Prognostic impact of soluble PD-L1 derived from tumor-associated macrophages in non-small-cell lung cancer.

Programmed cell death-ligand 1 (PD-L1) on tumor cells can be degraded to soluble form (sPD-L1) and enter circulation, however, the clinical significances of sPD-L1 in peripheral blood remains to be elucidated in non-small-cell lung cancer (NSCLC). We monitored plasma sPD-L1 levels during perioperative periods and evaluated PD-L1-positive cells in tumor tissues in patients with operable NSCLC. Then the correlation between preoperative plasma sPD-L1 levels and relapse-free survival (RFS) was analyzed retrospectively. In patients who underwent radical surgery (n = 61), plasma sPD-L1 levels (median; 63.5 pg/mL) significantly increased 1 month after surgery (72.2 pg/mL, P < 0.001). The combined score of PD-L1-positive cells including tumor cells and tumor-associated macrophages (TAMs) was significantly associated with preoperative plasma sPD-L1 levels. In patients with high levels of preoperative plasma sPD-L1, the probability of 5-year RFS was significantly poor for patients with low PD-L1 expression intensity of tumor cells (tcPD-L1) compared with those with high tcPD-L1 (33.3% vs. 87.5%, respectively, P = 0.016; 95% CI, 0.013-0.964). In former group, PD-L1-positive TAMs were markedly infiltrating compared with those from latter group (246.4 vs. 76.6 counts/mm2, respectively, P = 0.003). In NSCLC, plasma sPD-L1 can reflect the accumulation of PD-L1-posotive TAMs, not just PD-L1-positive tumor cells. In patients with high levels of preoperative plasma sPD-L1, the prognoses after surgery depends on which PD-L1-positive cells, tumor cells or TAMs, are the primary source of the sPD-L1. Thus, measuring both plasma sPD-L1 levels and PD-L1 expression status of tumor cells and TAMs is of benefit for assessment of postoperative prognosis in operable NSCLC.

Oncogenic epidermal growth factor receptor signal-induced histone deacetylation suppresses chemokine gene expression in human lung adenocarcinoma.

Epidermal growth factor receptor (EGFR)-mutated (mt) lung adenocarcinoma (LA) is refractory to immune checkpoint inhibitors (ICIs). However, the mechanisms have not been fully elucidated. CD8+ T cell infiltration was significantly lower in EGFR-mt than in EGFR-wild-type LA, which was associated with suppression of chemokine expression. Since this T cell-deserted tumor microenvironment may lead to the refractoriness of ICIs against EGFR-mt LA, we investigated the mechanism by focusing on the regulation of chemokine expression. The expression of C-X-C motif ligand (CXCL) 9, 10 and 11, which constitute a gene cluster on chromosome 4, was suppressed under EGFR signaling. The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) revealed open chromatin peaks near this gene cluster following EGFR-tyrosine kinase inhibitor (TKI) treatment. The histone deacetylase (HDAC) inhibitor recovered the expression of CXCL9, 10 and 11 in EGFR-mt LA. Nuclear HDAC activity, as well as histone H3 deacetylation, were dependent on oncogenic EGFR signaling. Furthermore, the Cleavage Under Targets and Tagmentation (CUT & Tag) assay revealed a histone H3K27 acetylation peak at 15 kb upstream of CXCL11 after treatment with EGFR-TKI, which corresponded to one of the open chromatin peaks detected by ATAC-seq. The data suggest that EGFR-HDAC axis mediates silencing of the chemokine gene cluster through chromatin conformational change, which might be relevant to the ICI resistance by creating T cell-deserted tumor microenvironment. Targeting this axis may develop a new therapeutic strategy to overcome the ICI resistance of EGFR-mt LA.

Factors Associated with Cancer-Related Pain Requiring High-Dose Opioid Use in Palliative Cancer Patients.

