Hirokazu Ohata

Apollon ubiquitinates SMAC and caspase-9, and has an essential cytoprotection function

Apollon (also known as BRUCE or BIRC6) is a large protein containing baculoviral-IAP-repeat (BIR) and ubiquitin-conjugating enzyme (UBC) domains at the amino- and carboxy termini, respectively. Apollon inhibits apoptosis, but its molecular and physiological function remains unclear. Here we report that Apollon binds to, ubiquitinates and facilitates proteasomal degradation of SMAC and caspase-9, which both contain IAP-binding motifs. Targeted disruption of Apollon in mice caused embryonic and neonatal lethality. Notably, SMAC induced apoptosis in Apollon-deficient cells, but not in Apollon-expressing cells. Furthermore, the IAP-binding motif of SMAC was required to induce apoptosis in Apollon-deficient cells. These results suggest that Apollon has an essential function in preventing SMAC-induced apoptosis.

miR‐493 induction during carcinogenesis blocks metastatic settlement of colon cancer cells in liver

Liver metastasis is a major lethal complication associated with colon cancer, and post‐intravasation steps of the metastasis are important for its clinical intervention. In order to identify inhibitory microRNAs (miRNAs) for these steps, we performed ‘dropout’ screens of a miRNA library in a mouse model of liver metastasis. Functional analyses showed that miR‐493 and to a lesser extent miR‐493* were capable of inhibiting liver metastasis. miR‐493 inhibited retention of metastasized cells in liver parenchyma and induced their cell death. IGF1R was identified as a direct target of miR‐493, and its inhibition partially phenocopied the anti‐metastatic effects. High levels of miR‐493 and miR‐493*, but not pri‐miR‐493, in primary colon cancer were inversely related to the presence of liver metastasis, and attributed to an increase of miR‐493 expression during carcinogenesis. We propose that, in a subset of colon cancer, upregulation of miR‐493 during carcinogenesis prevents liver metastasis via the induction of cell death of metastasized cells.

Induction of ZEB proteins by inactivation of RB protein is key determinant of mesenchymal phenotype of breast cancer.

Background: Inactivation of RB is a key event for induction of EMT in cancers. Results: ZEB proteins are markedly up-regulated through the reduction of miR-200 family of microRNAs in RB-inactive cancer cells. Conclusion: RB/ZEB pathway plays a pivotal role in mesenchymal and aggressive phenotype in breast cancers. Significance: Suppressing ZEB1 by cyclin-dependent kinase inhibitors provides a novel therapeutic strategy for RB-inactive breast cancers. We previously showed that depletion of the retinoblastoma protein (RB) induces down-regulation of the adhesion molecule E-cadherin and thereby triggers the epithelial-mesenchymal transition. To further characterize the effect of RB inactivation on the phenotype of cancer cells, we have now examined RB expression in human breast cancer cell lines and clinical specimens. We found that RB-inactive cells exhibit a mesenchymal-like morphology and are highly invasive. We also found that ZEB proteins, transcriptional repressors of the E-cadherin gene, are markedly up-regulated in these cells in a manner sensitive to the miR-200 family of microRNAs. Moreover, depletion of ZEB in RB-inactive cells suppressed cell invasiveness and proliferation and induced epithelial marker expression. These results implicate ZEB in induction of the epithelial-mesenchymal transition, as well as in maintenance of the mesenchymal phenotype in RB-inactive cells. We also developed a screening program for inhibitors of ZEB1 expression and thereby identified several cyclin-dependent kinase inhibitors that blocked both ZEB1 expression and RB phosphorylation. Together, our findings suggest that RB inactivation contributes to tumor progression not only through loss of cell cycle control but also through up-regulation of ZEB expression and induction of an invasive phenotype.

