A novel HIF1α-STIL-FOXM1 axis regulates tumor metastasis.

BACKGROUND: Metastasis is the major cause of morbidity and mortality in cancer that involves in multiple steps including epithelial-mesenchymal transition (EMT) process. Centrosome is an organelle that functions as the major microtubule organizing center (MTOC), and centrosome abnormalities are commonly correlated with tumor aggressiveness. However, the conclusive mechanisms indicating specific centrosomal proteins participated in tumor progression and metastasis remain largely unknown. METHODS: The expression levels of centriolar/centrosomal genes in various types of cancers were first examined by in silico analysis of the data derived from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and European Bioinformatics Institute (EBI) datasets. The expression of STIL (SCL/TAL1-interrupting locus) protein in clinical specimens was further assessed by Immunohistochemistry (IHC) analysis and the oncogenic roles of STIL in tumorigenesis were analyzed using in vitro and in vivo assays, including cell migration, invasion, xenograft tumor formation, and metastasis assays. The transcriptome differences between low- and high-STIL expression cells were analyzed by RNA-seq to uncover candidate genes involved in oncogenic pathways. The quantitative polymerase chain reaction (qPCR) and reporter assays were performed to confirm the results. The chromatin immunoprecipitation (ChIP)-qPCR assay was applied to demonstrate the binding of transcriptional factors to the promoter. RESULTS: The expression of STIL shows the most significant increase in lung and various other types of cancers, and is highly associated with patients' survival rate. Depletion of STIL inhibits tumor growth and metastasis. Interestingly, excess STIL activates the EMT pathway, and subsequently enhances cancer cell migration and invasion. Importantly, we reveal an unexpected role of STIL in tumor metastasis. A subset of STIL translocate into nucleus and associate with FOXM1 (Forkhead box protein M1) to promote tumor metastasis and stemness via FOXM1-mediated downstream target genes. Furthermore, we demonstrate that hypoxia-inducible factor 1α (HIF1α) directly binds to the STIL promoter and upregulates STIL expression under hypoxic condition. CONCLUSIONS: Our findings indicate that STIL promotes tumor metastasis through the HIF1α-STIL-FOXM1 axis, and highlight the importance of STIL as a promising therapeutic target for lung cancer treatment.

CEP120-mediated KIAA0753 recruitment onto centrioles is required for timely neuronal differentiation and germinal zone exit in the developing cerebellum.

Joubert syndrome (JS) is a recessive ciliopathy in which all affected individuals have congenital cerebellar vermis hypoplasia. Here, we report that CEP120, a JS-associated protein involved in centriole biogenesis and cilia assembly, regulates timely neuronal differentiation and the departure of granule neuron progenitors (GNPs) from their germinal zone during cerebellar development. Our results show that depletion of Cep120 perturbs GNP cell cycle progression, resulting in a delay of cell cycle exit in vivo. To dissect the potential mechanism, we investigated the association between CEP120 interactome and the JS database and identified KIAA0753 (a JS-associated protein) as a CEP120-interacting protein. Surprisingly, we found that CEP120 recruits KIAA0753 to centrioles, and that loss of this interaction induces accumulation of GNPs in the germinal zone and impairs neuronal differentiation. Importantly, the replenishment of wild-type CEP120 rescues the above defects, whereas expression of JS-associated CEP120 mutants, which hinder KIAA0753 recruitment, does not. Together, our data reveal a close interplay between CEP120 and KIAA0753 for the germinal zone exit and timely neuronal differentiation of GNPs during cerebellar development, and mutations in CEP120 and KIAA0753 may participate in the heterotopia and cerebellar hypoplasia observed in JS patients.

Author Correction: CEP120 interacts with C2CD3 and Talpid3 and is required for centriole appendage assembly and ciliogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Overexpression of Notch Signaling Induces Hyperosteogeny in Zebrafish.

Notch signaling is one of the evolutionarily conserved signaling pathways in multicellular organisms. It plays an important role in embryonic development. During skeletal development of vertebrates, it regulates bone homeostasis by manipulating both osteoblastogenesis and osteoclastogenesis through different mechanisms. However, due to the different nature of Notch signaling in mesenchymal stem cell and osteoblast, regulation of Notch signaling in bone-related diseases remains unsettled. Previous studies by cell culture and mouse models showed contradictory results regarding the role of Notch signaling in bone homeostasis. To clarify the role of Notch signaling in osteogenesis, we established a zebrafish model, in which Notch1a intracellular domain (N1aICD) was specifically expressed in the osteoblasts. We found that overexpression of N1aICD in osteoblasts caused hyperosteogeny in the column region of zebrafish with the morphology of narrowed neural/hemal canals. Moreover, increased metabolic activity of osteoblasts instead of augmenting osteoblast number led to hyperosteogeny in N1aICD-overexpressed zebrafish. In summary, we successfully established a transgenic zebrafish line overexpressing N1aICD to clarify the in-vivo function of Notch signaling during osteoblastogenesis. In the future, this fish line can serve as a valuable tool to test the therapeutic drugs for hyperosteogeny.

