Centrosome Clustering Research Articles

Kinesins regulate the heterogeneity in centrosome clustering after whole-genome duplication.

After whole-genome duplication (WGD), tetraploid cells can undergo multipolar mitosis or pseudo-bipolar mitosis with clustered centrosomes. Kinesins play a crucial role in regulating spindle formation. However, the contribution of kinesin expression levels to the heterogeneity in centrosome clustering observed across different cell lines after WGD remains unclear. We identified two subsets of cell lines: "BP" cells efficiently cluster extra centrosomes for pseudo-bipolar mitosis, and "MP" cells primarily undergo multipolar mitosis after WGD. Diploid MP cells contained higher levels of KIF11 and KIF15 compared with BP cells and showed reduced sensitivity to centrosome clustering induced by KIF11 inhibitors. Moreover, partial inhibition of KIF11 or depletion of KIF15 converted MP cells from multipolar to bipolar mitosis after WGD. Multipolar spindle formation involved microtubules but was independent of kinetochore-microtubule attachment. Silencing KIFC1, but not KIFC3, promoted multipolar mitosis in BP cells, indicating the involvement of specific kinesin-14 family members in counteracting the forces from KIF11/KIF15 after WGD. These findings highlight the collective role of KIF11, KIF15, and KIFC1 in determining the polarity of the mitotic spindle after WGD.

FOXM1 Transcriptionally Co-Upregulates Centrosome Amplification and Clustering Genes and Is a Biomarker for Poor Prognosis in Androgen Receptor-Low Triple-Negative Breast Cancer.

There are currently no approved targeted treatments for quadruple-negative breast cancer [QNBC; ER-/PR-/HER2-/androgen receptor (AR)-], a subtype of triple-negative breast cancer (TNBC). AR-low TNBC is more proliferative and clinically aggressive than AR-high TNBC. Centrosome amplification (CA), a cancer hallmark, is rampant in TNBC, where it induces spindle multipolarity-mediated cell death unless centrosome clustering pathways are co-upregulated to avert these sequelae. We recently showed that genes that confer CA and centrosome clustering are strongly overexpressed in AR-low TNBCs relative to AR-high TNBCs. However, the molecular mechanisms that index centrosome clustering to the levels of CA are undefined. We argue that FOXM1, a cell cycle-regulated oncogene, links the expression of genes that drive CA to the expression of genes that act at kinetochores and along microtubules to facilitate centrosome clustering. We provide compelling evidence that upregulation of the FOXM1-E2F1-ATAD2 oncogene triad in AR-low TNBC is accompanied by CA and the co-upregulation of centrosome clustering proteins such as KIFC1, AURKB, BIRC5, and CDCA8, conferring profound dysregulation of cell cycle controls. Targeting FOXM1 in AR-low TNBC may render cancer cells incapable of clustering their centrosomes and impair their ability to generate excess centrosomes. Hence, our review illuminates FOXM1 as a potential actionable target for AR-low TNBC.

Cell cycle-dependent centrosome clustering precedes proplatelet formation.

Platelet-producing megakaryocytes (MKs) primarily reside in the bone marrow, where they duplicate their DNA content with each cell cycle resulting in polyploid cells with an intricate demarcation membrane system. While key elements of the cytoskeletal reorganizations during proplatelet formation have been identified, what initiates the release of platelets into vessel sinusoids remains largely elusive. Using a cell cycle indicator, we observed a unique phenomenon, during which amplified centrosomes in MKs underwent clustering following mitosis, closely followed by proplatelet formation, which exclusively occurred in G1 of interphase. Forced cell cycle arrest in G1 increased proplatelet formation not only in vitro but also in vivo following short-term starvation of mice. We identified that inhibition of the centrosomal protein kinesin family member C1 (KIFC1) impaired clustering and subsequent proplatelet formation, while KIFC1-deficient mice exhibited reduced platelet counts. In summary, we identified KIFC1- and cell cycle-mediated centrosome clustering as an important initiator of proplatelet formation from MKs.

