Harunori Nakashima

Endothelial–Mesenchymal Transition in Bleomycin-Induced Pulmonary Fibrosis

The pathological hallmark lesions in idiopathic pulmonary fibrosis are the fibroblastic foci, in which fibroblasts are thought to be involved in the tissue remodeling, matrix deposition, and cross-talk with alveolar epithelium. Recent evidence indicates that some fibroblasts in fibrosis may be derived from bone marrow progenitors as well as from epithelial cells through epithelial-mesenchymal transition. To evaluate whether endothelial cells could represent an additional source for fibroblasts, bleomycin-induced lung fibrosis was established in Tie2-Cre/CAG-CAT-LacZ double-transgenic mice, in which LacZ was stably expressed in pan-endothelial cells. Combined X-gal staining and immunocytochemical staining for type I collagen and alpha-smooth muscle actin revealed the presence of X-gal-positive cells in lung fibroblast cultures from bleomycin-treated mice. To explore the underlying mechanisms, by which loss of endothelial-specific markers and gain of mesenchymal phenotypes could be involved in microvascular endothelial cells, the effects of activated Ras and TGF-beta on the microvascular endothelial cell line MS1 were analyzed. Combined treatment with activated Ras and TGF-beta caused a significant loss of endothelial-specific markers, while inducing de novo mesenchymal phenotypes. The altered expression of these markers in MS1 cells with activated Ras persisted after withdrawal of TGF-beta in vitro and in vivo. These findings are the first to show that lung capillary endothelial cells could give rise to significant numbers of fibroblasts through an endothelial-mesenchymal transition in bleomycin-induced lung fibrosis model.

Knockdown of ZEB1, a master epithelial-to-mesenchymal transition (EMT) gene, suppresses anchorage-independent cell growth of lung cancer cells

We found that among four master epithelial-to-mesenchymal transition (EMT)-inducing genes (ZEB1, SIP1, Snail, and Slug) ZEB1expression was most significantly correlated with the mesenchymal phenotype (high Vimentin and low E-cadherin expression) in non-small cell lung cancer (NSCLC) cell lines and tumors. Furthermore, ZEB1 knockdown with RNA interference in three NSCLC cell lines with high ZEB1 expression suppressed to varying degrees mass culture growth and liquid colony formation but in all cases dramatically suppressed soft agar colony formation. In addition, ZEB1 knockdown induced apoptosis in one of the three lines, indicating that the growth inhibitory effects of ZEB1 knockdown occurs in part through the activation of the apoptosis pathway. These results suggest that inhibiting ZEB1 function may be an attractive target for NSCLC therapeutic development.

Involvement of the transcription factor twist in phenotype alteration through epithelial–mesenchymal transition in lung cancer cells†

Epithelial–mesenchymal transition (EMT), which involves the persistent loss of epithelial markers and expression of mesenchymal markers, is assumed to have a critical role in not only tissue development during embryogenesis but also central mechanisms that enhance the invasive and metastatic ability of cancer cells. Twist has been identified to play an essential role in EMT‐mediated tumor invasion and metastasis. Although recent studies suggest that twist expression levels in tissue specimens of lung cancer might be associated with prognosis, the expression of twist in lung cancer cells itself and its effect have not been fully evaluated. Here, we evaluated twist expression and its effect on phenotype alteration in lung cancer cell lines. Twist expression varied among human lung cancer cell lines. The lung cancer cell lines with high twist expression also tended to show a high vimentin/E‐cadherin ratio, which was supported by a migration assay, in which high twist expression gave rise to high cell motility. Furthermore, in comparison to control cells, the lung cancer cells with ectopic expression of twist showed a significant phenotype alteration through EMT and an increasing ability to migrate in vitro, in part, due to a tenfold increase in matrix metalloproteinases activity and almost a 60% increase in modulation of focal adhesion kinase activity, although a contribution of microRNA appeared unlikely in our study. Our present analysis of twist expression in lung cancer provide clues to comprehensive understanding of the mechanisms, by which metastasis often develops in lung cancer.

Erythromycin-induced CXCR4 expression on microvascular endothelial cells

Although stromal-derived factor-1 (SDF-1) via its cognate receptor CXCR4 is assumed to play a critical role in migration of endothelial cells during new vessel formation after tissue injury, CXCR4 expression on endothelial cells is strictly regulated. Erythromycin (EM), a 14-membered ring macrolide, has an anti-inflammatory effect that may account for its clinical benefit in the treatment of chronic inflammatory diseases. However, the effects of EM on endothelial cells and especially their expression of CXCR4 have not been fully evaluated. In this study, we demonstrated that EM markedly induced CXCR4 surface expression on microvascular endothelial cells in vitro and lung capillary endothelial cells in vivo. This ability to induce CXCR4 surface expression on endothelial cells was restricted to 14-membered ring macrolides and was not observed in other antibiotics including a 16-membered ring macrolide, josamycin. Furthermore, this EM-induced expression of CXCR4 on endothelial cells was functionally significant as demonstrated by chemotaxis assays in vitro. These findings suggest that EM-induced CXCR4 surface expression on endothelial cells may promote migration of CXCR4-expressing endothelial cells into sites of tissue injury, which may be associated with the known anti-inflammatory activity of this macrolide.

