Transcriptional regulation of CCNO during the formation of multiple motile cilia.

Primary ciliary dyskinesia (PCD) is a group of genetically heterogeneous disorders characterized by clinical manifestations resulting from abnormal ciliary motility. Mutations in critical genes, such as Cyclin O (CCNO), have been associated with severe respiratory disease, though limited data are currently available. Here we show that CCNO deficient ciliated cells can only form a reduced number of fully functional centrioles that can mature into ciliated basal bodies, and their transport and anchoring to the top of the plasma membrane are abnormal. Furthermore, we observed that CCNO localizes not only in the cytoplasm but also in the nucleus during the early stages of ciliogenesis, and this dual localization persists into adulthood. Transcriptome analysis revealed downregulation of genes involved in cilia assembly and movement, along with altered transcription factors associated with ciliation upon CCNO depletion. These findings indicate that CCNO may serve as a key regulator in the transcriptional regulation of multiciliogenesis.

Aryl hydrocarbon receptor is required for optimal B-cell proliferation.

The aryl hydrocarbon receptor (AhR), a transcription factor known for mediating xenobiotic toxicity, is expressed in B cells, which are known targets for environmental pollutants. However, it is unclear what the physiological functions of AhR in B cells are. We show here that expression of Ahr in B cells is up-regulated upon B-cell receptor (BCR) engagement and IL-4 treatment. Addition of a natural ligand of AhR, FICZ, induces AhR translocation to the nucleus and transcription of the AhR target gene Cyp1a1, showing that the AhR pathway is functional in B cells. AhR-deficient (Ahr-/-) B cells proliferate less than AhR-sufficient (Ahr+/+) cells following in vitro BCR stimulation and in vivo adoptive transfer models confirmed that Ahr-/- B cells are outcompeted by Ahr+/+ cells. Transcriptome comparison of AhR-deficient and AhR-sufficient B cells identified cyclin O (Ccno), a direct target of AhR, as a top candidate affected by AhR deficiency.

RACK1 promotes miR-302b/c/d-3p expression and inhibits CCNO expression to induce cell apoptosis in cervical squamous cell carcinoma.

BACKGROUND: Cervical squamous cell carcinoma (CSCC) is one of the main causes of cancer-related deaths in women worldwide. The present study was conducted with the main objective of determining the potential role of receptor for activated protein kinase C1 (RACK1) in CSCC through regulation of microRNA (miR)-302b/c/d-3p and Cyclin O (CCNO). METHODS: The expression of RACK1, miR-302b/c/d-3p and CCNO in CSCC tissues and cells was measured by RT-qPCR and Western blot analysis. The interaction among RACK1, miR-302b/c/d-3p, and CCNO was determined by dual luciferase reporter assay. Subsequently, effects of RACK1, miR-302b/c/d-3p and CCNO on CSCC cell cycle entry, proliferation and apoptosis were investigated with the use of flow cytometry, EdU, and TUNEL assays. Furthermore, mouse xenograft model of CSCC cells was established to verify the function of RACK1 in vivo. RESULTS: RACK1 and miR-302b/c/d-3p were down-regulated and CCNO was overexpressed in CSCC. CCNO was identified as the target of miR-302b/c/d-3p. Importantly, overexpressed miR-302b-3p, miR-302c-3p or miR-302d-3p or RACK1 enhanced the apoptosis and suppressed the proliferation of CSCC cells in vitro, while inhibiting tumor growth in vivo by targeting CCNO. CONCLUSIONS: On all accounts, overexpressed RACK1 could dampen the progression of CSCC through miR-302b/c/d-3p-mediated CCNO inhibition.

The First Case of a Homozygous CCNO NM 021147.4 Mutation Associated With Primary Ciliary Dyskinesia in Two Indian Siblings.

Primary ciliary dyskinesia (PCD) is a heterogeneous autosomal recessive disease marked by organ lateralization in 50% of patients, chronic sinopulmonary disease, infertility in men, and neonatal respiratory distress. Respiratory control cells contain CCNO in their apical cytoplasm, which is necessary for the development of multiciliate cells, basal body amplification, and migration. Reduced generation of multiple motile cilia, a rare form of PCD, has been linked to CCNO gene abnormalities. Individuals with CCNO mutations have been reported to suffer from severe lower respiratory infections that cause progressive impairment of lung function. For the first time, we describe the CCNO NM 021147.4 (c.258 262dup.p, Gln88argfs*8 Homozygous) gene mutation in an Indian consanguineous family that resulted in severe PCD.

