Centrosome Function Research Articles

Proximity mapping of the microtubule plus-end tracking protein SLAIN2 using the BioID approach.

The centrosome is the main microtubule-organizing center of animal cells, which plays key roles in critical cellular processes ranging from cell division to cellular signaling. Accordingly, defects in the structure and function of centrosomes cause various human diseases such as cancer and primary microcephaly. To elucidate the molecular defects underlying these diseases, the biogenesis and functions of the centrosomes have to be fully understood. An essential step towards addressing these questions is the identification and functional dissection of the full repertoire of centrosome proteins. Here, we used high-resolution imaging and showed that the microtubule plus-end tracking protein SLAIN2 localizes to the pericentriolar material at the proximal end of centrioles. To gain insight into its cellular functions and mechanisms, we applied in vivo proximity-dependent biotin identification to SLAIN2 and generated its proximity interaction map. Gene ontology analysis of the SLAIN2 interactome revealed extensive interactions with centriole duplication, ciliogenesis, and microtubule-associated proteins, including previously characterized and uncharacterized interactions. Collectively, our results define SLAIN2 as a component of pericentriolar material and provide an important resource for future studies aimed at elucidating SLAIN2 functions at the centrosome.

Emerging roles of the centrosome in neuronal development.

The role of the centrosome-a microtubule-organizing center-in neuronal development has been under scrutiny and is controversial. The function and position of the centrosome have been shown to play an important role in selecting the position of axon outgrowth in cultured neurons and in situ. However, other studies have shown that axonal growth is independent of centrosomal functions. Recent discoveries define the centrosome as an F-actin organizing organelle in various cell types; thus, giving a whole new perspective to the role of the centrosome in lymphocyte polarity, cell division, and neuronal development. These discoveries compel the need to revisit centrosomal functions by investigating the fundamental mechanisms that regulate centrosomal F-actin remodeling during neuronal differentiation and polarization. In this review, we summarize the up-to-date knowledge regarding the function of the centrosome in neuronal differentiation. We put special emphasis on recent findings describing the centrosome as an F-actin organizing center. Additionally, with the available data regarding centrosome, microtubules and F-actin organization, we provide a model on how centrosomal F-actin could be modulating neuronal differentiation and polarity.

Centrosomal and Non-centrosomal Functions Emerged through Eliminating Centrosomes.

Centrosomes are highly conserved organelles that act as the major microtubule-organizing center (MTOC) in animal somatic cells. Through their MTOC activity, centrosomes play various roles throughout the cell cycle, such as supporting cell migration in interphase and spindle organization and positioning in mitosis. Various approaches for removing centrosomes from somatic cells have been developed and applied over the past few decades to understand the precise roles of centrosomes. Centrinone, a reversible and selective PLK4 (polo-like kinase 4) inhibitor, has recently emerged as an efficient approach to eliminate centrosomes. In this review, we describe the latest findings on centrosome function that have been revealed using various centrosome-eliminating approaches. In addition, we discuss our recent findings on the mechanism of centrosome-independent spindle bipolarization, discovered through the use of centrinone.Key words: centrosome, centrinone, mitotic spindle, bipolarity, NuMA.

CSAG1 maintains the integrity of the mitotic centrosome in cells with defective p53.

Centrosomes focus microtubules to promote mitotic spindle bipolarity, a critical requirement for balanced chromosome segregation. Comprehensive understanding of centrosome function and regulation requires a complete inventory of components. While many centrosome components have been identified, others yet remain undiscovered. We have used a bioinformatics approach, based on 'guilt by association' expression to identify novel mitotic components among the large group of predicted human proteins that have yet to be functionally characterized. Here, we identify chondrosarcoma-associated gene 1 protein (CSAG1) in maintaining centrosome integrity during mitosis. Depletion of CSAG1 disrupts centrosomes and leads to multipolar spindles, particularly in cells with compromised p53 function. Thus, CSAG1 may reflect a class of 'mitotic addiction' genes, whose expression is more essential in transformed cells.

Alpha-synuclein aggresomes inhibit ciliogenesis and multiple functions of the centrosome.