A novel role for KIFC1-MYH9 interaction in triple-negative breast cancer aggressiveness and racial disparity

African American (AA) women are twice as likely to develop triple-negative breast cancer (TNBC) as women of European descent. Additionally, AA women with TNBC present a much more aggressive disease course than their European American (EA) counterparts. Thus, there is an unmet clinical need to identify race-specific biomarkers and improve survival outcomes in AA patients with TNBC. The minus-end directed microtubule motor protein kinesin family member C1 (KIFC1) promotes centrosome clustering and chromosomal instability and is often overexpressed in TNBC. Previous findings suggest that KIFC1 plays a role in cell proliferation and migration in TNBC cells from AAs and that the levels of nuclear KIFC1 (nKIFC1) are particularly high in AA patients with TNBC. The nuclear localization of KIFC1 in interphase may underlie its previously unrecognized race-specific association. In this study, we found that in TNBC cells derived from AAs, nKIFC1 interacted with the tumor suppressor myosin heavy chain 9 (MYH9) over EA cells. Treatment of AA TNBC cells with commercial inhibitors of KIFC1 and MYH9 disrupted the interaction between KIFC1 and MYH9. To characterize the racial differences in the KIFC1-MYH9-MYC axis in TNBC, we established homozygous KIFC1 knockout (KO) TNBC cell lines. KIFC1 KO significantly inhibited proliferation, migration, and invasion in AA TNBC cells but not in EA TNBC cells. RNA sequencing analysis showed significant downregulation of genes involved in cell migration, invasion, and metastasis upon KIFC1 KO in TNBC cell lines from AAs compared to those from EAs. These data indicate that mechanistically, the role of nKIFC1 in driving TNBC progression and metastasis is stronger in AA patients than in EA patients, and that KIFC1 may be a critical therapeutic target for AA patients with TNBC.Graphical abstract

Loss of ninein interferes with osteoclast formation and causes premature ossification.

Ninein is a centrosome protein that has been implicated in microtubule anchorage and centrosome cohesion. Mutations in the human NINEIN gene have been linked to Seckel syndrome and to a rare form of skeletal dysplasia. However, the role of ninein in skeletal development remains unknown. Here, we describe a ninein knockout mouse with advanced endochondral ossification during embryonic development. Although the long bones maintain a regular size, the absence of ninein delays the formation of the bone marrow cavity in the prenatal tibia. Likewise, intramembranous ossification in the skull is more developed, leading to a premature closure of the interfrontal suture. We demonstrate that ninein is strongly expressed in osteoclasts of control mice, and that its absence reduces the fusion of precursor cells into syncytial osteoclasts, whereas the number of osteoblasts remains unaffected. As a consequence, ninein-deficient osteoclasts have a reduced capacity to resorb bone. At the cellular level, the absence of ninein interferes with centrosomal microtubule organization, reduces centrosome cohesion, and provokes the loss of centrosome clustering in multinucleated mature osteoclasts. We propose that centrosomal ninein is important for osteoclast fusion, to enable a functional balance between bone-forming osteoblasts and bone-resorbing osteoclasts during skeletal development.

MISP Is Overexpressed in Intestinal Metaplasia and Gastric Cancer.

Gastric cancer is the fifth most common cancer and the fourth cause of global cancer mortality. The identification of new biomarkers and drug targets is crucial to allow the better prognosis and treatment of patients. The mitotic spindle positioning (MISP) protein has the function of correcting mitotic spindle positioning and centrosome clustering and has been implicated in the cytokinesis and migration of cancer cells. The goal of this work was to evaluate the expression and clinical relevance of MISP in gastric cancer. MISP expression was evaluated by immunohistochemistry in a single hospital series (n = 286) of gastric adenocarcinomas and compared with normal gastric mucosa and intestinal metaplasia, a preneoplastic lesion. MISP was detected on the membrane in 83% of the cases, being overexpressed in gastric cancer compared to normal gastric mucosa (n = 10). Its expression was negatively associated with diffuse and poorly cohesive types. On the other hand, it was strongly expressed in intestinal metaplasia where it was associated with MUC2 and CDX2 expression. Furthermore, when we silenced MISP in vitro, a significant decrease in the viability of gastric carcinoma cells was observed. In conclusion, MISP is overexpressed in gastric cancer, being associated with an intestinal phenotype in gastric carcinogenesis and having a role in cellular proliferation.

