Notch2 integrates signaling by the transcription factors RBP-J and CREB1 to promote T cell cytotoxicity.

The acquisition of cytotoxic effector function by CD8(+) T cells is crucial for the control of intracellular infection and tumor invasion. However, it remains unclear which signaling pathways are required for the differentiation of CD8(+) cytotoxic T lymphocytes. We show here that Notch2-deficient T cells had impaired differentiation into cytotoxic T lymphocytes. In addition, dendritic cells with lower expression of the Notch ligand Delta-like 1 induced the differentiation of cytotoxic T lymphocytes less efficiently. We found that the intracellular domain of Notch2 interacted with a phosphorylated form of the transcription factor CREB1, and together these proteins bound the transcriptional coactivator p300 to form a complex on the promoter of the gene encoding granzyme B. Our results suggest that the highly regulated, dynamic control of T cell cytotoxicity depends on the integration of Notch2 and CREB1 signals.

[Patient-Reported Outcome(PRO)to Measure Health-Related QOL in Lung Cancer Patients].

Lung cancer as progressive disease is often associated with a poor quality of life(QOL)which is related to a poor prognosis. We review the impact of patient-reported outcome(PRO)as an indicator of health-related QOL on the management of lung cancer patients. Cancer-specific PRO measures, which are primarily applied in scientific research for the past 30 years to compare the therapy outcomes and in drug development with adverse events. Among several PRO measures developed, the EORTC QLQ-C30 and its lung cancer-specific module QLQ-LC13 are the most frequently used instruments in clinical trials with lung cancer patients. Interestingly, cancer-specific PRO measures have been also increasingly used as daily practice, which may provide positive effects on the communication with the patient, mutual decision making and the monitoring and managing of the patient. Moreover, PRO measures have an independent prognostic value to predict survival in lung cancer patients. PRO measures have also the potential to improve the quality of care. Electronic platforms are expected to simplify the implementation of PRO measure in the daily clinic. Nevertheless, the PRO measures have not been properly implemented in Japan.

A novel targeting therapy of malignant mesothelioma using anti-podoplanin antibody.

Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanin-induced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 and NZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73% (11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against podoplanin-positive MPM cells mediated by rat NK (CD161a(+)) cells, but not murine splenocytes or human mononuclear cells. Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanin-transfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8 preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated by human NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin with therapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM.

[Ethical Guidelines for Clinical Trials in Medical Research Involving Human Subjects].

In Japan, investigator-initiated clinical phase IV trials must follow the Ethical Guidelines for Medical and Health Research Involving Human Subjects, issued in December 2014. In addition, researchers must follow the Helsinki Declaration. In these clinical trials, academia-industry collaborations involving funding and technical support are required to develop better evidence- based medicine. Nevertheless, instances of publications reporting biases or misconduct in the research process occur frequently, which may lead to other problems due to lack of transparency. To address this issue, an institutional framework must be developed and maintained in which investigators maintain a high level of ethical adherence to protect the welfare of research subjects while carrying out research scientifically and appropriately under a conflict of interest (COI) disclosure. All authors must be seriously committed to greater responsibility, accountability, and transparency in collaborating with the industry.

Surfactant protein A suppresses lung cancer progression by regulating the polarization of tumor-associated macrophages.

Surfactant protein A (SP-A) is a large multimeric protein found in the lungs. In addition to its immunoregulatory function in infectious respiratory diseases, SP-A is also used as a marker of lung adenocarcinoma. Despite the finding that SP-A expression levels in cancer cells has a relationship with patient prognosis, the function of SP-A in lung cancer progression is unknown. We investigated the role of SP-A in lung cancer progression by introducing the SP-A gene into human lung adenocarcinoma cell lines. SP-A gene transduction suppressed the progression of tumor in subcutaneous xenograft or lung metastasis mouse models. Immunohistochemical analysis showed that the number of M1 antitumor tumor-associated macrophages (TAMs) was increased and the number of M2 tumor-promoting TAMs was not changed in the tumor tissue produced by SP-A-expressing cells. In addition, natural killer (NK) cells were also increased and activated in the SP-A-expressing tumor. Moreover, SP-A did not inhibit tumor progression in mice depleted of NK cells. Taking into account that SP-A did not directly activate NK cells, these results suggest that SP-A inhibited lung cancer progression by recruiting and activating NK cells via controlling the polarization of TAMs.