Background: There are no universal tools to predict the necessity of high-dose opioid use for cancer-related pain. Early recognition and interventions for intractable cancer pain could minimize the distress of palliative patients. Objective: We sought to identify the clinical factors associated with high-dose opioid use in advanced cancer patients to recognize palliative patients who would develop intractable cancer pain, as early as possible. Setting/Subjects: Among 385 in-hospital cancer patients from April 1, 2014 to July 31, 2019, who were referred to the palliative care team for cancer-related pain, clinical factors significantly correlated to high-dose opioid use were retrospectively analyzed. Measurements: We conducted a multiple logistic regression analysis to identify variables significantly related to high-dose opioid use (>120 mg/day oral morphine equivalent dose). Results: Independent factors of high-dose opioid use included younger age (odds ratio [OR] 0.965, 95% confidence interval [CI] 0.944-0.986, p = 0.001), respiratory cancers (OR 1.882, 95% CI 1.069-3.312, p < 0.001), and opioid switch (OR 2.869, 95% CI 1.497-5.497, p = 0.001). The percentage of correct classifications of the regression equation was 86.9%. Conclusions: Younger age, respiratory cancers, and opioid switch were related to high-dose opioid use. Our findings may help palliative caregivers to deal with intractable cancer pain in palliative patients, and thus relieve their distress.

Biphasic prognostic significance of PD-L1 expression status in patients with early- and locally advanced-stage non-small cell lung cancer.

Programmed cell death-ligand 1 (PD-L1) expression on tumor cells is induced by interferon-gamma, suggesting the induction of an anti-tumor immune response. In turn, binding of PD-L1 to programmed cell death 1 (PD-1) triggers an immune checkpoint pathway that contributes to tumor growth. Though it remains to be elucidated, the clinical significance of PD-L1 expression might vary with tumor progression in non-small-cell lung cancer (NSCLC). Immunohistochemical analysis of PD-L1 was done in tumor specimens from patients who underwent radical surgery for stage I-IIIA NSCLC (n = 228). Tumor PD-L1 expression intensity was semi-quantitatively scored and its correlation with various clinicopathological features and postoperative relapse-free survival (RFS) was assessed relative to pathological stage. In stage I, postoperative RFS was significantly prolonged in patients with a high PD-L1 score compared with a low PD-L1 score, exhibiting 5-year relapse-free probabilities of 94.1% and 75.1%, respectively (P = 0.031). A multivariate analysis revealed that a high PD-L1 score was a prognostic factor of longer postoperative RFS (hazard ratio: 0.111, P = 0.033). Conversely, in stages II and IIIA, patients with a high PD-L1 score tended to suffer from postoperative tumor recurrence. In early-stage NSCLC, high tumor PD-L1 expression status represents a biomarker to predict good prognosis after radical surgery and may reflect the induction of an antitumor immune response. However, in locally advanced stage NSCLC, tumor PD-L1 expression status may reflect the execution of an immune checkpoint pathway and predicts the incidence of postoperative tumor recurrence.

Detection of neoantigen-reactive T cell clones based on the clonal expansion using next-generation sequencing of T cell receptor ß complementarity-determining region 3.

Development of mechanism-driven biomarkers for immune checkpoint inhibitors (ICIs) in cancer immunotherapy requires sensitive and efficacious assays to identify tumor antigen (Ag)-specific T cells. We demonstrated the concept for a sensitive method to determine Ag-reactive T cell clones based on clonal expansion using model neoantigens (NeoAgs) rather than cytokine production. Sequential increase in T cell clonal frequencies following Ag stimulation was detected by next generation sequencing (NGS) of T cell receptor ß (TCR ß) complementarity-determining region 3 (CDR3), with a higher sensitivity than that of enzyme-linked immunospot (ELISPOT) by 100-fold. The TCRß CDR3 sequences could represent useful markers to track dynamic changes during immunotherapy. The TCRß NGS-based method could represent a novel platform both for the development of new biomarkers as well as several therapeutic options.

Clinical significance of PD-L1-positive cancer-associated fibroblasts in pN0M0 non-small cell lung cancer.

