Izumi Nagatomo

YAP modifies cancer cell sensitivity to EGFR and survivin inhibitors and is negatively regulated by the non-receptor type protein tyrosine phosphatase 14

The Yes-associated protein (YAP) is a transcriptional factor involved in tissue development and tumorigenesis. Although YAP has been recognized as a key element of the Hippo signaling pathway, the mechanisms that regulate YAP activities remain to be fully characterized. In this study, we demonstrate that the non-receptor type protein tyrosine phosphatase 14 (PTPN14) functions as a negative regulator of YAP. We show that YAP forms a protein complex with PTPN14 through the WW domains of YAP and the PPXY motifs of PTPN14. In addition, PTPN14 inhibits YAP-mediated transcriptional activities. Knockdown of YAP sensitizes cancer cells to various anti-cancer agents, such as cisplatin, the EGFR tyrosine kinase inhibitor erlotinib and the small-molecule antagonist of survivin, S12. YAP-targeted modalities may be used in combination with other cancer drugs to achieve maximal therapeutic effects.

Early [18F]Fluorodeoxyglucose Positron Emission Tomography at Two Days of Gefitinib Treatment Predicts Clinical Outcome in Patients with Adenocarcinoma of the Lung

Purpose: Positron emission tomography (PET) with [18F]fluorodeoxyglucose (FDG) is increasingly used in early assessment of tumor response after chemotherapy. We investigated whether a change in [18F]FDG uptake at 2 days of gefitinib treatment predicts outcome in patients with lung adenocarcinoma. Experimental Design: Twenty patients were enrolled. [18F]FDG-PET/computed tomographic (CT) scan was carried out before and 2 days after gefitinib treatment. Maximum standardized uptake values (SUV) were measured, and post-gefitinib percentage changes in SUV were calculated. Early metabolic response (SUV decline < −25%) was compared with morphologic response evaluated by CT scan and with progression-free survival (PFS). Results: At 2 days of gefitinib treatment, 10 patients (50%) showed metabolic response, 8 had metabolic stable disease, and 2 had progressive metabolic disease. Percentage changes of SUV at 2 days were correlated with those of tumor size in CT at 1 month (R2 = 0.496; P = 0.0008). EGFR gene was assessable in 15 patients, and of 12 patients with EGFR mutations, 8 showed metabolic response at 2 days and 6 showed morphologic response at 1 month. None of 3 patients with wild-type EGFR showed metabolic or morphologic response. Metabolic response at 2 days was not statistically associated with PFS (P = 0.095), but when a cutoff value of −20% in SUV decline was used, metabolic responders had longer PFS (P < 0.0001). Conclusion: Early assessment of [18F]FDG tumor uptake with PET at 2 days of gefitinib treatment could be useful to predict clinical outcome earlier than conventional CT evaluation in patients with lung adenocarcinoma. Clin Cancer Res; 18(1); 220–8. ©2011 AACR.

The role of HER2 in early breast cancer metastasis and the origins of resistance to HER2-targeted therapies.

The HER2 gene encodes the receptor tyrosine kinase HER2 and is often over-expressed or amplified in breast cancer. Up-regulation of HER2 contributes to tumor progression. Many aspects of tumor growth are favorably affected through activation of HER2 signaling. Indeed, HER2 plays a role in increasing proliferation and survival of the primary tumor and distant lesions which upon completion of full transformation cause metastases. P185(HER2/neu) receptors and signaling from them and associated molecules increase motility of both intravasating and extravasating cells, decrease apoptosis, enhance signaling interactions with the microenvironment, regulate adhesion, as well as a multitude of other functions. Recent experimental and clinical evidence supports the view that the spread of incompletely transformed cells occurs at a very early stage in tumor progression. This review concerns the identification and characterization of HER2, the evolution of the metastasis model, and the more recent cancer stem cell model. In particular, we review the evidence for an emerging mechanism of HER2(+) breast cancer progression, whereby the untransformed HER2-expressing cell shows characteristics of stem/progenitor cell, metastasizes, and then completes its final transformation at the secondary site.