ABSTRACTProtein aggregates are the pathogenic hallmarks of many different neurodegenerative diseases and include the accumulation of α-synuclein, the main component of Lewy bodies found in Parkinson's disease. Aggresomes are closely-related, cellular accumulations of misfolded proteins. They develop in a juxtanuclear position, adjacent to the centrosome, the microtubule organizing centre of the cell, and share some protein components. Despite the long-standing observation that aggresomes/Lewy bodies and the centrosome sit side-by-side in the cell, no studies have been done to see whether these protein accumulations impede organelle function. We investigated whether the formation of aggresomes affected key centrosome functions: its ability to organise the microtubule network and to promote cilia formation. We find that when aggresomes are present, neuronal cells are unable to organise their microtubule network. New microtubules are not nucleated and extended, and the cells fail to respond to polarity cues. Since neurons are polarised, ensuring correct localisation of organelles and the effective intracellular transport of neurotransmitter vesicles, loss of centrosome activity could contribute to functional deficits and neuronal cell death in Parkinson's disease. In addition, we provide evidence that many cell types, including dopaminergic neurons, cannot form cilia when aggresomes are present, which would affect their ability to receive extracellular signals.

BAG6 is a novel microtubule-binding protein that regulates ciliogenesis by modulating the cell cycle and interacting with γ-tubulin

Microtubule-binding proteins provide an alternative and vital pathway to the functional diversity of microtubules. Considerable work is still required to understand the complexities of microtubule-associated cellular processes and to identify novel microtubule-binding proteins. In this study, we identify Bcl2-associated athanogene cochaperone 6 (BAG6) as a novel microtubule-binding protein and reveal that it is crucial for primary ciliogenesis. By immunofluorescence we show that BAG6 largely colocalizes with intracellular microtubules and by co-immunoprecipitation we demonstated that it can interact with α-tubulin. Additionally, both the UBL and BAG domains of BAG6 are indispensable for its interaction with α-tubulin. Moreover, the assembly of primary cilia in RPE-1 cells is significantly inhibited upon the depletion of BAG6. Notably, BAG6 inhibition leads to an abnormal G0/G1 transition during the cell cycle. In addition, BAG6 colocalizes and interactes with the centrosomal protein γ-tubulin, suggesting that BAG6 might regulate primary ciliogenesis through its action in centrosomal function. Collectively, our findings suggest that BAG6 is a novel microtubule-bindng protein crucial for primary ciliogenesis.

CCDC57 Cooperates with Microtubules and Microcephaly Protein Cep63 and Regulates Centriole Duplication and Mitotic Progression

Centrosomes play critical roles in diverse cellular processes ranging from cell division to cellular signaling. At the core of the centrosomes are two centrioles, which duplicate only once per cell cycle and this duplication cycle is tightly regulated. Accordingly, their deregulation causes diseases such as cancer and developmental disorders like primary microcephaly. Complete understanding of the mechanisms that regulate centrosome biogenesis and function is required to elucidate the disease mechanisms. In this study, we identified a new centrosome protein CCDC57, which also localizes and binds to centriolar satellites and microtubules. Proximity-labeling and interaction studies showed that CCDC57 forms a complex with Cep63, centriolar satellites and microtubules. Importantly,characterization of CCDC57 deletion mutants revealed that its N-terminal 1-502 amino acid region mediated its centrosomal localization and interactions, whereas the C-terminal 606-916 amino acid region mediated its localization and binding to microtubules. Loss of CCDC57 causes defects in canonical centriole duplication and centriole amplification, and results in a failure to localize microcephaly-associated proteins Cep63 and Cep152 to the centriole during initiation of centriole duplication. Additionally, CCDC57 depletion resulted in prolonged mitosis, increased apoptosis and a higher ratio of mitotic defects such as misaligned chromosomes and multipolar spindles. Together, our results identify CCDC57 as dual regulator of the Cep63 centriole duplication module and microtubule-mediated mitotic processes and provide new mechanistic insight into defects underlying cancer and primary microcephaly.