Abstract PO4-24-04: Identifying the interactome of TACC3, a major driver in aggressive cancer cells with centrosome amplification

Abstract Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) is a centrosome- and spindle-associated protein that drives the growth of highly aggressive tumors, such as those with centrosome amplification (CA). In recent years, it has become apparent that TACC3 not only functions at the centrosomes and spindles but may also have many other non-canonical functions facilitating the growth of tumors. Given the multifaceted roles of TACC3 in driving tumor aggressiveness, the identification of novel interactors of TACC3 that are responsible for mediating key TACC3-driven processes is crucial. This will not only uncover novel drug targets but will also expand the patient subpopulations that can benefit from TACC3 inhibition. Here, we characterized the cancer cells with CA in terms of their dependency on TACC3 for executing distinct cellular processes depending on the cell cycle phase. We demonstrated that TACC3 is strongly upregulated in cancers with CA at mRNA and protein levels and associated with worse clinical outcome in highly aggressive patient subpopulations, especially in TNBC. We further demonstrated that in mitotic cancer cells with CA, TACC3 is localized at the centrosomes in a complex with KIFC1, thus mediating the clustering of extra centrosomes to enable bipolar spindle formation and faithful mitosis. We showed that inhibiting TACC3 blocks the interaction between TACC3 and KIFC1, thus leading to mitotic cell death in the highly TACC3-dependent cancer cells with CA. While TACC3-KIFC1 complex is needed for mitotic cancer cells with CA, we found that in CA-bearing interphase cells, TACC3 interacts with HDAC2 and MBD2, the two major components of the chromatin remodeling NuRD complex within the nucleus. Inhibiting TACC3 prevented its nuclear localization, thus unloading the NuRD complex from the chromatin, leading to transcription of key tumor suppressors driving G1 arrest and apoptosis. To obtain the proteome-wide interaction network of TACC3 in these highly TACC3-dependent mitotic and interphase cancer cells with CA, we mapped the TACC3 interactome using the state-of-the-art APEX2 method. This uncovered potentially novel partners of TACC3 that could be critical for the survival of mitotic and interphase cancer cells with CA. We performed an extensive bioinformatic analysis to identify the most clinically relevant interactors in the context of cancers with CA. We generated correlation matrices of the mitotic and interphase-specific interactors and coupled them with pathway enrichment analysis to identify the key processes that the interactors are potentially involved in. Altogether, our results map, for the first time, the novel cell cycle-dependent TACC3 interactome in cancer cells with CA. Our results support targeting this multi-functional protein and hold great promise to improve clinical outcome in these highly aggressive cancers given the availability of the first-in-class IND-approved TACC3 inhibitor. Citation Format: Ozge Saatci, Ozge Akbulut, Metin Cetin, Vitali Sikirzhytski, Meral Uner, Deniz Lengerli, Elizabeth C. O’Quinn, Martin J. Romeo, Burcu Caliskan, Erden Banoglu, Sercan Aksoy, Aysegul Uner, Ozgur Sahin. Identifying the interactome of TACC3, a major driver in aggressive cancer cells with centrosome amplification [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-04.

High Expression of KIFC1 in Glioma Correlates with Poor Prognosis.

Kinesin family member C1 (KIFC1), a non-essential kinesin-like motor protein, has been found to serve a crucial role in supernumerary centrosome clustering and the progression of several human cancer types. However, the role of KIFC1 in glioma has been rarely reported. Thus, the present study aimed to investigate the role of KIFC1 in glioma progression. Online bioinformatics analysis was performed to determine the association between KIFC1 expression and clinical outcomes in glioma. Immunohistochemical staining was conducted to analyze the expression levels of KIFC1 in glioma and normal brain tissues. Furthermore, KIFC1 expression was knocked in the glioma cell lines, U251 and U87MG, and the functional roles of KIFC1 in cell proliferation, invasion and migration were analyzed using cell multiplication, wound healing and Transwell invasion assays, respectively. The autophagic flux and expression levels matrix metalloproteinase-2 (MMP2) were also determined using imaging flow cytometry, western blotting and a gelation zymography assay. The results revealed that KIFC1 expression levels were significantly upregulated in glioma tissues compared with normal brain tissues, and the expression levels were positively associated with tumor grade. Patients with glioma with low KIFC1 expression levels had a more favorable prognosis compared with patients with high KIFC1 expression levels. In vitro, KIFC1 knockdown not only inhibited the proliferation, migration and invasion of glioma cells, but also increased the autophagic flux and downregulated the expression levels of MMP2. Upregulation of KIFC1 expression may promote glioma progression and KIFC1 may serve as a potential prognostic biomarker and possible therapeutic target for glioma.

