Jun Konishi

B7-H1 Expression on Non-Small Cell Lung Cancer Cells and Its Relationship with Tumor-Infiltrating Lymphocytes and Their PD-1 Expression

Purpose: B7-H1/PD-L1 (B7-H1) and B7-DC/PD-L2 (B7-DC) are ligands for the receptor PD-1, which is known to negatively regulate T-cell activation. In the present study, we investigated the expression of B7-H1 and B7-DC in tumor specimens of non-small cell lung cancer and their relationships with clinicopathological variables and postoperative survival. Furthermore, we examined the correlation between B7-H1 expression on tumor cells and the number of tumor-infiltrating lymphocytes (TILs) or PD-1 expression on TILs. Experimental Design: The expression of B7-H1 and B7-DC in 52 surgically resected specimens of non-small cell lung cancer was evaluated immunohistochemically. Results: Expression of B7-H1 and B7-DC was focally observed in all non-small cell lung cancer tumor specimens. No relationship was found between the expression of B7-H1 or B7-DC and clinicopathological variables or postoperative survival. However, in the same sections evaluated, significantly fewer TILs were identified in B7-H1-positive tumor regions than in B7-H1-negative tumor regions in a subset of five patients (P = 0.01). Moreover, the percentage of TILs expressing PD-1 was significantly lower in B7-H1-positive tumor regions than in B7-H1-negative tumor regions (P = 0.02). Conclusions: The expression of B7-H1 on tumor cells in local areas reciprocally correlated with the number of TILs, and this may contribute to negative regulation in antitumor immune responses in non-small cell lung cancer.

Skeletal isomerization of hydrocarbons over zirconium oxide promoted by platinum and sulfate ion

Abstract The genesis of the high activity of zirconium oxide promoted by platinum and sulfate ion (Pt/SO 4 2− --ZrO 2 ) for skeletal isomerization of butane and pentane in the presence of hydrogen is studied in terms of the interaction of the catalyst with molecular hydrogen. For skeletal isomerization of pentane at 523 K, Pt/SO 4 2− -ZrO 2 showed activity only in the presence of molecular hydrogen, and its activity persisted for a long period. For skeletal isomerization of butane at 523 K, the catalyst showed activity in the absence of hydrogen, but the activity was markedly enhanced in the presence of hydrogen. For pentane and butane skeletal isomerization, the products consisted exclusively of 2-methylbutane and 2-methylpropane, respectively. For a typical acid-catalyzed reaction of cyclopropane ring opening at 373 K, the presence of hydrogen enhanced the activity, but the hydrogen enhancement effect was small. The products consisted exclusively of propene even in the presence of hydrogen: hydrogenation of propene scarcely occurred. Infrared spectroscopic study of adsorbed pyridine showed that by heating the catalyst in the presence of hydrogen in the temperature range 423–623 K, protonic acid sites were formed with concomitant decrease in the number and strength of Lewis acid sites, demonstrating that the protonic acid sites originate from molecular hydrogen. The mechanisms of protonic acid site generation are discussed. It is suggested that molecular hydrogen dissociates on the platinum to hydrogen atoms which undergo spillover on the SO 4 2− -ZrO 2 and convert to an H+ and an e − or H − . The H+ acts as catalytic site for acid-catalyzed reactions.

γ-Secretase Inhibitor Prevents Notch3 Activation and Reduces Proliferation in Human Lung Cancers

Notch receptors are key regulators of development by controlling cell-fate determination in many multicellular organisms. Genes that are important for normal differentiation play a role in cancer when their normal functions became dysregulated. Notch signaling has been shown to promote and maintain survival of many types of cancers, and we previously have shown that Notch3 plays an important role in lung cancer. In this study, we showed that a high percentage of lung cancer lines expressed Jagged1, Notch receptors, and their transcriptional target genes (HES1, Hey1), suggesting that the Notch pathway plays an important role in lung cancer biology. Thus, inhibition of Notch receptor activation represents a compelling treatment strategy. Notch activation requires proteolytic cleavage of the receptor by gamma-secretase protein complex. In this study, we determined the ability of MRK-003, a gamma-secretase inhibitor, to inhibit Notch3 signaling, growth, and apoptosis of lung cancer cell lines in vitro and in vivo using mouse xenograft models. We also found that MRK-003 inhibited Notch3 signaling, reduced tumor cell proliferation, inhibited serum independence, and induced apoptosis. This drug had no effect when Notch3 expression was knocked down using small interfering RNA (siRNA), suggesting that the observed effects were mediated by specific action on this receptor. In conclusion, these results support the hypothesis that inhibition of Notch activation using a gamma-secretase inhibitor represents a potential new approach for the targeted therapy of lung cancer.