PL3.6 Targeting GSK-3 activity promotes mitotic catastrophe via centrosome destabilisation and enhances the effect of radiotherapy in glioma models

Abstract BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

524 Melanin localisation and transport in the human epidermis

Recent research has unraveled much about the control of melanosome movement in melanocytes (MC) and to a lesser extent how melanin granules are then transferred to neighboring keratinocytes (KC) in human epidermis. However, how melanin granules are distributed within KC is largely unknown despite this being critical for localised protection against UV damage. Here we examined how melanin is distributed within KC in human epidermis. Histocultures of human skin (photo-type II/III) were treated for 24h with nocodazole (microtubule polymerization inhibitor), monastrol (inhibitor of kinesin-5; important for spindle bipolarity) and cytochalasin B (actin filament polymerization inhibitor). Treatment effects were examined by expression of a) proliferation/mitosis (Ki67/pH3(Ser10)), b) differentiation (KRT10), c) centrosome localization (pericentrin), d) melanin movement (dynactin-1) and e) melanin localization (Warthin-Starry stain). The above treatments did not alter KC differentiation (KRT10). Nocodazole significantly increased mitosis detection in the surpabasal (but not basal) epidermis, and this concurred with distribution of total melanin predominating in the basal layer. Remarkably, the usual supranuclear distribution of melanin and centrosomes was reversed to a mostly subnuclear one, suggesting a previously-unreported association between the location of aggregated melanin and the centrosome. By contrast, monastrol increased mitosis detection in basal (but not suprabasal) epidermis. Now melanin was distributed suprabasally; still maintaining its supranuclear aggregation. This suggests that a) melanin can move between individual KC or b) MC can engage in more expansive melanin transfer beyond the basal layer. Cytochalasin-B did not alter mitosis detection but did disrupt aggregation of melanin granules, such that supranuclear melanin capping was now lost in the basal layer. Our results highlight a hitherto unappreciated role for centrosome, microtubule and actin function in the subcellular and intercellular melanin localization in human epidermis.

NudC-like protein 2 restrains centriole amplification by stabilizing HERC2

Centriole duplication is tightly controlled to occur once per cell cycle, and disruption of this synchrony causes centriole amplification, which is frequently observed in many cancers. Our previous work showed that nuclear distribution gene C (NudC)-like protein 2 (NudCL2) localizes to centrosomes; however, little is known about the role of NudCL2 in the regulation of centrosome function. Here, we find that NudCL2 is required for accurate centriole duplication by stabilizing the E3 ligase HECT domain and RCC1-like domain-containing protein 2 (HERC2). Knockout (KO) of NudCL2 using CRISPR/Cas9-based genome editing or depletion of NudCL2 using small interfering RNA causes significant centriole amplification. Overexpression of NudCL2 significantly suppresses hydroxyurea-induced centriole overduplication. Quantitative proteomic analysis reveals that HERC2 is downregulated in NudCL2 KO cells. NudCL2 is shown to interact with and stabilize HERC2. Depletion of HERC2 leads to the similar defects to that in NudCL2-downregulated cells, and ectopic expression of HERC2 effectively rescues the centriole amplification caused by the loss of NudCL2, whereas the defects induced by HERC2 depletion cannot be reversed by exogenous expression of NudCL2. Either loss of NudCL2 or depletion of HERC2 leads to the accumulation of ubiquitin-specific peptidase 33 (USP33), a centrosomal protein that positively regulates centriole duplication. Moreover, knockdown of USP33 reverses centriole amplification in both NudCL2 KO and HERC2-depleted cells. Taken together, our data suggest that NudCL2 plays an important role in maintaining the fidelity of centriole duplication by stabilizing HERC2 to control USP33 protein levels, providing a previously undescribed mechanism restraining centriole amplification.

