Kinase activity of histone chaperone APLF maintains steady state of centrosomes in mouse embryonic stem cells.

Our recent studies revealed the role of mouse Aprataxin PNK-like Factor (APLF) in development. Nevertheless, the comprehensive characterization of mouse APLF remains entirely unexplored. Based on domain deletion studies, here we report that mouse APLF's Acidic Domain and Fork Head Associated (FHA) domain can chaperone histones and repair DNA like the respective human orthologs. Immunofluorescence studies in mouse embryonic stem cells showed APLF co-localized with γ-tubulin within and around the centrosomes and govern the number and integrity of centrosomes via PLK4 phosphorylation. Enzymatic analysis established mouse APLF as a kinase. Docking studies identified three putative ATP binding sites within the FHA domain. Site-directed mutagenesis showed that R37 residue within the FHA domain is indispensable for the kinase activity of APLF thereby regulating the centrosome number. These findings might assist us comprehend APLF in different pathological and developmental conditions and reveal non-canonical kinase activity of proteins harbouring FHA domains that might impact multiple cellular processes.

Progress with polo-like kinase (PLK) inhibitors: a patent review (2018-present).

INTRODUCTION: Polo-like kinases (PLKs) have five isoforms, all of which play crucial roles in cell cycle and cell proliferation, offering opportunities for drug design and treatment of cancers and other related diseases. Notably, PLK1 and PLK4 have been extensively investigated as cancer drug targets. One distinctive feature of PLKs is the presence of a unique polo-box domain (PBD), which regulates kinase activity and subcellular localization. This provides possibilities for specifically targeting PLKs. AREA COVERED: This article provides an overview of the roles of PLKs in various cancers and related diseases, as well as the drug development involving PLKs, with a particular focus on PLK1 and PLK4. It summarizes the PLK1 and PLK4 inhibitors that have been disclosed in patents or literature (from 2018 - present), which were sourced from SciFinder and WIPO database. EXPERT OPINION: After two decades of drug development on PLKs, several drugs progressed into clinical trials for the treatment of many cancers; however, none of them has been approved yet. Further elucidating the mechanisms of PLKs and identifying and developing highly selective ATP-competitive inhibitors, highly potent drug-like PBD inhibitors, degraders, etc. may provide new opportunities for cancer therapy and the treatment for several nononcologic diseases. PLKs inhibition-based combination therapies can be another helpful strategy.

Spermatocytes have the capacity to segregate chromosomes despite centriole duplication failure.

Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a structurally ordered and dynamic pericentriolar matrix (PCM). Unlike in mitosis, where centrioles duplicate once per cycle, centrioles undergo two rounds of duplication during spermatogenesis. The first duplication is during early meiotic prophase I, and the second is during interkinesis. Using mouse mutants and chemical inhibition, we have blocked centriole duplication during spermatogenesis and determined that non-centrosomal MTOCs (ncMTOCs) can mediate chromosome segregation. This mechanism is different from the acentriolar MTOCs that form bipolar spindles in oocytes, which require PCM components, including gamma-tubulin and CEP192. From an in-depth analysis, we identified six microtubule-associated proteins, TPX2, KIF11, NuMA, and CAMSAP1-3, that localized to the non-centrosomal MTOC. These factors contribute to a mechanism that ensures bipolar MTOC formation and chromosome segregation during spermatogenesis when centriole duplication fails. However, despite the successful completion of meiosis and round spermatid formation, centriole inheritance and PLK4 function are required for normal spermiogenesis and flagella assembly, which are critical to ensure fertility.

Dissecting the Puzzling Roles of FAM46C: A Multifaceted Pan-Cancer Tumour Suppressor with Increasing Clinical Relevance.

