Yasunari Nakamoto

Hepatic ISG Expression Is Associated With Genetic Variation in Interleukin 28B and the Outcome of IFN Therapy for Chronic Hepatitis C

BACKGROUND & AIMS Multiple viral and host factors are related to the treatment response to pegylated-interferon and ribavirin combination therapy; however, the clinical relevance and relationship of these factors have not yet been fully evaluated. METHODS We studied 168 patients with chronic hepatitis C who received pegylated-interferon and ribavirin combination therapy. Gene expression profiles in the livers of 91 patients were analyzed using an Affymetrix genechip (Affymetrix, Santa Clara, CA). The expression of interferon-stimulated genes (ISGs) was evaluated in all samples by real-time polymerase chain reaction. Genetic variation in interleukin 28B (IL28B; rs8099917) was determined in 91 patients. RESULTS Gene expression profiling of the liver differentiated patients into 2 groups: patients with up-regulated ISGs and patients with down-regulated ISGs. A high proportion of patients with no response to treatment was found in the up-regulated ISGs group (P=.002). Multivariate logistic regression analysis showed that ISGs (<3.5) (odds ratio [OR], 16.2; P<.001), fibrosis stage (F1-F2) (OR, 4.18; P=.003), and ISDR mutation (>or=2) (OR, 5.09; P=.003) were strongly associated with the viral response. The IL28B polymorphism of 91 patients showed that 66% were major homozygotes (TT), 30% were heterozygotes (TG), and 4% were minor homozygotes (GG). Interestingly, hepatic ISGs were associated with the IL28B polymorphism (OR, 18.1; P<.001), and its expression was significantly higher in patients with the minor genotype (TG or GG) than in those with the major genotype (TT). CONCLUSIONS The expression of hepatic ISGs is strongly associated with treatment response and genetic variation of IL28B. The differential role of host and viral factors as predicting factors may also be present.

Discrete nature of EpCAM+ and CD90+ cancer stem cells in human hepatocellular carcinoma

Recent evidence suggests that hepatocellular carcinoma (HCC) is organized by a subset of cells with stem cell features (cancer stem cells; CSCs). CSCs are considered a pivotal target for the eradication of cancer, and liver CSCs have been identified by the use of various stem cell markers. However, little information is known about the expression patterns and characteristics of marker‐positive CSCs, hampering the development of personalized CSC‐targeted therapy. Here, we show that CSC markers EpCAM and CD90 are independently expressed in liver cancer. In primary HCC, EpCAM+ and CD90+ cells resided distinctively, and gene‐expression analysis of sorted cells suggested that EpCAM+ cells had features of epithelial cells, whereas CD90+ cells had those of vascular endothelial cells. Clinicopathological analysis indicated that the presence of EpCAM+ cells was associated with poorly differentiated morphology and high serum alpha‐fetoprotein (AFP), whereas the presence of CD90+ cells was associated with a high incidence of distant organ metastasis. Serial xenotransplantation of EpCAM+/CD90+ cells from primary HCCs in immune‐deficient mice revealed rapid growth of EpCAM+ cells in the subcutaneous lesion and a highly metastatic capacity of CD90+ cells in the lung. In cell lines, CD90+ cells showed abundant expression of c‐Kit and in vitro chemosensitivity to imatinib mesylate. Furthermore, CD90+ cells enhanced the motility of EpCAM+ cells when cocultured in vitro through the activation of transforming growth factor beta (TGF‐β) signaling, whereas imatinib mesylate suppressed TGFB1 expression in CD90+ cells as well as CD90+ cell‐induced motility of EpCAM+ cells. Conclusion: Our data suggest the discrete nature and potential interaction of EpCAM+ and CD90+ CSCs with specific gene‐expression patterns and chemosensitivity to molecular targeted therapy. The presence of distinct CSCs may determine the clinical outcome of HCC. (HEPATOLOGY 2013)

Aberrant expression of serine/threonine kinase Pim-3 in hepatocellular carcinoma development and its role in the proliferation of human hepatoma cell lines.