OBJECTIVES: Cancer-associated fibroblasts (CAFs) are a dominant cell type in tumor stroma and support the generation of pro-tumorigenic microenvironment. CAFs have frequent opportunities to interact with immune cells infiltrating the tumor stroma, but the process remains to be determined. In this study, we focused on immune checkpoint mechanism. We also examined the induction of programmed cell death-ligand 1 (PD-L1) on CAFs by immune cell, and the clinical significance of PD-L1-expressed CAFs in non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: CAFs were isolated from human NSCLC tissues, and PD-L1 expression levels in CAFs were analyzed by real-time polymerase chain reaction and flow-cytometry. Following immunohistochemical analysis of PD-L1 in surgically resected pN0M0 NSCLC (n = 125, including 88 invasive adenocarcinomas and 37 squamous cell carcinomas), the correlation of PD-L1-positive CAFs with clinicopathological features was investigated. RESULTS: PD-L1 mRNA and protein expression on CAFs was upregulated by exogenously supplemented interferon-gamma (IFN-γ) and downregulated through the depletion of IFN-γ. PD-L1 expression on CAFs was upregulated by co-culture with activated lymphocytes releasing IFN-γ. Immunohistochemistry revealed that PD-L1-positive CAFs were observed in 31 cases (24.8%). Postoperative relapse-free survival was significantly prolonged in patients with PD-L1-positive CAFs as compared with those with PD-L1-negative CAFs, with 5-year relapse-free probabilities of 84.5% and 66.3%, respectively (P = 0.031). Multivariate analysis revealed that PD-L1 expression on CAFs was an independent prognostic factor of longer relapse-free survival after surgery (hazard ratio: 3.225, P = 0.027). CONCLUSION: PD-L1 expression on CAFs is reversibly regulated by environmental stimuli including IFN-γ from activated lymphocytes. In the non-metastatic NSCLC, PD-L1 expression on CAFs suggests the induction of anti-tumor immune responses, contributing to better prognosis after surgery.

Sox9 regulates the luminal stem/progenitor cell properties of salivary glands.

Exocrine glands share a common morphology consisting of ductal, acinar, and basal/myoepithelial cells, but their functions and mechanisms of homeostasis differ among tissues. Salivary glands are an example of exocrine glands, and they have been reported to contain multipotent stem cells that differentiate into other tissues. In this study, we purified the salivary gland stem/progenitor cells of adult mouse salivary glands using the cell surface marker CD133 by flow cytometry. CD133+ cells possessed stem cell capacity, and the transplantation of CD133+ cells into the submandibular gland reconstituted gland structures, including functional acinar. CD133+ cells were sparsely distributed in the intercalated and exocrine ducts and expressed Sox9 at higher levels than CD133- cells. Moreover, we demonstrated that Sox9 was required for the stem cell properties CD133+ cells, including colony and sphere formation. Thus, the Sox9-related signaling may control the regeneration salivary glands.

RAS-Mitogen-Activated Protein Kinase Signal Is Required for Enhanced PD-L1 Expression in Human Lung Cancers.

Ectopic programmed cell death ligand 1 (PD-L1) expression in non-small cell lung cancers (NSCLCs) is related to immune evasion by cancer, and it is a molecular target of immune checkpoint therapies. Although some altered signals in NSCLCs are responsible for ectopic PD-L1 expression, the precise mechanisms remain obscure. Because we found a higher frequency of EGFR/KRAS mutations in NSCLC cell lines with high PD-L1 expression (p < 0.001), we evaluated the relationships between downstream signals and PD-L1 expression, particularly in three KRAS-mutant adenocarcinoma cell lines. The MEK inhibitor U0126 (20 µM) significantly decreased the surface PD-L1 levels by 50-60% compared with dimethyl sulfoxide (p < 0.0001). Phorbol 12-myristate 13-acetate stimulation (100 nM, 15 min) increased (p < 0.05) and two ERK2 siRNAs as well as KRAS siRNAs decreased (p < 0.05) PD-L1 expression. The transcriptional activity of the potential AP-1 site (+4785 to +5056 from the transcription start site) in the PD-L1 gene was demonstrated by luciferase assays, which was inhibited by U0126. The chromatin immunoprecipitation assay demonstrated the binding of cJUN to the AP-1 site. Two STAT3 siRNAs decreased PD-L1 expression by 10-32% in two of the three KRAS-mutant lung adenocarcinoma cell lines (p < 0.05), while the PI3K inhibitor LY294002 (40 µM) did not change the expression level. Supervised cluster analysis and gene set enrichment analysis between the PD-L1-high and -low NSCLCs revealed a correlation between PD-L1 expression and genes/pathways related to cell motility/adhesion. These results indicate that MAPK signaling is the dominant downstream signal responsible for ectopic PD-L1 expression, in which STAT3 is also involved to some extent. Furthermore, MAPK signaling may control the expression of PD-L1 and several genes related to enhanced cell motility. Our findings suggest that MAPK, along with STAT3, is important for determining PD-L1 expression, which could be useful for targeted therapies against lung cancers.