Abstract 2097: LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer

Abstract Currently, there are no targeted strategies to combat triple negative breast cancer, resulting in poor patient survival. This disease initially responds well to cytotoxic chemotherapies such as paclitaxel, yet resistance and metastatic recurrence are common. Taxanes cause defects in centrosome function and chromosome segregation leading to cell death. While greatly effective, they are also associated with high toxicity. Thus discovering new therapeutic targets that provide a selective vulnerability for TNBC should yield novel approaches for improving patient outcomes. We discovered that LIN9, a transcriptional regulator of mitosis, is overexpressed in 66% of TNBC and is associated with poor survival. We now report that both LIN9 mRNA and protein expression are upregulated in paclitaxel-resistant versus sensitive cells and directly correlates with paclitaxel IC50 values across eight breast cancer cell lines. In MDA-MB-231 and MDA-MB-468 cell lines, LIN9 silencing results in multi- and micronucleation, and supernumerary centrosomes. Moreover, LIN9 silencing increases sensitivity to paclitaxel in TNBC cells with intrinsic (BT549) or acquired (MDA-MB-231 and -468) resistance. We previously reported that Bromodomain and ExtraTerminal protein inhibitors (BETi) treatment reduces LIN9 expression, and thus determined if BETi could reverse paclitaxel resistance. Treatment with the BETi, JQ1, in conjunction with paclitaxel caused a greater induction of apoptosis, abnormal centrosomes, multi- and micronucleation compared to either drug alone. To identify the mechanism(s) by which suppression of LIN9 reverses paclitaxel resistance, we compared the transcriptomes of MDA-MB-231 and HCC70 TNBC cells transiently transfected with non-targeting or LIN9-targeted siRNAs. The resulting gene list was then filtered to include only genes whose expression correlates with LIN9 in breast cancer, are bound to LIN9 in a published ChIP-Seq dataset, and are associated with breast cancer survival. Using this approach, we identified NIMA-related Kinase 2 (NEK2), a serine/threonine kinase required for centrosome separation during mitosis, as a potential mediator of LIN9-associated paclitaxel resistance. NEK2 is overexpressed in 47% of basal breast cancers and is associated with poor patient outcomes. Additionally, NEK2 is upregulated in paclitaxel-resistant cells and LIN9 silencing decreases expression of NEK2. Importantly, silencing NEK2 expression also restores sensitivity to paclitaxel in resistant cells. Together, these data indicate that increased LIN9 expression in TNBC promotes paclitaxel resistance by upregulating NEK2 and that loss of LIN9 or NEK2 contributes to centrosome dysfunction, potentiating paclitaxel sensitivity. They also indicate that suppressing LIN9 expression using agents such as BET inhibitors may be a viable therapeutic approach for improving paclitaxel efficacy in TNBC patients. Citation Format: Melyssa S. Shively, Ruth A. Keri. LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2097.

Plk4 Regulates Centriole Asymmetry and Spindle Orientation in Neural Stem Cells

SummaryDefects in mitotic spindle orientation (MSO) disrupt the organization of stem cell niches impacting tissue morphogenesis and homeostasis. Mutations in centrosome genes reduce MSO fidelity, leading to tissue dysplasia and causing several diseases such as microcephaly, dwarfism, and cancer. Whether these mutations perturb spindle orientation solely by affecting astral microtubule nucleation or whether centrosome proteins have more direct functions in regulating MSO is unknown. To investigate this question, we analyzed the consequences of deregulating Plk4 (the master centriole duplication kinase) activity in Drosophila asymmetrically dividing neural stem cells. We found that Plk4 functions upstream of MSO control, orchestrating centriole symmetry breaking and consequently centrosome positioning. Mechanistically, we show that Plk4 acts through Spd2 phosphorylation, which induces centriole release from the apical cortex. Overall, this work not only reveals a role for Plk4 in regulating centrosome function but also links the centrosome biogenesis machinery with the MSO apparatus.

TUBG1 missense variants underlying cortical malformations disrupt neuronal locomotion and microtubule dynamics but not neurogenesis

De novo heterozygous missense variants in the γ-tubulin gene TUBG1 have been linked to human malformations of cortical development associated with intellectual disability and epilepsy. Here, we investigated through in-utero electroporation and in-vivo studies, how four of these variants affect cortical development. We show that TUBG1 mutants affect neuronal positioning, disrupting the locomotion of new-born neurons but without affecting progenitors’ proliferation. We further demonstrate that pathogenic TUBG1 variants are linked to reduced microtubule dynamics but without major structural nor functional centrosome defects in subject-derived fibroblasts. Additionally, we developed a knock-in Tubg1Y92C/+ mouse model and assessed consequences of the mutation. Although centrosomal positioning in bipolar neurons is correct, they fail to initiate locomotion. Furthermore, Tubg1Y92C/+ animals show neuroanatomical and behavioral defects and increased epileptic cortical activity. We show that Tubg1Y92C/+ mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of cortical malformations.