FAM46C is a well-established tumour suppressor with a role that is not completely defined or universally accepted. Although FAM46C expression is down-modulated in several tumours, significant mutations in the FAM46C gene are only found in multiple myeloma (MM). Consequently, its tumour suppressor activity has primarily been studied in the MM context. However, emerging evidence suggests that FAM46C is involved also in other cancer types, namely colorectal, prostate and gastric cancer and squamous cell and hepatocellular carcinoma, where FAM46C expression was found to be significantly reduced in tumoural versus non-tumoural tissues and where FAM46C was shown to possess anti-proliferative properties. Accordingly, FAM46C was recently proposed to function as a pan-cancer prognostic marker, bringing FAM46C under the spotlight and attracting growing interest from the scientific community in the pathways modulated by FAM46C and in its mechanistic activity. Here, we will provide the first comprehensive review regarding FAM46C by covering (1) the intracellular pathways regulated by FAM46C, namely the MAPK/ERK, PI3K/AKT, ß-catenin and TGF-ß/SMAD pathways; (2) the models regarding its mode of action, specifically the poly(A) polymerase, intracellular trafficking modulator and inhibitor of centriole duplication models, focusing on connections and interdependencies; (3) the regulation of FAM46C expression in different environments by interferons, IL-4, TLR engagement or transcriptional modulators; and, lastly, (4) how FAM46C expression levels associate with increased/decreased tumour cell sensitivity to anticancer agents, such as bortezomib, dexamethasone, lenalidomide, pomalidomide, doxorubicin, melphalan, SK1-I, docetaxel and norcantharidin.

PLK4 as a potential target to enhance radiosensitivity in triple-negative breast cancer.

Radioresistance is one of the barriers to developing more effective therapies against the most aggressive, triple-negative, breast cancer (TNBC) subtype. In our previous studies, we showed that inhibition of Polo-like Kinase 4 (PLK4) by a novel drug, CFI-400945 significantly enhances the anticancer effects of radiotherapy (RT) compared to single treatment alone. Here we further investigate the role of PLK4 in enhancing radiation effects in TNBC and explore mechanisms of PLK4 inhibition and radiation combinatorial antiproliferative effects. To assess cellular proliferation in response to treatments, we used colony formation assays in TNBC cell lines and patient-derived organoids (PDOs). Downregulation of PLK4 expression was achieved using siRNA silencing in TNBC cell lines. Immunofluorescence against centrin was used to assess the alteration of centriole amplification in response to treatments. We observed that inhibition of PLK4 by CFI-400945 or Centrinone B or its downregulation by siRNA, when combined with RT, resulted in a significant increase in antiproliferative effect in TNBC cells lines and PDOs compared to untreated or single-treated cells. Anticancer synergy was observed using a response matrix in PDOs treated with CFI-400945 and RT. We show that the overamplification of centrioles might be involved in the combined antiproliferative action of RT and PLK4 inhibition. Our data suggest that PLK4 is a promising target for enhancing the anticancer effects of RT in TNBC that, at least in part, is modulated by the overamplification of centrioles. These results support further mechanistic and translational studies of anti-PLK4 agents and RT as an anticancer combination treatment strategy.

Prolonged overexpression of PLK4 leads to formation of centriole rosette clusters that are connected via canonical centrosome linker proteins.

Centrosome amplification is a hallmark of cancer and PLK4 is one of the responsible factors for cancer associated centrosome amplification. Increased PLK4 levels was also shown to contribute to generation of cells with centriole amplification in mammalian tissues as olfactory neuron progenitor cells. PLK4 overexpression generates centriole rosette (CR) structures which harbor more than two centrioles each. Long term PLK4 overexpression results with centrosome amplification, but the maturation of amplified centrioles in CRs and linking of PLK4 induced amplified centrosomes has not yet been investigated in detail. Here, we show evidence for generation of large clustered centrosomes which have more than 2 centriole rosettes and define these structures as centriole rosette clusters (CRCs) in cells that have high PLK4 levels for 2 consecutive cell cycles. In addition, we show that PLK4 induced CRs follow normal centrosomal maturation processes and generate CRC structures that are inter-connected with canonical centrosomal linker proteins as C-Nap1, Rootletin and Cep68 in the second cell cycle after PLK4 induction. Increased PLK4 levels in cells with C-Nap1 and Rootletin knock-out resulted with distanced CRs and CRCs in interphase, while Nek2 knock-out inhibited separation of CRCs in prometaphase, providing functional evidence for the binding of CRC structures with centrosomal linker proteins. Taken together, these results suggest a cell cycle dependent model for PLK4 induced centrosome amplification which occurs in 2 consecutive cell cycles: (i) CR state in the first cell cycle, and (ii) CRC state in the second cell cycle.

Therapeutic potential of targeting polo-like kinase 4.