Diagnostic Accuracy of Noninvasive Genotyping of EGFR in Lung Cancer Patients by Deep Sequencing of Plasma Cell-Free DNA

BACKGROUND Genotyping of EGFR (epidermal growth factor receptor) mutations is indispensable for making therapeutic decisions regarding whether to use EGFR tyrosine kinase inhibitors (TKIs) for lung cancer. Because some cases might pose challenges for biopsy, noninvasive genotyping of EGFR in circulating tumor DNA (ctDNA) would be beneficial for lung cancer treatment. METHODS We developed a detection system for EGFR mutations in ctDNA by use of deep sequencing of plasma DNA. Mutations were searched in >100 000 reads obtained from each exon region. Parameters corresponding to the limit of detection and limit of quantification were used as the thresholds for mutation detection. We conducted a multi-institute prospective study to evaluate the detection system, enrolling 288 non-small cell lung cancer (NSCLC) patients. RESULTS In evaluating the performance of the detection system, we used the genotyping results from biopsy samples as a comparator: diagnostic sensitivity for exon 19 deletions, 50.9% (95% CI 37.9%-63.9%); diagnostic specificity for exon 19 deletions, 98.0% (88.5%-100%); sensitivity for the L858R mutation, 51.9% (38.7%-64.9%); and specificity for L858R, 94.1% (83.5%-98.6%). The overall sensitivities were as follows: all cases, 54.4% (44.8%-63.7%); stages IA-IIIA, 22.2% (11.5%-38.3%); and stages IIIB-IV, 72.7% (60.9%-82.1%). CONCLUSIONS Deep sequencing of plasma DNA can be used for genotyping of EGFR in lung cancer patients. In particular, the high specificity of the system may enable a direct recommendation for EGFR-TKI on the basis of positive results with plasma DNA. Because sensitivity was low in early-stage NSCLC, the detection system is preferred for stage IIIB-IV NSCLC.

Involvement of Endothelial Apoptosis Underlying Chronic Obstructive Pulmonary Disease―like Phenotype in Adiponectin-null Mice: Implications for Therapy

RATIONALE Chronic obstructive pulmonary disease is frequently complicated with comorbidities, such as cardiovascular disease, osteoporosis, and body weight loss, but the causal link remains unclear. OBJECTIVES To investigate the role of adiponectin in the pathogenesis of chronic obstructive pulmonary disease and its potential use in therapy. METHODS Adiponectin localization and dynamics in the lung were analyzed in an elastase-induced emphysema model. Next, the lung of adiponectin-knockout mice, extrapulmonary effects, and the underlying mechanism were investigated. Finally, we tested whether exogenous adiponectin could ameliorate the emphysematous change in adiponectin-knockout mice. MEASUREMENTS AND MAIN RESULTS Adiponectin expression in lung vasculature and plasma concentration of adiponectin were reduced after elastase-instillation. Notably, adiponectin-knockout mice showed progressive alveolar enlargement and increased lung compliance. They further exhibited not only systemic inflammation, but also extrapulmonary phenotype, such as body weight loss, fat atrophy, and osteoporosis. Moreover, endothelial apoptosis was enhanced in the lungs of adiponectin-knockout mice, as evidenced by caspase-3 activity. Consistent with this, expressions of vascular endothelial growth factor receptor-2 and platelet endothelial cell adhesion molecule-1 on endothelial cells were decreased in the adiponectin-knockout mice. Finally, adenovirus-mediated adiponectin supplementation ameliorated the emphysematous phenotype. CONCLUSIONS Adiponectin-knockout mice develop progressive chronic obstructive pulmonary disease-like phenotype with systemic inflammation and extrapulmonary phenotypes. Hypoadiponectinemia could thus play a critical role in the progression of chronic obstructive pulmonary disease and concomitant comorbidities through endothelial dysfunction. Together, adiponectin could be a novel target for chronic obstructive pulmonary disease therapy.

