Takahiko Sakaue

Older Age Is a Risk Factor for the Development of Cardiovascular Sequelae in Kawasaki Disease

Objectives. To clarify the characteristics of Kawasaki disease (KD) in children 6 years and older and to determine whether age is a risk factor for cardiovascular abnormalities. Methods. Patients who had KD and were reported between 1999 and 2000 in the 16th nationwide survey of KD in Japan (n = 15 314) were analyzed. Patients who were aged 6 years or older (older group) were matched with patients who were aged 6 months to 3 years and were treated at the same hospital (younger groups). The total number of analyzed patients was 1498 (749 matched pairs). Results. The proportion of complete KD in the older group was similar to that in the younger group. Recurrent cases in the older group were significantly more common than those in the younger group (9% vs 2%). The proportion of patients who were treated with intravenous γ-globulin in the older group was significantly lower than that in the younger group (82% vs 87%). The proportion of older group patients who were treated with intravenous γ-globulin at or after 7 days of illness was significantly higher than that in the younger group (35% vs 14%). There was a higher prevalence of cardiovascular abnormalities in the older group than in the younger group (20% vs 15%). Multivariate logistic regression analysis showed that older age was an independent risk factor for cardiovascular sequelae (odds ratio: 1.58; 95% confidence interval: 1.01–2.46). Conclusions. In children older than 6 years, age is an independent risk factor for cardiovascular sequelae in KD.

Antiangiogenic and Antitumor Activities of Aflibercept, a Soluble VEGF Receptor-1 and -2, in a Mouse Model of Hepatocellular Carcinoma

BACKGROUND & AIM: Aflibercept known as ziv-aflibercept in the United States is a soluble decoy receptor of both vascular endothelial growth factor (VEGF) receptor-1 and -2 known to inhibit the binding of VEGF and placental growth factor (PlGF) to VEGF receptor-1 and -2. Here, we analyzed the mechanisms of the antitumor effects of aflibercept in mouse hepatoma models. METHODS: In in vitro studies, we determined the effects of aflibercept on human umbilical vein cell (HUVEC) proliferation and bone marrow (BM) cell differentiation to endothelial progenitor cells (EPCs). In in vivo experiments, aflibercept was injected intraperitoneally in hepatoma cell tumor-bearing mice, and its inhibitory effects on tumor growth and BM cell migration to tumor tissues were evaluated. RESULTS: Aflibercept suppressed phosphorylation of VEGF receptor-1 and -2 in HUVEC and dose-dependently inhibited VEGF-induced HUVEC proliferation. It suppressed the differentiation of BM cells to EPCs and migration of BM cells to tumor tissues. It also suppressed tumor growth and prolonged survival time of tumor-bearing mice without side effects. In tumor tissues, aflibercept upregulated the expression of hypoxia inducible factor1-α, VEGF, PlGF, fibroblast growth factor-2, platelet derived growth factor-BB, and transforming growth factor-α and reduced microvascular density. It also reduced sinusoidal density in noncancerous liver tissues. CONCLUSIONS: Our results demonstrated potent antitumor activity for aflibercept in a mouse model of hepatocellular carcinoma. These effects were mediated through inhibition of neovascularization, caused by inhibition of endothelial cell proliferation, EPC differentiation, and BM cell migration to tumor tissues.

Ex vivo expansion of circulating CD34+ cells enhances the regenerative effect on rat liver cirrhosis