Antifibrotic effects of focal adhesion kinase inhibitor in bleomycin-induced pulmonary fibrosis in mice.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in various biological functions, including cell survival, proliferation, migration, and adhesion. FAK is an essential factor for transforming growth factor ß to induce myofibroblast differentiation. In the present study, we investigated whether the targeted inhibition of FAK by using a specific inhibitor, TAE226, has the potential to regulate pulmonary fibrosis. TAE226 showed inhibitory activity of autophosphorylation of FAK at tyrosine 397 in lung fibroblasts. The addition of TAE226 inhibited the proliferation of lung fibroblasts in response to various growth factors, including platelet-derived growth factor and insulin-like growth factor I, in vitro. TAE226 strongly suppressed the production of type I collagen by lung fibroblasts. Furthermore, treatment of fibroblasts with TAE226 reduced the expression of α-smooth muscle actin induced by transforming growth factor ß, indicating the inhibition of differentiation of fibroblasts to myofibroblasts. Administration of TAE226 ameliorated the pulmonary fibrosis induced by bleomycin in mice even when used late in the treatment. The number of proliferating mesenchymal cells was reduced in the lungs of TAE226-treated mice. These data suggest that FAK signal plays a significant role in the progression of pulmonary fibrosis and that it can become a promising target for therapeutic approaches to pulmonary fibrosis.

Dual inhibition of Met kinase and angiogenesis to overcome HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer.

Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a serious problem in the management of EGFR mutant lung cancer. We recently reported that hepatocyte growth factor (HGF) induces resistance to EGFR-TKIs by activating the Met/PI3K pathway. HGF is also known to induce angiogenesis in cooperation with vascular endothelial growth factor (VEGF), which is an important therapeutic target in lung cancer. Therefore, we hypothesized that dual inhibition of HGF and VEGF may be therapeutically useful for controlling HGF-induced EGFR-TKI-resistant lung cancer. We found that a dual Met/VEGF receptor 2 kinase inhibitor, E7050, circumvented HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer cell lines by inhibiting the Met/Gab1/PI3K/Akt pathway in vitro. HGF stimulated VEGF production by activation of the Met/Gab1 signaling pathway in EGFR mutant lung cancer cell lines, and E7050 showed an inhibitory effect. In a xenograft model, tumors produced by HGF-transfected Ma-1 (Ma-1/HGF) cells were more angiogenic than vector control tumors and showed resistance to gefitinib. E7050 alone inhibited angiogenesis and retarded growth of Ma-1/HGF tumors. E7050 combined with gefitinib induced marked regression of tumor growth. Moreover, dual inhibition of HGF and VEGF by neutralizing antibodies combined with gefitinib also markedly regressed tumor growth. These results indicate the therapeutic rationale of dual targeting of HGF-Met and VEGF-VEGF receptor 2 for overcoming HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer.

Surfactant protein-D regulates effector cell function and fibrotic lung remodeling in response to bleomycin injury.

RATIONALE: Surfactant protein (SP)-D and SP-A have been implicated in immunomodulation in the lung. It has been reported that patients with idiopathic pulmonary fibrosis (IPF) often have elevated serum levels of SP-A and SP-D, although their role in the disease is not known. OBJECTIVES: The goal of this study was to test the hypothesis that SP-D plays an important role in lung fibrosis using a mouse model of fibrosis induced by bleomycin (BLM). METHODS: Triple transgenic inducible SP-D mice (iSP-D mice), in which rat SP-D is expressed in response to doxycycline (Dox) treatment, were administered BLM (100 U/kg) or saline subcutaneously using miniosmotic pumps. MEASUREMENTS AND MAIN RESULTS: BLM-treated iSP-D mice off Dox (SP-D off) had increased lung fibrosis compared with mice on Dox (SP-D on). SP-D deficiency also increased macrophage-dominant cell infiltration and the expression of profibrotic cytokines (transforming growth factor [TGF]-ß1, platelet-derived growth factor-AA). Alveolar macrophages isolated from BLM-treated iSP-D mice off Dox (SP-D off) secreted more TGF-ß1. Fibrocytes, which are bone marrow-derived mesenchymal progenitor cells, were increased to a greater extent in the lungs of the BLM-treated iSP-D mice off Dox (SP-D off). Fibrocytes isolated from BLM-treated iSP-D mice off Dox (SP-D off) expressed more of the profibrotic cytokine TGF-ß1 and more CXCR4, a chemokine receptor that is important in fibrocyte migration into the lungs. Exogenous SP-D administered intratracheally attenuated BLM-induced lung fibrosis in SP-D(-/-) mice. CONCLUSIONS: These data suggest that alveolar SP-D regulates numbers of macrophages and fibrocytes in the lungs, profibrotic cytokine expression, and fibrotic lung remodeling in response to BLM injury.

