Kazutsugu Uematsu

SOCS-3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer

Lung cancer is the leading cause of cancer death in the world, but the molecular mechanisms for its development have not been well characterized. The suppressors of cytokine signaling (SOCS) are inhibitors of cytokine signaling that function via the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Eight SOCS proteins with similar structures have been identified so far. SOCS family members, however, have distinct mechanisms of inhibition of JAK/STAT signaling. Abnormalities of the JAK/STAT pathway are associated with cancer. Inhibition of signaling results in growth suppression in various cell types. Recently, the involvement of SOCS-1 in carcinogenesis has been reported. Here, we report identification of frequent hypermethylation in CpG islands of the functional SOCS-3 promoter that correlates with its transcription silencing in cell lines (lung cancer, breast cancer, and mesothelioma) and primary lung cancer tissue samples. Restoration of SOCS-3 in lung cancer cells where SOCS-3 was methylation-silenced resulted in the down-regulation of active STAT3, induction of apoptosis, and growth suppression. Our results suggest that methylation silencing of SOCS-3 is one of the important mechanisms of constitutive activation of the JAK/STAT pathway in cancer pathogenesis. The data also suggest that SOCS-3 therapy may be useful in the treatment of cancer.

Activation of the Wnt pathway in non small cell lung cancer: evidence of dishevelled overexpression

Non small cell lung cancer (NSCLC) is the leading cause of cancer deaths in the United States and worldwide. Unfortunately, standard therapies remain inadequate. An increased understanding of the molecular biology of lung cancer biology is required to develop more effective new therapies. In this report, we show that the Wnt pathway is activated through Dishevelled (Dvl) overexpression in NSCLC. Analysis of freshly resected tumors and lung cancer cell lines demonstrate that Dvl-3, a critical mediator of Wnt signaling, is overexpressed. Specifically, Dvl-3 was overexpressed significantly in 75% of fresh NSCLC microdissected samples compared to control paired matched normal lung samples. To evaluate the biological significance of Wnt signaling and, in particular, Dvl function in lung cancer, we transfected siRNA (designed to inhibit selectively human Dvl-1, -2, and -3), to the NSCLC cell line H1703, which is known to have β-catenin-mediated Tcf-dependent transcriptional activity. Here, we demonstrate that Dvl-specific siRNA treatment in H1703 decreases significantly Dvl and β-catenin expression, resulting in reduction of Tcf-dependent transcriptional activity, and, importantly, growth inhibition. Taken together, these data support the novel hypothesis that Dvl overexpression is critical to Wnt signaling activation and cell growth in NSCLC.

Phase II Trial of Amrubicin for Treatment of Refractory or Relapsed Small-Cell Lung Cancer: Thoracic Oncology Research Group Study 0301

PURPOSE This multicenter, phase II study was conducted to evaluate the activity of amrubicin, a topoisomerase II inhibitor, against refractory or relapsed small-cell lung cancer (SCLC). PATIENTS AND METHODS SCLC patients with measurable disease who had been treated previously with at least one platinum-based chemotherapy regimen and had an Eastern Cooperative Oncology Group performance status of 0 to 2 were eligible. Two groups of patients were selected: patients who experienced first-line treatment failure less than 60 days from treatment discontinuation (refractory group), and patients who responded to first-line treatment and experienced disease progression > or = 60 days after treatment discontinuation (sensitive group). Amrubicin was administered as a 5-minute daily intravenous injection at a dose of 40 mg/m2 for 3 consecutive days, every 3 weeks. RESULTS Between June 2003 and December 2004, 60 patients (16 refractory and 44 sensitive) were enrolled. The median number of treatment cycles was four (range, one to eight). Grade 3 or 4 hematologic toxicities comprised neutropenia (83%), thrombocytopenia (20%), and anemia (33%). Febrile neutropenia was observed in three patients (5%). Nonhematologic toxicities were mild. No treatment-related death was observed. The overall response rates were 50% (95% CI, 25% to 75%) in the refractory group, and 52% (95% CI, 37% to 68%) in the sensitive group. The progression-free survival, overall survival, and 1-year survival in the refractory group and the sensitive group were 2.6 and 4.2 months, 10.3 and 11.6 months, and 40% and 46%, respectively. CONCLUSION Amrubicin exhibits significant activity against SCLC, with predictable and manageable toxicities; this agent deserves to be studied more extensively in additional trials.

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.

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.

Frequency of and variables associated with the EGFR mutation and its subtypes.

Mutation in the epidermal growth factor receptor (EGFR) is frequently seen in non‐small cell lung cancers (NSCLCs), especially in Asian females with adenocarcinoma. The frequency of mutation and the factors associated requires to be elucidated by analyzing a large number of consecutive clinical samples. We summarized the result of the EGFR mutation analysis for 1,176 patients performed at the time of diagnosis or relapse. The PNA‐LNA PCR clamp, a highly sensitive detection method for the EGFR mutation, was employed. For fresh cases a portion of samples isolated to establish the diagnosis of lung cancer was used. For cases with a relapsed disease archival tissue were tested. The variables associated with the EGFR mutation after removing the confound factors were investigated by the logistic analysis using the samples collected in our university (n = 308) where detailed information on patients were available. The frequency of the EGFR mutation and its subtypes were investigated using all samples (n = 1,176). The EGFR mutation was significantly associated with adenocarcinoma (p = 0.006) and light‐smoking (p < 0.0001), but not gender. The deletions in exon 19 were more frequently associated with male gender while exon 21 deletions were with female gender (p = 0.0011). The overall frequency of the EGFR mutation was 31%. Our result suggests that the female predominance in the EGFR mutation rate is a reflection of a higher frequency of adenocarcinoma in females. The gender difference in the mutation subtypes may provide a clue for the mechanism of the occurrence of the EGFR mutation.

