Iwao Mikami

First-Line Gefitinib for Patients With Advanced Non-Small-Cell Lung Cancer Harboring Epidermal Growth Factor Receptor Mutations Without Indication for Chemotherapy

PURPOSE This multicenter phase II study was undertaken to investigate the efficacy and feasibility of gefitinib for patients with advanced non-small-cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations without indication for chemotherapy as a result of poor performance status (PS). PATIENTS AND METHODS Chemotherapy-naïve patients with poor PS (patients 20 to 74 years of age with Eastern Cooperative Oncology Group PS 3 to 4, 75 to 79 years of age with PS 2 to 4, and >or= 80 years of age with PS 1 to 4) who had EGFR mutations examined by the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method were enrolled and received gefitinib (250 mg/d) alone. RESULTS Between February 2006 and May 2007, 30 patients with NSCLC and poor PS, including 22 patients with PS 3 to 4, were enrolled. The overall response rate was 66% (90% CI, 51% to 80%), and the disease control rate was 90%. PS improvement rate was 79% (P < .00005); in particular, 68% of the 22 patients improved from >or= PS 3 at baseline to <or= PS 1. The median progression-free survival, median survival time, and 1-year survival rate were 6.5 months, 17.8 months, and 63%, respectively. No treatment-related deaths were observed. CONCLUSION This is the first report indicating that EGFR mutation-positive patients with extremely poor PS benefit from first-line gefitinib. Because there previously has been no standard treatment for these patients with short life expectancy other than best supportive care, examination of EGFR mutations as a biomarker is recommended in this patient population.

Wnt Inhibitory Factor-1 Is Silenced by Promoter Hypermethylation in Human Lung Cancer

Aberrant activation of the Wingless-type (Wnt) signaling pathway is associated with a variety of human cancers, and we recently reported the importance of aberrant Wnt signaling in lung cancer. On the other hand, inhibition of Wnt signaling suppresses growth in numerous cell types. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind Wnt in the extracellular space and inhibit Wnt signaling. Recently, down-regulation of WIF-1 has been reported in several human cancers. To discover the mechanism of WIF-1 silencing in lung cancer, we first identified the human WIF-1 promoter and subsequently examined the methylation status in the CpG islands. By using methylation-specific PCR and sequence analysis after bisulfite treatment, we demonstrate here frequent CpG island hypermethylation in the functional WIF-1 promoter region. This hypermethylation correlates with its transcriptional silencing in human lung cancer cell lines. Moreover, treatment with 5-aza-2′-deoxycytidine restores WIF-1 expression. We then studied WIF-1 expression in 18 freshly resected lung cancers, and we show a down-regulation in 15 of them (83%). This silencing also correlates with WIF-1 promoter methylation. Our results suggest that methylation silencing of WIF-1 is a common and likely important mechanism of aberrant activation of the Wnt signaling pathway in lung cancer pathogenesis, raising its therapeutic interest.

Inhibition of Wnt-2-mediated signaling induces programmed cell death in non-small-cell lung cancer cells

In this report, we have demonstrated that Wnt-2 protein is overexpressed in freshly resected human non-small-cell lung cancer (NSCLC) tissues. We have also developed a monoclonal antibody against the N-terminus of human Wnt-2 protein. This monoclonal antibody induces apoptosis in human NSCLC cell lines that overexpress Wnt-2 protein. Incubation of this antibody with normal human airway cells lacking Wnt-2 expression does not induce apoptosis. Wnt-2 signaling blockade by the anti-Wnt-2 antibody is confirmed by downregulation of cytosolic β-catenin and reduction in TCF-dependent transcriptional activity (TOPFLASH assay). In addition, Wnt-2-specific small interfering RNA (siRNA) treatment in the NSCLC cell line A549 also downregulated cytosolic β-catenin and induced apoptosis. Moreover, downregulation of an inhibitor of apoptosis family protein, Survivin, was noticed both in the Wnt-2 antibody- and siRNA-treated NSCLC cells, suggesting that inhibition of Wnt-2-mediated signaling induces apoptosis through inactivating Survinin.

