Centrobin-centrosomal protein 4.1-associated protein (CPAP) interaction promotes CPAP localization to the centrioles during centriole duplication.

Centriole duplication is the process by which two new daughter centrioles are generated from the proximal end of preexisting mother centrioles. Accurate centriole duplication is important for many cellular and physiological events, including cell division and ciliogenesis. Centrosomal protein 4.1-associated protein (CPAP), centrosomal protein of 152 kDa (CEP152), and centrobin are known to be essential for centriole duplication. However, the precise mechanism by which they contribute to centriole duplication is not known. In this study, we show that centrobin interacts with CEP152 and CPAP, and the centrobin-CPAP interaction is critical for centriole duplication. Although depletion of centrobin from cells did not have an effect on the centriolar levels of CEP152, it caused the disappearance of CPAP from both the preexisting and newly formed centrioles. Moreover, exogenous expression of the CPAP-binding fragment of centrobin also caused the disappearance of CPAP from both the preexisting and newly synthesized centrioles, possibly in a dominant negative manner, thereby inhibiting centriole duplication and the PLK4 overexpression-mediated centrosome amplification. Interestingly, exogenous overexpression of CPAP in the centrobin-depleted cells did not restore CPAP localization to the centrioles. However, restoration of centrobin expression in the centrobin-depleted cells led to the reappearance of centriolar CPAP. Hence, we conclude that centrobin-CPAP interaction is critical for the recruitment of CPAP to procentrioles to promote the elongation of daughter centrioles and for the persistence of CPAP on preexisting mother centrioles. Our study indicates that regulation of CPAP levels on the centrioles by centrobin is critical for preserving the normal size, shape, and number of centrioles in the cell.

Centrobin: a novel daughter centriole-associated protein that is required for centriole duplication.

In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle. Centrosomes play a pivotal role in orchestrating the formation of the bipolar spindle during mitosis. Recent studies have linked centrosomal activity on centrioles or centriole-associated structures to cytokinesis and cell cycle progression through G1 into the S phase. In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole. The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication. Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.

Identification of Angiogenesis Inhibitors Using a Co-culture Cell Model in a High-Content and High-Throughput Screening Platform.

Angiogenesis is an important hallmark of cancer, contributing to tumor formation and metastasis. In vitro angiogenesis models for analyzing tube formation serve as useful tools to study these processes. However, current in vitro co-culture models using primary cells have limitations in usefulness and consistency. Therefore, in the present study, an in vitro co-culture assay system was optimized in a 1536-well format for high-throughput screening using human telomerase reverse transcriptase (hTERT)-immortalized mesenchymal stem cells and aortic endothelial cells. The National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (NPC) library containing 2816 drugs was evaluated using the in vitro co-culture assay. From the screen, 35 potent inhibitors (IC50 ≤1 µM) were identified, followed by 15 weaker inhibitors (IC50 1-50 µM). Moreover, many known angiogenesis inhibitors were identified, such as topotecan, docetaxel, and bortezomib. Several potential novel angiogenesis inhibitors were also identified from this study, including thimerosal and podofilox. Among the inhibitors, some compounds were proved to be involved in the hypoxia-inducible factor-1α (HIF-1α) and the nuclear factor-kappa B (NF-κB) pathways. The co-culture model developed by using hTERT-immortalized cell lines described in this report provides a consistent and robust in vitro system for antiangiogenic drug screening.

BRCA2 suppresses cell proliferation via stabilizing MAGE-D1.

Germ line mutations in BRCA2 gene predispose women to early-onset familial breast and ovarian cancer. BRCA2 is a protein of multiple functions. In addition to its role in DNA double-strand break repair, BRCA2 also plays a role in stabilization of stalled DNA replication forks, cytokinesis, transcription regulation, mammalian gametogenesis, centrosome duplication, and suppression of cell proliferation. However, how BRCA2 mutations predispose women specifically to breast and ovarian cancer remains undefined. Here we found that BRCA2 binds and stabilizes MAGE-D1, a member of the MAGE gene family of proteins. Expression of BRCA2 and MAGE-D1 synergistically suppresses cell proliferation independently of the p53 pathway. Using two MAGE-D1 RNA interferences and two cell lines expressing low or undetectable levels of MAGE-D1, we further showed that the expression of MAGE-D1 is required for BRCA2-mediated suppression of cell proliferation, indicating that MAGE-D1 is a downstream target of BRCA2 and that BRCA2 suppresses cell proliferation via stabilizing MAGE-D1. Importantly, MAGE-D1 protein expression was reduced in 6 of 16 breast carcinoma cell lines tested as compared with untransformed immortal mammary epithelial cell lines, suggesting that suppression of MAGE-D1 expression may be involved in the tumorigenesis of a subset of sporadic breast cancers.

Identification of approved and investigational drugs that inhibit hypoxia-inducible factor-1 signaling.

One of the requirements for tumor development is blood supply, most often driven by hypoxia-induced angiogenesis. Hypoxia induces the stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), which induces expression of an angiogenic factor, vascular endothelial growth factor (VEGF). The purpose of this study is to validate a new screening platform combined with orthogonal assays to rapidly identify HIF-1 inhibitors and to evaluate the effectiveness of approved drugs on modulating HIF-1 signaling. We generated an endogenous HIF-1α-NanoLuc luciferase reporter allele in the human HCT116 colon cancer cell line using genome editing and screened a panel of small interfering RNAs (siRNAs) to 960 druggable targets and approximately 2,500 drugs on a quantitative high-throughput screening (qHTS) platform. Selected compounds were further investigated with secondary assays to confirm their anti-HIF activity and to study their mode of action. The qHTS assay identified over 300 drugs that inhibited HIF-1α-NanoLuc expression. The siRNA screening results supported the effectiveness of several target-specific inhibitors. Moreover, the identified HIF-1 inhibitors, such as mycophenolate mofetil, niclosamide, and trametinib, were able to suppress cancer cell proliferation and angiogenesis. Our study indicates that blocking the mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways effectively inhibits hypoxia-induced HIF-1α accumulation and HIF-1α transactivation and that proteasome inhibitors induce accumulation and decrease transcriptional activity of HIF-1α. These findings underline the importance of developing a battery of robust assay platforms and confirmation studies that focus on endogenous protein targets so that only relevant and reliable data will be taken into pre-clinical and clinical studies.

Centrobin-tubulin interaction is required for centriole elongation and stability.

Centrobin is a daughter centriole protein that is essential for centrosome duplication. However, the molecular mechanism by which centrobin functions during centriole duplication remains undefined. In this study, we show that centrobin interacts with tubulin directly, and centrobin-tubulin interaction is pivotal for the function of centrobin during centriole duplication. We found that centrobin is recruited to the centriole biogenesis site via its interaction with tubulins during the early stage of centriole biogenesis, and its recruitment is dependent on hSAS-6 but not centrosomal P4.1-associated protein (CPAP) and CP110. The function of centrobin is also required for the elongation of centrioles, which is likely mediated by its interaction with tubulin. Furthermore, disruption of centrobin-tubulin interaction led to destabilization of existing centrioles and the preformed procentriole-like structures induced by CPAP expression, indicating that centrobin-tubulin interaction is critical for the stability of centrioles. Together, our study demonstrates that centrobin facilitates the elongation and stability of centrioles via its interaction with tubulins.