Improvement of cancer immunotherapy by combining molecular targeted therapy.

In human cancer cells, a constitutive activation of MAPK, STAT3, ß-catenin, and various other signaling pathways triggers multiple immunosuppressive cascades. These cascades result in the production of immunosuppressive molecules (e.g., TGF-ß, IL-10, IL-6, VEGF, and CCL2) and induction of immunosuppressive immune cells (e.g., regulatory T cells, tolerogenic dendritic cells, and myeloid-derived suppressor cells). Consequently, immunosuppressive conditions are formed in tumor-associated microenvironments, including the tumor and sentinel lymph nodes. Some of these cancer-derived cytokines and chemokines impair immune cells and render them immunosuppressive via the activation of signaling molecules, such as STAT3, in the immune cells. Thus, administration of signal inhibitors may inhibit the multiple immunosuppressive cascades by acting simultaneously on both cancer and immune cells at the key regulatory points in the cancer-immune network. Since common signaling pathways are involved in manifestation of several hallmarks of cancer, including cancer cell proliferation/survival, invasion/metastasis, and immunosuppression, targeting these shared signaling pathways in combination with immunotherapy may be a promising strategy for cancer treatment.

Cancer-induced immunosuppressive cascades and their reversal by molecular-targeted therapy.

Immunological status in tumor tissues varies among patients. Infiltration of memory-type CD8(+) T cells into tumors correlates with prognosis of patients with various cancers. However, the mechanism of the differential CD8(+) T cell infiltration has not been well investigated. In general, tumor-associated microenvironments, including tumor and sentinel lymph nodes, are under immunosuppressive conditions such that the immune system is not able to eliminate cancer cells without immune-activating interventions. Constitutive activation of various signaling pathways in human cancer cells triggers multiple immunosuppressive cascades that involve various cytokines, chemokines, and immunosuppressive cells. Signaling pathway inhibitors could inhibit these immunosuppressive cascades by acting on either cancer or immune cells, or both. In addition, common signaling mechanisms are often utilized for multiple hallmarks of cancer (e.g., cell proliferation/survival, invasion/metastasis, and immunosuppression). Therefore, targeting these common signaling pathways may be an attractive strategy for cancer therapy including immunotherapy.

Suppression of myeloid cell leukemia-1 (Mcl-1) enhances chemotherapy-associated apoptosis in gastric cancer cells.

BACKGROUND: Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein that regulates apoptosis sensitivity in a variety of cell types. Here we evaluate the roles of Mcl-1 in chemotherapy-associated apoptosis in gastric cancer cells. In addition, our study examined whether Mcl-1 contributed to apoptosis resistance in so-called cancer stem cell (CSC)-like populations in gastric cancer. METHODS: Seven gastric cancer cell lines were used. The expression of Mcl-1 was assessed by either real-time polymerase chain reaction or Western blot analysis. Apoptosis was quantitated by morphological observation and caspase activity measurement. Adenovirus-mediated RNA interference (RNAi) technology was used to knockdown the expression of Mcl-1. The release of cytochrome c was evaluated by subcellular fractionation and immunoblot analysis. To identify and isolate the CSC-like populations, we used the CSC-associated cell surface marker CD44 and flow cytometry. RESULTS: Six out of the 7 gastric cancer cell lines overexpressed Mcl-1 protein. These Mcl-1-expressing cell lines were relatively resistant to chemotherapeutic agents such as 5-fluorouracil (5-FU) and cisplatin (CDDP). Depletion of Mcl-1 protein by RNAi technology effectively sensitized the cells to anticancer drug-induced mitochondrial cytochrome c release, caspase activation, and apoptosis. In addition, vast amounts of Mcl-1 mRNA were expressed in CD44-positive CSC-like cells. Mcl-1 suppression enhanced the apoptosis in CD44-positive cells to a level equivalent to that in CD44-negative cells, suggesting that Mcl-1 mediates chemotherapy resistance in CSC-like populations. CONCLUSION: These results suggest that Mcl-1 mediates the resistance to apoptosis in gastric cancer cells by blocking the mitochondrial pathway of cell death. Mcl-1 depletion appears to be an attractive strategy to overcome chemotherapy resistance in gastric cancer cells.