Polo-like kinase 4 (PLK4), a highly conserved serine/threonine kinase, masterfully regulates centriole duplication in a spatiotemporal manner to ensure the fidelity of centrosome duplication and proper mitosis. Abnormal expression of PLK4 contributes to genomic instability and associates with a poor prognosis in cancer. Inhibition of PLK4 is demonstrated to exhibit significant efficacy against various types of human cancers, further highlighting its potential as a promising therapeutic target for cancer treatment. As such, numerous small-molecule inhibitors with distinct chemical scaffolds targeting PLK4 have been extensively investigated for the treatment of different human cancers, with several undergoing clinical evaluation (e.g., CFI-400945). Here, we review the structure, distribution, and biological functions of PLK4, encapsulate its intricate regulatory mechanisms of expression, and highlighting its multifaceted roles in cancer development and metastasis. Moreover, the recent advancements of PLK4 inhibitors in patent or literature are summarized, and their therapeutic potential as monotherapies or combination therapies with other anticancer agents are also discussed.

Cadmium promotes the binding and centrosomal translocation of CCDC85C and PLK4 via ROS-GCLM pathway to trigger centrosome amplification in colon cancer cells.

Cadmium (Cd) is a prevalent heavy metal contaminant that can cause centrosome amplification (CA) and cancer. Since CA can initiate tumorigenesis, it is plausible that cadmium initiates tumorigenesis via CA. The present study investigated the signaling pathways underlying CA by Cd. Our findings confirmed that sub-toxic concentrations of Cd could induce CA in the HCT116 colon cancer cells, and revealed that reactive oxygen species (ROS), GCLM, CCDC85C and PLK4 were the signaling molecules that formed a pathway of ROS-GCLM-CCDC85C-PLK4. Cd not only increased the protein levels of CCDC85C and PLK4, but also promoted their distribution to the centrosomes. Molecular docking analysis revealed that CCDC85C and PLK4 had the binding potential. Indeed, antibodies against CCDC85C and PLK4 were able to pull down PLK4 and CCDC85C, respectively. Knockdown of CCDC85C decreased the Cd-promoted centrosomal distribution of PLK4. Similarly, knockdown of PLK4 reduced the centrosomal distribution of CCDC85C. Our results suggest that Cd activates ROS-GCLM pathway that triggers the expression of and binding between CCDC85C and PLK4, and promotes the translocation of CCDC85C-PLK4 complex to the centrosomes, which eventually leads to CA.

Polo-like kinase 4 promotes tumorigenesis and glucose metabolism in glioma by activating AKT1 signaling.

Glioblastoma (GBM) is an extremely aggressive tumor associated with a poor prognosis that impacts the central nervous system. Increasing evidence suggests an inherent association between glucose metabolism dysregulation and the aggression of GBM. Polo-like kinase 4 (PLK4), a highly conserved serine/threonine protein kinase, was found to relate to glioma progression and unfavorable prognosis. As revealed by the integration of proteomics and phosphoproteomics, PLK4 was found to be involved in governing metabolic processes and the PI3K/AKT/mTOR pathway. For the first time, this study supports evidence demonstrating that PLK4 activated PI3K/AKT/mTOR signaling through direct binding to AKT1 and subsequent phosphorylating AKT1 at S124, T308, and S473 to promote tumorigenesis and glucose metabolism in glioma. In addition, PLK4-mediated phosphorylation of AKT1 S124 significantly augmented the phosphorylation of AKT1 S473. Therefore, PLK4 exerted an influence on glucose metabolism by stimulating PI3K/AKT/mTOR signaling. Additionally, the expression of PLK4 protein exhibited a positive correlation with AKT1 phosphorylation in glioma patient tissues. These findings highlight the pivotal role of PLK4-mediated phosphorylation of AKT1 in glioma tumorigenesis and dysregulation of glucose metabolism.

NAMPT mediates PDGF-induced pulmonary arterial smooth muscle cell proliferation by TLR4/NF-κB/PLK4 signaling pathway.