HSP70 is required for the proper assembly of pericentriolar material and function of mitotic centrosomes

BackgroundAt the onset of mitosis, the centrosome expands and matures, acquiring enhanced activities for microtubule nucleation and assembly of a functional bipolar mitotic spindle. However, the mechanisms that regulate centrosome expansion and maturation are largely unknown. Previously, we demonstrated in an immortalized human cell line CGL2 and cancer cell line HeLa that the inducible form of heat shock protein 70 (HSP70) accumulates at the mitotic centrosome and is required for centrosome maturation and bipolar spindle assembly.ResultsIn this study, we further show that HSP70 accumulated at the spindle pole in a PLK1-dependent manner. HSP70 colocalized with pericentrin (PCNT), CEP215 and γ-tubulin at the spindle pole and was required for the 3D assembly of these three proteins, which supports mitotic centrosome function. Loss of HSP70 disrupted mitotic centrosome structure, reduced pericentriolar material recruitment and induced fragmentation of spindle poles. In addition, HSP70 was necessary for the interaction between PCNT and CEP215 and also facilitated PLK1 accumulation and function at the spindle pole. Furthermore, we found that HSP70 chaperone activity is required for PCNT accumulation at the mitotic centrosome and assembly of mitotic spindles.ConclusionOur current results demonstrate that HSP70 is required for the accurate assembly of the pericentriolar material and proper functioning of mitotic centrosomes.

Molecular determinants regulating selective binding of autophagy adapters and receptors to ATG8 proteins

Autophagy is an essential recycling and quality control pathway. Mammalian ATG8 proteins drive autophagosome formation and selective removal of protein aggregates and organelles by recruiting autophagy receptors and adaptors that contain a LC3-interacting region (LIR) motif. LIR motifs can be highly selective for ATG8 subfamily proteins (LC3s/GABARAPs), however the molecular determinants regulating these selective interactions remain elusive. Here we show that residues within the core LIR motif and adjacent C-terminal region as well as ATG8 subfamily-specific residues in the LIR docking site are critical for binding of receptors and adaptors to GABARAPs. Moreover, rendering GABARAP more LC3B-like impairs autophagy receptor degradation. Modulating LIR binding specificity of the centriolar satellite protein PCM1, implicated in autophagy and centrosomal function, alters its dynamics in cells. Our data provides new mechanistic insight into how selective binding of LIR motifs to GABARAPs is achieved, and elucidate the overlapping and distinct functions of ATG8 subfamily proteins.

EGFR Controls Hair Shaft Differentiation in a p53-Independent Manner

SummaryEpidermal growth factor receptor (EGFR) signaling controls skin development and homeostasis in mice and humans, and its deficiency causes severe skin inflammation, which might affect epidermal stem cell behavior. Here, we describe the inflammation-independent effects of EGFR deficiency during skin morphogenesis and in adult hair follicle stem cells. Expression and alternative splicing analysis of RNA sequencing data from interfollicular epidermis and outer root sheath indicate that EGFR controls genes involved in epidermal differentiation and also in centrosome function, DNA damage, cell cycle, and apoptosis. Genetic experiments employing p53 deletion in EGFR-deficient epidermis reveal that EGFR signaling exhibits p53-dependent functions in proliferative epidermal compartments, as well as p53-independent functions in differentiated hair shaft keratinocytes. Loss of EGFR leads to absence of LEF1 protein specifically in the innermost epithelial hair layers, resulting in disorganization of medulla cells. Thus, our results uncover important spatial and temporal features of cell-autonomous EGFR functions in the epidermis.

