Keisuke Kohga

Sorafenib inhibits the shedding of major histocompatibility complex class I-related chain A on hepatocellular carcinoma cells by down-regulating a disintegrin and metalloproteinase 9.

The ectodomain of major histocompatibility complex class I–related chain A (MICA) is shed from tumor cells, and may be an important means of evading antitumor immunity. This study investigated the roles of a disintegrin and metalloproteinase 9 (ADAM9) in the shedding of MICA in human hepatocellular carcinoma (HCC). Small interfering RNA–mediated knockdown (KD) of ADAM9 resulted in up‐regulation of membrane‐bound MICA expression on the HepG2 and PLC/PRF/5 cellular surfaces and down‐regulation of soluble MICA levels in their culture supernatant. ADAM9 was cleaved at a site between Gln347 and Val348 of MICA in vitro. We constructed a plasmid of the MICA gene with mutation or deletion of the ADAM9 cleavage site to examine the detailed mechanism of MICA shedding by ADAM9 protease. The results suggested that MICA might be cleaved at the intracellular ADAM9‐recognized cleavage site and was further cleaved at the extracellular ADAM9‐independent cleavage site in HCC cells, resulting in the production of soluble MICA. Immunohistochemical analysis revealed that ADAM9 was overexpressed in human HCC compared to normal liver tissues. The cytolytic activity of natural killer (NK) cells against ADAM9KD‐HCC cells was higher than that against control cells, and the enhancement of this cytotoxicity depended on the MICA/B and NK group 2, member D pathway. Sorafenib treatment resulted in decreased expression of ADAM9, increased expression of membrane‐bound MICA expression, and decreased levels of soluble MICA in HCC cells. Adding sorafenib enhanced the NK sensitivity of HCC cells via increased expression of membrane‐bound MICA. Conclusion: ADAM9 is involved in MICA ectodomain shedding in HCC cells, and sorafenib can modulate ADAM9 expression. Sorafenib therapy may have a previously unrecognized effect on antitumor immunity in patients with HCC. (HEPATOLOGY 2010.)

Anticancer Chemotherapy Inhibits MHC Class I-Related Chain A Ectodomain Shedding by Downregulating ADAM10 Expression in Hepatocellular Carcinoma

MHC class I-related chain A (MICA) is a ligand for the NKG2D-activating immunoreceptor that mediates activation of natural killer (NK) cells. The ectodomain of MICA is shed from tumor cells, which may be an important means of evading antitumor immunity. We previously reported that patients with hepatocellular carcinoma (HCC) display high levels of soluble MICA in circulation, which could be downregulated by chemotherapy. The present study shows that anti-HCC drugs suppress MICA ectodomain shedding by inhibiting expression of a disintegrin and metalloproteinase 10 (ADAM10). Both ADAM10 and CD44, a typical substrate of the ADAM10 protease, were expressed in human HCC tissues and HCC cells but not in normal liver tissues or cultured hepatocytes. Small interfering RNA-mediated knockdown experiments revealed that ADAM10 is a critical sheddase for both MICA and CD44 in HCC cells. Of interest is the finding that epirubicin clearly downregulated ADAM10 expression and MICA shedding in HCC cells; its suppressive effect on MICA shedding was abolished in ADAM10-depleted cells. Epirubicin treatment also enhanced the NKG2D-mediated NK sensitivity of HCC cells. Patients with HCC had significantly higher levels of serum-soluble CD44, which correlated well with serum-soluble MICA levels, thus suggesting a close link between ADAM10 activity and MICA shedding in these patients. Soluble MICA and CD44 levels were downregulated with a significant correlation in patients treated by transarterial chemoembolization using epirubicin. In conclusion, anticancer drugs can modulate expression of ADAM10, which is critically involved in MICA ectodomain shedding. Epirubicin therapy may have a previously unrecognized effect on antitumor immunity in HCC patients.