Cell Surface Tetraspanin CD9 Mediates Chemoresistance in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive malignancy with extremely high mortality due to the appearance of widespread metastases early in its clinical course and rapid acquisition of chemoresistance after initial therapy. A theory of cell adhesion-mediated drug resistance is thought to be a principal mechanism in which extracellular matrix proteins provide a survival advantage against cytotoxic drug-induced apoptosis. We found that the tetraspanin family member CD9 was expressed preferentially in SCLC tumors and metastases from three of seven relapsed patients, whereas chemonaïve primary tumors from 16 patients were CD9 negative with only one exception. Additionally, CD9 was highly expressed on SCLC cell lines rendered resistant to cisplatin or etoposide, and was upregulated in parental chemosensitive cells within 48 hours after exposure to either of these compounds. CD9-expressing chemoresistant SCLC cells adhered more tightly to fibronectin via β1 integrin, but they were less motile than the respective chemosensitive parental lines. Notably, treatment of the chemoresistant cells with chemokine CXCL12 downregulated CD9 and transiently restored motility. Moreover, selective targeting of CD9 by treatment with specific monoclonal antibody ALB6 or a small interfering RNA triggered apoptosis in the chemoresistant cells. Taken together, our findings implicate CD9 in the cell adhesion-mediated drug resistance mechanism, highlighting CD9 as an attractive therapeutic target to improve therapeutic outcomes in SCLC.

Tetraspanin CD151 Protects against Pulmonary Fibrosis by Maintaining Epithelial Integrity

RATIONALE Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disorder of unknown etiology with few treatment options. Although tetraspanins are involved in various diseases, their roles in fibrosis have not been determined. OBJECTIVES To investigate the role of tetraspanin CD151 in pulmonary fibrosis. METHODS CD151 knockout (KO) mice were studied by histological, biochemical, and physiological analyses and compared with wild-type mice and CD9 KO mice. Further mechanistic analyses were performed in vitro, in vivo, and on samples from patients with IPF. MEASUREMENTS AND MAIN RESULTS A microarray study identified an enrichment of genes involved in connective tissue disorders in the lungs of CD151 KO mice, but not in CD9 KO mice. Consistent with this, CD151 KO mice spontaneously exhibited age-related pulmonary fibrosis. Deletion of CD151 did not affect pulmonary fibroblast functions but instead degraded epithelial integrity via attenuated adhesion strength on the basement membrane; CD151-deleted alveolar epithelial cells exhibited increased α-SMA expression with activation of p-Smad2, leading to fibrotic changes in the lungs. This loss of epithelial integrity in CD151 KO lungs was further exacerbated by intratracheal bleomycin exposure, resulting in severe fibrosis with increased mortality. We also observed decreased numbers of CD151-positive alveolar epithelial cells in patients with IPF. CONCLUSIONS CD151 is essential for normal function of alveolar epithelial cells; loss of CD151 causes pulmonary fibrosis as a result of epithelial disintegrity. Given that CD151 may protect against fibrosis, this protein represents a novel target for the treatment of fibrotic diseases.

Inhibitory Roles of Signal Transducer and Activator of Transcription 3 in Antitumor Immunity during Carcinogen-Induced Lung Tumorigenesis

Stat3 mediates a complex spectrum of cellular responses, including inflammation, cell proliferation, and apoptosis. Although evidence exists in support of a positive role for Stat3 in cancer, its role has remained somewhat controversial because of insufficient study of how its genetic deletion may affect carcinogenesis in various tissues. In this study, we show using epithelium-specific knockout mice (Stat3(Δ/Δ)) that Stat3 blunts rather than supports antitumor immunity in carcinogen-induced lung tumorigenesis. Although Stat3(Δ/Δ) mice did not show any lung defects in terms of proliferation, apoptosis, or angiogenesis, they exhibited reduced urethane-induced tumorigenesis and increased antitumor inflammation and natural killer (NK) cell immunity. Comparative microarray analysis revealed an increase in Stat3(Δ/Δ) tumors in proinflammatory chemokine production and a decrease in MHC class I antigen expression associated with NK cell recognition. Consistent with these findings, human non-small cell lung cancer (NSCLC) cells in which Stat3 was silenced displayed an enhancement of proinflammatory chemokine production, reduced expression of MHC class I antigen, and increased susceptibility to NK cell-mediated cytotoxicity. In addition, supernatants from Stat3-silenced NSCLC cells promoted monocyte migration. Collectively, our findings argue that Stat3 exerts an inhibitory effect on antitumor NK cell immunity in the setting of carcinogen-induced tumorigenesis.