An Anti-Wnt-2 Monoclonal Antibody Induces Apoptosis in Malignant Melanoma Cells and Inhibits Tumor Growth

Activation of the Wnt/β-catenin signaling pathway has been associated with human cancers. To test whether Wnt-2 signal is a survival factor in human melanoma cells and thus represents a potential therapeutic target, we investigated the effects of inhibition of Wnt-2 signaling in human melanoma cell lines. We have developed a novel monoclonal antibody against the NH2 terminus of the human Wnt-2 ligand that induces apoptosis in human melanoma cells overexpressing Wnt-2. Whereas incubation of this antibody with normal cells lacking Wnt-2 expression does not induce apoptosis, Wnt-2 signaling blockade by the ligand-binding antibody is confirmed by down-regulation of Dishevelled and β-catenin. Wnt-2 small interfering RNA treatment in these cells yielded similar apoptotic effects and downstream changes. Down-regulation of an inhibitor of apoptosis family protein, survivin, was observed in both the Wnt-2 antibody-treated and small interfering RNA-treated melanoma cell lines, suggesting that the antibody induces apoptosis by inactivating survivin. In an in vivo study, this monoclonal anti-Wnt-2 antibody suppresses tumor growth in a xenograft model. These findings suggest that the anti-Wnt-2 monoclonal antibody may be useful for the treatment of patients with malignant melanoma.

Expression of the secreted frizzled-related protein gene family is downregulated in human mesothelioma.

Secreted frizzled-related proteins (sFRPs) comprise a family of five secreted glycoproteins that antagonize Wnt signaling. Aberrant activation and upregulation of the Wnt pathway is a key feature of many cancers. Thus, role of sFRP as a negative regulator of Wnt signaling may have important implications in tumorigenesis, and its downregulation has been correlated with human cancers. Recently, we reported Wnt signaling and dishevelled (Dvl) overexpression in malignant pleural mesothelioma (MM). Here, we report significant transcriptional downregulation of the SFRP gene family in MM primary tissues and cell lines as well as several other cancer cell lines (breast, lung, glioma, and cervical) compared to normal cells. One or more SFRPs were downregulated in approximately 85% (18 of 21) of primary MM tumor specimens compared to normal pleural tissue. Eight of the nine cancer cell lines we examined showed silencing of the SFRP family. Methylation-specific PCR (MSP) analysis showed that SFRP1, SFRP4, and SFRP5 gene promoters are frequently methylated in MM primary tissue (>80%). Furthermore, transfection of the SFRP gene construct into MM cell lines lacking SFRP expression resulted in apoptosis and growth suppression. Our results suggest that methylation silencing of SFRPs may be one of the important mechanisms of aberrant Wnt signaling activation in MM.

An Antagonist of Dishevelled Protein-Protein Interaction Suppresses β-Catenin–Dependent Tumor Cell Growth

Recent progress in the development of inhibitors of protein-protein interactions has opened the door for developing drugs that act by novel and selective mechanisms. Building on that work, we designed a small-molecule inhibitor of the Wnt signaling pathway, which is aberrantly activated across a wide range of human tumors. The compound, named FJ9, disrupts the interaction between the Frizzed-7 Wnt receptor and the PDZ domain of Dishevelled, down-regulating canonical Wnt signaling and suppressing tumor cell growth. The antitumorigenic effects of FJ9 were pronounced, including induction of apoptosis in human cancer cell lines and tumor growth inhibition in a mouse xenograft model. FJ9 is thus among the first non-peptide inhibitors to show therapeutic efficacy through disruption of PDZ protein-protein interactions.

Blockade of Wnt-1 signaling induces apoptosis in human colorectal cancer cells containing downstream mutations

Aberrant Wnt signaling, mainly through mutations of APC and in some cases of CTNNB1 or AXIN2, has been found in the majority of colorectal cancers. Recently, frequent promoter hypermethylation was identified to cause silencing of the secreted frizzled-related protein (sFRP) family in colorectal cancer. Restoration of sFRP in colorectal cancer cells attenuates Wnt signaling even in the presence of downstream mutations. Here we show that Wnt inhibitory factor-1 (WIF-1), a different secreted antagonist of Wnt signaling, is also silenced by promoter hypermethylation in colorectal cancer cells. Restoration of WIF-1 function, Wnt-1 siRNA, or a monoclonal anti-Wnt-1 antibody that we developed attenuates Wnt-1 signaling and induces significant apoptosis in these cells containing downstream mutations and expressing Wnt-1. In addition, this monoclonal anti-Wnt-1 antibody showed synergistic effects with docetaxel in treating these colorectal cancer cells and great efficacy in treating primary colorectal cancer cultures freshly prepared from patients. Therefore, our data support the hypothesis that constitutive Wnt signaling may be required to complement downstream mutations in the evolution of colorectal cancer. Furthermore, our results suggest that blockade of the Wnt signal may have a therapeutic role in the treatment of colorectal cancer.