Expression of CD133 confers malignant potential by regulating metalloproteinases in human hepatocellular carcinoma

BACKGROUND & AIMS Although CD133 expression is identified as a cancer stem cell marker of hepatocellular carcinoma (HCC), the detailed characteristics of HCC cells expressing CD133 remain unclear. METHODS We examined the malignant characteristics of CD133-expressing HCC cells. RESULTS CD133-expressing cells could be detected with low frequency in 5 HCC tissues. We derived two different HCC cell lines by (1) transfection of CD133 siRNA in PLC/PRF/5 cells in (CD133si-PLC/PRF/5), and (2) by a magnetic cell sorting method that allowed to divide Huh7 cells into two CD133 positive (+) and negative (-) groups. CD133 knockdown in PLC/PRF/5 cells resulted in a decrease of the mRNA and protein expressions of matrix metalloproteinase (MMP)-2 and a disintegrin and metalloproteinase (ADAM)9. We next examined the malignant characteristics related to decreasing MMP-2 and ADAM9 in HCC cells. In CD133si-PLC/PRF/5 cells and CD133- Huh7 cells, invasiveness and vascular endothelial growth factor (VEGF) production, which are both related to the activity of MMP-2, were inhibited compared CD133-expressing HCC cells. We previously demonstrated that ADAM9 protease plays critical roles in the shedding of MHC class I-related chain A (MICA) which regulates the sensitivity of tumor cells to natural killer cells (NK). Decreasing ADAM9 expression in CD133si-PLC/PRF/5 cells and CD133- Huh7 cells resulted in an increase in membrane-bound MICA and a decrease in soluble MICA production. Both CD133si-PLC/PRF/5 cells and CD133- Huh7 cells were susceptible to NK activity, depending on the expression levels of membrane-bound MICA, but CD133-expressing HCC cells were not. CONCLUSION These results demonstrate that CD133 expression in HCC cells confers malignant potential which may contribute to the survival of HCC cells.

Serum levels of soluble major histocompatibility complex (MHC) class I-related chain A in patients with chronic liver diseases and changes during transcatheter arterial embolization for hepatocellular carcinoma

Soluble forms of major histocompatibility complex (MHC) class I‐related chain A and B (MICA/B) are increased in the sera of patients with malignancy and impair the antitumor immune response by downregulating expression of their cognate immunoreceptor natural killer group 2, member D (NKG2D). Recently, soluble MICA/B were reported to appear even in some premalignant diseases, raising questions about the impact of soluble MICA/B produced from tumors on the expression of NKG2D. The present study examined soluble MICA/B in chronic liver disease and hepatocellular carcinoma (HCC) and their involvement in the immune‐cell expression of NKG2D during transcatheter arterial embolization for HCC. The levels of soluble MICA/B were significantly higher in chronic liver disease and HCC patients than in healthy volunteers. The progression of liver disease and that of the tumor were independent determinants for soluble MICA/B levels. Immunohistochemistry revealed that MICA/B were expressed not only in HCC tissue but also on hepatocytes in cirrhotic livers. The transcatheter arterial embolization therapy significantly decreased serum levels of soluble MICA, but not soluble MICB, and increased the NKG2D expression on natural killer cells and CD8‐positive T cells; there was an inverse correlation between changes in soluble MICA levels and in NKG2D expression. In conclusion, although soluble MICA/B are produced from both HCC and premalignant cirrhotic livers, therapeutic intervention for HCC can reduce the levels of soluble MICA and thereby upregulate the expression of NKG2D. Cancer therapy may have a beneficial effect on NKG2D‐mediated antitumor immunity. (Cancer Sci 2008; 99: 1643–1649)

Altered interferon-α-signaling in natural killer cells from patients with chronic hepatitis C virus infection

BACKGROUND & AIMS Natural killer (NK) cells play an important role in the immune response against virus infection. Interferon (IFN)-alpha, an essential component in therapy against hepatitis C virus (HCV) infection, regulates NK cell function. However, it remains obscure how chronic HCV infection (CHC) modifies intracellular IFN-alpha signaling in NK cells. We investigated IFN-alpha signaling in NK cells in patients with CHC. METHODS Peripheral blood mononuclear cells were obtained from patients with CHC and healthy subjects (HS) as controls. RESULTS The expression level of signal transducer and activator of transcription (STAT) 1, a key molecule of IFN-alpha signaling, was clearly higher in NK cells from the CHC patients than in those from HS. The phosphorylation level of STAT1 with IFN-alpha stimulation was significantly greater in NK cells from the CHC patients than in those from the HS, while that of STAT4 was significantly less. These phosphorylation levels of STAT1 and STAT4 positively and negatively correlated with the STAT1 level in NK cells, respectively. The IFN-alpha induced messenger RNA level of the suppressor of cytokine signaling 1, which is a downstream gene of phosphorylated-STAT1, was clearly greater in NK cells from the CHC patients than in those from the HS, while that of IFN-gamma, which is a downstream gene of phosphorylated-STAT4, was clearly lower. CONCLUSIONS These results indicate altered IFN-alpha signaling in NK cells in CHC patients, suggesting that this alteration is associated with the persistence of HCV infection and resistance to IFN-alpha therapy.