Deletion of tetraspanin CD9 diminishes lymphangiogenesis in vivo and in vitro.

Background: The molecular mechanisms regulating lymphangiogenesis remain unclear. Results: Tetraspanin CD9 modulates molecular organization of integrins in LEC, thereby supporting several functions required for lymphangiogenesis. Conclusion: Deletion of CD9 diminished lymphangiogenesis in mice and humans. Significance: Given that CD9 mediates inflammation and tumor progression, CD9 might be a key component not only in tumor metastasis, but also in inflammation. Tetraspanins have emerged as key players in malignancy and inflammatory diseases, yet little is known about their roles in angiogenesis, and nothing is known about their involvement in lymphangiogenesis. We found here that tetraspanins are abundantly expressed in human lymphatic endothelial cells (LEC). After intrathoracic tumor implantation, metastasis to lymph nodes was diminished and accompanied by decreased angiogenesis and lymphangiogenesis in tetraspanin CD9-KO mice. Moreover, lymphangiomas induced in CD9-KO mice were less pronounced with decreased lymphangiogenesis compared with those in wild-type mice. Although mouse LEC isolated from CD9-KO mice showed normal adhesion, lymphangiogenesis was markedly impaired in several assays (migration, proliferation, and cable formation) in vitro and in the lymphatic ring assay ex vivo. Consistent with these findings in mouse LEC, knocking down CD9 in human LEC also produced decreased migration, proliferation, and cable formation. Immunoprecipitation analysis demonstrated that deletion of CD9 in LEC diminished formation of functional complexes between VEGF receptor-3 and integrins (α5 and α9). Therefore, knocking down CD9 in LEC attenuated VEGF receptor-3 signaling, as well as downstream signaling such as Erk and p38 upon VEGF-C stimulation. Finally, double deletion of CD9/CD81 in mice caused abnormal development of lymphatic vasculature in the trachea and diaphragm, suggesting that CD9 and a closely related tetraspanin CD81 coordinately play an essential role in physiological lymphangiogenesis. In conclusion, tetraspanin CD9 modulates molecular organization of integrins in LEC, thereby supporting several functions required for lymphangiogenesis.

Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

Tetraspanins organize protein complexes in tetraspanin-enriched membrane microdomains that are distinct from lipid rafts. Our previous studies suggested that reduction in the levels of tetraspanins CD9 and CD81 may be involved in the progression of inflammatory lung diseases, especially COPD. To search for agents that increase the levels of these tetraspanins, we screened 1,165 drugs in clinical use and found that statins upregulate CD9 and CD81 in RAW264.7 macrophages. The lipophilic statins, fluvastatin and simvastatin, reversed LPS-induced downregulation of CD9 and CD81, simultaneously preventing TNF-α and matrix metalloproteinase-9 production and spreading of RAW264.7 cells. These statins exerted anti-inflammatory effects in vitro in wild-type macrophages but not in CD9 knockout macrophages, and decreased lung inflammation in vivo in wild-type mice but not in CD9 knockout mice, suggesting that their effects are dependent on CD9. Mechanistically, the statins promoted reverse transfer of the LPS-signaling mediator CD14 from lipid rafts into CD9-enriched microdomains, thereby preventing LPS receptor formation. Finally, upregulation of CD9/CD81 by statins was related to blockade of GTPase geranylgeranylation in the mevalonate pathway. Our data underscore the importance of the negative regulator CD9 in lung inflammation, and suggest that statins exert anti-inflammatory effects by upregulating tetraspanin CD9 in macrophages.