Pleural mesothelioma instigates tumor-associated fibroblasts to promote progression via a malignant cytokine network.

The tumor microenvironment is crucial to the progression of various malignancies. Malignant pleural mesothelioma (MPM), which originates from the pleura, grows aggressively in the thoracic cavity. Here we describe an orthotopic implantation SCID mouse model of MPM and demonstrate that α-SMA-positive fibroblast-like cells accumulate in the tumors produced by the human MPM cell lines MSTO-211H and Y-Meso-14. We assessed the interaction between MPM cells and their microenvironments, focusing on tumor-associated fibroblasts. MSTO-211H and Y-Meso-14 cells produced fibroblast growth factor-2 (FGF-2) and/or platelet-derived growth factor-AA (PDGF-AA); they also enhanced growth, migration, and production of hepatocyte growth factor (HGF) by human lung fibroblast MRC-5 cells. MRC-5 cells stimulated HGF-mediated growth and migration of MSTO-211H and Y-Meso-14 cells in an in vitro coculture system. In the orthotopic model, tumor formation by MSTO-211H and Y-Meso-14 cells was significantly inhibited by TSU-68, an inhibitor of FGF, VEGF, and PDGF receptors; imatinib, an inhibitor of PDGF receptors; and NK4, an antagonist of HGF. Histological analyses of clinical specimens from 51 MPM patients revealed considerable tumor-associated fibroblasts infiltration and expression of HGF, together with FGF-2 or PDGF-AA, in tumors. These findings indicate that MPM instigates tumor-associated fibroblasts, promoting tumor progression via a malignant cytokine network. Regulation of this cytokine network may be therapeutically useful for controlling MPM.

SU6668, a multiple tyrosine kinase inhibitor, inhibits progression of human malignant pleural mesothelioma in an orthotopic model.

BACKGROUND AND OBJECTIVE: Malignant pleural mesothelioma (MPM) is an aggressive neoplasm of the mesothelium with high chemotherapeutic resistance. In this study, the preclinical therapeutic activity of the multiple tyrosine kinase inhibitor, SU6668, against MPM was examined. METHODS: Two human MPM cell lines with different pro-angiogenic cytokine expression, Y-MESO-14 cells that express high levels of vascular endothelial growth factor (VEGF) and MSTO-211H cells that express high levels of basic fibroblast growth factor (bFGF), were orthotopically inoculated into the thoracic cavities of mice with severe combined immunodeficiency. The mice with MPM were treated or not treated with SU6668 (200 mg/kg/day). RESULTS: SU6668 abrogated the proliferation of endothelial cells stimulated by VEGF or bFGF, but did not directly affect the growth of human MPM cells in vitro. In this orthotopic implantation model, treatment with SU6668 effectively reduced tumour weight and pleural effusion volumes, in association with inhibition of the growth of tumour vasculature. More importantly, treatment with SU6668 significantly prolonged survival time in mice with MPM. CONCLUSIONS: These findings suggest that SU6668 has a promising therapeutic effect on the progression of MPM in vivo through its anti-angiogenic effects.

Genetically engineered humanized anti-ganglioside GM2 antibody against multiple organ metastasis produced by GM2-expressing small-cell lung cancer cells.

Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC.

Expression of soluble vascular endothelial growth factor receptor-1 in human monocyte-derived mature dendritic cells contributes to their antiangiogenic property.

The soluble form of vascular endothelial growth factor receptor-1 (sVEGFR-1) is produced from endothelial cells by alternative splicing of VEGFR-1 mRNA, and can inhibit angiogenesis by blocking the biological effects of VEGF. In this study, we show the expression of a large amount of sVEGFR-1 in human monocyte-derived mature dendritic cells (mDCs). As compared with monocytes and immature DCs, mDCs generated by TNF-alpha or soluble CD40L with IFN-gamma, but not LPS or other stimuli, preferentially produce sVEGFR-1. We also detected the mRNA of sVEGFR-1 generated by alternative splicing of VEGFR-1 mRNA in mDCs induced by TNF-alpha. The production of sVEGFR-1 showed a distinct contrast to those of VEGF in each DC matured with various stimuli. The supernatant of DCs matured with TNF-alpha or soluble CD40L with IFN-gamma showed inhibition of the tube formation of HUVECs, which was neutralized by anti-VEGFR-1 Ab, indicating that sVEGFR-1 secreted from mDCs was biologically active. Interestingly, the supernatant of mDCs generated with LPS increased HUVEC capillary-like formation in vitro. The ratio of sVEGFR-1 to VEGF clearly reflected the net angiogenic property of mDCs. Administration of mDCs induced by TNF-alpha into the s.c. tumor of PC-14 cells implanted in SCID mice demonstrated the inhibition of tumor growth via reduction of the number of CD31-positive vessels, indicating their in vivo antiangiogenic potential. These results suggest that sVEGFR-1 produced by mDCs contribute to their antiangiogenic property, and the ratio of sVEGFR-1 to VEGF might be a useful tool for evaluating their ability to regulate angiogenesis mediated by VEGF.