Injection of IL-12 gene-transduced dendritic cells into mouse liver tumor lesions activates both innate and acquired immunity.

Dendritic cell (DC)-based vaccines have been applied clinically in the setting of advanced-stage cancer. To date, the clinical efficacy of these vaccines has been limited, possibly owing to the impairment of transferred DC function in cancer-bearing patients. In this study, we examined the therapeutic efficacy of interleukin-12 (IL-12) gene-transfected DCs isolated from tumor-bearing hosts against liver tumor. The endogenous DCs isolated from subcutaneous (s.c.) CMS4 tumor-bearing mice (CMS4DC) exhibited decreased expression levels of antigen-presenting molecules and low-allostimulatory capacity. CMS4DC produced less IL-12p70 than DCs isolated from normal mice. Adenoviral transfection of IL-12 gene into CMS4DC (AdIL12DC) restored the expression of antigen-presenting molecules and allostimulatory capacity. Intratumoral (i.t.) delivery of AdIL12DC resulted in complete rejection of intrahepatic CMS4 tumors and activation of innate and acquired immune cells. Antibody depletion studies revealed that both CD4+ and CD8+ T cells as well as natural killer cells play critical roles in mediating liver tumor rejection. I.t. treatment of AdIL12DC resulted in long-term protection against s.c. rechallenge with CMS4 tumor cells. These results revealed that IL-12 gene transfer is capable of improving the impaired functions of DC isolated from tumor-bearing hosts, and support the preclinical therapeutic efficacy of intrahepatic injection of AdIL12DC.

Intrahepatic delivery of α -galactosylceramide -pulsed dendritic cells suppresses liver tumor

Alpha‐galactosylceramide, a glycosphingolipid, mediates interaction of dendritic cells (DCs) and NKT cells, leading to activation of both innate and acquired immunity. For cancer treatment, conventional DC‐based vaccine has been tried, but its clinical efficacy is limited against liver cancer. Intrahepatic injection of α‐Galactosylceramide‐pulsed DCs (αGCDC) has not yet been tested in the liver that contains abundant immune cells such as NK, NKT, and T cells. In the present study, we examined the efficacy of αGCDC administration in comparison with p53 peptide‐pulsed DCs using a well‐established murine CMS4 tumor model. Injection of αGCDC into CMS4 liver tumors resulted in complete tumor rejection and established long‐term survival of the animals, while injection of p53232‐240 peptide‐pulsed DCs (pepDC) only partially suppressed tumor growth in the liver. The levels of IFN‐γ in sera of αGCDC‐treated mice were significantly higher than those of pepDC‐treated mice. Hepatic NK cells were efficiently activated by αGCDC injection and played a critical role in liver tumor rejection as evidenced by an in vivo antibody‐mediated NK cell depletion study. Injection of αGCDC into liver tumor led to higher p53232‐240 peptide‐specific CD8+ T cell response than that of pepDC. The mice that had been protected from CMS4 liver tumor by αGCDC injection became resistant to subcutaneous CMS4 rechallenge, but not to Colon26 rechallenge. Conclusion: These results demonstrate that αGCDC injection into the liver can efficiently activate NK cells that in turn reject liver tumors to establish potent acquired immunity against the original tumor. (HEPATOLOGY 2007;45:22–30.)

Dendritic cell-based vaccines suppress metastatic liver tumor via activation of local innate and acquired immunity