Most cases of human hepatocellular carcinoma develop after persistent chronic infection with human hepatitis B virus or hepatitis C virus, and host responses are presumed to have major roles in this process. To recapitulate this process, we have developed the mouse model of hepatocellular carcinoma using hepatitis B virus surface antigen transgenic mice. To identify the genes associated with hepatocarcinogenesis in this model, we compared the gene expression patterns between pre‐malignant lesions surrounded by hepatocellular carcinoma tissues and control liver tissues by using a fluorescent differential display analysis. Among the genes that were expressed differentially in the pre‐malignant lesions, we focused on Pim‐3, a member of a proto‐oncogene Pim family, because its contribution to hepatocarcinogenesis remains unknown. Moreover, the unavailability of the nucleotide sequence of full‐length human Pim‐3 cDNA prompted us to clone it from the cDNA library constructed from a human hepatoma cell line, HepG2. The obtained 2,392 bp human Pim‐3 cDNA encodes a predicted open reading frame consisting of 326 amino acids. Pim‐3 mRNA was selectively expressed in human hepatoma cell lines, but not in normal liver tissues. Moreover, Pim‐3 protein was detected in human hepatocellular carcinoma tissues and cell lines but not in normal hepatocytes. Furthermore, cell proliferation was attenuated and apoptosis was enhanced in human hepatoma cell lines by the ablation of Pim‐3 gene with RNA interference. These observations suggest that aberrantly expressed Pim‐3 can cause autonomous cell proliferation or prevent apoptosis in hepatoma cell lines.

Comparative analysis of various tumor-associated antigen-specific t-cell responses in patients with hepatocellular carcinoma.

Many tumor‐associated antigens (TAAs) recognized by cytotoxic T cells (CTLs) have been identified during the last two decades and some of them have been used in clinical trials. However, there are very few in the field of immunotherapy for hepatocellular carcinoma (HCC) because there have not been comparative data regarding CTL responses to various TAAs. In the present study, using 27 peptides derived from 14 different TAAs, we performed comparative analysis of various TAA‐specific T‐cell responses in 31 HCC patients to select useful antigens for immunotherapy and examined the factors that affect the immune responses to determine a strategy for more effective therapy. Twenty‐four of 31 (77.4%) HCC patients showed positive responses to at least one TAA‐derived peptide in enzyme‐linked immunospot assay. The TAAs consisting of cyclophilin B, squamous cell carcinoma antigen recognized by T cells (SART) 2, SART3, p53, multidrug resistance‐associated protein (MRP) 3, alpha‐fetoprotein (AFP) and human telomerase reverse transcriptase (hTERT) were frequently recognized by T cells and these TAA‐derived peptides were capable of generating peptide‐specific CTLs in HCC patients, which suggested that these TAAs are immunogenic. HCC treatments enhanced TAA‐specific immune responses with an increased number of memory T cells and induced de novo T‐cell responses to lymphocyte‐specific protein tyrosine kinase, human epidermal growth factor receptor type 2, p53, and hTERT. Blocking cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) resulted in unmasking of TAA‐specific immune responses by changing cytokine and chemokine profiles of peripheral blood mononuclear cells stimulated by TAA‐derived peptides. Conclusion: Cyclophilin B, SART2, SART3, p53, MRP3, AFP, and hTERT were immunogenic targets for HCC immunotherapy. TAA‐specific immunotherapy combined with HCC treatments and anti‐CTLA‐4 antibody has the possibility to produce stronger tumor‐specific immune responses. (HEPATOLOGY 2011;)

Cytotoxic T cell responses to human telomerase reverse transcriptase in patients with hepatocellular carcinoma