Surviving the Storm: The Role of Poly- and Depolyploidization in Tissues and Tumors.

Polyploidization and depolyploidization are critical processes in the normal development and tissue homeostasis of diploid organisms. Recent investigations have revealed that polyaneuploid cancer cells (PACCs) exploit this ploidy variation as a survival strategy against anticancer treatment and for the repopulation of tumors. Unscheduled polyploidization and chromosomal instability in PACCs enhance malignancy and treatment resistance. However, their inability to undergo mitosis causes catastrophic cellular death in most PACCs. Adaptive ploid reversal mechanisms, such as multipolar mitosis, centrosome clustering, meiosis-like division, and amitosis, counteract this lethal outcome and drive cancer relapse. The purpose of this work is to focus on PACCs induced by cytotoxic therapy, highlighting the latest discoveries in ploidy dynamics in physiological and pathological contexts. Specifically, by emphasizing the role of "poly-depolyploidization" in tumor progression, the aim is to identify novel therapeutic targets or paradigms for combating diseases associated with aberrant ploidies.

Abstract 3347: KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe

Abstract Cancer cells often have more than two centrosomes (supernumerary centrosomes) and this is one of the hallmarks of cancer. We previously demonstrated that CDK2 antagonism inhibits clustering of supernumerary centrosomes at mitosis leading to multipolar cell division and apoptotic death in non-small cell lung cancer. This antineoplastic process was designated anaphase catastrophe. Anaphase catastrophe can preferentially affect cancer cells sparing normal cells with two centrosomes. On the other hand, as CDK2 functions in normal cell cycle progression, its inhibition can also affect normal and malignant cells. Identification of a novel drug target that would preferentially cause anaphase catastrophe is needed. The motor protein KIFC1 affects centrosome clustering. Small cell lung cancer (SCLC), known for its poor prognosis with limited therapeutic options, displays disruptions in the cell cycle checkpoint mechanism due to inactivation of p53 and RB. We hypothesized SCLC was susceptible to mitotic abnormalities, including anaphase catastrophe. In this study, we explored the possibility of KIFC1 as a novel therapeutic target that triggers anaphase catastrophe in SCLC. In-silico GEO database analysis revealed elevated levels of KIFC1 and PLK4 in SCLCs compared with other cancers and normal tissues. PLK4 plays a key role in centrosome amplification. Our in-silico analysis results imply that centrosome amplification and their clustering frequently occur in SCLC. We performed in-vitro functional analysis of KIFC1 inhibition in SCLC cell lines (NCI-H146, SHP77, NCI-H209, and NCI-H524 cells), using siRNAs and CRISPR-Cas9 system for genetic inhibition and AZ82, a specific KIFC1 inhibitor, for pharmacologic inhibition of KIFC1. Cell proliferation was measured by the CellTiter-Glo Luminescent Assay and apoptosis was scored by annexin V and PI staining followed by flow cytometry analysis. Cell growth was reduced and apoptosis was statistically significantly increased after genetic inhibition of KIFC1 by siRNAs and by use of the CRISPR-Cas9 system in SCLC cell lines. These antineoplastic effects also occurred after AZ82 treatments, in a dose-dependent manner in these SCLC cell lines. To examine the mitotic status of SCLC cells after KIFC1 inhibition, SCLC cells were stained with α-tubulin antibody and DAPI before analysis with a fluorescence microscope. Besides normal bipolar mitotic cells, multipolar mitotic cells were observed, and their population was increased after KIFC1 knockdown with siRNAs. This indicated that clustering of supernumerary centrosomes was inhibited by targeting KIFC1 in SCLC cells. Taken together, KIFC1 is a potential therapeutic target to induce anaphase catastrophe in cancer cells. Targeting KIFC1 is a novel therapeutic strategy to consider for SCLC that currently has limited treatment options. Citation Format: Natsuki Nakagawa, Masakatsu Tokunaga, Mirei Ka, Yuriko Sugiura, Takahiro Iida, Takahiro Ando, Kousuke Watanabe, Xi Liu, Ethan Dmitrovsky, Hidenori Kage, Masanori Kawakami. KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3347.