Nicotinamide phosphoribosyltransferase (NAMPT), a pleiotropic protein, promotes the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), which is associated with the genesis and progression of pulmonary arterial hypertension (PAH). NAMPT is highly increased in PAH patient's plasma and highly relevant to PAH severity. The mRNA and protein levels of NAMPT are elevated in PAH animal models. However, the underlying molecular mechanisms how NAMPT mediated platelet-derived growth factor (PDGF)-induced PASMCs proliferation are still unclear. The present study aimed to address these issues. Primary cultured PASMCs were attained from male Sprague-Dawley (SD) rats. Western blotting, RT-PCR, ELISA, cell transfection, Cell Counting Kit-8 (CCK-8) and EdU incorporation assays were used in the experiments. We showed that PDGF upregulated NAMPT expression through the activation of signal transducers and activators of transcription 5 (STAT5), and elevated extracellular NAMPT further promoted the activation of NF-κB through Toll-like receptor 4 (TLR4), which ultimately upregulated polo-like kinase 4 (PLK4) expression leading to PASMCs proliferation. Knockdown of STAT5, NAMPT or PLK4, and inhibition of TLR4 or NF-κB suppressed PDGF-induced PASMCs proliferation. Our study suggests that NAMPT plays an essential role in PDGF-induced PASMCs proliferation via TLR4/NF-κB/PLK4 pathway, suggesting that targeting NAMPT might be valuable in ameliorating pulmonary arterial hypertension.

Polo-like Kinase 4: A Multifaceted Marker Linking Tumor Aggressiveness and Unfavorable Prognosis, and Insights into Therapeutic Strategies.

(1) Background: This study investigated whether polo-like kinase 4 (PLK4) is a suitable therapeutic target or biomarker for lung adenocarcinoma (LUAD). (2) Methods: We acquired LUAD data from The Cancer Genome Atlas (TCGA) database through the UCSC Xena data portal. Gene expression, clinical, survival, and mutation data from multiple samples were analyzed. Gene enrichment analysis, unsupervised clustering of PLK4-related pathways, and differential gene expression analyses were performed. Additionally, correlations, t-tests, survival analyses, and statistical analyses were performed. (3) Results: PLK4 expression was higher in LUAD tissues than in normal tissues and was associated with poor prognosis for both overall and progression-free survival in LUAD. PLK4 was highly correlated with cell-proliferation-related pathways using Gene Ontology (GO) biological process terms. PLK4 expression and pathways that were highly correlated with PLK4 expression levels were upregulated in patients with LUAD with the TP53 mutation. (4) Conclusions: PLK4 expression affects the survival of patients with LUAD and is a potential therapeutic target for LUAD with TP53 mutations.

Autophagy Inhibition Contributes to Apoptosis of PLK4 Downregulation-induced Dormant Cells in Colorectal Cancer.

Dormant cancer cells account for cancer recurrence, distant metastasis and drug resistance which lead to poor prognosis in colorectal cancer (CRC). However, little is known about the molecular mechanisms regulating tumor cell dormancy and how to eliminate dormant cancer cells. Recent studies indicate autophagy affects dormant tumor cell survival. Here, we found that polo-like kinases 4 (PLK4), a central regulator of the cell cycle and proliferation, plays a crucial role in regulating CRC cells dormancy both in vitro and in vivo. Downregulation of PLK4 induced dormancy and inhibited migration and invasion in different CRC cell lines. Clinically, PLK4 expression was correlated with the dormancy markers (Ki67, p-ERK, p-p38) and late recurrence in CRC tissues. Mechanistically, downregulation of PLK4 induced autophagy contributed to restoring phenotypically aggressive tumor cells to a dormant state through the MAPK signaling pathway, and inhibition of autophagy would trigger apoptosis of dormant cells. Our findings reveal that downregulation of PLK4-induced autophagy contributes to tumor dormancy and autophagy inhibition leads to apoptosis of CRC dormant cells. Our study is the first to report that downregulation PLK4 induced autophagy is an early event in CRC dormancy and highlights autophagy inhibitor as a potential therapeutic target for dormant cell elimination.

PLK4 inhibitor plus bortezomib exhibits a synergistic effect on treating multiple myeloma via inactivating PI3K/AKT signaling.

