Susumu Nakata

LGR5 is a marker of poor prognosis in glioblastoma and is required for survival of brain cancer stem-like cells

In various types of cancers including glioblastoma, accumulating evidence show the existence of cancer stem‐like cells (CSCs), characterized by stem cell marker expression, capability of differentiation and self‐renewal, and high potential for tumor propagation in vivo. LGR5, whose expression is positively regulated by the Wnt signaling pathway, is a stem cell marker in intestinal mucosa and hair follicle in the skin. As Wnt signaling is also involved in brain development, the function of LGR5 in the maintenance of brain CSCs is to be assessed. Our study showed that the LGR5 transcript level was increased in CSCs. Co‐immunofluorescence staining demonstrated the co‐localization of CD133‐ and LGR5‐positive cells in glioblastoma tissue sections. Functionally, silencing of LGR5 by lentiviral shRNA‐mediated knockdown induced apoptosis in brain CSCs. Moreover, LGR5 depletion led to a downregulation of L1 cell adhesion molecule expression. In line with an important function in glioma tumorigenesis, LGR5 expression increased with glioma progression and correlated with an adverse outcome. Our findings suggest that LGR5 plays a role in maintenance and/or survival of brain CSCs.

15-Deoxy-Δ12,14-prostaglandin J2 induces death receptor 5 expression through mRNA stabilization independently of PPARγ and potentiates TRAIL-induced apoptosis

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), the terminal derivative of the PGJ series, is emerging as a potent antineoplastic agent among cyclopentenone prostaglandins derivatives and also known as the endogenous ligand of peroxisome proliferator-activated receptor γ (PPARγ). On the other hand, death receptor 5 (DR5) is a specific receptor for tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), which is one of the most promising candidates for new cancer therapeutics. Here, we report that 15d-PGJ2 induces DR5 expression at both mRNA and protein levels, resulting in the synergistic sensitization of TRAIL-induced apoptosis in human neoplastic cells, such as Jurkat human leukemia cells or PC3 human prostate cancer cells. 15d-PGJ2 significantly increased DR5 mRNA stability, whereas it did not activate DR5 promoter activity. Synthetic PPARγ agonists, such as pioglitazone or rosiglitazone, did not mimic the DR5-inducing effects of 15d-PGJ2, and a potent PPARγ inhibitor GW9662 failed to block DR5 induction by 15d-PGJ2, suggesting PPARγ-independent mechanisms. Cotreatment with 15d-PGJ2 and TRAIL enhanced the sequential activation of caspase-8, caspase-10, caspase-9, caspase-3, and Bid. DR5/Fc chimera protein, zVAD-fmk pancaspase inhibitor, and caspase-8 inhibitor efficiently blocked the activation of these apoptotic signal mediators and the induction of apoptotic cell death enhanced by cotreatment with 15d-PGJ2 and TRAIL. Moreover, a double-stranded small interfering RNA targeting DR5 gene, which suppressed DR5 up-regulation by 15d-PGJ2, significantly attenuated apoptosis induced by cotreatment with 15d-PGJ2 and TRAIL. These results suggest that 15d-PGJ2 is a potent sensitizer of TRAIL-mediated cancer therapeutics through DR5 up-regulation. [Mol Cancer Ther 2006;5(7):1827–35]

Emerging role for leucine-rich repeat-containing G-protein-coupled receptors LGR5 and LGR4 in cancer stem cells

The concept of cancer stem cells has gained considerable interest in the last few decades, partly because of their potential implication in therapy resistance. However, the lack of specific cellular surface markers for these cells has impeded their isolation, making the characterization of this cellular subpopulation technically challenging. Recent studies have indicated that leucine-rich repeat-containing G-protein-coupled receptor 4 and 5 (LGR4 and LGR5) expression in multiple organs may represent a global marker of adult stem cells. This review aims to give an overview of LGR4 and LGR5 as cancer stem cell markers and their function in development.

Extract of Cordyceps militaris inhibits angiogenesis and suppresses tumor growth of human malignant melanoma cells