Mediastinal Lymph Node Staging by FDG-PET in Patients with Non-Small Cell Lung Cancer: Analysis of False-Positive FDG-PET Findings

Background: Accurate staging of mediastinal and hilar lymph nodes is a critical factor determining operability in patients with non-small cell lung cancer (NSCLC). Positron emission tomography with 2-[18F] fluoro-2-deoxy-D-glucose as a tracer (FDG-PET) has recently been reported to be more effective in detecting tumor involvement in mediastinal and hilar lymph nodes than computed tomography (CT). Objective: In this study, we analyzed the accuracy of FDG-PET in mediastinal and hilar lymph node staging in patients with NSCLC and the factors associated with false-positive or false-negative FDG-PET findings in mediastinal and hilar lymph node staging. Methods: Fifty-four patients with NSCLC who underwent preoperative analysis including chest CT and whole-body FDG-PET were evaluated retrospectively. Using FDG-PET, lesions were considered to be positive if a definite, localized area of higher uptake, excluding physiologic uptake, than in surrounding normal tissue was present. On CT findings, lymph nodes were considered to be positive if they were >10 mm in short-axis diameter, except subcarinal lymph nodes (#7), which were considered to be positive if they were >15 mm in short-axis diameter. All patients underwent surgical resection of primary tumors and mediastinal and hilar lymph nodes between 1999 and 2001 in our institute. Resected lymph nodes were histologically examined for the existence of tumor cells. Results: A total of 306 lymph nodes were resected and used for analysis. The sensitivity, specificity, positive predictive value and negative predictive value of FDG-PET were 73, 98, 70 and 98%, while those of CT were 55, 96, 55 and 96%, respectively. When pre-operative nodal staging was compared with post-operative histopathological staging, 44 patients (81%) were correctly staged, 7 (13%) were overstaged and 3 (6%) were understaged by FDG-PET, while 39 patients (72%) were correctly staged, 8 (15%) were overstaged and 7 (13%) were understaged by CT. All 7 overstaged patients by FDG-PET had other pulmonary complications, including interstitial pneumonitis (n = 2), previous pulmonary tuberculosis (n = 3), silicosis (n = 1) and emphysema (n = 1), although they were not in the active stage. In 3 understaged patients by FDG-PET, lymph nodes were also undetectable by CT. Conclusion: FDG-PET is superior to CT in mediastinal and hilar lymph node staging of patients with NSCLC. However, care should be taken in lymph node staging for patients who have other pulmonary complications, including interstitial pneumonitis, previous pulmonary tuberculosis and silicosis.

Clinical benefit of readministration of gefitinib for initial gefitinib-responders with non-small cell lung cancer

BackgroundGefitinib, an oral agent of epidermal growth factor receptor tyrosine kinase inhibitor, has a certain efficacy against non-small cell lung cancer (NSCLC). Several predictive factors of gefitinib sensitivity have been well described. However, few studies have investigated the clinical features of gefitinib-responders. In the present study, we analyzed the response and disease progression of primary and metastatic lesions to gefitinib in responders and the results of gefitinib readministration following temporary cessation of gefitinib upon progression of initial gefitinib treatment and other treatments.MethodWe retrospectively evaluated the clinical courses of 27 NSCLC patients who received gefitinib and achieved either a complete or partial response.ResultsThe best-response rate and disease-control rate against the initial chemotherapy for the gefitinib-responders were 27.3% and 77.3%, respectively. Favorable efficacy was observed in the primary lesion and metastases to the lung, liver and brain, while there was no obvious effect on bone metastasis. The primary lesion and intrapulmonary metastasis were the sites of major recurrence. Median progression-free survival was 13.8 months, median duration of gefitinib treatment was 17.0 months and median overall survival was 29.2 months. Some of the patients who experienced disease progression after responding to gefitinib were again sensitive to readministration of gefitinib following temporary cessation of gefitinib and other treatments.ConclusionPatients may still be expected to have prolonged survival if they once responded to gefitinib and then underwent various subsequent treatments followed by readministration of gefitinib. These findings might provide valuable information for the management of gefitinib-responders.