Soy isoflavone equol perpetuates dextran sulfate sodium-induced acute colitis in mice.

The effects of the soy isoflavones, genistein, daidzein and equol, on experimental colitis were examined. Equol severely perpetrated dextran sulfate sodium (DSS)-induced colitis as evaluated by the weight loss. Production of the anti-inflammatory cytokine, IL-10, from T cells was decreased in the equol-treated mice. The results show that the soy isoflavone, equol, played an important role in the inflammatory response in the gastrointestinal tract.

[A case of primary pulmonary leiomyosarcoma with multiple nodular lesions in the lungs].

Multiple pulmonary nodular lesions were noted on a chest X-ray film of a 56-year-old man. Partial resection of the left lung via thoracoscopy was performed, yielding a pathological diagnosis of leiomyosarcoma. Positron emission tomography/computed tomography (PET/CT) and other examinations revealed that the lesions originated in the lung. Chemotherapy with adriamycin and ifosfamide was performed but was ineffective. It is reported that leiomyosarcoma with multiple pulmonary nodules often derives from a uterine primary lesion. Primary pulmonary leiomyosarcoma with multiple nodular lesions is rare, but it should be considered in the differential diagnosis in cases with multiple lung nodules.

[Clinical analysis of elderly patients with primary lung cancer].

We clinically reviewed 33 surgery patients and 15 non-surgery patients aged 80 years or older with primary lung cancer treated at our hospital. The surgery group consisted of 21 males and 12 females (82.0 +/- 1.9 years old). The surgical procedures were 1 pneumonectomy, 19 lobectomies (1 bronchoplasty), 4 segmentectomies and 9 partial resections. The cancer types were 17 adenocarcinomas, 14 squamous cell carcinomas and 2 others. The stagings were 24 in stage I, 4 in stage II and 5 in stage III. There were no direct surgical deaths within 30 days post operatively. There have been 9 other disease-related deaths to date (27%). The non-surgery group consisted of 9 males and 6 females (81.7 +/- 1.5 years old). Treatment procedures consisted of radiationtherapy in 11, chemotherapy in 2 and best supportive care in 3. The cancer types were 2 adenocarcinomas, 11 squamous cell carcinomas and 2 others. The stagings were 7 in stage I, 4 in stage II and 4 in stage III. There have been 3 other disease-related deaths to date (20%). We must carefully select the therapeutic approach for elderly lung cancer patients, because the other disease-related death rates of both groups were high. The 5-year survival rate of stage I patients in the surgical group was relatively good (60.2%). There were long-term survival (7 5-year survivors) in the surgical group. Since there were some cases of radiation pneumonia in the group receiving radiation therapy, it would be better to perform surgery for elderly patients with lung cancer, especially those with stage I disease. For elderly patients, it is important to consider quality of life as well as the survival rate.

[A case of severe pulmonary tuberculosis with septic shock and ARDS].

A 69-year-old man visited another hospital due to dyspnea and bloody sputum. A diagnosis of pulmonary tuberculosis was given because of multiple nodular shadows and consolidation on chest radiography and positive testing for acid-fast bacilli in his sputum; he was then referred to our hospital for treatment. Despite antituberculosis chemotherapy with isoniazid, rifampicin and ethambutol, his general condition worsened. On the 10th hospital day he needed mechanical ventilation and circulatory control in the intensive care unit. We diagnosed pulmonary tuberculosis complicated with septic shock, acute respiratory distress syndrome and disseminated intravascular coagulation based on his clinical course and laboratory data. After he recovered from shock, the antituberculosis chemotherapy was restarted. Intensive care resulted in the improvement of his general condition and the reduction of his chest abnormal shadows, and he was discharged 8 months after admission. No obvious recurrence was observed even after the cessation of antituberculosis chemotherapy, although pulmonary nodules on chest radiography remained.