How Many Passes Are Needed for Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration for Sarcoidosis? A Prospective Multicenter Study

Background: While endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is widely used as an initial diagnostic procedure for pathological confirmation of sarcoidosis, it is unclear how many passes are required to obtain diagnostic materials. Objectives: The aim of this study was to determine the number of needle passes needed for the diagnosis of stage I/II sarcoidosis using EBUS-TBNA. Methods: At three institutions, 109 patients with suspected stage I/II sarcoidosis were recruited and underwent 6 passes of EBUS-TBNA for the main target lesion. Additional EBUS-TBNA for other lesions was permitted. The cumulative yields of needle passes for detecting noncaseating epithelioid cell granulomas were analyzed. Results: A total of 109 patients underwent EBUS-TBNA for 184 lesions. EBUS-TBNA identified specimens containing granulomas in 81 of 92 patients (88%) with a final diagnosis of sarcoidosis. The cumulative yields through the first, second, third, fourth, fifth, and sixth passes for the main target lesion were 63, 75, 82, 85, 86 and 88%, respectively. In the 55 patients that underwent EBUS-TBNA for multiple lesions, the cumulative yields of 2 passes per lesion for 2 lesions (total of 4 passes) and of 4 passes for single lesions were 86 and 84%, respectively (p = 1.00). Conclusions: If rapid on-site cytological evaluation is not available, we recommend at least 4 passes per patient for either single or multiple lesions with EBUS-TBNA for pathological diagnosis of stage I/II sarcoidosis.

Abstract 2295: Knockdown of ZEB1, a master EMT gene, suppresses anchorage-independent cell growth of lung cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Epithelial-to-mesenchymal transition (EMT), an essential process during embryonic development and in wound healing, has been shown to be a key event in tumor migration, invasion and metastasis. EMT program is controlled by several master regulators including Twist, ZEB1, SIP1, Snail, Slug, and Goosecoid which are called master EMT genes1),2). ZEB1 protein binds E-boxes within the promoter region of E-cadherin gene, leading to its transcriptional repression. Also, it directly represses many other genes encoding components of the epithelial junctional complex and cell polarity factors. ZEB1 was shown to promote metastasis in colorectal cancer and breast cancer3), and the association between ZEB1 and tumor progression has been studied in several human cancers. Purpose: To evaluate the association between ZEB1 expression and mesenchymal phenotype in lung cancer, and to test the effect of ZEB1 knockdown with RNA interference on the growth of lung cancer cells. Methods: We analyzed the expression of E-cadherin (epithelial marker), Vimentin (mesenchymal marker), and four master EMT genes (Snail, Slug, ZEB1, SIP1) in 19 NSCLC cell lines and 32 NSCLC tumor tissues. Transient knockdown of ZEB1 with RNA interference was done in three NSCLC cell lines with high expression of ZEB1: H157, H1299, and H460. Quantitative real-time RT-PCR and western blot of ZEB1, E-cadherin and Vimentin were done. Cellular proliferation was measured by WST-1 assay and clonogenic growth was measured by liquid (anchorage-dependent) and soft agar (anchorage-independent) colony formation assays. Apoptosis was evaluated by FACS and western blot of cleaved caspase-3. Results: In cell lines, of four master EMT genes, only ZEB1 expression significantly correlated with both E-cadherin and Vimentin expression. Most EGFR mutant lines showed epithelial phenotype (ratio of Vimentin to E-cadherin (RVE) < 1.0). ZEB1 expression was also inversely correlated with miR-200c and miR-205. In tumor tissues, ZEB1 and Snail expression correlated with the ratio of Vimentin to E-cadherin, and ZEB1 expression highly significantly correlated with Vimentin expression. After ZEB1 knockdown E-cadherin protein was reexpressed in H460 but not in H1299 and H157 cells. ZEB1 knockdown suppressed cell proliferation and liquid colony formation significantly in H1299 and modestly in H157 and H460. Notably, ZEB1 knockdown dramatically suppressed growth of soft agar in the all three cell lines studied. FACS and western blot of cleaved caspase-3 showed apoptosis induction by ZEB1 knockdown in H460, suggesting that growth inhibitory effect of ZEB1 knockdown in lung cancer was caused in part by apoptosis. Conclusions: ZEB1 has the dominant role in maintaining mesencymal phenotype in NSCLC and removal of ZEB1 in lung cancer cell lines induces inhibition of soft agar growth. These results suggest that ZEB1 could be a therapeutic target for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2295.