OBJECTIVE: The anti-tumor effect of polo-like kinase 4 (PLK4) inhibitor has been explored in several neoplasms, while its synergy with bortezomib in multiple myeloma (MM) remains elusive. Hence, the present study aimed to investigate the effect of PLK4 inhibitor on the sensitivity of MM to bortezomib treatment and its underlying mechanism. METHODS: MM cell lines (RPMI-8226 and U266) were cultured in different concentrations of CFI-400945 (PLK4 inhibitor), bortezomib, or their combination. Subsequently, 740 Y-P (PI3K activator) was added in the combination of CFI-400945 and bortezomib. Besides, cell viability and apoptosis were measured by CCK-8 reagent and TUNEL apoptosis kit, separately; meanwhile, western blot was carried out for detecting PLK4, p-PI3K, PI3K, p-AKT, and AKT. RESULTS: CFI-400945 and bortezomib decreased the cell viability in dose-dependent manners in MM cell lines, respectively. The combination of different concentrations of CFI-400945 and bortezomib reduced cell viability compared with monotherapy in MM cell lines (all P < 0.05). Interestingly, 200 nM CFI-400945 and 4 nM bortezomib showed the maximum synergy in MM cell lines. Furthermore, 200 nM CFI-400945 plus 4 nM bortezomib showed a better effect on decreasing cell viability and promoting cell apoptosis than CFI-400945 or bortezomib monotherapy in MM cells cell lines (all P < 0.05). Moreover, 740 Y-P alleviated the effect of bortezomib and CFI-400945 on PI3K/AKT signaling, cell viability, and apoptosis in MM cell lines. CONCLUSIONS: PLK4 inhibitor plus bortezomib shows synergy in decreasing cell viability and enhancing cell apoptosis via repressing PI3K/AKT signaling in MM.

Discovery of Polo-like Kinase 4 Inhibitors for the Treatment of Cancer: A Mini Patent Review.

Polo-like kinase 4 (PLK4), a serine/threonine kinase, is a member of the PLK family. As a key regulator of the cell cycle, PLK4 controls centrosome duplication and mitosis. Abnormal PLK4's function can induce centrosome amplification, leading to tumorigenesis, therefore, PLK4 has been regarded as a promising target for cancer therapy, and PLK4 inhibitors have potentials to treat multiple cancers and other PLK4-associated human disorders, such as myelodysplastic syndrome. In addition, PLK4 may function as a DNA-damage sensitizer, therefore improving the efficacy of chemotherapy. To date, some small-molecule inhibitors with different chemical scaffolds targeting PLK4 have been reported, among which, CFI-400945 has entered clinical trials for the treatment of various solid tumors, myeloid leukemia, and myelodysplastic syndrome. In this review, the structure and biological functions of PLK4 with other homologous PLKs are compared; the roles of PLK4 in different cancers are reviewed; and PLK4 inhibitors disclosed in patent or literature are summarized. Used alone or in combination with other anticancer drugs in preclinical and clinical studies, PLK4 inhibitors have shown significant efficacy in the treatment of different cancers, demonstrating that PLK4 could be a critical target for cancer diagnosis and therapy. However, our understanding of PLK4 is still limited, and novel mechanisms of PLK4 should be identified in future studies.

Design, synthesis, and biological evaluation of a potent PLK4 inhibitor WY29 with 1H-pyrazolo[3,4-d]pyrimidine scaffold.

Centriole duplication occurs once per cell cycle and is regulated by Polo-like kinase 4 (PLK4). Overexpression of PLK4 in somatic cells can lead to the excessive formation of centrioles, directly causing chromosome segregation errors and tumorigenesis. In this study, we described our efforts to develop a series of PLK4 inhibitors with 1H-pyrazolo[3,4-d]pyrimidine core, and further structure- and receptor-based design and optimization resulted in a potent inhibitor WY29 (IC50 = 0.027 µM), which exhibited good selectivity to other PLK family members (PLK1-3). At the cellular level, compound WY29 showed excellent antiproliferative activity against three breast cancer cell lines (MCF-7, BT474, and MDA-MB-231) while weak inhibitory activity was found on normal cell line HUVECs. In addition, the in vitro preliminary drug-like properties evaluation of compound WY29 showed outstanding stability in human plasma and liver microsomes, and weak inhibitory activity against the major subtypes of human cytochrome P450. Also, the drug-like properties prediction of compound WY29 displayed remarkable drug-like properties (drug-likeness mode score: 1.06). In conclusion, these results support the further development of compound WY29 as a lead compound for PLK4-targeted anticancer drug discovery.