Ex vivo expansion of autologous cells is indispensable for cell transplantation therapy of patients with liver cirrhosis. The aim of this study was to investigate the efficacy of human ex vivo-expanded CD34+ cells for treatment of cirrhotic rat liver. Recipient rats were intraperitoneally injected with CCl4 twice weekly for 3 weeks before administration of CD34+ cells. CCl4 was then re-administered twice weekly for 3 more weeks, and the rats were sacrificed. Saline, nonexpanded or expanded CD34+ cells were injected via the spleen. After 7 days, CD34+ cells were effectively expanded in a serum-free culture medium. Expanded CD34+ cells were also increasingly positive for cell surface markers of VE-cadherin, VEGF receptor-2, and Tie-2. The expression of proangiogenic growth factors and adhesion molecules in expanded CD34+ cells increased compared with nonexpanded CD34+ cells. Expanded CD34+ cell transplantation reduced liver fibrosis, with a decrease of αSMA+ cells. Assessments of hepatocyte and sinusoidal endothelial cell proliferative activity indicated the superior potency of expanded CD34+ cells over non-expanded CD34+ cells. The inhibition of integrin αvβ3 and αvβ5 disturbed the engraftment of transplanted CD34+ cells and aggravated liver fibrosis. These findings suggest that expanded CD34+ cells enhanced the preventive efficacy of cell transplantation in a cirrhotic model.

Pancreatic Neuroendocrine Tumors and EMT Behavior Are Driven by the CSC Marker DCLK1

Doublecortin-like kinase 1 (DCLK1), a marker for intestinal and pancreatic cancer stem cells, is highly expressed in neuroblastomas. This study was conducted to assess DCLK1 expression levels in pancreatic neuroendocrine tumor (PNET) tissues and to explore the roles of this molecule in clinical tissue from multiple PNET patients, cells (BON1, QGP1, and CM) and tumor xenografts. Immunohistochemically, all PNET tissues highly and diffusely expressed DCLK1 as a full-length isoform, identical to that detected in primary liver NETs. A DCLK1-overexpressing PNET cell line (QGP1-DCLK1) exhibited epithelial–mesenchymal transition (EMT)-related gene signatures, and robust upregulation of Slug (SNAI2), N-Cadherin (CDH2), and Vimentin (VIM) was validated by real-time PCR and immunoblotting. QGP1-DCLK1 cells had increased cell migration in a wound-healing assay and formed significantly larger xenograft tumors in nude mice. The factors involved in the formation of the fast-growing tumors included p-FAK (on Tyr925), p-ERK1/2, p-AKT, Paxillin, and Cyclin D1, which upon knockdown or pharmacologic inhibition of DCLK1 abolished the expression of these molecules. In conclusion, robust and ubiquitous expression of DCLK1 was first demonstrated here in human PNET tissue specimens and cells. DCLK1 characterized the PNET cell behavior, inducing p-FAK/SLUG-mediated EMT. These findings suggest the possibility of developing novel therapeutic strategies against PNETs by targeting DCLK1. Implications: Evidence here reveals that human PNETs diffusely and robustly express the cancer stem cell marker DCLK1, which drives SLUG-mediated EMT, and suggests that NETs share biological features for druggable targets with other tumors, including neuroblastoma that also highly expresses DCLK1. Mol Cancer Res; 15(6); 744–52. ©2017 AACR.

mFOLFOX6 Chemotherapy after Resection of Anal Canal Mucinous Adenocarcinoma

Because of their rarity, there are no clear guidelines for the treatment of anal carcinomas; such tumors are normally subjected to the same modalities as recommended for rectal cancer. We report a patient with anal canal mucinous adenocarcinoma, with metastases in the pararectal and right inguinal lymph nodes, who was treated with abdominoperineal resection followed by mFOLFOX6 chemotherapy for 6 months (12 cycles). The patient has remained recurrence-free thus far, approximately 2 years since the surgery. As the optimal treatments for anal carcinomas have not been fully elucidated, we present this case to highlight a possible course of action for such patients that appears to be effective and promising.