Inhibition of Wnt16 in human acute lymphoblastoid leukemia cells containing the t(1; 19) translocation induces apoptosis

The Wnt family of secreted glycoproteins is widely involved in cell proliferation, differentiation and oncogenesis. Many Wnt signaling genes are upregulated and activated in chronic lymphocytic leukemia. Less is known concerning acute leukemia. One subtype of acute lymphoblastoid leukemia (ALL) is characterized by a t(1;19) chromosomal translocation resulting in a fusion protein E2A–Pbx1 that promotes transformation and leukemogenesis. Wnt16 has been shown to be targeted by E2A–Pbx1. We performed a differential gene expression array in acute leukemia cell lines displaying or not displaying the t(1;19) translocation. We found that Wnt16 and many Wnt signaling-related genes were upregulated in the translocation-containing cells. As two isoforms of Wnt16, Wnt16a and Wnt16b, have been recently identified, we demonstrated by using RT–PCR and Western blot that Wnt16b (and not Wnt16a) is overexpressed in t(1;19)-containing cell lines. We then directly addressed the role played by both isoforms in this type of leukemia. Using specific short interfering RNA (siRNA) and an anti-Wnt16 antibody, we showed that targeted-Wnt16b inhibition leads to apoptotic cell death. We also demonstrated that Wnt16b mediates its effect through the canonical Wnt pathway involving dishevelled-2, β-catenin and survivin. We thus propose that Wnt16 plays an important role in leukemogenesis, raising its therapeutic interest.

Wnt2 as a new therapeutic target in malignant pleural mesothelioma

Malignant mesothelioma of the pleura (MPM) is a highly aggressive neoplasm with a poor prognosis and limited treatment options. A better understanding of its pathogenesis is essential to developing alternative therapeutic strategies. We previously demonstrated that the Wnt signaling pathway is activated in MPM through the overexpression of disheveled proteins. To extend our knowledge of Wnt signaling activation in MPM, we performed Wnt‐specific microarrays in normal pleura and MPM. We found that the most common event in MPM was the upregulation of Wnt2. We inhibited Wnt2 by siRNA and a monoclonal anti‐Wnt2 antibody and analyzed their effects on apoptosis and downstream signaling effectors. We then assessed the antiproliferative effects of the Wnt2 antibody and Alimta, one of the current standard treatments of MPM. We confirmed Wnt2 overexpression at the mRNA and protein level in MPM cell lines and tissues. We then demonstrated that inhibition of Wnt2 by siRNA or a monoclonal antibody induces programmed cell death in MPM cells. We next analyzed the effects of the anti‐Wnt2 antibody and of Alimta on MPM cell proliferation. We found that although Wnt2 antibody by itself had less antiproliferative potency than Alimta, the two in combination had substantially more activity than Alimta alone. We thus propose that inhibition of Wnt2 is of therapeutic interest in the development of more effective treatments for MPM.

Serum levels of nicotinamide N-methyltransferase in patients with lung cancer

PurposeThe aim of this study was to determine if nicotinamide N-methyltransferase (NNMT) is useful as a biomarker of lung cancer (stages I–III).MethodsWe established an ELISA system for NNMT. We determined the levels of NNMT and carcinoembryonic antigen (CEA) in sera of 113 non-small cell lung cancer (NSCLC) patients undergoing surgery and sera of 50 non-neoplastic lung disease patients with chronic obstructive pulmonary disease (COPD) and of 24 healthy donors.ResultsThe serum levels of NNMT were significantly higher in lung cancer patients than in COPD patients and healthy donors. The relationship between the specificity and sensitivity of NNMT and CEA measurements for the detection of lung cancer was analyzed by means of receiver-operating characteristic curves. The corresponding areas under the curves were 0.703 for NNMT and 0.621 for CEA, indicating slightly better sensitivity of NNMT. With 90% specificity, the sensitivities of NNMT and CEA as lung cancer markers were 25 and 24%, respectively. There was no significant correlation between NNMT and CEA. Therefore, the sensitivity of NSCLC detection at 90% specificity increased from 25 to 32% when NNMT was used in combination with CEA.ConclusionThe NNMT serum level may have significance in the early detection of NSCLC patients.