Distinctive expression of the polycomb group proteins Bmi1 polycomb ring finger oncogene and enhancer of zeste homolog 2 in nonsmall cell lung cancers and their clinical and clinicopathologic significance

The polycomb group genes Bmi1 polycomb ring finger oncogene (Bmi1) and enhancer of zeste homolog 2 (EZH2) function as transcriptional repressors involved in gene silencing and in the malignant transformation and biologic aggressiveness of several human carcinomas. In the current study, the authors evaluated Bmi1 and EZH2 protein expression in specimens of human nonsmall cell lung cancer (NSCLC).

Minichromosome maintenance (MCM) protein 4 as a marker for proliferation and its clinical and clinicopathological significance in non-small cell lung cancer

BACKGROUND Minichromosome maintenance (MCM) proteins 2-7 form a complex essential for the initiation of DNA replication. In the process to screen expression changes related to growth suppression of non-small cell lung cancer (NSCLC) cells by a cJun dominant-negative mutant, we found that reduced expression of MCM4 was correlated with this growth suppression. METHOD We determined the relevance of MCM4 in proliferation of NSCLC by downregulating its expression with small-interfering RNA in three NSCLC cell lines. We then immunohistochemically analyzed MCM4 expression in 156 surgically resected NSCLCs to correlate clinicopathologic characteristics. RESULTS MCM4 downregulation reduced proliferation in two cell lines. MCM4 expression was higher in cancer cells than in adjacent normal bronchial epithelial cells (p<0.001). High MCM4 expression was correlated with male gender, heavy smoking, poorer differentiation and non-adenocarcinoma histology (p<0.001, respectively). High MCM4 expression was also correlated with proliferation markers, Ki-67 and cyclin E expression (p<0.001, respectively). MCM4 expression was not associated with survival. CONCLUSION MCM4 may play an essential role in the proliferation of some NSCLC cells. Taken together with higher expression in NSCLCs and its correlation with clinicopathologic characteristics such as non-adenocarcinoma histology, MCM4 may have potential as a therapeutic target in certain population with NSCLCs.

Different influence of macrophage migration inhibitory factor (MIF) in signal transduction pathway of various T cell subsets.

It has been shown that macrophage migration inhibitory factor (MIF) modulates not only macrophage functions, but also T cell functions. However, detailed analysis of the MIF function on responses of various T cell subpopulations remained to be elucidated. In this report, using a neutralizing anti-MIF monoclonal antibody (mAb) we examined MIF functions on various T cell lineages. It was shown that anti-MIF mAb inhibited antigen-specific responses of both IFN-gamma producing and IL-4 producing T cells. The inhibition appeared to be related to blockade of the signal pathway via T cell receptor (TCR) but not that via IL-2 receptor (IL-2R). However, the anti-MIF mAb showed no inhibitory effect on NK-T cell responses stimulated through TCR. These results suggest that MIF is involved in the signal pathway via TCR in mainstream T cells but not in NK-T cells.

Granulocyte-Macrophage Colony-Stimulating Factor Gene-Transduced Tumor Cells Combined with Tumor-Derived gp96 Inhibit Tumor Growth in Mice

