Novel Antibody Exerts Antitumor Effect through Downregulation of CD147 and Activation of Multiple Stress Signals.

CD147 is an immunoglobulin-like receptor that is highly expressed in various cancers and involved in the growth, metastasis, and activation of inflammatory pathways via interactions with various functional molecules, such as integrins, CD44, and monocarboxylate transporters. Through screening of CD147-targeting antibodies with antitumor efficacy, we discovered a novel rat monoclonal antibody #147D. This humanized IgG4-formatted antibody, h4#147D, showed potent antitumor efficacy in xenograft mouse models harboring the human PDAC cell line MIA PaCa-2, HCC cell line Hep G2, and CML cell line KU812, which featured low sensitivity to the corresponding standard-of-care drugs (gemcitabine, sorafenib, and imatinib, respectively). An analysis of tumor cells derived from MIA PaCa-2 xenograft mice treated with h4#147D revealed that cell surface expression of CD147 and its binding partners, including CD44 and integrin α3ß1/α6ß1, was significantly reduced by h4#147D. Inhibition of focal adhesion kinase (FAK), activation of multiple stress responsible signal proteins such as c-JunN-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38MAPK), and expression of SMAD4, as well as activation of caspase-3 were obviously observed in the tumor cells, suggesting that h4#147D induced tumor shrinkage by inducing multiple stress responsible signals. These results suggest that the anti-CD147 antibody h4#147D offers promise as a new antibody drug candidate.

Clinical and immunologic evaluation of dendritic cell-based immunotherapy in combination with gemcitabine and/or S-1 in patients with advanced pancreatic carcinoma.

OBJECTIVES: In the current study, we have evaluated the clinical and immunological responses in patients with advanced pancreatic carcinoma who received dendritic cell (DC)-based immunotherapy in combination with gemcitabine and/or S-1. METHODS: Dendritic cell-based immunotherapy (DC vaccine alone or DC vaccine plus lymphokine-activated killer [LAK] cell therapy) in combination with gemcitabine and/or S-1 has been carried out in 49 patients with inoperable pancreatic carcinoma refractory to standard treatment. RESULTS: Of 49 patients, 2 patients had complete remission, 5 had partial remission, and 10 had stable disease. Prolongation of survival in this cohort was highly likely (median survival, 360 days). Survival of patients receiving DC vaccine and chemotherapy plus LAK cell therapy was longer than those receiving DC vaccine in combination with chemotherapy but no LAK cells. Increased numbers of cancer antigen-specific cytotoxic T cells and decreased regulatory T cells were observed in several patients on immunotherapy, but increased overall survival time tended to be associated only with the latter. None of the patients experienced grade 3 or worse adverse events during the treatment period. CONCLUSIONS: Dendritic cell vaccine-based immunotherapy combined with chemotherapy was shown to be safe and possibly effective in patients with advanced pancreatic cancer refractory to standard treatment.

Langerhans cell histiocytosis of the petrous bone with sudden sensorineural hearing loss. Case report.

A 56-year-old woman with Langerhans cell histiocytosis of the petrous bone presented with sudden onset of sensorineural hearing loss and vertigo without other neurological impairment, mimicking idiopathic sudden sensorineural hearing loss. Differential diagnosis was difficult until neuroimaging demonstrated a lesion of the petrous bone. The patient eventually underwent removal of the lesion via the transpetrosal approach, and received postoperative chemotherapy consisting of vinblastine, methotrexate, 6-mercaptopurine, and prednisolone. Although her hearing did not recover, complete remission was achieved, and the patient is currently free from disease. Physicians need to be aware that patients with sudden onset of hearing loss may have an unexpected and hidden disease which requires neuroimaging and histological examinations for definitive diagnosis and appropriate treatment.

The galanin signaling cascade is a candidate pathway regulating oncogenesis in human squamous cell carcinoma.

To identify putative biomarkers in squamous cell carcinoma (SCC), a survey of parallel chromosomal alterations and gene expression studies in 10 SCC cell lines were performed using array-comparative genomic hybridization (CGH) and oligo-microarray techniques. The most frequent changes were gains of 11q13.1-13.3 and losses of 18q12.1-23 in SCC. Furthermore, the expression levels of the sets of genes at both these loci in SCC were measured using microarray analysis. By combining the array-CGH with the microarray data, 10 genes at 11q13.1-13.3 and 6 genes at 18q12.1-23 whose expression correlated with chromosomal alterations were identified. To verify the expression levels of the identified genes, we used expression analysis data derived from our earlier study of clinical specimens. In clinical samples, six genes (GAL, GSTP1, MRPL11, MRPL21, SF3B2, and YIF1A) at 11q13.1-13.3 and one gene (GALR1) at 18q23 showed a significant difference between normal and tumor samples. GAL, coding for the neuropeptide galanin, and GALR1, a galanin receptor, were identified as candidate genes of oncogenesis in SCC. The expression levels of GAL, GALR1, GALR2, and GALR3 were confirmed by real-time PCR. The expression ratio between GAL and GALR1 showed a significant negative correlation. GALR1 is a G-protein-coupled receptor that activates GTP-binding proteins to trigger signaling cascades such as the mitogen-activated protein kinase pathway, and is a well-established mitogenic pathway. This further supports the hypothesis that the genes involved in the GAL signaling cascade are candidates for regulation of oncogenesis in SCC.