Polo-like kinase 4 maintains centriolar satellite integrity by phosphorylation of centrosomal protein 131 (CEP131)

The centrosome, consisting of two centrioles surrounded by a dense network of proteins, is the microtubule-organizing center of animal cells. Polo-like kinase 4 (PLK4) is a Ser/Thr protein kinase and the master regulator of centriole duplication, but it may play additional roles in centrosome function. To identify additional proteins regulated by PLK4, we generated an RPE-1 human cell line with a genetically engineered "analog-sensitive" PLK4AS, which genetically encodes chemical sensitivity to competitive inhibition via a bulky ATP analog. We used this transgenic line in an unbiased multiplex phosphoproteomic screen. Several hits were identified and validated as direct PLK4 substrates by in vitro kinase assays. Among them, we confirmed Ser-78 in centrosomal protein 131 (CEP131, also known as AZI1) as a direct substrate of PLK4. Using immunofluorescence microscopy, we observed that although PLK4-mediated phosphorylation of Ser-78 is dispensable for CEP131 localization, ciliogenesis, and centriole duplication, it is essential for maintaining the integrity of centriolar satellites. We also found that PLK4 inhibition or use of a nonphosphorylatable CEP131 variant results in dispersed centriolar satellites. Moreover, replacement of endogenous WT CEP131 with an S78D phosphomimetic variant promoted aggregation of centriolar satellites. We conclude that PLK4 phosphorylates CEP131 at Ser-78 to maintain centriolar satellite integrity.

Abstract P5-03-01: LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer

Abstract Currently, there are no targeted strategies to combat triple negative breast cancer, resulting in poor patient survival. TNBC initially respond well to cytotoxic chemotherapies such as paclitaxel, yet resistance and metastatic recurrence are common. Paclitaxel causes defects in mitotic spindle formation and centrosome function, as well as improper chromosome segregation leading to cell death. While being some of the most effective drugs for this disease, taxanes are associated with high toxicity. Thus discovering new therapeutic targets that are selective for TNBC should yield novel approaches for improving patient outcomes. We discovered that LIN9, a transcriptional regulator of mitosis, is overexpressed in 66% of TNBC and associated with poor survival. We further found that both LIN9 mRNA and protein expression are upregulated in paclitaxel-resistant versus sensitive cells and directly correlates with paclitaxel IC50 values across nine breast cancer cell lines. In MDA-MB-231 and MDA-MB-468 cell lines, enforced overexpression of LIN9 increases multi- and micronucleation, indicators of chromosomal instability. Conversely, LIN9 silencing also results in multi- and micronucleation, and supernumerary centrosomes. Most importantly, LIN9 silencing increases sensitivity to paclitaxel in TNBC cells with intrinsic (BT549) or acquired (MDA-MB-231 and MDA-MB-468) resistance. We have previously reported that treating TNBC cells with Bromodomain and ExtraTerminal protein inhibitors (BETi) reduces LIN9 expression, thus we determined if BETi could reverse paclitaxel resistance. Treatment with the BETi, JQ1, and paclitaxel caused a greater induction of apoptosis compared to either drug alone. Dual treatment also resulted in a potentiation of abnormal centrosomes, multinucleation, and micronucleation compared to that caused by either BETi or JQ1 alone. To identify the mechanism(s) by which genetic or therapeutic suppression of LIN9 reverses paclitaxel resistance, we compared the transcriptomes of TNBC cells transiently transfected with non-targeting or LIN9-targeted siRNAs in MDA-MB-231 and HCC70 cell lines. We further narrowed the list of candidate LIN9 targets by identifying genes that were bound by LIN9 in a published ChIP-Seq dataset from HeLa cells whose expression is also correlated with LIN9 expression and associated with reduced breast cancer patient survival. Using this approach, we identified NIMA-related Kinase 2 (NEK2), a serine/threonine kinase required for centrosome separation during mitosis as a potential mediator of the effects of LIN9 suppression. NEK2 is overexpressed in 47% of basal breast cancers and is associated with poor survival. In addition, NEK2 is upregulated in paclitaxel-resistant cells and LIN9 silencing decreases expression of NEK2. Silencing NEK2 expression also restores sensitivity to paclitaxel in resistant cells. Together, these data indicate that increased LIN9 expression in TNBC promotes paclitaxel resistance by contributing to centrosome dysfunction through upregulation of NEK2. They also indicate that targeting LIN9 expression in addition to paclitaxel treatment may be a viable therapeutic approach for TNBC patients. Citation Format: Shively MS, Gayle SS, Sahni JM, Keri RA. LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-01.

A method of quantifying centrosomes at the single-cell level in human normal and cancer tissue.