Angiogenesis is essential for tumor development and metastasis. Among several angiogenic factors, vascular endothelial growth factor receptor (VEGF) is important for tumor-derived angiogenesis and commonly overexpressed in solid tumors. Thus, many antitumor strategies targeting VEGF have been developed to inhibit cancer angiogenesis, offering insights into the successful treatment of solid cancers. However, there are a number of issues such as harmful effects on normal vascularity in clinical trials. Taking this into consideration, we employed Cordyceps militaris as an antitumor approach due to its biological safety in vivo. The herbal medicinal mushroom Cordyceps militaris has been reported to show potential anticancer properties including anti-angiogenic capacity; however, its concrete properties have yet to be fully demonstrated. In this study, we aimed to elucidate the biological role of Cordyceps militaris extract in tumor cells, especially in regulating angiogenesis and tumor growth of a human malignant melanoma cell line. We demonstrated that Cordyceps militaris extract remarkably suppressed tumor growth via induction of apoptotic cell death in culture that links to the abrogation of VEGF production in melanoma cells. This was followed by mitigation of Akt1 and GSK-3β activation, while p38α phosphorylation levels were increased. Extract treatment in mouse model xenografted with human melanoma cells resulted in a dramatic antitumor effect with down-regulation of VEGF expression. The results suggest that suppression of tumor growth by Cordyceps militaris extract is, at least, mediated by its anti-angiogenicity and apoptosis induction capacities. Cordyceps militaris extract may be a potent antitumor herbal drug for solid tumors.

A gene signature distinguishing CD133hi from CD133- colorectal cancer cells: essential role for EGR1 and downstream factors

Aims: In colorectal cancer (CRC), CD133 expression is an independent prognostic marker associated with adverse clinical outcome. The CD133 epitope AC133 allowed isolating stem cells from normal and cancerous tissues, although its use in colon was questioned. We aimed to identify differences between AC133hi and AC133− cells. Methods: We analysed the gene expression profiles of EpCAM+/CEA+/AC133hi and EpCAM+/CEA+/AC133− cells from primary CRC and liver metastasis tissues (n = 5). Immunohistochemistry confirmed these results in a validation set. Results: We identified 68 genes differentially expressed between both populations, including genes of notorious importance in CRC pathogenesis, and several candidates not previously shown to play a major role in CRC. Notably, EGR1 belonged to the most highly expressed genes in AC133hi cells. In the validation set, the presence of EGR1 and CD133 correlated (r = 0.625). Since EGR1 regulates Wnt through up-regulation of TCF4, which induces stem cell marker LGR5, the potential association between LGR5, EGR1 and CD133 was investigated. The presence of LGR5 correlated with the presence of EGR1 and CD133. Strong signals for LGR5 were detected throughout tumour invasion fronts. Conclusions: The study suggests a connection between CD133 and EGR1 and emphasises the importance of the EGR1/TCF4/CD133/LGR5 network in CRC.

Polycomb group protein Ezh2 regulates hepatic progenitor cell proliferation and differentiation in murine embryonic liver.

In embryonic liver, hepatic progenitor cells are actively proliferating and generate a fundamental cellular pool for establishing parenchymal components. However, the molecular basis for the expansion of the progenitors maintaining their immature state remains elusive. Polycomb group proteins regulate gene expression throughout the genome by modulating of chromatin structure and play crucial roles in development. Enhancer of zeste homolog 2 (Ezh2), a key component of polycomb group proteins, catalyzes tri-methylation of lysine 27 of histone H3 (H3K27me3), which trigger the gene suppression. In the present study, we investigated a role of Ezh2 in the regulation of the expanding hepatic progenitor population in vivo. We found that Ezh2 is highly expressed in the actively proliferating cells at the early developmental stage. Using a conditional knockout mouse model, we show that the deletion of the SET domain of Ezh2, which is responsible for catalytic induction of H3K27me3, results in significant reduction of the total liver size, absolute number of liver parenchymal cells, and hepatic progenitor cell population in size. A clonal colony assay in the hepatic progenitor cells directly isolated from in vivo fetal livers revealed that the bi-potent clonogenicity was significantly attenuated by the Ezh2 loss of function. Moreover, a marker expression based analysis and a global gene expression analysis showed that the knockout of Ezh2 inhibited differentiation to hepatocyte with reduced expression of a number of liver-function related genes. Taken together, our results indicate that Ezh2 is required for the hepatic progenitor expansion in vivo, which is essential for the functional maturation of embryonic liver, through its activity for catalyzing H3K27me3.

Aclarubicin enhances tumor necrosis factor‐related apoptosis‐inducing ligand‐induced apoptosis through death receptor 5 upregulation

Anthracycline drugs are potent anti‐tumor agents. Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is a death ligand with promising anti‐cancer effects. However, some tumor types develop resistance to TRAIL. We examined the effect of aclarubicin (ACR), an anthracycline, in combination with TRAIL. The combination of TRAIL and ACR synergistically induced apoptosis in human acute lymphoblastic leukemia Jurkat cells and human lung cancer A549 cells. In contrast, another anthracycline, doxorubicin (DOX), only slightly sensitized Jurkat cells and A549 cells to TRAIL‐induced apoptosis, with weaker enhancement of death receptor 5 (DR5) expression than ACR. The RNase protection assay, real time RT‐PCR and western blot demonstrated that ACR upregulated the expression of a TRAIL receptor, DR5. Caspase inhibitors and dominant negative DR5 efficiently reduced the apoptotic response to the treatment with ACR and TRAIL, indicating that the combined effect depends on caspase activities and the interaction between TRAIL and its receptor. ACR but not DOX increased the activity of the DR5 gene promoter in Jurkat cells carrying a mutation in the p53 gene, suggesting that ACR upregulates DR5 expression through p53‐independent transcription. These results suggest the combination of TRAIL and ACR to be a promising treatment for malignant tumors. (Cancer Sci 2012; 103: 282–287)