Abstract 3955: Expression of cancer stem cell-associated proteins in exosomes derived from ascites of patients with advanced pancreatic cancer

Background: The number of patients with pancreatic cancer is rapidly growing, and the disease is the fifth leading cause of cancer-related death in Japan. At end stage of the disease, the patients are prone to suffer peritonitis carcinomatosa with chemorefractory ascites. It is known that cancer cells surviving in ascites show cancer stem cell (CSC)-like features (PLoS One 2012). The CSC-like cells robustly secrete extracellular vesicle called exosome, which plays important roles in tumorigenesis, tumor growth, metastasis, angiogenesis, pre-metastatic niche formation, immunosuppression, drug resistance, and epithelial-mesenchymal transition. The AIM of this prospective study was to assess whether exosomes taken from malignant ascites of patients with advanced pancreatic cancer included the CSC-associated proteins, that might be predictive markers for chemoresistance and prognosis. Methods: The malignant ascites was collected from the cancer patients who underwent abdominocentesis and/or cell-free and concentrated ascites reinfusion therapy (CART) in Kurume University Hospital. Ascites derived from patients with benign diseases, including decompensated liver cirrhosis (d-LC), was used as control. Informed consent was obtained from all of the patients. Exosomes in ascites were purified by using ExoQuick Exosome Precipitation Solution (System Biosciences) according to the manufacturer9s protocol. Western blot analysis was performed to detect CSC-associated proteins, including CD133, CD44, CD44v9, xCT, CD24, and Dclk1. Results: Successful purification of exosomes from both the malignant ascites and the benign one was confirmed by monitoring exosome-specific proteins such as CD68, CD9, CD81, and HSP70. Among the CSC-associated proteins examined, CD133 was predominantly expressed in exosomes obtained from ascites of the pancreatic cancer patients compared with those obtained from ascites of the d-LC patients. Other molecules were faintly expressed or absent even in the malignant ascites. Conclusions: We first demonstrated abundant expression of CD133, a human pancreatic CSC marker, in exosomes derived from ascites of patients with the disease. The finding suggests that exosomal CD133 might be a potential biomarker for chemoresistance and prognosis of the patients. Citation Format: Takahiko Sakaue, Hironori Koga, Masaru Fukahori, Yasuko Imamura, Toru Nakamura, Yoshinobu Okabe, Yu Ikezono, Fumitaka Wada, Hideki Iwamoto, Atsutaka Masuda, Tomoyuki Ushijima, Keisuke Miwa, Tatsuyuki Kakuma, Osamu Tsuruta, Takuji Torimura. Expression of cancer stem cell-associated proteins in exosomes derived from ascites of patients with advanced pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3955.

Inhibition of hypoxia-inducible factor via upregulation of von Hippel-Lindau protein induces “angiogenic switch off” in a hepatoma mouse model

“Angiogenic switch off” is one of the ideal therapeutic concepts in the treatment of cancer. However, the specific molecules which can induce “angiogenic switch off” in tumor have not been identified yet. In this study, we focused on von Hippel-Lindau protein (pVHL) in hepatocellular carcinoma (HCC) and investigated the effects of sulfoquinovosyl-acylpropanediol (SQAP), a novel synthetic sulfoglycolipid, for HCC. We examined mutation ratio of VHL gene in HCC using 30 HCC samples and we treated the HCC-implanted mice with SQAP. Thirty clinical samples showed no VHL genetic mutation in HCC. SQAP significantly inhibited tumor growth by inhibiting angiogenesis in a hepatoma mouse model. SQAP induced tumor “angiogenic switch off” by decreasing hypoxia-inducible factor (HIF)-1, 2α protein via pVHL upregulation. pVHL upregulation decreased HIFα protein levels through different multiple mechanisms: (i) increasing pVHL-dependent HIFα protein degradation; (ii) decreasing HIFα synthesis with decrease of NF-κB expression; and (iii) decrease of tumor hypoxia by vascular normalization. We confirmed these antitumor effects of SQAP by the loss-of-function experiments. We found that SQAP directly bound to and inhibited transglutaminase 2. This study provides evidence that upregulation of tumor pVHL is a promising target, which can induce “angiogenic switch off” in HCC.