Inhibition of Wnt-1 Signaling Induces Apoptosis in β-Catenin-Deficient Mesothelioma Cells

It is known that Wnt-1 signaling inhibits apoptosis by activating β-catenin/tcf-mediated transcription. Here, we show that blocking Wnt-1 signaling in β-catenin-deficient mesothelioma cell lines H28 and MS-1 induces apoptotic cell death. Both Wnt-1 small interfering RNA (siRNA) and Dishevelled siRNA induced significant apoptosis in these cell lines. A small molecule inhibitor of c-Jun NH2-terminal kinase inhibited the apoptotic cell killing induced by either Wnt-1 siRNA or Dishevelled siRNA in these cells. Our data suggest that β-catenin-independent noncanonical pathway(s), i.e., Wnt/JNK pathway, may play a role in the apoptotic inhibition caused by Wnt-1 signaling.

Preventive effect of erythromycin on experimental bleomycin-induced acute lung injury in rats

BACKGROUND Erythromycin has been reported to have an inhibitory effect on chronic inflammatory airway disease and chronic infiltration of neutrophils into the airway. Bleomycin (BLM) often induces interstitial lung fibrosis following acute lung injury. A study was undertaken to investigate the effects of erythromycin (EM) on experimental bleomycin-induced acute lung injury in rats. METHODS Bleomycin-induced lung injury was assessed by light microscopic examination, measurement of neutrophil elastase activity and of the interleukin 8 (IL-8) content in bronchoalveolar lavage (BAL) fluid. The potential inhibitory effect of erythromycin was assessed by overall comparison of erythromycin untreated (BLM alone), concurrently treated (BLM + EM), and pretreated (BLM + pre-EM) groups. RESULTS The neutrophil count and concentration of neutrophil-derived elastase in BAL fluid were significantly different in the three groups. The morphological changes of lung injury were also less extensive in rats pretreated with erythromycin. However, these protective effects were not marked in the group concurrently treated with erythromycin. Moreover, the concentration of IL-8 in the BAL fluid tended to be less in the erythromycin treated groups; however, there were no significant differences between the bleomycin-treated groups. CONCLUSION Erythromycin exhibits a prophylactic effect on acute lung injury induced by intratracheal administration of bleomycin, which is possibly associated with a downregulation of neutrophil-derived elastase.

Gefitinib‐induced lung injury successfully treated with high‐dose corticosteroids

Abstract:  A 55‐year‐old man was treated with gefitinib for disseminated pleural lesions, 1 year after resection of the left lower lobe for non‐small cell lung cancer. After 6 weeks of continuous daily treatment with oral gefitinib, he developed dyspnoea on exertion and a non‐productive cough. CXR and CT revealed focal areas of ground‐glass opacity (GGO) in the right upper lobe. Despite gefitinib being discontinued, high‐resolution CT revealed extension of GGO and restructuring of lung parenchyma, suggesting acute interstitial pneumonia. Transbronchial biopsy revealed acute‐phase diffuse alveolar damage. After administration of methylprednisolone pulse therapy (1 g/day intravenously) for three consecutive days, the areas of GGO shrank on high‐resolution CT and symptoms resolved. Diffuse alveolar damage caused by gefitinib can be successfully treated in the early phase with high‐dose corticosteroids. Patients receiving gefitinib should be carefully examined for symptoms and undergo CT if their condition deteriorates.

Reduced transcription of the Smad4 gene during pulmonary carcinogenesis in idiopathic pulmonary fibrosis.

Patients with idiopathic pulmonary fibrosis (IPF) have an increased risk of developing lung cancer. To identify key molecules involved in malignant transformation in IPF, we analyzed the expression profiles of lung and lung tumor tissue from patients with lung cancer and IPF (lung cancer/IPF) using cDNA arrays and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Reduced expression of the Smad4 gene was identified in all eight tumor samples from the lung cancer/IPF patients using real-time RT-PCR. Expression levels of Smad4 were significantly lower in tumors from lung cancer/IPF patients than in those from lung cancer patients without IPF or in lung cancer cell lines (p<0.01). Mutational analysis of TGF-β type II receptor and Smad4 was performed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The methylation status of the Smad4 promoter was analyzed using methylation-specific PCR with subsequent sequence analysis. No mutations were detected in the eight tumor samples, but hypermethylated regions were detected in the Smad4 promoter in two of the eight tumors with reduced Smad4 expression. Promoter reporter assays showed that the activity of the Smad4 promoter containing the sequence of the methylated region was significantly stronger than that of the Smad4 promoter with a deleted methylated region (p<0.002). Our findings indicate that the loss of the growth inhibitory response to TGF-β signaling may be crucial in pulmonary carcinogensis or in the progression of lung cancer in IPF patients in whom TGF-β is overexpressed; hypermethylation of the Smad4 promoter region may be one mechanism by which this occurs. These findings are useful for the development of preventive measures or treatment for lung cancer patients with IPF.