Ring1B promotes hepatic stem/progenitor cell expansion through simultaneous suppression of Cdkn1a and Cdkn2a in mice

Polycomb‐group (PcG) proteins play crucial roles in self‐renewal of stem cells by suppressing a host of genes through histone modifications. Identification of the downstream genes of PcG proteins is essential for elucidation of the molecular mechanisms of stem cell self‐renewal. However, little is known about the PcG target genes in tissue stem cells. We found that the PcG protein, Ring1B, which regulates expression of various genes through monoubiquitination of histone H2AK119, is essential for expansion of hepatic stem/progenitor cells. In mouse embryos with a conditional knockout of Ring1B, we found that the lack of Ring1B inhibited proliferation and differentiation of hepatic stem/progenitor cells and thereby inhibited hepatic organogenesis. These events were characterized by derepression of cyclin‐dependent kinase inhibitors (CDKIs) Cdkn1a and Cdkn2a, known negative regulators of cell proliferation. We conducted clonal culture experiments with hepatic stem/progenitor cells to investigate the individual genetic functions of Ring1B, Cdkn1a, and Cdkn2a. The data showed that the cell‐cycle inhibition caused by Ring1B depletion was reversed when Cdkn1a and Cdkn2a were suppressed simultaneously, but not when they were suppressed individually. Conclusion: Our results show that expansion of hepatic stem/progenitor cells requires Ring1B‐mediated epigenetic silencing of Cdkn1a and Cdkn2a, demonstrating that Ring1B simultaneously regulates multiple CDKIs in tissue stem/progenitor cells. (Hepatology 2014;60:323‐333)

Intimo-Intimal Intussusception: A Rare Form of Common Carotid Artery Dissection

clinical indications of the EOS device include management of venous (eg, varicocele, ovarian varices, varicose veins) and arterial (eg, emergency arterial embolization in trauma patients) conditions. As no late imaging was obtained in the present case, durable occlusion with the EOS device could be assessed in future studies. To conclude, the EOS may provide a safe and reliable method of immediate vessel occlusion in the peripheral arterial circulation. Its shortand long-term clinical efficacy in patients remains a subject for future research.

Abstract 1079: COX-2 blockade immunologically suppresses brain metastasis of lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Epidemiological studies have highlighted associations between the regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) and reduced cancer risks in human. In addition, NSAIDs reduce VEGF-D production levels, which in turn inhibit lymphatic vessel dilation and eventually cancer metastasis. Most NSAIDs function as cyclooxygenase-2 (COX-2) inhibitors that prevent the production of prostaglandin E2 (PGE2). The PGE2 induces myeloid-derived suppressor cells (MDSCs) in the bone marrow of cancer-bearing hosts, which is known to be responsible for T-cell suppression in chronic inflammation. Based on these findings, we hypothesized that COX-2 blockade would suppress brain metastasis of cancers by inhibiting MDSC development and their chemokine-mediated accumulation in the tumor microenvironment (TME). In both mouse and human brain metastasis cases, the expression levels of COX-2 and CCL2, a chemokine primarily attracting MDSCs, were increased in the brain tumor tissues compared with normal brain or peripheral bloods. Concomitantly, the infiltration of MDSCs (CD11+Gr-1+ for mice and CD14-CD15+HLA-DRlow for human, respectively) was observed at high levels in the tumor tissues. In the mouse brain tumor models using LL/2 murine lung cancer cell line, treatment with NSAIDs inhibited the PGE2 production levels and the formation of brain metastases. Furthermore, the NSAID treatment reduced the CCL2 in the TME, which was associated with a reduction in Ly6ChighLy6Glow monocytic and Ly6ClowLy6Ghigh granulocytic MDSCs in both the bone marrow and the TME. Mice genetically deficient for CCR2, a receptor for CCL2, exhibited low penetration of brain metastases along with reduced infiltration of MDSCs. Collectively, our findings show that the COX-2 pathway promotes brain metastases of lung cancers by supporting systemic development of MDSC and their accumulation in the TME mediated by the CCL2-CCR2 chemokine axis. Citation Format: Mitsugu Fujita, Susumu Nakata, Takeshi Okuda, Amami Kato, Osamu Yoshie. COX-2 blockade immunologically suppresses brain metastasis of lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1079. doi:10.1158/1538-7445.AM2014-1079