Effects of the PLK4 inhibitor Centrinone on the biological behaviors of acute myeloid leukemia cell lines.

Polo-like kinase 4 (PLK4), a key regulator of centriole biogenesis, is frequently overexpressed in cancer cells. However, roles and the mechanism of PLK4 in the leukemiagenesis of acute myeloid leukemia (AML) remain unclear. In this study, the PLK4 inhibitor Centrinone and the shRNA knockdown were used to investigate roles and the mechanism of PLK4 in the leukemiagenesis of AML. Our results indicated that Centrinone inhibited the proliferation of AML cells in a dose- and time-dependent manner via reduced the expression of PLK4 both in the protein and mRNA levels. Moreover, colony formation assay revealed that Centrinone reduced the number and the size of the AML colonies. Centrinone induced AML cell apoptosis by increasing the activation of Caspase-3/poly ADP-ribose polymerase (PARP). Notably, Centrinone caused the G2/M phase cell cycle arrest by decreasing the expression of cell cycle-related proteins such as Cyclin A2, Cyclin B1, and Cyclin-dependent kinase 1 (CDK1). Consistent with above results, knockdown the expression of PLK4 also inhibited cell proliferation and colony formation, induced cell apoptosis, and caused G2/M phase cell cycle arrest without affecting cell differentiation. All in all, this study suggested that PLK4 inhibited the progression of AML in vitro, and these results herein may provide clues in roles of PLK4 in the leukemiagenesis of AML.

One master to rule them all.

Multiciliated cells rely on the same master regulator as dividing cells to amplify the number of centrioles needed to generate the hair-like structures that coat their cell surface.

Structure-based discovery of 1-(3-fluoro-5-(5-(3-(methylsulfonyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)phenyl)-3-(pyrimidin-5-yl)urea as a potent and selective nanomolar type-II PLK4 inhibitor.

Polo-like kinase 4 (PLK4) is a serine/threonine protein kinase involved in regulating cell mitosis and centriole duplication, and has emerged as a therapeutic target for treating multiple cancers. At first, the design and in vitro validation of PLK4 inhibitors (12a-12e, 17a-17f, 22a-22e) bearing 1H-pyrazolo[3,4-b]pyridine scaffold was described and lead compound 22a (IC50 = 0.106 µM) was identified. Then, selectivity- and activity-guided development of a series of potent and selective type-II PLK4 inhibitors using a homology model approach was carried out. Further structure-based optimization resulted in a potent type-II PLK4 inhibitor 29u (IC50 = 0.026 µM), which exhibited outstanding selectivity in a panel of 47 kinases at a single concentration of 1.0 µM. Furthermore, compound 29u significantly inhibited the proliferation of breast cancer cell line MCF-7 with an IC50 value of 1.52 µM, while it exhibited no inhibitory effect on normal cell lines (L02 and HUVECs). Meanwhile, the clone formation, senescence and migration abilities of compound 29u were evaluated using MCF-7 cells. The detailed biological evaluation revealed that compound 29u could arrest cell division in S/G2 phase by inhibiting PLK4, and then affect the expression of downstream signalling pathway proteins regulated by PLK4. Moreover, the in vitro preliminary evaluation of the drug-like properties of compound 29u exhibited outstanding plasma stability, moderate liver microsomal stability, and low risk of drug-drug interactions (DDIs). The current discovery will support the further development of compound 29u as a lead compound for PLK4-targeted anticancer drug discovery and as a useful chemical probe for the further biological research of PLK4.

PLK4 is a key molecule in the formation of PGCCs and promotes invasion and migration of progeny cells derived from PGCCs.