Abstract 527: Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification

Abstract Therapy resistance is one of the most common underlying causes of poor prognosis in cancer patients, ultimately driving fatal outcome. We have previous demonstrated that following chemotherapeutic stress, a subset of cancer cells undergo a mitotic skip and enter an endocycle, causing whole-genome duplication without division. This endocycling cell state is resistant to cytotoxic therapies and thus is an actuator of therapy resistance. Cells in the endocycling state eventually undergo depolyploidization and repopulate the tumor, observed clinically as a recurrence. We also demonstrated that the minus-end directed motor protein KIFC1, a mediator of centrosome clustering, has higher expression in cells following treatment. Centrosome amplification (CA) involves aberrations in centrosome number, where cells have 3 centrosomes. Cells with extra centrosomes create a multi-polar spindle during mitosis, which presents high risk for cell death and arrest, but cancer cells with CA avoid this by clustering extra centrosomes to create a pseudo-bipolar spindle. Based upon this data, we hypothesize that studying centrosome dynamics and abnormalities presents a window into better understanding endocycle biology and this novel mechanism of therapy resistance. Cancer cells were treated with LD50 dose of cisplatin and released from treatment for varying lengths of time. CA in endocycling cells increased from 59.2% to 100% during the treatment recovery period while maintaining identical levels of clustering. Endocycling cancer cells have a nearly 5-fold increase in declustered centrosomes compared to proliferative cells. Centrosomes in endocycling cells are displaced from the perinuclear region and toward the cell periphery. We hypothesize that centrosome number may indicate the number of times a cell has endocycled and provide insight into the timing and mechanism of mitotic bypass. Centrosomes play a major role in microtubule nucleation, affecting cell motility and polarity, and act as signaling platforms, enhancing signaling activation and specificity. Because of this, we hypothesize that CA may be a critical mechanism for endocycling cancer cells to survive and maintain this resistant cell state. Citation Format: Madison Purkerson, Sarah Amend, Kenneth J. Pienta. Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 527.

Abstract 2117: Elucidation of the fates of CDK2 inhibited aneuploid and residual lung cancers

Abstract Genomic instability is a hallmark of cancer. Unlike normal diploid cells, cancer cells exhibit abnormal centrosome numbers and chromosomal instability. Mitosis with supernumerary centrosomes yields progeny with aberrant chromosome segregation and aneuploidy. While most progeny cells with abnormal chromosomes are eliminated through apoptosis, some have distinct fates with a growth advantage. These cancer cells can acquire resistance to antineoplastic agents. This confers an aggressive tumor biology and unfavorable clinical outcomes. Cyclin-dependent kinase 2 (CDK2) regulates cell cycle progression and the centrosome cycle. We previously reported that CDK2 inhibition prevents supernumerary centrosome clustering, causing multipolar mitosis and anaphase catastrophe in lung and other aneuploid cancers. This was associated with residual transplanted tumors in mice. To elucidate cell fates of progeny with multipolar mitosis after CDK2/9 inhibition with CYC065 (0.2 µM) treatment, a panel of murine and human lung cancer cells underwent time-lapse fluorescent microscopy. This elucidated multipolar mitotic events within the progeny. Intriguingly, distinct outcomes occurred after mitosis including formation of multipolar and multinucleated cells. Cell death of progeny is the predominant fate, but some progeny survive despite continuous CDK2 inhibition. Surprisingly, some of these cells fuse together. Those multinucleated cells can undergo multiple cell cycles without successful cytokinesis. To confirm these effects were through CDK2 inhibition, the selective CDK2 inhibitor Tagtociclib (PF-07104091, at the 2 µM dosage) was used. This treatment statistically-significantly increased multipolar lung cancer cells and increased apoptotic death during time-lapse fluorescent microscopy. Findings were independently validated by CDK2 shRNA knockdown. Focused ion beam scanning electron microscopy (FIB-SEM) and immunofluorescent staining revealed the ultrastructure of multinucleated cells. Insights into tumor biology came from intravital imaging of transplanted lung cancer cells in mice. Multipolar and multinuclear cells followed CDK2 antagonism. Incucyte® Live-Cell Analysis System and Artificial Intelligence-based imaging determined distinct growth responses in aneuploid as compared to non-aneuploid lung cancer cells after CDK2 inhibitor treatments. In summary, these findings are translationally relevant. CDK2 inhibition of aneuploid lung cancers yields distinct cell fates. This is linked to resistance to CDK2 antagonism and to formation of residual in vivo tumors. Citation Format: Liliya Tyutyunyk, Zibo Chen, Xiuxia Liu, Yeap Ng, Aayush Bhatawadekar, Kedar Narayan, Roberto Weigert, Xi Liu, Ethan Dmitrovsky. Elucidation of the fates of CDK2 inhibited aneuploid and residual lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2117.

