Tomoatsu Hayashi

Role of the Kinesin-2 Family Protein, KIF3, during Mitosis

During mitosis, kinesin and dynein motor proteins play critical roles in the equal segregation of chromosomes between two daughter cells. Kinesin-2 is composed of two microtubule-based motor subunits, KIF3A/3B, and a kinesin-associated protein known as KAP3, which links KIF3A/3B to cargo that is carried to cellular organelles along microtubules in interphase cells. We have shown here that the kinesin-2 complex is localized with components of the mitotic apparatus such as spindle microtubules and centrosomes. Furthermore, we found that expression of a mutant KIF3B, which is able to associate with KIF3A but not KAP3 in NIH3T3 cells, caused chromosomal aneuploidy and abnormal spindle formation. Our data suggested that the kinesin-2 complex plays an important role not only in interphase but also in mitosis.

Role of the Rho GTPase-activating protein RICS in neurite outgrowth.

The Rho family of small GTPases, including RhoA, Rac1 and Cdc42, are critical regulators of the actin cytoskeleton. In neuronal systems, Rho GTPase‐activating proteins (RhoGAPs) and their substrates, Rho GTPases, have been implicated in regulating multiple processes in the morphological development of neurons, including axonal growth and guidance, dendritic elaboration and formation of synapses. RICS is mainly expressed in the brain and functions as a RhoGAP protein for Cdc42 and Rac1 in vitro. To examine the biological function of RICS, we disrupted the RICS gene in mice. RICS knockout mice developed normally and were fertile. However, when cultured in vitro, Cdc42 activity in RICS–/– neurons was higher than that in wild‐type neurons. Consistent with this finding, hippocampal and cerebellar granule neurons derived from RICS–/– mice bore longer neurites than those from wild‐type mice. These findings suggest that RICS plays an important role in neurite extension by regulating Cdc42 in vivo.

The miR-363-GATA6-Lgr5 pathway is critical for colorectal tumourigenesis

Aberrant activation of Wnt signalling results in colorectal tumours. Lgr5 is specifically expressed in stem cells of the intestine and has an essential role in maintaining tissue homeostasis. Lgr5-positive stem cells are responsible for the intestinal adenoma initiated by mutations in adenomatous polyposis coli. Furthermore, Lgr5 interacts with R-spondins and thereby activates Wnt signalling. However, the function of Lgr5 in colorectal tumourigenesis is unclear. Here we show that LGR5 is required for the tumourigenicity of colorectal cancer cells. We show that the transcription factor GATA6 directly enhances the expression of LGR5. We further demonstrate that GATA6 is upregulated in colorectal cancer cells due to the downregulation of miR-363, which directly targets GATA6. Moreover, we show that overexpression of miR-363 suppresses the tumourigenicity of colorectal cancer cells. These results suggest that the miR-363-GATA6-LGR5 pathway is critical for colorectal tumourigenesis and would be a promising target for the treatment of colorectal cancer.

PX-RICS mediates ER-to-Golgi transport of the N-cadherin/β-catenin complex

Cadherins mediate Ca2+-dependent cell-cell adhesion. Efficient export of cadherins from the endoplasmic reticulum (ER) is known to require complex formation with beta-catenin. However, the molecular mechanisms underlying this requirement remain elusive. Here we show that PX-RICS, a beta-catenin-interacting GTPase-activating protein (GAP) for Cdc42, mediates ER-to-Golgi transport of the N-cadherin/beta-catenin complex. Knockdown of PX-RICS expression induced the accumulation of the N-cadherin/beta-catenin complex in the ER and ER exit site, resulting in a decrease in cell-cell adhesion. PX-RICS was also required for ER-to-Golgi transport of the fibroblast growth factor-receptor 4 (FGFR4) associated with N-cadherin. PX-RICS-mediated ER-to-Golgi transport was dependent on its interaction with beta-catenin, phosphatidylinositol-4-phosphate (PI4P), Cdc42, and its novel binding partner gamma-aminobutyric acid type A receptor-associated protein (GABARAP). These results suggest that PX-RICS ensures the efficient entry of the N-cadherin/beta-catenin complex into the secretory pathway, and thereby regulates the amount of N-cadherin available for cell adhesion and FGFR4-mediated signaling.

A member of the ETS family, EHF, and the ATPase RUVBL1 inhibit p53-mediated apoptosis

Tumour cells are known to be dependent on, or ‘addicted to’, not only oncogenes, but also some non‐oncogenes. However, the mechanisms by which tumour cells are addicted to these genes have not been fully explained. Here, we show that overexpression of a member of the ETS family, EHF, is required for the survival of colon tumour cells that contain wild‐type p53. We found that EHF directly activates the transcription of RUVBL1, an ATPase associated with chromatin‐remodelling complexes. RUVBL1 blocks p53‐mediated apoptosis by repressing the expression of p53 and its target genes. Moreover, we found that RUVBL1 represses p53 transcription by binding to the p53 promoter, interfering with RNF20/hBRE1‐mediated histone H2B monoubiquitination and promoting PAF1‐mediated histone H3K9 trimethylation. These results indicate that EHF‐mediated RUVBL1 expression allows colon tumour cells to avoid p53‐mediated apoptosis. Thus, EHF and RUVBL1 might be promising molecular targets for the treatment of colon tumours.