Human telomerase reverse transcriptase, hTERT, has been identified as the catalytic enzyme required for telomere elongation. hTERT is expressed in most tumor cells but seldom expressed in most human adult cells. It has been reported that 80% to 90% of hepatocellular carcinomas (HCCs) express hTERT, making the enzyme a potential target in immunotherapy for HCC. In the current study, we identified hTERT‐derived, HLA‐A*2402–restricted cytotoxic T cell (CTL) epitopes and analyzed hTERT‐specific CTL responses in patients with HCC. Peptides containing the epitopes showed high affinity to bind HLA‐A*2402 in a major histocompatibility complex binding assay and were able to induce hTERT‐specific CTLs in both hTERT cDNA‐immunized HLA‐A*2402/Kb transgenic mice and patients with HCC. The CTLs were able to kill hepatoma cell lines depending on hTERT expression levels in an HLA‐A*2402–restricted manner and induced irrespective of hepatitis viral infection. The number of single hTERT epitope‐specific T cells detected by ELISPOT assay was 10 to 100 specific cells per 3 × 105 PBMCs, and positive T cell responses were observed in 6.9% to 12.5% of HCC patients. hTERT‐specific T cell responses were observed even in the patients with early stages of HCC. The frequency of hTERT/tetramer+CD8+ T cells in the tumor tissue of patients with HCC was quite high, and they were functional. In conclusion, these results suggest that hTERT is an attractive target for T‐cell–based immunotherapy for HCC, and the identified hTERT epitopes may be valuable both for immunotherapy and for analyzing host immune responses to HCC. (HEPATOLOGY 2006;43: 1284–1294.)

Crucial Contribution of Thymic Sirpα+ Conventional Dendritic Cells to Central Tolerance against Blood-Borne Antigens in a CCR2-Dependent Manner

Thymic dendritic cells (DCs) as well as thymic epithelial cells are presumed to be major sentinels in central tolerance by inducing the apoptosis of autoreactive T progenitor cells. The thymic DC population is composed of heterogeneous subsets including CD11c+B220+ plasmacytoid DCs, CD11c+B220−CD8α+ signal regulatory protein α (Sirpα)− and CD11c+B220−CD8α−Sirpα+ conventional DCs (cDCs). However, the distinctive role of each DC subset remains undefined. We show herein that Sirpα+ cDCs, a minor subpopulation, was disseminated in the thymic cortical area with some of them uniquely localized inside perivascular regions and nearby small vessels in the thymus. The Sirpα+ but not Sirpα− cDC subset can selectively capture blood-circulating Ags. Moreover, in CCR2-deficient mice, the thymic Sirpα+ cDC subset, but not other thymic cell components, was moderately decreased especially in the perivascular regions. Concomitantly, these mice exhibited a modest impairment in intrathymic negative selection against blood-borne Ags, with the reduced capacity to uptake blood-borne Ags. Given their intrathymic cortical localization, CD11c+B220−CD8α−Sirpα+ cDCs can have a unique role in the development of central tolerance against circulating peripheral Ags, at least partially in a CCR2-dependent manner.

Impact of diabetes on recurrence of hepatocellular carcinoma after surgical treatment in patients with viral hepatitis.

OBJECTIVES: Consensus has been reached that diabetes is a risk factor for development of HCC, but the impact on postoperative recurrence is still controversial. To clarify this point, we analyzed the relationship of postoperative recurrence rate of HCC and coexistence of diabetes in the patients with viral hepatitis.METHODS: A total of 90 patients who had undergone curative resection for HCC were analyzed. They were divided into two groups with and without diabetes, and the recurrence-free survival rates after surgical treatment and the factors contributing to recurrence were examined.RESULTS: Kaplan-Meier survival analysis showed the recurrence-free survival rates in the diabetic group were significantly lower than those in the nondiabetic group (P = 0.005) and overall survival rates in the diabetic group were significantly lower than those in the nondiabetic group (P = 0.005). These results were emphasized in the analysis of patients infected with hepatitis C virus. Univariate and multivariate analyses showed diabetes was a significant factor contributing to HCC recurrence after treatment. Furthermore, multivariate analysis in HCC patients with diabetes showed Child-Pugh classification B (P = 0.001) and insulin therapy (P = 0.049) were significant factors contributing to HCC recurrence after treatment.CONCLUSIONS: The results of the present study suggest that diabetes is a risk factor for the recurrence of HCV-related HCC and decreases the overall survival rates after surgical treatment. HCV-related HCC patients with diabetes should be closely followed for postoperative recurrence.

