Hidetoshi Sumimoto

The BRAF-MAPK signaling pathway is essential for cancer-immune evasion in human melanoma cells

The mitogen-activated protein kinase (MAPK) pathway is frequently activated in human cancers, leading to malignant phenotypes such as autonomous cellular proliferation. Here, we demonstrate a novel role of the activated MAPK pathway in immune evasion by melanoma cells with the mutation of BRAF, which encodes a MAPKKs, (BRAFV600E). MEK inhibitor U0126 or RNA interference (RNAi) for BRAFV600E decreased production of the immunosuppressive soluble factors interleukin (IL)-10, VEGF, or IL-6 from melanoma cells to levels comparable to those after signal transducer and activator of transcription (STAT)3 inactivation. The suppressive activity of the culture supernatants from the melanoma cells on the production of inflammatory cytokines IL-12 and tumor necrosis factor α by dendritic cells upon lipopolysaccharide stimulation was markedly reduced after transduction with BRAFV600E RNAi, comparable to the effects observed with STAT3 RNAi transduction. No additive or synergistic effects were observed by the simultaneous transduction of RNAi for both BRAFV600E and STAT3. Furthermore, specific DNA binding and transcriptional activity of STAT3 were not affected by down-regulation of the MAPK signaling with the BRAF RNAi. These results indicate that the MAPK signal, along with the STAT3 signal, is essential for immune evasion by human melanomas that have constitutively active MAPK signaling and is a potential molecular target for overcoming melanoma cell evasion of the immune system.

Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference.

Oncogenic mutations of molecules involved in the mitogen-activated protein kinase (MAPK) pathways provide signals mediating both tumor growth and invasion in various cancers including melanomas. BRAF somatic mutations, found in 66% of melanomas, have NIH3T3 transforming ability with the elevated kinase activity in vitro. We attempted to mediate RNA interference (RNAi) with HIV lentiviral vectors specific for either wild type or the most frequently mutated form of BRAF (V599E) in 10 melanoma cell lines, and found that RNAi inhibited the growth of most melanoma cell lines in vitro as well as in vivo, which was accompanied by decrease of both BRAF protein and ERK phosphorylation. Interestingly, the mutated BRAF (V599E)-specific siRNA inhibited the growth and MAPK activity of only melanoma cell lines with this mutation. Furthermore, BRAF RNAi inhibited matrigel invasion of melanoma cells accompanied with a decrease of matrix metalloproteinase activity and β1 integrin expression. These results clarify that the mutated BRAF (V599E) is essentially involved in malignant phenotype of melanoma cells through the MAPK activation and is an attractive molecular target for melanoma treatment. The lentivirus-mediated RNAi specific for oncogenic mutations may be a powerful technique for gene therapy of cancer.

Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells

RNA interference (RNAi) is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double‐stranded RNA, one complementary to the gene, into cells. This phenomenon can be observed in mammalian cells when small interfering RNAs (siRNAs) are used, and is receiving attention as the most powerful tool for reverse genetics in the post genome era. Several groups have developed vector‐based siRNA‐expression systems that can induce RNAi in living cells.

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

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)‐α 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)‐β1 at all stages of NASH; adiponectin/AdipoR2 signaling ameliorated TGF‐β–induced ROS accumulation in primary cultured hepatocytes, by enhancing PPAR‐α activity and catalase expression. Conclusion: The adiponectin resistance and sensitivity mediated by AdipoR2 in hepatocytes regulated steatohepatitis progression by changing PPAR‐α activity and ROS accumulation, a process in which TGF‐β signaling is implicated. Thus, the liver AdipoR2 signaling pathway could be a promising target in treating NASH. (HEPATOLOGY 2008;48:458–473.)

Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference

Increase of Skp-2, which is involved in the degradation of cell cycle regulators including p27Kip1, p21 and c-myc, is one of the important mechanisms for dysregulation of cell cycles in various cancers. We applied RNA interference (RNAi) for Skp-2 by using HIV-lentiviral or adenoviral vectors for a human small-cell lung carcinoma cell line with increased Skp-2 to evaluate RNAi strategy for cancer gene therapy. HIV-lentivirus-mediated RNAi for Skp-2 resulted in efficient inhibition of the in vitro cell growth of cancer cells with increased Skp-2 through the increase of p27Kip1 and p21, but no significant effect on the growth of cells without high Skp-2 expression. Furthermore, intratumoral administration of adenovirus siRNA vector for Skp-2 efficiently inhibited growth of established subcutaneous tumor on NOD/SCID mice. These results indicate that the Skp-2 RNAi may be a useful strategy for gene therapy of cancers with high Skp-2 expression.

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/β-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.

Effective inhibition of cell growth and invasion of melanoma by combined suppression of BRAF (V599E) and Skp2 with lentiviral RNAi

p27Kip1 that regulates the G1/S transition of cell cycle and inhibits Rho A signaling is frequently lost in several cancers leading to the deregulation of cell growth and cell motility. Mitogen‐activated protein kinases (MAPK) regulate the export of p27Kip1 from nucleus to cytoplasm, followed by the degradation with proteases. Skp‐2, a subunit of an SCF ubiquitin‐protein ligase complex responsible for the ubiquitination of p27Kip1, is upregulated frequently in several cancers, leading to the decrease of p27Kip1. We applied human immunodeficiency virus (HIV) lentivirus‐mediated RNA interference (RNAi) to melanoma cells with the BRAF mutation (V599E) and overexpressed Skp‐2 and found that the simultaneous suppression of these activated oncogenes resulted in the effective inhibition of in vitro cell growth and invasive ability of melanoma cells accompanied by the additional increase of p27Kip1. Our results suggest that gene therapy against melanoma with the enhanced MAPK and ubiquitin‐proteasomal pathways could be a specific and effective therapeutic strategy for cancers.

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/STAT3flox/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.

Isolation of a new melanoma antigen, MART-2, containing a mutated epitope recognized by autologous tumor-infiltrating T lymphocytes

Using cDNA expression cloning, a cDNA encoding a novel human melanoma Ag, MART-2 (melanoma Ag recognized by T cells-2), recognized by HLA-A1-restricted CD8+ T cells from tumor-infiltrating lymphocytes (TIL1362) was isolated from an autologous melanoma cell line, 1362 mel. Homologous sequences to the cDNA had been registered in the EST database. This gene encoded an uncharacterized protein expressed ubiquitously in most normal and cancer cells. A mutation (A to G transition) was found in the cDNA obtained from the1362 mel melanoma cell line in the sequences encoding the phosphate binding loop (P-loop) that resulted in loss of the ability to bind GTP. Transfection of NIH-3T3 with the mutated MART-2 did not result in the development of significant foci. By screening 36 various cancer cell lines using single-strand conformation polymorphism, a possible mutation in the P-loop of MART-2 was found in one squamous cell lung cancer cell line, EBC1. The T cell epitope for TIL1362, FLEGNEVGKTY, was identified to be encoded by the mutated sequence of the MART-2 Ag. The mutation substituted glycine in the normal peptide with glutamic acid at the third amino acid of the epitope, which is an important primary anchor amino acid for HLA-A1 peptide binding. The normal peptide, FLGGNEVGKTY, was not recognized by TIL1362, suggesting that this T cell response was specific for the autologous tumor. Although transforming activity was not detected in the NIH-3T3 assay, MART-2 with the mutation in the P-loop may be involved in the generation of melanoma through a loss of GTP binding activity.

Immune Suppression and Resistance Mediated by Constitutive Activation of Wnt/β-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/β-catenin pathways in human melanoma for dendritic cells (DCs) and CTLs. IL-10 expression was associated with β-catenin accumulation in human melanoma cell lines and tissues and was induced by direct β-catenin/TCF binding to the IL-10 promoter. Culture supernatants from β-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 β-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 β-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/β-catenin signaling in human melanoma. This in vivo DC suppression was restored by the administration of a β-catenin inhibitor, PKF115-584. β-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/β-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/β-catenin–activated melanoma.