Atsutaka Masuda

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.

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.

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.

Abstract 1728: DCLK1 promotes tumor growth and invasion through Slug-mediated epithelial-mesenchymal transition in pancreatic neuroendocrine tumors

Introduction: Accumulating evidence suggests that Doublecortin-like kinase 1 (DCLK1) is a putative marker for intestinal and pancreatic cancer stem cells. (Nakanishi et al., Nat Genet 2013, Bailey et al., Gastroenterology 2013). Recently, we have reported that DCLK1 was highly and diffusely expressed in human rectal neuroendocrine tumors (NETs) (Ikezono et al., Oncol Lett, 2015). However, the function of DCLK1 has not yet been investigated in detail. Aims: The aims of the present study were to assess expression levels of DCLK1 in pancreatic NET (PNET) tissues and to identify critical functions of this molecule in PNET cells. Methods: Fifteen patients (8 male, 7 female; mean age, 56) with PNETs were enrolled in this study. The tumors were surgically resected between 1997 and 2012 in Kurume University Hospital. Immunohistochemistry (IHC) was employed to assess expression levels of DCLK1. QGP1, a human PNET cell line, was used in this study, and the cells were transfected with dclk1 cDNA to establish the DCLK1-overexpressing (QGP1-DOE) cells. The QGP1-DOE cells were subjected to dclk1 silencing to confirm acquired cellular characteristics by DCLK1 overexpression. Protein and mRNA expression levels were analyzed by Western blot and real-time PCR (ABI PRISM 7700), respectively. Results: In IHC, all of the 15 PNETs clearly and diffusely expressed DCLK1 in the tumor areas. QGP1-DOE cells robustly expressed full length of DCLK1, showing epithelial-mesenchymal transition (EMT)-like appearance. Of note, extremely high expression of the EMT regulator Slug was found in QGP1-DOE cells compared with control cells at both protein and mRNA levels. Expressions of N-cadherin and Vimentin were also upregulated, in concert with those of phospholylated (p-) FAK, Paxillin, and cyclin D1, in the QGP1-DOE cells. These cells increased cellular motility and proliferation. DCLK1 knockdown restored the above-mentioned expression profile of the EMT-associated molecules. Conclusion: We demonstrated robust expression of DCLK1 in human PNET tissues and PNET cells for the first time. Enforced expression of DCLK1 induced EMT via upregulating Slug expression and promoted proliferation and invasion probably through increasing expression levels of cyclin D1, pFAK, and Paxillin. Therefore, it is speculated that inhibition of DCLK1 expression is a novel therapeutic strategy for PNETs. Citation Format: Yu Ikezono, Hironori Koga, Jun Akiba, Mitsuhiko Abe, Fumitaka Wada, Toru Nakamura, Hideki Iwamoto, Atsutaka Masuda, Takahiko Sakaue, Hirohisa Yano, Osamu Tsuruta, takuji Torimura. DCLK1 promotes tumor growth and invasion through Slug-mediated epithelial-mesenchymal transition in pancreatic neuroendocrine tumors. [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 1728.

Abstract 4601: Regulation of Hes1 expression by the Wnt transcription factor T-cell factor-4

Background: The Notch effector Hes1 plays a critical role in stemness of cancer stem cells (CSCs). T-cell factor (TCF)-4 is a key transcription factor in Wnt signaling, which is suggested to be linked with Notch signaling. Among our TCF-4 isoforms cloned previously, the TCF-4J and K pair have been characterized based on the presence (K) or absence (J) of SxxSS motif (Exp Cell Res 2010). TCF-4J was highly expressed in poorly differentiated human hepatocellular carcinoma (HCC) tissues, and the TCF-4J-overexpressing HCC cells (J cells) exhibited high tumor-initiating potential, which is one of the features of CSCs (PLoS One 2013). Thus, the AIM of this study was to investigate whether the SxxSS motif of TCF-4 was involved in the regulation of Hes1 expression in HCC cells. Methods: TCF-4K mutants were prepared by site-directed mutagenesis. Sphere formation assay was used to condense CSC-like cells. Results: Hes1 was strikingly expressed in spheres of J cells and K-mutant cells in both protein and mRNA levels, while its expression level was 70% inhibited in K cells. Consistently, protein expression levels of Jagged1 and Notch1 were highest in J cells under both attached and floating conditions. The Notch inhibitor DAPT, a γ-secretase inhibitor, clearly decreased the expression levels of Hes1, suggesting that the Notch-Hes1 axis was functional. Conclusion: Lack of SxxSS motif in TCF-4 robustly upregulated Hes1 expression in HCC cell spheres. The finding strongly suggests that TCF-4 directly regulates Hes1 in HCC spheres, where CSCs abundantly exist. Citation Format: Hironori Koga, Yasuko Imamura, Yu Ikezono, Fumitaka Wada, Toru Nakamura, Hideki Iwamoto, Atsutaka Masuda, Takahiko Sakaue, Hirohisa Yano, Takuji Torimura. Regulation of Hes1 expression by the Wnt transcription factor T-cell factor-4. [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 4601.