The Eμ-Ret mouse is a novel model of hyperdiploid B-cell acute lymphoblastic leukemia

The presence of supernumerary chromosomes is the only abnormality shared by all patients diagnosed with high-hyperdiploid B cell acute lymphoblastic leukemia (HD-ALL). Despite being the most frequently diagnosed pediatric leukemia, the lack of clonal molecular lesions and complete absence of appropriate experimental models have impeded the elucidation of HD-ALL leukemogenesis. Here, we report that for 23 leukemia samples isolated from moribund Eμ-Ret mice, all were characterized by non-random chromosomal gains, involving combinations of trisomy 9, 12, 14, 15, and 17. With a median gain of three chromosomes, leukemia emerged after a prolonged latency from a preleukemic B cell precursor cell population displaying more diverse aneuploidy. Transition from preleukemia to overt disease in Eμ-Ret mice is associated with acquisition of heterogeneous genomic abnormalities affecting the expression of genes implicated in pediatric B-ALL. The development of abnormal centrosomes in parallel with aneuploidy renders both preleukemic and leukemic cells sensitive to inhibitors of centrosome clustering, enabling targeted in vivo depletion of leukemia-propagating cells. This study reveals the Eμ-Ret mouse to be a novel tool for investigating HD-ALL leukemogenesis, including supervision and selection of preleukemic aneuploid clones by the immune system and identification of vulnerabilities that could be targeted to prevent relapse.

Nek2A prevents centrosome clustering and induces cell death in cancer cells via KIF2C interaction

Unlike normal cells, cancer cells frequently exhibit supernumerary centrosomes, leading to formation of multipolar spindles that can trigger cell death. Nevertheless, cancer cells with supernumerary centrosomes escape the deadly consequences of unequal segregation of genomic material by coalescing their centrosomes into two poles. This unique trait of cancer cells presents a promising target for cancer therapy, focusing on selectively attacking cells with supernumerary centrosomes. Nek2A is a kinase involved in mitotic regulation, including the centrosome cycle, where it phosphorylates linker proteins to separate centrosomes. In this study, we investigated if Nek2A also prevents clustering of supernumerary centrosomes, akin to its separation function. Reduction of Nek2A activity, achieved through knockout, silencing, or inhibition, promotes centrosome clustering, whereas its overexpression results in inhibition of clustering. Significantly, prevention of centrosome clustering induces cell death, but only in cancer cells with supernumerary centrosomes, both in vitro and in vivo. Notably, none of the known centrosomal (e.g., CNAP1, Rootletin, Gas2L1) or non-centrosomal (e.g., TRF1, HEC1) Nek2A targets were implicated in this machinery. Additionally, Nek2A operated via a pathway distinct from other proteins involved in centrosome clustering mechanisms, like HSET and NuMA. Through TurboID proximity labeling analysis, we identified novel proteins associated with the centrosome or microtubules, expanding the known interaction partners of Nek2A. KIF2C, in particular, emerged as a novel interactor, confirmed through coimmunoprecipitation and localization analysis. The silencing of KIF2C diminished the impact of Nek2A on centrosome clustering and rescued cell viability. Additionally, elevated Nek2A levels were indicative of better patient outcomes, specifically in those predicted to have excess centrosomes. Therefore, while Nek2A is a proposed target, its use must be specifically adapted to the broader cellular context, especially considering centrosome amplification. Discovering partners such as KIF2C offers fresh insights into cancer biology and new possibilities for targeted treatment.