[Historical aspect of molecular-targeted therapy for cancer].

Recently, discovery and identification of critical molecules responsible for cancer progression lead to development of specific target-directed therapies, including monoclonal antibodies and small molecular compounds. For the past 15 years, the advent of target-specific therapeutics has remarkably improved the clinical outcomes of certain patients with various malignancies, and it was followed by the understanding of mechanism of the drug resistance. Moreover, useful biomarkers were developed to further increase the power of patient selection for molecular-targeted therapy. From the historical point of view, we review the progress in developing new molecular-targeted drugs for personalized medicine.

[Molecular targeted therapy for cancer].

Recent insights into the molecular mechanism of cancer progression have given rise to specific target-directed therapies, including monoclonal antibodies and small molecular compounds, and the advent of target-specific therapeutics has remarkably improved the outcomes of patients with various malignancies. Recent advance also lead to the identification of prognostic biomarkers as predictive factors in determining response to molecular targeted drugs. Future studies also need to develop biomarkers to further increase the power of patient selection for molecular targeted therapy. Here we review the recent progress in developing new molecular targeted drugs and the resistance to treatments as well as the importance of measuring the QOL by patient-reported outcome, for personalized therapy.

Intensification therapy with anti-parathyroid hormone-related protein antibody plus zoledronic acid for bone metastases of small cell lung cancer cells in severe combined immunodeficient mice.

Bone metastases occur in more than one-third of patients with advanced lung cancer and are difficult to treat. We showed previously the therapeutic effect of a third-generation bisphosphonate, minodronate, and anti-parathyroid hormone-related protein (PTHrP) neutralizing antibody on bone metastases induced by the human small cell lung cancer cell line, SBC-5, in natural killer cell-depleted severe combined immunodeficient mice. The purpose of our current study was to examine the effect of the combination of PTHrP antibody and zoledronic acid, which has been approved to treat bone metastases, against bone metastases produced by SBC-5 cells expressing PTHrP. Treatment with PTHrP antibody and/or zoledronic acid did not affect the proliferation of SBC-5 cells in vitro. Repeated treatments with either PTHrP antibody or zoledronic acid inhibited the formation of osteolytic bone metastases of SBC-5 cells but had no effect on metastases to visceral organs. Importantly, combined treatment with PTHrP antibody and zoledronic acid further inhibited the formation of bone metastases. Histologic assays showed that, compared with either PTHrP antibody or zoledronic acid alone, their combination decreased the number of tumor-associated osteoclasts and increased the number of apoptotic tumor cells. These findings suggest that this novel dual-targeting therapy may be useful for controlling bone metastases in a subpopulation of small cell lung cancer patients.

HM1.24 (CD317) is a novel target against lung cancer for immunotherapy using anti-HM1.24 antibody.

HM1.24 antigen (CD317) was originally identified as a cell surface protein that is preferentially overexpressed on multiple myeloma cells. Immunotherapy using anti-HM1.24 antibody has been performed in patients with multiple myeloma as a phase I study. We examined the expression of HM1.24 antigen in lung cancer cells and the possibility of immunotherapy with anti-HM1.24 antibody which can induce antibody-dependent cellular cytotoxicity (ADCC). The expression of HM1.24 antigen in lung cancer cells was examined by flow cytometry as well as immunohistochemistry using anti-HM1.24 antibody. ADCC was evaluated using a 6-h (51)Cr release assay. Effects of various cytokines on the expression of HM1.24 and the ADCC were examined. The antitumor activity of anti-HM1.24 antibody in vivo was examined in SCID mice. HM1.24 antigen was detected in 11 of 26 non-small cell lung cancer cell lines (42%) and four of seven (57%) of small cell lung cancer cells, and also expressed in the tissues of lung cancer. Anti-HM1.24 antibody effectively induced ADCC in HM1.24-positive lung cancer cells. Interferon-beta and -gamma increased the levels of HM1.24 antigen and the susceptibility of lung cancer cells to ADCC. Treatment with anti-HM1.24 antibody inhibited the growth of lung cancer cells expressing HM1.24 antigen in SCID mice. The combined therapy with IFN-beta and anti-HM1.24 antibody showed the enhanced antitumor effects even in the delayed treatment schedule. HM1.24 antigen is a novel immunological target for the treatment of lung cancer with anti-HM1.24 antibody.