Identification of a novel therapeutic target for head and neck squamous cell carcinomas: a role for the neurotensin-neurotensin receptor 1 oncogenic signaling pathway.

Distant metastasis is a major factor associated with poor prognosis in head and neck squamous cell carcinomas (HNSCC), but little is known of its molecular mechanisms. New markers that predict clinical outcome, in particular the ability of primary tumors to develop metastatic tumors, are urgently needed. Based on a genome-wide gene expression analysis using clinical specimens of HNSCC, we narrowed our focus to the analysis of the neurotensin (NTS) and neurotensin receptor 1 (NTSR1) oncogenic signal pathways. Kaplan-Meier curves and log rank tests revealed that high mRNA expression levels of NTS and NTSR1 had a significant adverse effect on metastasis-free survival rate, suggesting a contribution of this pathway in HNSCC cancer progression. In HNSCC cells, which expressed NTSR1, a NTS agonist promoted cellular invasion, migration and induction of several mRNAs, such as interleukin 8 and matrix metalloproteinase 1 transcripts. In addition, knock down of NTSR1 expression with small interfering RNAs resulted in reduction of cellular invasion and migration in HNSCC cell lines. Our findings suggest a critical role for the NTS and NTSR1 oncogenic pathways in invasion and migration of HNSCC cells during the metastatic process. Our study raises the possibility that NTS and NTSR1 could be a useful predictive marker of poor prognosis in patients with HNSCC and a molecular therapeutic target in antimetastatic strategies for HNSCCs.

The interleukin-4 enhancer CNS-2 is regulated by Notch signals and controls initial expression in NKT cells and memory-type CD4 T cells.

Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses.

Computer simulation of the role of SOCS family protein in helper T cell differentiation.

The Th1/Th2 balance determines the nature of an immune response, and particular cytokines, IL-4 and IL-12, determine the direction at the initial stage of activation through TCRs. To investigate how cytokine networks and related signaling pathways impact upon the Th1/Th2 balance, we have developed a computer model for the simulation of Th differentiation. The model includes the IL-4, IL-12 and IFN-gamma signal transduction pathways, a positive and negative feedback mechanism for cytokine signaling and cytokine-induced negative regulators such as suppressors of cytokine signaling (SOCS)1, SOCS3 and SOCS5. In the present study, we propose a 'Th0 model', in which naive T cells differentiate neither into Th1 nor into Th2 states in unskewed cytokine conditions. The model was found to be consistent with experimental results in BALB/c mice. The results of in silico analysis in the condition with SOCS- and signal transducer and activator of transcription (STAT) family-deficient and transgenic states were well fitted to ex vivo experimental results for Th1 and Th2 differentiation profiles in the deficient and transgenic mice. The Th0 model suggested the possibility that dominant Th1 differentiation in STAT4/STAT6 double-deficient mice may be due to a positive feedback effect of initial IFN-gamma production from T cells. The in silico assessment of beneficial effects of inhibitory drugs by simulation analysis with our Th0 model indicated that Janus kinase 3-specific inhibitors might be suitable candidates for the modification of Th2-dominant immune responses. Our results demonstrate that models for the simulation of signaling network, such as our Th0 model, are useful tools for the in silico evaluation of novel drug candidates.

The role of suppressor of cytokine signaling 1 as a negative regulator for aberrant expansion of CD8alpha+ dendritic cell subset.

The suppressor of cytokine signaling (SOCS) 1 is a negative regulator in multiple cytokine-related aspects to maintain immunological homeostasis. Here, we studied a role of SOCS1 on dendritic cell (DC) maturation in the mice lacking either TCRalpha chain or CD28 in SOCS1-deficient background, and found that the SOCS1 could restore acute phase of inflammatory response in SOCS1-deficient mice. The CD11c+ CD8- DC population in freshly isolated splenic DCs from normal mice highly expressed SOCS1. However, in SOCS1-deficient environment, the proportion of CD8alpha+ DCs (CD8 DCs) noticeably increased without affecting the cell number of conventional and plasmacytoid DC populations. This population revealed the CD11cdull CD8alpha+ CD11b- CD45RA- B220- phenotype, which is a minor population in normal mice. Localization of the abnormal CD8 DCs in splenic microenvironments was mainly restricted to deep within red pulp. The CD8 DCs secrete a large amount of IFN-gamma, IL-12 and B lymphocyte stimulator/B cell activation factor of the tumor necrosis factor family in response to LPS and CpG stimulation. This is responsible for the development of DC-mediated systemic autoimmunity in the old age of SOCS1-deficient mice. Moreover, the CD8 DC subsets expressed more indoleamine 2,3-dioxygenase and IL-10, and hence inhibit the allogeneic proliferative T cell response and antigen-induced Th1 responses. Therefore, SOCS1 expression during DC maturation plays a role in surveillance in controlling the aberrant expansion of abnormal DC subset to maintain homeostasis of immune system.