Immune suppression and resistance mediated by constitutive activation of Wnt/ß-catenin signaling in human melanoma cells.

Cancer-induced immunosuppression is a major problem reducing antitumor effects of immunotherapies, but its molecular mechanism has not been well understood. We evaluated immunosuppressive roles of activated Wnt/ß-catenin pathways in human melanoma for dendritic cells (DCs) and CTLs. IL-10 expression was associated with ß-catenin accumulation in human melanoma cell lines and tissues and was induced by direct ß-catenin/TCF binding to the IL-10 promoter. Culture supernatants from ß-catenin-accumulated melanoma have activities to impair DC maturation and to induce possible regulatory DCs. Those immunosuppressive culture supernatant activities were reduced by knocking down ß-catenin in melanoma cells, partly owing to downregulation of IL-10. Murine splenic and tumor-infiltrating DCs obtained from nude mice implanted with human mutant ß-catenin-overexpressed melanoma cells had less ability to activate T cells than did DCs from mice with control melanoma cells, showing in vivo suppression of DCs by activated Wnt/ß-catenin signaling in human melanoma. This in vivo DC suppression was restored by the administration of a ß-catenin inhibitor, PKF115-584. ß-catenin-overexpressed melanoma inhibited IFN-γ production by melanoma-specific CTLs in an IL-10-independent manner and is more resistant to CTL lysis in vitro and in vivo. These results indicate that Wnt/ß-catenin pathways in human melanoma may be involved in immunosuppression and immunoresistance in both induction and effector phases of antitumor immunoresponses partly through IL-10 production, and they may be attractive targets for restoring immunocompetence in patients with Wnt/ß-catenin-activated melanoma.

Transplantation of side population cells restores the function of damaged exocrine glands through clusterin.

Stem cell-based therapy has been proposed as a promising strategy for regenerating tissues lost through incurable diseases. Side population (SP) cells have been identified as putative stem cells in various organs. To examine therapeutic potential of SP cells in hypofunction of exocrine glands, SP cells isolated from mouse exocrine glands, namely, lacrimal and salivary glands, were transplanted into mice with irradiation-induced hypofunction of the respective glands. The secretions from both glands in the recipient mice were restored within 2 months of transplantation, although the transplanted cells were only sparsely distributed and produced no outgrowths. Consistent with this, most SP cells were shown to be CD31-positive endothelial-like cells. In addition, we clarified that endothelial cell-derived clusterin, a secretory protein, was an essential factor for SP cell-mediated recovery of the hypofunctioning glands because SP cells isolated from salivary glands of clusterin-deficient mice had no therapeutic potential, whereas lentiviral transduction of clusterin restored the hypofunction. In vitro and in vivo studies showed that clusterin had an ability to directly inhibit oxidative stress and oxidative stress-induced cell damage. Thus, endothelial cell-derived clusterin possibly inhibit oxidative stress-induced hypofunction of these glands.

Enhanced cancer immunotherapy using STAT3-depleted dendritic cells with high Th1-inducing ability and resistance to cancer cell-derived inhibitory factors.

