Satoshi Kohmo

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.

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.

HER2 As Therapeutic Target for Overcoming ATP-Binding Cassette Transporter–Mediated Chemoresistance in Small Cell Lung Cancer

Small cell lung cancer (SCLC) easily acquires multidrug resistance after successful initial therapy. Overexpression of ATP-binding cassette (ABC) transporters is important for the multidrug resistance. Among them, ABCB1 and ABCG2 are known to be upregulated in chemoresistant SCLC cells. We found that human epidermal growth factor receptor 2 (HER2) expressions are also upregulated in chemoresistant SBC-3/ETP, SBC-3/SN-38, and SBC-3/CDDP cells, compared with chemosensitive SBC-3 cells. Lapatinib, a tyrosine kinase inhibitor of HER2, could not suppress proliferation of these HER2-positive SCLC cells alone but successfully restored chemosensitivity to etoposide and SN-38 with a clinically applicable concentration. The reversal effect of lapatinib was thought to be caused by inhibition of drug efflux pump functions of ABC transporters, although lapatinib itself has been reported to be a substrate for them. Moreover, knocking down of HER2 by an short interfering RNA weakened the effect of lapatinib on ABCB1, indicating the involvement of HER2 in the inhibitory mechanisms. Notably, we showed that caveolin-1 and Src play key roles in modulating ABCB1 function via HER2 inactivation. In SBC-3/ETP cells, dephosphorylation of HER2 by lapatinib activates Src and successively leads to increased caveolin-1 phosphorylation. Through this process, caveolin-1 dissociates from HER2 and strengthens association with ABCB1, and finally impairs the pump functions. Furthermore, we showed that treatment by lapatinib in combination with etoposide or irinotecan significantly suppresses the growth of subcutaneous SBC-3/ETP and SBC-3/SN-38 tumors in mice, respectively. Collectively, these results indicate that combination therapy with lapatinib and cytotoxic agents could conquer ABC transporter–mediated chemoresistance especially in HER2-positive SCLC. Mol Cancer Ther; 11(4); 830–41. ©2012 AACR.

Overcoming chemoresistance of small-cell lung cancer through stepwise HER2-targeted antibody-dependent cell-mediated cytotoxicity and VEGF-targeted antiangiogenesis

Small-cell lung cancer (SCLC) easily recurs with a multidrug resistant phenotype. However, standard therapeutic strategies for relapsed SCLC remain unestablished. We found that human epidermal growth factor receptor 2 (HER2) is not only expressed in pretreated human SCLC specimens, but is also upregulated when HER2-positive SCLC cells acquire chemoresistance. Trastuzumab induced differential levels of antibody-dependent cell-mediated cytotoxicity (ADCC) to HER2-positive SCLC cells. Furthermore, as a mechanism of the differential levels of ADCC, we have revealed that coexpression of intracellular adhesion molecule (ICAM)-1 on SCLC cells is essential to facilitate and accelerate the trastuzumab-mediated ADCC. Although SN-38–resistant SCLC cells lacking ICAM-1 expression were still refractory to trastuzumab, their in vivo growth was significantly suppressed by bevacizumab treatment due to dependence on their distinctive and abundant production of vascular endothelial growth factor. Collectively, stepwise treatment with trastuzumab and bevacizumab is promising for the treatment of chemoresistant SCLC.

Suppression of metastases of small cell lung cancer cells in mice by a peptidic CXCR4 inhibitor TF14016

CXCL12 is a chemokine essential for the organ‐specific spread of a variety of cancers including small cell lung cancer (SCLC). Here, we examined the anti‐metastatic efficacy of TF14016, a small peptidic inhibitor of CXCL12 receptor CXCR4, in SCLC. Treatment of mice with TF14016 significantly suppressed pulmonary metastases of CXCR4‐expressing SCLC in size and number. Furthermore, histological examination revealed that the expression of vascular endothelial cell growth factor and the density of CD31‐positive microvessels in metastatic foci were both significantly reduced in TF14016‐treated mice. Collectively, CXCR4 could be an attractive target for anti‐metastatic and anti‐angiogenic therapy in SCLC.

Echinoderm Microtubule-Associated Protein-Like 4 (EML4)–Anaplastic Lymphoma Kinase (ALK) Rearrangement in Congenital Pulmonary Airway Malformation

Address for correspondence: Takashi Kijima, MD, PhD, Department of Respiratory Medicine, Allergy and Rheumatic Diseases, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565–0871, Japan Clinical Pra ● Echinoderm microtubule-associated protein-like 4 (EML4)–anaplastic lymphoma kinase (ALK) (EML4ALK) fusion-type oncoprotein causes approximately 5% of all non–small-cell lung cancer (NSCLC) and is targetable by ALK-specific inhibitors. ● Crizotinib has become clinically available for the treatment of patients with ALK fusion–positive NSCLC. ● Congenital pulmonary airway malformation (CPAM) is an airway developmental disorder and should be considered as a differential diagnosis in children or young adults presenting with pulmonary cysts.

Validation of Noninvasive Morphological and Diffusion Imaging in Mouse Emphysema by Micro–Computed Tomography and Hyperpolarized 129Xe Magnetic Resonance Imaging

Animal disease models are pivotal in investigating the pathogenesis of emphysema and developing novel drugs, but the modalities to evaluate murine emphysema models have been of limited validity and sensitivity. In this study, we evaluated hyperpolarized (129)Xe magnetic resonance imaging (MRI) and micro-computed tomography (micro-CT) compared with traditional methods, such as plethysmography and histology. Elastase-treated mice and adiponectin knockout mice were used as murine emphysema models to evaluate these modalities. Three weeks after elastase administration, significant and heterogeneous emphysema was evaluated according to the mean linear intercept and plethysmography parameters. Notably, the distribution of low-density areas, as examined by micro-CT, correlated with the mean linear intercept and plethysmography parameters in whole lungs. These correlations were also observed in regional areas. Furthermore, we introduced hyperpolarized (129)Xe MRI, which can evaluate gas exchange between the alveoli and blood during spontaneous breathing. Parameters of gas exchange (fD) and alveolar size (Vs/Va) were significantly decreased in elastase-treated mice, and moderately correlated with the plethysmography parameters. Of importance, we could detect a decrease of the fD value in low-density areas with micro-CT, suggesting that gas exchange decreased in emphysematous lesions. Likewise, these parameters (fD and Vs/Va) were also decreased in adiponectin knockout mice, which exhibit emphysema with a homogeneous distribution. We demonstrated the feasibility of (129)Xe MRI and micro-CT in combination with traditional modalities. These noninvasive modalities provide complementary data that can be used for repeated estimations of regional gas exchange and lung morphology.

Calretinin mediates apoptosis in small cell lung cancer cells expressing tetraspanin CD9

A majority of small cell lung cancer (SCLC) cells lack a metastasis suppressor, tetraspanin CD9, and CD9 expression promotes their apoptosis. By a proteomics‐based approach, we compared an SCLC cell line with its CD9 transfectant and found that a calcium‐binding neuronal protein, calretinin, is upregulated in CD9‐positive SCLC cells. Ectopic or anticancer drug‐induced CD9 expression upregulated calretinin, whereas CD9 knockdown down‐regulated calretinin in SCLC cells. When calretinin was knocked down, CD9‐positive SCLC cells revealed increased Akt phosphorylation and decreased apoptosis. These results suggest that CD9 positively regulates the expression of calretinin that mediates proapoptotic effect in SCLC cells.