The PX-RICS/14-3-3ζ/θ complex couples N-cadherin/β-catenin with dynein/dynactin to mediate its export from the endoplasmic reticulum

We have recently shown that β-catenin-facilitated export of cadherins from the endoplasmic reticulum requires PX-RICS, a β-catenin-interacting GTPase-activating protein for Cdc42. Here we show that PX-RICS interacts with isoforms of 14-3-3 and couples the N-cadherin-β-catenin complex to the microtubule-based molecular motor dynein-dynactin. Similar to knockdown of PX-RICS, knockdown of either 14-3-3ζ or -θ resulted in the disappearance of N-cadherin and β-catenin from the cell-cell boundaries. Furthermore, we found that PX-RICS and 14-3-3ζ/θ are present in a large multiprotein complex that contains dynein-dynactin components as well as N-cadherin and β-catenin. Both RNAi- and dynamitin-mediated inhibition of dynein-dynactin function also led to the absence of N-cadherin and β-catenin at the cell-cell contact sites. Our results suggest that the PX-RICS-14-3-3ζ/θ complex links the N-cadherin-β-catenin cargo with the dynein-dynactin motor and thereby mediates its endoplasmic reticulum export.

PX‐RICS, a novel splicing variant of RICS, is a main isoform expressed during neural development

In our previous study, we identified RICS, a novel β‐catenin‐interacting protein with the GAP activity toward Cdc42 and Rac1, and found that RICS plays an important role in the regulation of neural functions, including postsynaptic NMDA signaling and neurite outgrowth. Here we report the characterization of an N‐terminal splicing variant of RICS, termed PX‐RICS, which has additional phox homology (PX) and src homology 3 (SH3) domains in its N‐terminal region. The PX domain of PX‐RICS interacted specifically with phosphatidylinositol 3‐phosphate [PtdIns(3)P], PtdIns(4)P and PtdIns(5)P. Consistent with this binding affinity, PX‐RICS was found to be localized at the endoplasmic reticulum (ER), Golgi and endosomes. We also found that wild‐type PX‐RICS possessed much lower GAP activity than RICS, whereas a mutant form of PX‐RICS whose PX domain lacks the binding ability to phosphoinositides (PIs) exhibited the GAP activity comparable to that of RICS. However, PX‐RICS and RICS exhibited similar inhibitory effects on neurite elongation of Neuro‐2a cells. Furthermore, we demonstrate that PX‐RICS is a main isoform expressed during neural development. Our results suggest that PX‐RICS is involved in early brain development including extension of axons and dendrites, and postnatal remodeling and fine‐tuning of neural circuits.

SOX9-mediated upregulation of LGR5 is important for glioblastoma tumorigenicity

LGR5 plays an important role in the self-renewal of stem cells and is used as a marker identifying self-renewing stem cells in small intestine and hair follicles. Moreover, LGR5 has been reported to be overexpressed in several cancers. SOX9 is a transcription factor that plays a key role in development, differentiation and lineage commitment in various tissues. It has also been reported that SOX9 is overexpressed in a variety of cancers and contributes to their malignant phenotype. Here we show that LGR5 is required for the tumorigenicity of glioblastoma cells. We further show that SOX9 is upregulated in glioblastoma cells and directly enhances the expression of LGR5. We also demonstrate that knockdown of SOX9 suppresses the proliferation and tumorigenicity of glioblastoma cells. These results suggest that SOX9-mediated transcriptional regulation of LGR5 is critical for the tumorigenicity of glioblastoma cells. We speculate that the SOX9-LGR5 pathway could be a potentially promising target for the therapy of glioblastoma.

Design, synthesis and structure-activity relationship studies of novel sirtuin 2 (SIRT2) inhibitors with a benzamide skeleton.

Human sirtuin 2 (SIRT2) is an attractive target molecule for development of drugs to treat neurodegenerative diseases and cancer, because SIRT2 inhibitors have a protective effect against neurodegeneration and an anti-proliferative effect on cancer stem cells. We designed and synthesized a series of benzamide derivatives as SIRT2 inhibitor candidates. Among them, compound 17k showed the most potent SIRT2-inhibitory activity (IC50=0.60μM), with more than 150-fold selectivity over SIRT1 and SIRT3 isoforms (IC50 >100μM).

PCDH10 is required for the tumorigenicity of glioblastoma cells

Protocadherin10 (PCDH10)/OL-protocadherin is a cadherin-related transmembrane protein that has multiple roles in the brain, including facilitating specific cell-cell connections, cell migration and axon guidance. It has recently been reported that PCDH10 functions as a tumor suppressor and that its overexpression inhibits proliferation or invasion of multiple tumor cells. However, the function of PCDH10 in glioblastoma cells has not been elucidated. In contrast to previous reports on other tumors, we show here that suppression of the expression of PCDH10 by RNA interference (RNAi) induces the growth arrest and apoptosis of glioblastoma cells in vitro. Furthermore, we demonstrate that knockdown of PCDH10 inhibits the growth of glioblastoma cells xenografted into immunocompromised mice. These results suggest that PCDH10 is required for the proliferation and tumorigenicity of glioblastoma cells. We speculate that PCDH10 may be a promising target for the therapy of glioblastoma.