Induction of the Stem-like Cell Regulator CD44 by Rho Kinase Inhibition Contributes to the Maintenance of Colon Cancer–Initiating Cells

The difficulty in expanding cancer-initiating cells in vitro is one of major obstacles for their biochemical characterization. We found that Rho kinase (ROCK) inhibitors as well as blebbistatin, a myosin II inhibitor, greatly facilitated the establishment of spheroids from primary colon cancer. The spheroid cells expressed cancer stem cell markers, showed the ability to differentiate, and induced tumors in mice. The spheroids were composed of cells that express various levels of CD44, whereas CD44(high) cells were associated with increased sphere-forming ability, expression of the activating form of β-catenin, and elevated levels of glycolytic genes, CD44(-/low) cells showed increased levels of differentiation markers and apoptotic cells. The spheroid cells expressed variant forms of CD44 including v6, and the induction of the variants was associated with the activating phosphorylation of c-Met. As expected from the predicted hierarchy, CD44(high) cells differentiated into CD44(-/low) cells. Unexpectedly, a fraction of CD44(-/low) cells generated CD44(high) cells, and the ROCK inhibitor or blebbistatin primed the transition by inducing CD44 expression. We propose that the transition from CD44(-/low) to CD44(high) state helps to maintain a CD44(high) fraction and the tumorigenic diversity in colon cancer.

Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system

Haploid embryonic stem cells (ESCs) are useful for studying mammalian genes because disruption of only one allele can cause loss-of-function phenotypes. Here, we report the use of haploid ESCs and the CRISPR RNA-guided Cas9 nuclease gene-targeting system to manipulate mammalian genes. Co-transfection of haploid ESCs with vectors expressing Cas9 nuclease and single-guide RNAs (sgRNAs) targeting Tet1, Tet2, and Tet3 resulted in the complete disruption of all three genes and caused a loss-of-function phenotype with high efficiency (50%). Co-transfection of cells with vectors expressing Cas9 and sgRNAs targeting two loci on the same chromosome resulted in the creation of a large chromosomal deletion and a large inversion. Thus, the use of the CRISPR system in combination with haploid ESCs provides a powerful platform to manipulate the mammalian genome.

Differential expression of nanog1 and nanogp8 in colon cancer cells

Nanog, a homeodomain transcription factor, is an essential regulator for promotion of self-renewal of embryonic stem cells and inhibition of their differentiation. It has been demonstrated that nanog1 as well as nanogp8, a retrogene of nanog1, is preferentially expressed in advanced stages of several types of cancer, suggesting their involvement during cancer progression. Here, we investigated the expression of Nanog in well-characterized colon cancer cell lines. Expression of Nanog was detectable in 5 (HCT116, HT29, RKO, SW48, SW620) out of seven cell lines examined. RNA expression analyses of nanog1 and nanogp8 indicated that, while nanog1 was a major form in SW620 as well as in teratoma cells Tera-2, nanogp8 was preferentially expressed in HT29 and HCT116. In accordance with this, shRNA-mediated knockdown of nanog1 caused the reduction of Nanog in SW620 but not in HT29. Inhibition of Nanog in SW620 cells negatively affected cell proliferation and tumor formation in mouse xenograft. Biochemical subcellular fractionation and immunostaining analyses revealed predominant localization of Nanog in cytoplasm in SW620 and HT29, while it was mainly localized in nucleus in Tera-2. Our data indicate that nanog1 and nanogp8 are differentially expressed in colon cancer cells, and suggest that their expression contributes to proliferation of colon cancer cells.

Tumor-derived spheroids: Relevance to cancer stem cells and clinical applications

Recently, many types of in vitro 3‐D culture systems have been developed to recapitulate the in vivo growth conditions of cancer. The cancer 3‐D culture methods aim to preserve the biological characteristics of original tumors better than conventional 2‐D monolayer cultures, and include tumor‐derived organoids, tumor‐derived spheroids, organotypic multicellular spheroids, and multicellular tumor spheroids. The 3‐D culture methods differ in terms of cancer cell sources, protocols for cell handling, and the required time intervals. Tumor‐derived spheroids are unique because they are purposed for the enrichment of cancer stem cells (CSCs) or cells with stem cell‐related characteristics. These spheroids are grown as floating spheres and have been used as surrogate systems to evaluate the CSC‐related characteristics of solid tumors in vitro. Because eradication of CSCs is likely to be of clinical importance due to their association with the malignant nature of cancer cells, such as tumorigenicity or chemoresistance, the investigation of tumor‐derived spheroids may provide invaluable clues to fight against cancer. Spheroid cultures have been established from cancers including glioma, breast, colon, ovary, and prostate cancers, and their biological and biochemical characteristics have been investigated by many research groups. In addition to the investigation of CSCs, tumor‐derived spheroids may prove to be instrumental for a high‐throughput screening platform or for the cultivation of CSC‐related tumor cells found in the circulation or body fluids.