Centrosome abnormalities are emerging hallmarks of cancer. The overproduction of centrosomes (known as centrosome amplification) has been reported in a variety of cancers and is currently being explored as a promising target for therapy. However, to understand different types of centrosome abnormalities and their impact on centrosome function during tumor progression, as well as to identify tumor subtypes that would respond to the targeting of a centrosome abnormality, a reliable method for accurately quantifying centrosomes in human tissue samples is needed. Here, we established a method of quantifying centrosomes at a single-cell level in different types of human tissue samples. We tested multiple anti-centriole and pericentriolar-material antibodies to identify bona fide centrosomes and multiplexed these with cell border markers to identify individual cells within the tissue. High-resolution microscopy was used to generate multiple Z-section images, allowing us to acquire whole cell volumes in which to scan for centrosomes. The normal cells within the tissue serve as internal positive controls. Our method provides a simple, accurate way to distinguish alterations in centrosome numbers at the level of single cells.

SA102 - A COGNITIVE AND MOLECULAR ANALYSIS OF SDCCAG8, A SCHIZOPHRENIA RISK GENE THAT FUNCTIONS IN THE CENTROSOME

Background Schizophrenia affects 1% of adults and is a major global health problem. Although aetiology is complex, genetics represents a vital tool for probing the neurobiology of the disorder in the absence of biomarkers. We undertook an interdisciplinary project investigating the role of centrosome genes in schizophrenia. The centrosome, an organelle within cells, plays a crucial role in brain development where it directs cell shape, polarity and motility. The centrosome also seeds the growth of antenna-like signalling structures called primary cilia. Rare mutations in centrosome genes cause disorders that present with severe cognitive deficits and variable neuropsychiatric phenotypes. Methods We mined online databases to generate a list of genes encoding proteins with centrosome function. These were cross-referenced with schizophrenia risk genes identified in the 2014 PGC GWAS. Neurocognitive analysis: prioritised candidate genes were taken forward to analysis in a sample of Irish psychosis patients and controls (n=1,200). Participants were grouped by genotype at genome-wide significant SNPs and linear regression was used to test if genotype at schizophrenia risk SNPs was significantly associated with different domains of cognitive function. Molecular analysis: CRISPR-Cas9 genome editing technology was used to generate SDCCAG8 knockout cell lines in hTERT-RPE1 epithelial cells and in SH-SY5Y cells, a neuronal cell line. After serum starvation, cilia and centrosome markers were used to perform ciliation counts by immunofluorescence. Centrosome amplification was induced by exposing cells to 5 Gy ionising radiation and the number of centrosomes was counted after 72 hours by immunofluorescence. Results We identified 7 schizophrenia risk genes that have centrosomal functions: SDCCAG8, NEK4, MPHOSPH9, MAD1L1, PRKD1, MAPK3 and GIGYF2. Neurocognitive analysis of schizophrenia risk SNPs within these seven genes identified nominally significant associations with at least one domain of cognition for 5 of these 7 genes. Most of the significant associations were identified for social cognition, with the effect of SDCCAG8 on this domain being the most significant finding (p=0.001). SDCCAG8 is also associated with educational attainment and the genome-wide significant SNPs for the two phenotypes are in high linkage disequilibrium indicating a pleiotropic effect. We successfully generated SDCCAG8 knockout in RPE1 cells and in SH-SY5Y cells. Preliminary data shows that SDCCAG8 knockout clones, while viable in a p53 wildtype background, have slower proliferation rates. The capacity of the clones to ciliate is also reduced, by 50% in RPE1 clones and 75% in SH-SY5Y clones. In response to DNA damage there was an increase in centrosome amplification in RPE1 knockout clones when compared to wildtype cells. Discussion SDCCAG8 is a schizophrenia risk gene that we find is also associated with social cognition and educational attainment. Our data suggest that loss of SDCCAG8 impairs cells’ ability to make primary cilia and that their capacity to repair genome damage may be reduced. Further work will address whether SDCCAG8 affects activities that may contribute to schizophrenia, including cell migration and cell signalling. This could identify molecular mechanisms by which SDCCAG8 mutations contribute to schizophrenia risk and cognition, and help uncover the biological processes that implicate centrosome genes in various neurodevelopmental phenotypes.