STAT3 signaling constitutes an important negative feedback mechanism for the maintenance of immune homeostasis, a suppressive signal for the Th1 immune response in murine macrophages, and a cancer immune evasion signal in various immune cells. The strategy for STAT3 signal inhibition should be considered, because these features could impede effective cancer immunotherapy. We have evaluated the effects of STAT3 inactivation in dendritic cells (DCs) on immune responses in mice and humans. DCs derived from LysMcre/STAT3(flox/flox) mice displayed higher cytokine production in response to TLR stimulation, activated T cells more efficiently, and were more resistant to the suppression of cytokine production by cancer-derived immunosuppressive factors compared with DCs from control littermates. Antitumor activities of STAT3-depleted and control DCs were compared by intratumoral administration of gp70 Ag peptide-pulsed DCs in the therapeutic MC38 tumor model. Intratumoral administration of STAT3-depleted DCs significantly inhibited MC38 tumor growth of both injected and nontreated remote tumors. The inhibition was accompanied by an increase in gp70-specific T cell response as well as in systemic Th1 immune response. STAT3-depleted human DCs with adenoviral STAT3 short hairpin RNA were also capable of producing more cytokines with TLR stimulation and more resistant to cancer-derived factors, and they induced tumor Ag-specific T cells more efficiently than control DCs. The identified role of DC STAT3 signaling in both in vivo therapeutic tumor models in mice and in vitro-specific T cell induction in humans indicates that STAT3-inactivated DCs may be a promising approach for cancer immunotherapy.

The mechanisms of cancer immunoescape and development of overcoming strategies.

Cancer-induced immunosuppression is a major problem as it reduces the anti-tumor effects of immunotherapies. In cancer tissues, cancer cells, immune cells, and other stromal cells interact and create an immunosuppressive microenvironment through a variety of immunosuppressive factors. Some cancer subpopulations such as cancer cells undergoing epithelial-mesenchymal transition and cancer stem-like cells have immunosuppressive and immunoresistant properties. The production of immunosuppressive factors by cancer cells is mechanistically attributed to oncogenic signals frequently activated in cancer cells, including the STAT3, MAPK, NF-κB, and Wnt/ß-catenin signals, which are upstream events leading to immunosuppressive cascades. Moreover, some of these signals are also activated in immunosuppressive immune cells stimulated by cancer-derived factors and contribute to their immunosuppressive activities. Therefore, targeting these signals both in cancer cells and immunosuppressive immune cells may result in the restoration of immunocompetence in cancer patients and improve current immunotherapy.

Blast phase of chronic myeloid leukemia presenting lymphoid phenotype with a chronic phase of extremely short duration.

Chronic myeloid leukemia (CML) is generally diagnosed in the chronic phase. We have recently encountered two CML-blastic phase (BP) cases, a 71-year-old woman and a 74-year-old man, who resembled de novo acute leukemia. The complete blood count was normal at least 11 and 13 months before the presentation, respectively. The leukemic cells showed predominant lymphoid phenotype. The blasts and granulocytes were positive for BCR-ABL, indicative of CML-BP. Both patients were successfully treated with prednisone and vincristine, followed by Imatinib. Our cases indicate rare presentations of CML-BP with an extremely short chronic phase. Ph-positive de novo acute leukemia should be carefully distinguished from CML-BP.

The RNA silencing technology applied by lentiviral vectors in oncology.

Since the discovery of RNA interference (RNAi) in Caenorhabditis elegans in 1998, this mechanism has been found to be conserved in a wide variety of species, including insects, plants, and mammals. In mammals, small (or short) interfering RNA (siRNA) or short hairpin RNA (shRNA) can be expressed by using several expression vectors including lentiviral vectors. The lentiviral vector has several useful characteristics for RNAi experiments including broad host tropism and stable gene transduction to both dividing and nondividing cells, which permits stable depletion of target genes. This technology can be useful for several applications, including basic cancer research.

Simultaneous suppression of MITF and BRAF V600E enhanced inhibition of melanoma cell proliferation.

Microphthalmia-associated transcription factor (MITF) is a master gene regulating differentiation of melanocytes, and a lineage survival oncogene mediating pro-proliferative function in malignant melanoma. However, high expression of MITF also has an anti-proliferative effect. To clarify the therapeutic implication of MITF as a molecular target for human melanoma, we evaluated the role of MITF in cell proliferation in a panel of human melanoma cell lines which express different levels of MITF. We found that both MITF depletion and forced expression of MITF significantly inhibited proliferation, suggesting that endogenous MITF is regulated at an appropriate level for melanoma cell proliferation, and could be a molecular target for melanoma. However, half of the melanoma cell lines in this study were relatively resistant to MITF depletion, indicating other treatment strategies are required for therapy. Our microarray analysis indicated that regulation of several cell growth-associated molecules may be independent of MITF and dependent on BRAF(V600E). Thus to enhance the anti-proliferative effect of MITF down-regulation, we combined shRNA-mediated MITF depletion with BRAF(V600E) inactivation, another known molecular target for melanoma. Indeed, simultaneous depletion of both MITF and BRAF(V600E) significantly inhibited melanoma growth even for the melanoma cell lines resistant to MITF depletion. These results suggest MITF may be an important molecular target for human melanoma and simultaneous inhibition of MITF and MAPK signaling may be an attractive strategy for melanoma treatment.