Essential contribution of a chemokine, CCL3, and its receptor, CCR1, to hepatocellular carcinoma progression

We previously observed that a chemokine, macrophage inflammatory protein‐1 α/CCL3, and its receptor, CCR1, were aberrantly expressed in human hepatocellular carcinoma (HCC) tissues. Here, we show that CCL3 and CCR1 are also expressed in 2 different models of this cancer; N‐nitrosodiethylamine (DEN)‐induced HCC and HCC induced by hepatitis B virus surface (HBs) antigen‐primed splenocyte transfer to myelo‐ablated syngeneic HBs antigen transgenic mice. At 10 months after DEN treatment, foci number and sizes were remarkably reduced in CCR1‐ and CCL3‐deficient mice, compared with those of wild‐type (WT) mice, although tumor incidence were marginally, but significantly, higher in CCR1‐ and CCL3‐deficient mice than in WT mice. Of note is that tumor angiogenesis was also markedly diminished in CCL3‐ and CCR1‐deficient mice, with a concomitant reduction in the number of intratumoral Kupffer cells, a rich source of growth factors and matrix metalloproteinases (MMPs). Among growth factors and MMPs that we examined, only MMP9 and MMP13 gene expression was augmented progressively in liver of WT mice after DEN treatment. Moreover, MMP9, but not MMP13, gene expression was attenuated in CCR1‐ and CCL3‐deficient mice, compared with that of WT mice. Furthermore, MMP9 was expressed mainly by mononuclear cells but not hepatoma cells, and MMP9‐expressing cell numbers were decreased in CCR1‐ or CCL3‐deficient mice, compared with WT mice. These observations suggest the contribution of the CCR1‐CCL3 axis to HCC progression.

Prevention of Hepatocellular Carcinoma Development Associated with Chronic Hepatitis by Anti-Fas Ligand Antibody Therapy

A persistent immune response to hepatitis viruses is a well-recognized risk factor for hepatocellular carcinoma. However, the molecular and cellular basis for the procarcinogenic potential of the immune response is not well defined. Here, using a unique animal model of chronic hepatitis that induces hepatocellular carcinogenesis, we demonstrate that neutralization of the activity of Fas ligand prevented hepatocyte apoptosis, proliferation, liver inflammation, and the eventual development of hepatocellular carcinoma. The results indicate that Fas ligand is involved not only in direct hepatocyte killing but also in the process of inflammation and hepatocellular carcinogenesis in chronic hepatitis. This is the first demonstration that amelioration of chronic inflammation by some treatment actually caused reduction of cancer development.

Potential interaction between CCR1 and its ligand, CCL3, induced by endogenously produced interleukin-1 in human hepatomas

Hepatoma cell lines can produce a massive amount of chemokines in response to various stimuli including hepatitis viruses and their products. However, it remains elusive on the types of chemokine receptor(s) expressed in the hepatoma tissues and its roles in hepatoma development. To clarify these points, we examined the chemokine receptor expression in six human hepatoma cell lines. All of the hepatoma cell lines constitutively and exclusively expressed CCR1 mRNA and its protein on their cell surface. CCR1 expression was also detected on hepatoma cells and to a lesser degree, on endothelial cells in hepatoma tissues but not in normal liver tissues. Furthermore, CCL3 expression was detected in hepatoma cells, endothelial cells, and to a lesser degree, fibroblast-like cells in hepatoma tissue, whereas only occasional vascular endothelial cells and inflammatory cells in normal liver tissues were weakly positive for CCL3. Moreover, the forskolin-mediated increases in intracellular cAMP concentrations were inhibited by the ligands for CCR1, CCL3, CCL4, and CCL5, suggesting that the expressed CCR1 was functional. Four hepatoma cell lines produced CCL3 only in response to interleukin (IL)-1 alpha and IL-1 beta. Finally, IL-1 alpha and IL-1 beta were detected abundantly in hepatoma tissues but not in normal liver tissues. Thus, IL-1 may enhance the local production of CCL3, which may interact with CCR1 expressed on hepatoma cells, in an autocrine and/or paracrine manner.