Purpose: Cancer stem cells (CSCs) are the evil source of tumor metastasis and recurrence. Polyploid giant cancer cells (PGCCs) that exhibit the characteristics of CSCs produced daughter cells via asymmetric division. The molecular mechanisms of daughter cells derived from PGCCs with high migration, invasion, and proliferation abilities in colorectal cancer (CRC) are explored in this paper based on the bioinformatics analysis. Materials and Methods: We characterized the expression of CSC-related genes in CRCs by analyzing the mRNAsi of The Cancer Genome Atlas and survival time. Weighted gene co-expression network analysis was performed to identify the modules of the hub and key genes. The migration, invasion, and proliferation abilities of cells, the expression of epithelial-mesenchymal transition (EMT)-related proteins and polo-like kinase 4 (PLK4) were compared in LoVo and Hct116 cells with and without bufalin treatment. In addition, the expression and subcellular location of cell division cycle 25C (CDC25C) in cells before and after PLK4 knockdown were assessed. Results: Eight hub genes were screened out and positively association with mRNAsi in CRCs based on bioinformatic analysis. Among them, checkpoint Kinase-1 (CHEK1), budding uninhibited by benzimidazoles 1 Homolog Beta (BUB1B) and PLK4 were closely associated with the prognosis of CRC patients. Bufalin could induce the formation of PGCCs in LoVo and Hct116 cell lines. PLK4 was overexpressed in PGCCs with progeny cells and progeny cells derived from PGCCs had strong migration and invasion abilities by expressing epithelial-mesenchymal transition (EMT)-related proteins. PLK4 could interact with CDC25C and promote CDC25C phosphorylation which was associated with the formation of PGCCs. Decreasing CDC25C expression in both LoVo and Hct116 PGCCs with progeny cells, while levels of pCDC25C-ser216 and pCDC25C-ser198 were increased in LoVo and decreased in Hct116 PGCCs with progeny cells. pCDC25C-ser216 located in the cytoplasm and pCDC25C-ser198 located in the nucleus in cells after bufalin treatment. Furthermore, expression of CDC25C, pCDC25C-ser216, and pCDC25C-ser198 was downregulated after PLK4 knockdown. Furthermore, the expression level of PLK4 was associated with differentiated degree, and lymph node metastasis in human CRC tissues. Conclusion: PLK4 contributes to the formation of PGCCs by regulating the expression of CDC25C and is associated with the expression and subcellular location of CDC25C, pCDC25C-ser216 and pCDC25C-ser198.

PLK4 drives centriole amplification and apical surface area expansion in multiciliated cells.

Multiciliated cells (MCCs) are terminally differentiated epithelia that assemble multiple motile cilia used to promote fluid flow. To template these cilia, MCCs dramatically expand their centriole content during a process known as centriole amplification. In cycling cells, the master regulator of centriole assembly Polo-like kinase 4 (PLK4) is essential for centriole duplication; however recent work has questioned the role of PLK4 in centriole assembly in MCCs. To address this discrepancy, we created genetically engineered mouse models and demonstrated that both PLK4 protein and kinase activity are critical for centriole amplification in MCCs. Tracheal epithelial cells that fail centriole amplification accumulate large assemblies of centriole proteins and do not undergo apical surface area expansion. These results show that the initial stages of centriole assembly are conserved between cycling cells and MCCs and suggest that centriole amplification and surface area expansion are coordinated events.

Every day, we inhale thousands of viruses, bacteria and pollution particles. To protect against these threats, cells in our airways produce mucus that traps inhaled particles before they reach the lungs. This mucus then needs to be removed to prevent it from becoming a breeding ground for microbes that may cause a respiratory infection. This is the responsibility of cells covered in tiny hair-like structures called cilia that move together to propel the mucus-trapped particles out of the airways. These specialized cells can have up to 300 motile cilia on their surface, which grow from structures called centrioles that then anchor the cilia in place. Multiciliated cells are generated from precursor cells that only have two centrioles. Therefore, as these precursors develop, they must produce large numbers of centrioles, considerably more than other cells that only need a couple of extra centrioles during cell division. However, recent studies have questioned whether the precursors of multiciliated cells rely on the same regulatory proteins to produce centrioles as dividing cells. To help answer this question, LoMastro et al. created genetically engineered mice that lacked or had an inactive form of PLK4, a protein which controls centriole formation in all cell types lacking multiple cilia. This showed that multiciliated cells also need this protein to produce centrioles. LoMastro et al. also found that multiciliated cells became larger while building centrioles, suggesting that this amplification process helps control the cell's final size. Defects in motile cilia activity can lead to fluid build-up in the brain, respiratory infections and infertility. Unfortunately, these disorders are difficult to diagnose currently and there is no cure. The findings of LoMastro et al. further our understanding of how motile cilia are built and maintained, and may help future scientists to develop better diagnostic tools and treatments for patients.