BackgroundDendritic cell (DC)-based vaccines have been applied clinically in the setting of cancer, but tumor-associated antigens (TAAs) have not yet been enough identified in various cancers. In this study, we investigated whether preventive vaccination with unpulsed DCs or peptide-pulsed DCs could offer anti-tumor effects against MC38 or BL6 liver tumors.MethodsMice were subcutaneously (s.c.) immunized with unpulsed DCs or the recently defined TAA EphA2 derived peptide-pulsed dendritic cells (Eph-DCs) to treat EphA2-positive MC38 and EphA2-negative BL6 liver tumors. Liver mononuclear cells (LMNCs) from treated mice were subjected to 51Cr release assays against YAC-1 target cells. In some experiments, mice were injected with anti-CD8, anti-CD4 or anti-asialo GM1 antibody to deplete each lymphocyte subsets.ResultsImmunization with unpulsed DCs displayed comparable efficacy against both MC38 and BL6 liver tumors when compared with Eph-DCs. Both DC-based vaccines significantly augmented the cytotoxicity of LMNCs against YAC-1 cells. In vivo antibody depletion studies revealed that NK cells, as well as, CD4+ and CD8+ T cells play critical roles in the anti-tumor efficacy associated with either DC-based modality.Tumor-specific cytotoxic T lymphocyte (CTL) activity was generally higher if mice had received Eph-DCs versus unpulsed DCs. Importantly, the mice that had been protected from MC38 liver tumor by either unpulsed DCs or Eph-DCs became resistant to s.c. MC38 rechallenge, but not to BL6 rechallenge.ConclusionsThese results demonstrate that unpulsed DC vaccines might serve as an effective therapy for treating metastatic liver tumor, for which TAA has not yet been identified.

Fibroblast growth factor‐2 enhances NK sensitivity of hepatocellular carcinoma cells

The roles of fibroblast growth factor‐2 (FGF‐2) in the hepatocellular carcinoma (HCC) development are still controversial. In this study, we investigated the expression of FGF‐2 in chronic hepatitis (CH) type C patients with or without HCC and the immunoregulation of FGF‐2 in NK sensitivity of HCC cells. The FGF‐2 expressions were detected in the liver tissues of patients, but not in normal liver. The serum FGF‐2 levels of the patients with CH, liver cirrhosis (LC) or HCC were significantly higher than those of healthy volunteers. The serum FGF‐2 levels of patients decreased with the progression of chronic liver disease. HCC occurrence of LC patients with high levels of serum FGF‐2 was significantly lower than that with low levels of serum FGF‐2. Proinflammatory cytokines, such as IL‐1β and IL‐6, induced FGF‐2 expressions in HCC cells and normal hepatocytes. FGF‐2 stimulation resulted in increasing the expression of the membrane‐bound major histocompatibility complex class I‐related chain A (MICA), an NK activating molecule, and decreasing that of human leukocyte antigen (HLA) class I, an NK inhibitory molecule, on HCC cells. This did not occur with normal hepatocytes. Adding anti‐FGF receptor‐2 neutralizing antibody resulted in inhibiting the change of MICA and HLA class I expressions on FGF‐2 stimulated HCC cells. FGF‐2 stimulation on HCC cells resulted in increasing NK sensitivity against HCC cells. These findings indicate that FGF‐2 produced by HCC cells or normal hepatocytes of chronic liver disease may play critical roles in eliminating HCC cells by innate immunity.

Supportive Role Played by Precore and PreS2 Genomic Changes in the Establishment of Lamivudine-Resistant Hepatitis B Virus

BACKGROUND Hepatitis B virus (HBV) establishes lamivudine resistance via the resistance-causative rtM204V/I mutation and the replication-compensatory rtL180M mutation. However, both lamivudine-resistant viruses with and those without rtL180M can exist in clinical settings. To elucidate the differences between viruses with and those without rtL180M, we conducted full-length sequencing analysis of HBV derived from patients with type B chronic hepatitis showing lamivudine resistance. METHODS The full-length HBV DNA sequences derived from 44 patients showing lamivudine resistance were determined by polymerase chain reaction direct sequencing. Viral replicative competence was examined by in vitro transfection analysis using various HBV-expressing plasmids. RESULTS Throughout the HBV genome, a precore-defective A1896 mutation and a short deletion in the preS2 gene were detected more frequently in viruses without rtL180M than in those with it (64% vs. 17% [P < .005] and 50% vs. 10% [P < .01], respectively). In vitro transfection analysis revealed that the level of reduction in intracellular viral replication caused by the introduction of lamivudine resistance-associated mutations was lower in precore-defective and preS2-deleted viruses than in wild-type virus. CONCLUSIONS Both the precore-defective mutation and the preS2 deletion may play a supportive role in the replication of lamivudine-resistant HBV, which may be a reason for there being no need for the compensatory rtL180M mutation in lamivudine-resistant HBV possessing the precore and preS2 genomic changes.