Successful treatment of a patient with chyluria due to lymphangioleiomyomatosis using sirolimus

Lymphangioleiomyomatosis (LAM) is a rare and progressive neoplastic disease of young woman, characterized by the proliferation of abnormal smooth muscle-like cells (LAM cells) in the lungs and axial lymphatics. A 44-year-old woman was referred to our hospital because pleural effusion was detected during a health checkup. She had chylothorax, chylous ascites, and chyluria, and her computed tomography scan showed a solid tumor in the pelvis. Surgical biopsy was performed; she was diagnosed as having LAM. We could not control the fluid collection and chyluria using standard medical treatments. Therefore, we chose to administer sirolimus, and her symptoms dramatically improved. The mechanism of chyluria presumably involved LAM cell infiltrates in the ureter via the lymphatic vessel flow, which causes LAM to develop because of ureter wall exposure.

High expression of CD44v9 and xCT in chemoresistant hepatocellular carcinoma: Potential targets by sulfasalazine

CD44v9 is expressed in cancer stem cells (CSC) and stabilizes the glutamate‐cystine transporter xCT on the cytoplasmic membrane, thereby decreasing intracellular levels of reactive oxygen species (ROS). This mechanism confers ROS resistance to CSC and CD44v9‐expressing cancer cells. The aims of the present study were to assess: (i) expression status of CD44v9 and xCT in hepatocellular carcinoma (HCC) tissues, including those derived from patients treated with hepatic arterial infusion chemoembolization (HAIC) therapy with cisplatin (CDDP); and (ii) whether combination of CDDP with sulfasalazine (SASP), an inhibitor of xCT, was more effective on tumor cells than CDDP alone by inducing ROS‐mediated apoptosis. Twenty non‐pretreated HCC tissues and 7 HCC tissues administered HAIC therapy with CDDP before surgical resection were subjected to immunohistochemistry analysis of CD44v9 and xCT expression. Human HCC cell lines HAK‐1A and HAK‐1B were used in this study; the latter was also used for xenograft experiments in nude mice to assess in vivo efficacy of combination treatment. CD44v9 positivity was significantly higher in HAIC‐treated tissues (5/7) than in non‐pretreated tissues (2/30), suggesting the involvement of CD44v9 in the resistance to HAIC. xCT was significantly expressed in poorly differentiated HCC tissues. Combination treatment effectively killed the CD44v9‐harboring HAK‐1B cells through ROS‐mediated apoptosis and significantly decreased xenografted tumor growth. In conclusion, the xCT inhibitor SASP augmented ROS‐mediated apoptosis in CDDP‐treated HCC cells, in which the CD44v9‐xCT system functioned. As CD44v9 is typically expressed in HAIC‐resistant HCC cells, combination treatment with SASP with CDDP may overcome such drug resistance.

STAT3 deficiency prevents hepatocarcinogenesis and promotes biliary proliferation in thioacetamide-induced liver injury

AIM To elucidate the role of STAT3 in hepatocarcinogenesis and biliary ductular proliferation following chronic liver injury. METHODS We investigated thioacetamide (TAA)-induced liver injury, compensatory hepatocyte proliferation, and hepatocellular carcinoma (HCC) development in hepatic STAT3-deficient mice. In addition, we evaluated TAA-induced biliary ductular proliferation and analyzed the activation of sex determining region Y-box9 (SOX9) and Yes-associated protein (YAP), which regulate the transdifferentiation of hepatocytes to cholangiocytes. RESULTS Both compensatory hepatocyte proliferation and HCC formation were significantly decreased in hepatic STAT3-deficient mice as compared with control mice. STAT3 deficiency resulted in augmentation of hepatic necrosis and fibrosis. On the other hand, biliary ductular proliferation increased in hepatic STAT3-deficient livers as compared with control livers. SOX9 and YAP were upregulated in hepatic STAT3-deficient hepatocytes. CONCLUSION STAT3 may regulate hepatocyte proliferation as well as transdifferentiation into cholangiocytes and serve as a therapeutic target for HCC inhibition and biliary regeneration.