Transcriptional regulation of the human ABO histo-blood group genes is dependent on the N box upstream of the proximal promoter.

BACKGROUND: Our previous studies of the transcriptional regulation of the human ABO gene indicated that negative regulatory elements are present in the sequence just upstream from the proximal promoter. The role of the -275 to -118 region in regulation of ABO gene transcription is further characterized. STUDY DESIGN AND METHODS: Transient transfection experiments into various cells were performed with luciferase reporter plasmids carrying ABO upstream sequences, and electrophoretic mobility shift assay was carried out with a nuclear extract prepared from the human gastric cancer KATOIII cells. RESULTS: It is shown that the -202 to -118 region is involved in the negative regulation of ABO gene transcription in all cell lines examined. Transient transfection experiments in KATOIII cells with a reporter plasmid carrying mutated N box at -196 to -191 demonstrate that the N box is a negative regulatory element in the -202 to -118 sequence. Electrophoretic mobility shift assays indicate that the N box binds with a nuclear factor from KATOIII cells. CONCLUSION: These results indicate that repression of transcription from the ABO proximal promoter is partially dependent upon the N box. Therefore, reduced binding of the protein with the N box might play a direct role in ABO gene expression.

Regulation of alphabeta/gammadelta T cell lineage commitment and peripheral T cell responses by Notch/RBP-J signaling.

RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells.

Suppressor of cytokine signaling-1 is essential for suppressing dendritic cell activation and systemic autoimmunity.

Suppressor of cytokine signaling-1 (SOCS1/JAB) negatively regulates not only the cytokine-signaling pathway but also lipopolysaccharide (LPS)-induced macrophage activation. We found that SOCS1-deficient dendritic cells (DCs) were also hyperresponsive to interferon-gamma and interleukin-4. To define the role of SOCS1-deficient DCs in vivo, we generated mice in which the SOCS1 expression was restored in T and B cells on a SOCS1(-/-) background. In these mice, DCs were accumulated in the thymus and spleen and produced high levels of BAFF/BLyS and APRIL, resulting in the aberrant expansion of B cells and autoreactive antibody production. SOCS1-deficient DCs efficiently stimulated B cell proliferation in vitro and autoantibody production in vivo. These results indicate that SOCS1 plays an essential role in the normal DC functions and suppression of systemic autoimmunity.

Expression of the suppressor of cytokine signaling-5 (SOCS5) negatively regulates IL-4-dependent STAT6 activation and Th2 differentiation.

The development of helper T (Th) cell subsets, which secrete distinct cytokines, plays an important role in determining the type of immune response. The IL-4-mediated Janus kinase-signal transducer and activator of transcription signaling pathway is crucial for mediating Th2 cell development. Notably, this pathway is selectively impaired in Th1 cells, although the molecular basis of this impairment remains unclear. We show here that during Th1 differentiation a reduction in the association of Janus kinase 1 with the IL-4 receptor (IL-4R) correlated with the appearance of the suppressor of cytokine signaling-5 (SOCS5). SOCS5 protein was preferentially expressed in committed Th1 cells and interacted with the cytoplasmic region of the IL-4Ralpha chain irrespective of receptor tyrosine phosphorylation. This unconventional interaction of SOCS5 protein with the IL-4R resulted in the inhibition of IL-4-mediated signal transducer and activator of transcription-6 activation. T cells from transgenic mice constitutively expressing SOCS5 exhibited a significant reduction of IL-4-mediated Th2 development. Therefore, the induced SOCS5 protein in Th1 differentiation environment may play an important role by regulating Th1 and Th2 balance.

Alternative promoter identified between a hypermethylated upstream region of repetitive elements and a CpG island in human ABO histo-blood group genes.

We have studied the expression of human histo-blood group ABO genes during erythroid differentiation, using an ex vivo culture of AC133(-)CD34(+) cells obtained from peripheral blood. 5'-Rapid amplification of cDNA ends analysis of RNA from those cells revealed a novel transcription start site, which appeared to mark an alternative starting exon (1a) comprising 27 bp at the 5'-end of a CpG island in ABO genes. Results from reverse transcription-PCR specific to exon 1a indicated that the cells of both erythroid and epithelial lineages utilize this exon as the transcription starting exon. Transient transfection experiments showed that the region just upstream from the transcription start site possesses promoter activity in a cell type-specific manner when placed 5' adjacent to the reporter luciferase gene. Results from bisulfite genomic sequencing and reverse transcription-PCR analysis indicated that hypermethylation of the distal promoter region correlated with the absence of transcripts containing exon 1a, whereas hypermethylation in the interspersed repeats 5' adjacent to the distal promoter was commonly observed in all of the cell lines examined. These results suggest that a functional alternative promoter is located between the hypermethylated region of repetitive elements and the CpG island in the ABO genes.