Two Japanese Pediatric Patients With Primary Ciliary Dyskinesia Caused by Loss-of-Function Variants in the CCNO gene.

Primary ciliary dyskinesia (PCD) is a rare congenital disorder caused by pathogenic variants of genes related to cilia. Here, we report two Japanese pediatric patients with PCD caused by pathogenic compound heterozygous variants in the cyclin O (CCNO) gene (Case 1, NM_021147.4:c.[262C>T];[781delC], p.[Gln88Ter];[Leu261fs]; Case 2, c.[262C>T];[c.248_252dupTGCCC], p.[Gln88Ter];[Gly85fs]). The clinical symptoms of the patients were varied. Neither of the patients had situs inversus. Transmission electron microscopy of the respiratory cilia from the nasal mucosa in Case 1 showed a remarkable reduction of cilia and the few residual cilia had central pair defects and microtubular disorganization.

Cyclin O regulates germinal vesicle breakdown in mouse oocytes.

It is well accepted that oocyte meiotic resumption is mainly regulated by the maturation-promoting factor (MPF), which is composed of cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDC2). Maturation-promoting factor activity is regulated by the expression level of CCNB1, phosphorylation of CDC2, and their germinal vesicle (GV) localization. In addition to CCNB1, cyclin O (CCNO) is highly expressed in oocytes, but its biological functions are still not clear. By employing short interfering RNA microinjection of GV-stage oocytes, we found that Ccno knockdown inhibited CDC2 (Tyr15) dephosphorylation and arrested oocytes at the GV stage. To rescue meiotic resumption, cell division cycle 25 B kinase (Cdc25b) and Ccnb1 were overexpressed in the Ccno knockdown oocytes. Unexpectedly, we found that Ccno knockdown did not affect CDC25B entry into the GV, and overexpression of CDC25B was not able to rescue resumption of oocyte meiosis. However, GV breakdown (GVBD) was significantly increased after overexpression of Ccnb1 in Ccno knockdown oocytes, indicating that GVBD block caused by cyclin O knockdown can be rescued by cyclin B1 overexpression. We thus conclude that cyclin O, as an upstream regulator of MPF, plays an important role in oocyte meiotic resumption in mouse oocytes.

Cyclin O promotes lung cancer progression and cetuximab resistance via cell cycle regulation and CDK13 interaction.

Background: Cyclin O (CCNO) is a novel cyclin family protein containing a cyclin-like domain, which plays a role in cell cycle regulation. Recent research suggests that inhibition of CCNO leads to cell apoptosis in gastric cancer, cervical squamous cell carcinoma, and post-operative lung cancer. Methods: The protein expression and signal transduction were detected by Western blot (WB) and immunohistochemistry (IHC). Overexpression or lacking CCNO stable cell lines were transfected with lentiviruses and selected with puromycin. The tumor behaviors of lung adenocarcinoma (LUAD) cells were assessed: cell proliferation by 5-Ethynyl-2'-deoxyuridine (EdU) staining and Cell Counting Kit-8 (CCK8) assay, cell cycle and by flow cytometry analysis, and migration and invasion using wound healing and Transwell system. Co-immunoprecipitation was used to detect protein-protein interactions. Xenograft models for evaluating tumor growth and anti-tumor drug efficacy. Results: A higher expression of CCNO was observed in LUAD cancer tissues and predicted the overall survival of LUAD patients. Moreover, CCNO expression was negatively correlated with cancer cell proliferation, migration, and invasion. Co-immunoprecipitation and western blot indicated that CCNO interacted with CDK13 to promote cancer cell proliferation signaling activation. Furthermore, CCNO promoted tumor cell growth and cetuximab resistance in vivo, and a CDK13 inhibitor effectively inhibited the oncological effect of CCNO. Conclusions: The current study suggests that CCNO may be a driver in the development of LUAD and that its function is related to CDK13 interaction that promotes proliferation signaling activation.

Knockdown of CCNO decreases the tumorigenicity of gastric cancer by inducing apoptosis.