TNIK inhibition abrogates colorectal cancer stemness

Canonical Wnt/β-catenin signalling is essential for maintaining intestinal stem cells, and its constitutive activation has been implicated in colorectal carcinogenesis. We and others have previously identified Traf2- and Nck-interacting kinase (TNIK) as an essential regulatory component of the T-cell factor-4 and β-catenin transcriptional complex. Consistent with this, Tnik-deficient mice are resistant to azoxymethane-induced colon tumorigenesis, and Tnik−/−/Apcmin/+ mutant mice develop significantly fewer intestinal tumours. Here we report the first orally available small-molecule TNIK inhibitor, NCB-0846, having anti-Wnt activity. X-ray co-crystal structure analysis reveals that NCB-0846 binds to TNIK in an inactive conformation, and this binding mode seems to be essential for Wnt inhibition. NCB-0846 suppresses Wnt-driven intestinal tumorigenesis in Apcmin/+ mice and the sphere- and tumour-forming activities of colorectal cancer cells. TNIK is required for the tumour-initiating function of colorectal cancer stem cells. Its inhibition is a promising therapeutic approach.

NuMA Is Required for the Selective Induction of p53-Target Genes

ABSTRACT The p53 tumor suppressor protein is a transcription factor controlling various outcomes, such as growth arrest and apoptosis, through the regulation of different sets of target genes. The nuclear mitotic apparatus protein (NuMA) plays important roles in spindle pole organization during mitosis and in chromatin regulation in the nucleus during interphase. Although NuMA has been shown to colocalize with several nuclear proteins, including high-mobility-group proteins I and Y and GAS41, the role of NuMA during interphase remains unclear. Here we report that NuMA binds to p53 to modulate p53-mediated transcription. Acute and partial ablation of NuMA attenuates the induction of the proarrested p21 gene following DNA damage, subsequently causing impaired cell cycle arrest. Interestingly, NuMA knockdown had little effect on the induction of the p53-dependent proapoptotic PUMA gene. Furthermore, NuMA is required for the recruitment of cyclin-dependent kinase 8 (Cdk8), a component of the Mediator complex and a promoter of p53-mediated p21 gene function. These data demonstrate that NuMA is critical for the target selectivity of p53-mediated transcription.

APOLLON Protein Promotes Early Mitotic CYCLIN A Degradation Independent of the Spindle Assembly Checkpoint

Background: CYCLIN A is degraded in early mitosis independent of spindle assembly checkpoint. Results: APOLLON interacts with CYCLIN A and promotes its degradation in mitosis. Conclusion: APOLLON is a novel regulator of CYCLIN A degradation in early mitosis. Significance: This study expands our knowledge on the huge APOLLON protein known to regulate apoptosis and cytokinesis. In the mammalian cell cycle, both CYCLIN A and CYCLIN B are required for entry into mitosis, and their elimination is also essential to complete the process. During mitosis, CYCLIN A and CYCLIN B are ubiquitylated by the anaphase-promoting complex/cyclosome (APC/C) and then subjected to proteasomal degradation. However, CYCLIN A, but not CYCLIN B, begins to be degraded in the prometaphase when APC/C is inactivated by the spindle assembly checkpoint (SAC). Here, we show that APOLLON (also known as BRUCE or BIRC6) plays a role in SAC-independent degradation of CYCLIN A in early mitosis. APPOLON interacts with CYCLIN A that is not associated with cyclin-dependent kinases. APPOLON also interacts with APC/C, and it facilitates CYCLIN A ubiquitylation. In APPOLON-deficient cells, mitotic degradation of CYCLIN A is delayed, and the total, but not the cyclin-dependent kinase-bound, CYCLIN A level was increased. We propose APPOLON to be a novel regulator of mitotic CYCLIN A degradation independent of SAC.