientation phenotype. The role of Kin1 in spindle orientation was also evident in vitro. Moreover, data from our lab revealed showed reduction in spindle ac-tub following Kin1 depletion, mirroring our in vivo observation. Additionally to orientation defects, in vitro depletion of Kin1 led to enhanced chromosome missegregation, which is likely to result from reduced microtubule stability due to low levels of ac-tub. We showed that role of Kin1 in spindle orientation and chromosome segregation is dependent on HDAC6, a known inhibitor of ac-tub. Overall, our results uncover an in vitro and in vivo role of Kin1 in mitotic spindle fidelity that could be crucial to skin homeostasis, and, when disturbed, may lead to reduced keratinocyte proliferation. Interestingly, our in vitro studies also revealed that in mitosis Kin1 and Kindlin-2 (Kin2) had overlapping, but also distinct roles, which is in line with various reports that show different biological functions for the two protein isoforms. Our next and final aim was to explore the roles of Kin1 in the development and progression of SCC, which would help us comprehend the reason behind the cancer's aggressive nature in KS. By employing in vitro and in vivo SCC growth assays and tumour immunohistochemical staining we found that absence of Kin1 in SCC cells and tumours enhanced proliferation and growth, and enhanced tumour vascularisation. RNA sequencing of tumour material revealed that lack of Kin1 increased expression of matrix metalloproteinases and chemokines, which have been implicated in tumour progression via promotion of angiogenesis and invasion in a plethora of studies, and of various angiogenesis markers. Together this provides an insight into the mechanisms via which Kin1 controls tumour microenvironment and, ultimately, SCC tumour growth and development. Overall, we report an in vitro and in vivo role for Kin1 in mitotic spindle stability, which affects a variety of mitotic processes and may be linked to reduced keratinocyte proliferation observed in epidermis of KS patients, thus contributing to skin homeostasis. Moreover, we describe a role for Kin1 in regulation of SCC tumour growth and progression, which may ultimately offer an explanation for the aggressive and life-threatening nature of SCC developed in KS.

The chromosomal passenger complex (CPC: Aurora B-INCENP-Survivin-Borealin) is a key regulator of cell division whose localisation at centromeres is required for stable kinetochore-microtubule attachments and proper chromosomal segregation (Ruchaud et al. 2007; Carmena et al. 2012; van der Waal et al. 2012). Shugoshin1 (hSgo1) (via Borealin) and Histone H3 (via Survivin) have been implicated in centromeric targeting of CPC (Wang et al. 2010; Jeyaprakash et al. 2011; Tsukahara et al. 2010; Kawashima et al. 2010). Although the Survivin-Histone H3 pathway has been extensively studied, the intermolecular interactions dictating CPC-hSgo1 interactions remain unclear. My PhD work focused on characterising the molecular framework of the CPC-hSgo1 interaction using biochemical, biophysical and structural biology methods. I optimised and improved human CPC and hSgo1 recombinant protein production in an E. coli system. Post optimisation, I used Size-Exclusion Chromatography to successfully reconstitute the CPC-hSgo1 complex in vitro and further confirmed that hSgo1 possessing no modification or extra amino acids on its N-terminus can interact with Survivin and Borealin-Survivin-INCENP1-57. This suggested that the hSgo1 N-terminal tail interaction with Survivin is crucial for CPC-hSgo1 interaction. Furthermore, I conducted calorimetric binding studies to molecularly dissect the individual contributions of CPC components and their domains towards CPC-hSgo1 interaction. Towards this aim, I expressed and purified different versions of CPC and analysed their binding energetics with hSgo1. The results from these experiments clearly suggested the contribution of Borealin and INCENP towards CPC-hSgo1 interaction.

Cell division relies on many steps, precisely synchronised, to ensure the fidelity of chromosome segregation. To achieve such complex and multiple functions, cells have evolved mechanisms by which one protein can participate in numerous events on the cell life. Over the past few years, an increasing number of functions have been assigned to the proteins of the nuclear pore complex (NPC) also called nucleoporins. NPCs are large complexes studded in the nuclear envelope, which control the nucleocytoplasmic transport. It is now known that nucleoporins participate in spindle assembly, kinetochore organisation, spindle assembly checkpoint, and all processes important for genome integrity maintenance. This work demonstrates that the nucleoporin ALADIN participates in mitosis, meiosis and in cilia. In both mitosis and meiosis, ALADIN is important for proper spindle assembly. In mitosis, it was also discovered that ALADIN is a novel factor in the spatial regulation of the mitotic regulator Aurora A kinase. Without ALADIN, active Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. Interestingly, mutations in ALADIN causes triple A syndrome and some of the mitotic phenotypes observed after ALADIN depletion also occur in cells from triple A syndrome patients. In meiosis, ALADIN contributes to trigger the resumption of meiosis in female mouse. Impairment of ALADIN from mouse oocyte slows spindle assembly, migration and reduces oocytes ability to extrude polar bodies during meiosis I, which concomitantly affects the robustness of oocyte maturation and impairs mouse embryo development. Nucleoporins were also found at the base of the cilia, a centriole-derived organelle that participates in differentiation, migration, cell growth from development to adulthood. Here it is shown that ALADIN is also localised at the base of the cilia. With this work, new ALADIN’s functions have been identified across cell division, as well as uncovered an unexpected relation between triple A syndrome and cell division.

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