PURPOSE: Recently, Cyclin O (CCNO) has been reported to be a novel protein of the cyclin family. However, the clinical significance and functional roles of CCNO in human cancer, including gastric cancer (GC), remain largely unexplored. In this study, we investigated the clinical and functional roles of CCNO in GC. METHODS: We analyzed CCNO expression patterns in GC patients. To investigate the role of CCNO in malignancy of GC, we used lentivirus-delivered short hairpin RNA to knockdown CCNO expression in GC cell lines. Then multiparametric high-content screening and MTT incorporation assay were used to assess the cell proliferation capability. Cell apoptosis was detected by flow cytometry and Caspase 3/7 assays. Furthermore, the effect of CCNO on tumorigenicity of GC was also determined in vivo. Finally, microarray analysis was performed to elucidate the molecular mechanisms by which shCCNO inhibited the malignancy of GC cells. RESULTS: The analysis from The Cancer Genome Atlas database revealed elevated CCNO mRNA expression in GC tissue than in the adjacent normal tissue. Immunohistochemical studies also showed that stronger cytoplasmic staining of CCNO was detected in GC tissues. Downregulation of CCNO in GC cells efficiently, through infection with the lentivirus-mediated specific short hairpin RNA, could significantly induce cell apoptosis and inhibit the proliferative properties both in vitro and in vivo. Microarray analysis further revealed 652 upregulated genes and 527 downregulated genes in the shCCNO group compared with control, and indicated that CCNO knockdown could inhibit the malignancy of GC cells through inducing genome-wide gene expression changes. CONCLUSION: Our work is the first to reveal that elevated CCNO expression is closely associated with human GC development and that CCNO knockdown could efficiently inhibit the malignant properties of GC cells by inducing cell apoptosis. Therefore, CCNO could be used as a potential biomarker for prognosis or even as a therapeutic target in human GC.

Constitutive Cyclin O deficiency results in penetrant hydrocephalus, impaired growth and infertility.

Cyclin O (encoded by CCNO) is a member of the cyclin family with regulatory functions in ciliogenesis and apoptosis. Homozygous CCNO mutations have been identified in human patients with Reduced Generation of Multiple Motile Cilia (RGMC) and conditional inactivation of Ccno in the mouse recapitulates some of the pathologies associated with the human disease. These include defects in the development of motile cilia and hydrocephalus. To further investigate the functions of Ccno in vivo, we have generated a new mouse model characterized by the constitutive loss of Ccno in all tissues and followed a cohort during ageing. Ccno-/- mice were growth impaired and developed hydrocephalus with high penetrance. In addition, some Ccno+/- mice also developed hydrocephalus and affected Ccno-/- and Ccno+/- mice exhibited additional CNS defects including cortical thinning and hippocampal abnormalities. In addition to the CNS defects, both male and female Ccno-/- mice were infertile and female mice exhibited few motile cilia in the oviduct. Our results further establish CCNO as an important gene for normal development and suggest that heterozygous CCNO mutations could underlie hydrocephalus or diminished fertility in some human patients.

Phosphorylation of cyclin O, a novel cyclin family protein containing a cyclin-like domain, is involved in the activation of cyclin-dependent kinase 2.

Cell cycles, ordered series of events modulating cell growth and division, are tightly regulated by complexes containing cyclin-dependent kinases (CDKs) and cyclins. Cyclin O is a novel cyclin family protein which interacts with CDK2. However, the molecular effects of cyclin O on the activity of CDK2 have not been fully evaluated. In this study, an interaction between cyclin O and CDK2 was identified by co-immunoprecipitation and the effect of cyclin O on the kinase activity of CDK2 was investigated using cyclin O point mutants. Co-immunoprecipitation was achieved using using HEK293 human embryonic kidney cells which were transiently transfected with vectors expressing cyclin O and CDK2, which revealed that cyclin O interacted with CDK2, particularly with the active form of endogenous CDK2. Cyclin O was expressed as several different bands with molecular weights between 45 and 50 kDa, possibly due to different post-translational modifications. When co-expressed with CDK2, cyclin O appeared as a band with a molecular weight of 50 kDa. Treatment with calf intestinal phosphatase reduced the intensity of the uppermost band. Mass spectroscopic analysis of cyclin O co-expressed with CDK2 revealed that the 81st serine residue of cyclin O was phosphorylated. The in vitro kinase activity of CDK2 phosphorylating histone H1 was markedly increased in the cells overexpressing cyclin O. This activity was reduced in cells overexpressing cyclin O, in which the 81st serine had been replaced with alanine (S81A). These results suggest that cyclin O is a novel cyclin family protein that regulates CDK2 kinase activity, which is mediated by the phosphorylation of the 81st serine residue of cyclin O.