Inhibiting centrosome clustering reduces cystogenesis and improves kidney function in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder accounting for approximately 5% of patients with renal failure, yet therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display abnormalities in the biogenesis of the centrosome, a defect that can cause genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via "centrosome clustering," a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that preventing centrosome clustering with 2 inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibiting centrosome clustering activates a p53-mediated surveillance mechanism leading to apoptosis, reduced cyst expansion, decreased interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results demonstrate that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.

Inhibition of the YAP-MMB interaction and targeting NEK2 as potential therapeutic strategies for YAP-driven cancers.

YAP activation in cancer is linked to poor outcomes, making it an attractive therapeutic target. Previous research focused on blocking the interaction of YAP with TEAD transcription factors. Here, we took a different approach by disrupting YAP's binding to the transcription factor B-MYB using MY-COMP, a fragment of B-MYB containing the YAP binding domain fused to a nuclear localization signal. MY-COMP induced cell cycle defects, nuclear abnormalities, and polyploidization. In an AKT and YAP-driven liver cancer model, MY-COMP significantly reduced liver tumorigenesis, highlighting the importance of the YAP-B-MYB interaction in tumor development. MY-COMP also perturbed the cell cycle progression of YAP-dependent uveal melanoma cells but not of YAP-independent cutaneous melanoma cell lines. It counteracted YAP-dependent expression of MMB-regulated cell cycle genes, explaining the observed effects. We also identified NIMA-related kinase (NEK2) as a downstream target of YAP and B-MYB, promoting YAP-driven transformation by facilitating centrosome clustering and inhibiting multipolar mitosis.

CDK2 inhibition disorders centrosome stoichiometry and alters cellular outcomes in aneuploid cancer cells

ABSTRACT Cyclin-dependent Kinase 2 (CDK2) inhibition prevents supernumerary centrosome clustering. This causes multipolarity, anaphase catastrophe and apoptotic death of aneuploid cancers. This study elucidated how CDK2 antagonism affected centrosome stoichiometry. Focused ion beam scanning electron microscopy (FIB-SEM) and immunofluorescent imaging were used. Studies interrogated multipolar mitosis after pharmacologic or genetic repression of CDK2. CDK2/9 antagonism with CYC065 (Fadraciclib)-treatment disordered centrosome stoichiometry in aneuploid cancer cells, preventing centrosome clustering. This caused ring-like chromosomes or multipolar cancer cells to form before onset of cell death. Intriguingly, CDK2 inhibition caused a statistically significant increase in single centrioles rather than intact centrosomes with two centrioles in cancer cells having chromosome rings or multipolarity. Statistically significant alterations in centrosome stoichiometry were undetected in other mitotic cancer cells. To confirm this pharmacodynamic effect, CDK2 but not CDK9 siRNA-mediated knockdown augmented cancer cells with chromosome ring or multipolarity formation. Notably, engineered gain of CDK2, but not CDK9 expression, reversed emergence of cancer cells with chromosome rings or multipolarity, despite CYC065-treatment. In marked contrast, CDK2 inhibition of primary human alveolar epithelial cells did not confer statistically significant increases of cells with ring-like chromosomes or multipolarity. Hence, CDK2 antagonism caused differential effects in malignant versus normal alveolar epithelial cells. Translational relevance was confirmed by CYC065-treatment of syngeneic lung cancers in mice. Mitotic figures in tumors exhibited chromosome rings or multipolarity. Thus, CDK2 inhibition preferentially disorders centrosome stoichiometry in cancer cells. Engaging this disruption is a strategy to explore against aneuploid cancers in future clinical trials.