CEP120 interacts with C2CD3 and Talpid3 and is required for centriole appendage assembly and ciliogenesis.

Centrosomal protein 120 (CEP120) was originally identified as a daughter centriole-enriched protein that participates in centriole elongation. Recent studies showed that CEP120 gene mutations cause complex ciliopathy phenotypes in humans, including Joubert syndrome and Jeune asphyxiating thoracic dystrophy, suggesting that CEP120 plays an additional role in ciliogenesis. To investigate the potential roles of CEP120 in centriole elongation and cilia formation, we knocked out the CEP120 gene in p53-deficient RPE1 cells using the CRISPR/Cas9 editing system, and performed various analyses. We herein report that loss of CEP120 produces short centrioles with no apparent distal and subdistal appendages. CEP120 knockout was also associated with defective centriole elongation, impaired recruitment of C2CD3 and Talpid3 to the distal ends of centrioles, and consequent defects in centriole appendage assembly and cilia formation. Interestingly, wild-type CEP120 interacts with C2CD3 and Talpid3, whereas a disease-associated CEP120 mutant (I975S) has a low affinity for C2CD3 binding and perturbs cilia assembly. Together, our findings reveal a novel role of CEP120 in ciliogenesis by showing that it interacts with C2CD3 and Talpid3 to assemble centriole appendages and by illuminating the molecular mechanism through which the CEP120 (I975S) mutation causes complex ciliopathies.

Live Fluorescent Staining Platform for Drug-Screening and Mechanism-Analysis in Zebrafish for Bone Mineralization.

Currently, drug screening relies on cell-based experiments or on animal models to confirm biological effects. The mammalian system is considered too time-consuming, expensive and complex to perform high-throughput drug screening. There is a gap between in vitro cell-based models and the in vivo mammalian models. The zebrafish is an ideal model that could link preclinical toxicity screening with the drug development pipeline. Taking advantage of a highly conservative genomic, rapid development, large number of offspring, low cost and easy manipulation, zebrafish has been considered an excellent animal model for disease-based drug screening. In this study, zebrafish embryos were incubated with small molecular compounds that potentially affected bone mineralization in microplates. Two compounds of alendronate and dorsomorphin were used as positive and negative controls, respectively. The level of osteogenic mineralization was measured and quantified by using ImageJ software with fluorescent calcein-staining images. Among twenty-four tested compounds from the kinase inhibitor library, we identified two compounds, pentamidine and BML-267, which showed increased embryonic mineralization; while six compounds, RWJ-60475, levamisole HCL, tetramisole HCL, fenvalerate, NSC-663284, and BML-267ester, were inhibitory to bone mineralization. In addition, real time quantitative PCR (RT-qPCR) was performed to evaluate the biological pathways involved in bone metabolism at the molecular level. We confirmed that alendronate enhanced the level of bone mineralization by inhibiting osteoclast-related genes. In summary, our research established a simple method to screen potential bone metabolic drugs and to perform mechanism analysis for bone mineralization in vivo.

CEP295 interacts with microtubules and is required for centriole elongation.

Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295 (Drosophila Ana1) is not essential for initial cartwheel assembly, but is required to build distal half centrioles during S and G2. Using super-resolution and immunogold electron microscopy, we demonstrate that CEP295 is recruited to the proximal end of procentrioles in early S phase, when it is also localized at the centriolar microtubule wall that surrounds the human SAS6 cartwheel hub. Interestingly, depletion of CEP295 not only inhibits the recruitments of POC5 and POC1B to the distal half centrioles in G2, resulting in shorter centrioles, it also blocks the post-translational modification of centriolar microtubules (e.g. acetylation and glutamylation). Importantly, our results indicate that CEP295 directly interacts with microtubules, and that excess CEP295 could induce the assembly of overly long centrioles. Furthermore, exogenous expression of the N-terminal domain of CEP295 exerts a dominant-negative effect on centriole elongation. Collectively, these findings suggest that CEP295 is essential for building the distal half centrioles and for post-translational modification of centriolar microtubules.