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-based cancer cell vaccines have been shown to be potent inducers of antitumor immunity in several murine models, but the antitumor effects on established tumors have been minimal. Conversely, the major role of the heat shock protein gp96, localized in the endoplasmic reticulum (ER), is to act as a molecular chaperone to assist the folding of nascent polypeptide chains in the ER. gp96 derived from tumor cells elicits specific protective immunity against parental tumors, presumably through the transport of tumor-specific peptides to antigen-presenting cells and the maturation of dendritic cells (DCs). However, the therapeutic effects of tumor-derived gp96 on established tumors have not been promising. The present study analyzes the therapeutic effects of GM-CSF gene-transduced Lewis lung cancer (LLC/GM) cells combined with LLC-derived gp96 on established wild-type LLC tumors in immunocompetent C57BL/6 mice. Therapy with either irradiated LLC/GM cells or LLC-derived gp96 barely affected established LLC tumor growth. The antitumor effect was significantly enhanced when 1 microg of LLC-derived gp96 was administered together with 1 x 10(6) irradiated LLC/GM cells (p < 0.05). The antitumor effects of irradiated LLC/GM cells and LLC-derived gp96 required mainly CD8(+) T cells. Spleen cells obtained from mice vaccinated with irradiated LLC/GM cells and LLC-derived gp96 showed specific CD8 cytotoxic activities against LLC cells (specific lysis rate of approximately 28%). This antibody response was not associated with a synergic effect of the combination therapy. Moreover, draining lymph nodes from mice immunized with irradiated LLC/GM cells and LLC-derived gp96 contained more migrating mature CD11c(+) cells (higher levels of CD86 and major histocompatibility complex [MHC] class II molecules) compared with those from any other immunization protocols. These results suggest that the combination of irradiated LLC/GM cells and tumor-derived gp96 has potential as a new immunogene therapeutic strategy against lung cancer.

Abstract 5162: Effects of combined epigenetic therapy with histone methyltransferase EZH2 inhibitor 3-deazaneplanocin and histone deacetylase inhibitor SAHA on non-small cell lung cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA [Background] EZH2, a polycomb group protein, has histone methyltransferase activity, and is involved in malignant transformation of several cancers. We have previously shown that an EZH2 inhibitor, DZNep, inhibits growth of non-small cell lung cancer(NSCLC) cell lines via G1 arrest and apoptosis. Recently, cotreatment with DZNep and an histone deacetylase (HDAC) inhibitor has been shown to induce apoptosis synergistically in AML, ovarian cancers and renal cancers. Therefore, we determined the combined effect of DZNep and an HDAC inhibitor, SAHA, on NSCLC cells. [Methods] We evaluated the effect of combined therapy with DZNep and SAHA against four human NSCLC cell lines_H1299, H1975, A549 and PC-3. Cell proliferation was measured by MTT assay. Percentage of apoptotic cells was measured using Annexin V-FITC with a flow cytometer. Western blot analysis was performed on total cell lysates. For in vivo assay, H1975 cells were subcutaneously implanted into the flank of BALB/cAJcl nu/nu nude mice. When the average tumor volume reached approximately 50∼100mm3, the following treatments were intraperitoneally administered twice per week for 6 weeks; vehicle alone (5% DM SO), DZNep (4mg/kg), SAHA (40mg/kg), and DZNep (4mg/kg) plus SAHA (40mg/kg). [Results] Co-treatment with DZNep and SAHA inhibited cell proliferation synergistically, and reduced EZH2 expression and histone H3 lysine 27 trimethylation more effectively compared with each agent alone. The co-treatment greatly induced accumulation of p27 Kip1 and decrease in cyclin A expression. Flow cytometry analysis demonstrated that the apoptotic fraction was increased in an additive or synergistic manner by the combination therapy. These effects were more evident in H1975 and PC-3 cells with EGFR mutation, in which the co-treatment markedly reduced phosphorylation of EGFR, AKT and ERK1/2. The combined therapy reduced the levels of β-catenin, and its downstream pro-proliferative targets cyclin D1 and EGFR. The co-treatment with DZNep and SAHA also caused significantly greater inhibition of tumor growth of H1975 xenografts than each agent alone in nude mice without visible toxicity. [Conclusion] Combined epigenetic therapy with an EZH2 inhibitor and an HDAC inhibitor may represent an effective approach for NSCLCs. Citation Format: Taichi Takashina, Ichiro Kinoshita, Junko Kikuchi, Yasushi Shimizu, Jun Sakakibara Konishi, Satoshi Oizumi, Masaharu Nishimura, Hirotoshi Dosaka Akita. Effects of combined epigenetic therapy with histone methyltransferase EZH2 inhibitor 3-deazaneplanocin and histone deacetylase inhibitor SAHA on non-small cell lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5162. doi:10.1158/1538-7445.AM2014-5162