Mechanisms underlying Myosin 10's contribution to the maintenance of mitotic spindle bipolarity.

Myosin 10 (Myo10) couples microtubules and integrin-based adhesions to movement along actin filaments via its microtubule-binding MyTH4 domain and integrin-binding FERM domain, respectively. Here we show that Myo10-depleted HeLa cells and mouse embryo fibroblasts (MEFs) both exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in Myo10-depleted MEFs and in Myo10-depleted HeLa cells lacking supernumerary centrosomes is pericentriolar material (PCM) fragmentation, which creates y-tubulin-positive acentriolar foci that serve as extra spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing the clustering of the extra spindle poles. Complementation experiments show that Myo10 must interact with both microtubules and integrins to promote PCM/pole integrity. Conversely, Myo10 only needs interact with integrins to promote supernumerary centrosome clustering. Importantly, images of metaphase Halo-Myo10 knockin cells show that the myosin localizes exclusively to the spindle and the tips of adhesive retraction fibers. We conclude that Myo10 promotes PCM/pole integrity in part by interacting with spindle microtubules, and that it promotes supernumerary centrosome clustering by supporting retraction fiber-based cell adhesion, which likely serves to anchor the microtubule-based forces driving pole focusing.

Centrosome clustering control in osteoclasts through CCR5-mediated signaling

Osteoclasts uniquely resorb calcified bone matrices. To exert their function, mature osteoclasts maintain the cellular polarity and directional vesicle trafficking to and from the resorbing bone surface. However, the regulatory mechanisms and pathophysiological relevance of these processes remain largely unexplored. Bone histomorphometric analyses in Ccr5-deficient mice showed abnormalities in the morphology and functional phenotype of their osteoclasts, compared to wild type mice. We observed disorganized clustering of nuclei, as well as centrosomes that organize the microtubule network, which was concomitant with impaired cathepsin K secretion in cultured Ccr5-deficient osteoclasts. Intriguingly, forced expression of constitutively active Rho or Rac restored these cytoskeletal phenotypes with recovery of cathepsin K secretion. Furthermore, a gene-disease enrichment analysis identified that PLEKHM1, a responsible gene for osteopetrosis, which regulates lysosomal trafficking in osteoclasts, was regulated by CCR5. These experimental results highlighted that CCR5-mediated signaling served as an intracellular organizer for centrosome clustering in osteoclasts, which was involved in the pathophysiology of bone metabolism.

Kinesin Family Member C1 (KIFC1/HSET) Underlies Aggressive Disease in Androgen Receptor-Low and Basal-Like Triple-Negative Breast Cancers.

Quadruple-negative breast cancer (QNBC) lacks traditional actionable targets, including androgen receptor (AR). QNBC disproportionately afflicts and impacts patients of African genetic ancestry. Kinesin family member C1 (KIFC1/HSET), a centrosome clustering protein that prevents cancer cells from undergoing centrosome-amplification-induced apoptosis, has been reported to be upregulated in TNBCs and African-American (AA) TNBCs. Herein, we analyzed KIFC1 RNA levels and their associations with clinical features and outcomes among AR-low and AR-high TNBC tumors in three distinct publicly available gene expression datasets and in the breast cancer gene expression database (bc-GenExMiner). KIFC1 levels were significantly higher in AR-low and basal-like TNBCs than in AR-high and non-basal-like TNBCs, irrespective of the stage, grade, tumor size, and lymph node status. KIFC1 levels were also upregulated in AR-low tumors relative to AR-high tumors among Black and premenopausal women with TNBC. High KIFC1 levels conferred significantly shorter overall survival, disease-free survival, and distant metastasis-free survival among AR-low and basal-like TNBC patients in Kaplan-Meier analyses. In conclusion, KIFC1 levels may be upregulated in AR-low tumors and, specifically, in those of African descent, wherein it may promote poor outcomes. KIFC1 may be an actionable cancer-cell-specific target for the AR-low TNBC subpopulation and could aid in alleviating racial disparities in TNBC outcomes.