Tumor growth suppression by adenovirus-mediated introduction of a cell-growth-suppressing gene tob in a pancreatic cancer model.

TOB (transducer of ErbB-2) is a tumor suppressor that interacts with protein-tyrosine kinase receptors, including ErbB-2. Introduction of the tob gene into NIH3T3 cells results in cell growth suppression. In this study, we evaluated the effect of tob expression in pancreatic cell lines (AsPC-1, BxPC-3, SOJ) and discuss the tumor-suppressing effects of adenoviral vector expressing tob cDNA. We first measured the levels of endogenous tob mRNA being expressed in all pancreatic cancer cell lines. Then, we examined the effect of adenoviral vector containing tob cDNA (Ad-tob vector) on cancer cell lines. The viral vector was expanded with transfection in 293 cells. The titer of the vector was 350x10(6) pfu/ml. These cancer cells were able to be transfected with MOI 20 without adenoviral toxicity. The transfection of Ad-tob vector results in growth suppression of SOJ and AsPC-1 cell lines. The magnitude of the expression of the Ad-tob gene in cancer is correlated to tumor suppressive activity. We prepared pancreatic cancer peritonitis models using a peritoneal injection of AsPC-1 cells. In this model, bloody ascites and multiple tumor nodules were seen at the mesentery after 16 days. AdCAtob (50x10(6) pfu/day) was administered from day 5 to day 9 after 4 days of peritoneal injection of 2x10(6) AsPC-1 cells. Tumor growth suppression occurred 10 days after peritoneal injection of AdCAtob compared with the control group. There were no tumor nodules in the abdomen and no bloody ascites. These results suggest that the peritoneal injection of AdCAtob has potential to suppress the formation of pancreatic cancer peritonitis, and can be applied for chemotherapy-resistant cancer peritonitis.

Hepatic AdipoR2 signaling plays a protective role against progression of nonalcoholic steatohepatitis in mice.

UNLABELLED: It is unclear how hepatic adiponectin resistance and sensitivity mediated by the adiponectin receptor, AdipoR2, contributes to the progression of nonalcoholic steatohepatitis (NASH). The aim of this study was to examine the roles of hepatic AdipoR2 in NASH, using an animal model. We fed C57BL/6 mice a methionine-deficient and choline-deficient (MCD) diet for up to 8 weeks and analyzed changes in liver pathology caused by either an AdipoR2 short hairpin RNA-expressing adenovirus or an AdipoR2-overexpressing adenovirus. Inhibition of hepatic AdipoR2 expression aggravated the pathological state of NASH at all stages: fatty changes, inflammation, and fibrosis. In contrast, enhancement of AdipoR2 expression in the liver improved NASH at every stage, from the early stage to the progression of fibrosis. Inhibition of AdipoR2 signaling in the liver diminished hepatic peroxisome proliferator activated receptor (PPAR)-alpha signaling, with decreased expression of acyl-CoA oxidase (ACO) and catalase, leading to an increase in lipid peroxidation. Hepatic AdipoR2 overexpression had the opposite effect. Reactive oxygen species (ROS) accumulation in liver increases hepatic production of transforming growth factor (TGF)-beta1 at all stages of NASH; adiponectin/AdipoR2 signaling ameliorated TGF-beta-induced ROS accumulation in primary cultured hepatocytes, by enhancing PPAR-alpha activity and catalase expression. CONCLUSION: The adiponectin resistance and sensitivity mediated by AdipoR2 in hepatocytes regulated steatohepatitis progression by changing PPAR-alpha activity and ROS accumulation, a process in which TGF-beta signaling is implicated. Thus, the liver AdipoR2 signaling pathway could be a promising target in treating NASH.