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Activated Leukocyte Cell-Adhesion Molecule (ALCAM) Promotes Malignant Phenotypes of Malignant Mesothelioma  Futoshi Ishiguro, MD, Hideki Murakami, MD,

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Presentation on theme: "Activated Leukocyte Cell-Adhesion Molecule (ALCAM) Promotes Malignant Phenotypes of Malignant Mesothelioma  Futoshi Ishiguro, MD, Hideki Murakami, MD,"— Presentation transcript:

1 Activated Leukocyte Cell-Adhesion Molecule (ALCAM) Promotes Malignant Phenotypes of Malignant Mesothelioma  Futoshi Ishiguro, MD, Hideki Murakami, MD, PhD, Tetsuya Mizuno, MD, Makiko Fujii, DDS, PhD, Yutaka Kondo, MD, PhD, Noriyasu Usami, MD, PhD, Kohei Yokoi, MD, PhD, Hirotaka Osada, MD, PhD, Yoshitaka Sekido, MD, PhD  Journal of Thoracic Oncology  Volume 7, Issue 5, Pages (May 2012) DOI: /JTO.0b013e31824af2db Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

2 FIGURE 1 Expression of ALCAM in MM cell lines. (A) Quantitative RT-PCR analysis of ALCAM in 20 MM cell lines, 1 immortalized cell line, MeT-5A, and 2 primary normal mesothelial cultures, OV-M1 and GAS-M1. Relative expression of MeT-5A was arbitrarily set at 1.0, (B) Western blot analysis of ALCAM in 20 MM cell lines. Expression of b-actin was used as the control, (C) Immunocytochemical analysis. Strong, membranous signals of ALCAM were observed in NCI-H290 cells, but no signals in MeT-5A cells. b-catenin staining was used as the marker of the cell-to-cell interaction region. ALCAM, Activated leukocyte cell-adhesion molecule; MM, malignant mesothelioma; RT-PCR, reverse transcription polymerase chain reaction. Journal of Thoracic Oncology 2012 7, DOI: ( /JTO.0b013e31824af2db) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

3 FIGURE 2 Immunohistochemical analysis of ALCAM in primary MM specimens. ALCAM was positive in KD1048 (3+) (A), KD1050 (2+) (B), KD1053 (3+) (C), and KD1056 (3+) (D). These samples were the origins of Y-MESO-9 cell line (20.2 for relative ALCAM mRNA level), Y-MESO-12 (4.7), Y-MESO-14 (35.2), and Y-MESO-21 (51.6), respectively, as shown in Figure 1A. Although KD1062 showed faint staining (1+) (E), its corresponding cell line, Y-MESO-26B, showed high ALCAM expression (30.4). Normal pleural mesothelial cells (arrows) (F) showed faint staining (1+) of ALCAM. ALCAM, Activated leukocyte cell-adhesion molecule; MM, malignant mesothelioma; mRNA, messengerRNA. Journal of Thoracic Oncology 2012 7, DOI: ( /JTO.0b013e31824af2db) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

4 FIGURE 3 Lentiviral shRNA-mediated ALCAM knockdown in two MM cell lines, NCI-H290 and Y-MESO-27. (A) Western blot analysis demonstrates efficiency of ALCAM knockdown. Both ALCAM-Sh vectors, ALCAM Sh1 and ALCAM Sh2, showed effective suppression of the level of ALCAM protein, whereas the control vectors, ALCAM Scr1 and ALCAM Scr2, showed no inhibition. Cells were lysed 96 hours after infection. (B) ALCAM knockdown inhibited migration and invasion of two MM cell lines. (C) Inhibition of ALCAM with shRNA-mediated knockdown in MM cell lines suppressed anchorage-independent colony formation. Representative results of the NCI-H290 and Y-MESO-27 cell lines are shown (top) with higher magnifications of their representative colonies (bottom). The results of the triplicate experiments are presented. Columns, mean; bars, SD. ALCAM, Activated leukocyte cell-adhesion molecule; MM, malignant mesothelioma; shRNA, short hairpin RNA. *p < 0.05 vs. control. Journal of Thoracic Oncology 2012 7, DOI: ( /JTO.0b013e31824af2db) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

5 FIGURE 4 Enhancement of cell migration and anchorage-independent growth of immortalized mesothelial cells, MeT-5A, with infection of ALCAM expressing virus. (A) Western blot analysis. Whole cell lysates of MeT-5A cells were obtained 96 hours after infection of wild-type ALCAM or green fluorescent protein virus. (B) Immunocytochemical analysis. Positive signals of ALCAM were detected mainly at the cell-cell junction. (C) Cell migration and colony formation analysis of MeT-5A cells. Exogenous ALCAM enhanced migration and anchorage-independent cell growth of the MeT-5A cells. Columns, mean; bars, SD. ALCAM, Activated leukocyte cell-adhesion molecule; MM, malignant mesothelioma. *p < 0.05 versus GFP control. Journal of Thoracic Oncology 2012 7, DOI: ( /JTO.0b013e31824af2db) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

6 FIGURE 5 Purification of sALCAM and its inhibitory effects on MM cells. (A) Western blot analysis using anti-V5 antibody. Secreted sALCAM in the conditioned medium as well as in the whole cell lysates was confirmed. (B) Silver staining. Purity of synthesized sALCAM was confirmed with no obvious contaminated proteins. (C) Cell migration and invasion assays. In the presence of sALCAM protein at the indicated final concentration in the media, both migration and invasion abilities of MM cells were significantly inhibited. (D) Anchorage-independent colony formation assay. Anchorage-independent growth of NCI-H290 and Y-MESO-27 cell lines was suppressed with addition of sALCAM. Columns, mean; bars, SD. sALCAM, soluble activated leukocyte cell-adhesion molecule; MM, malignant mesothelioma. *p < 0.05 vs. control. Journal of Thoracic Oncology 2012 7, DOI: ( /JTO.0b013e31824af2db) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

7 FIGURE 6 MM cells with sALCAM expression showed longer survival in vivo. Nude mice were injected with MM cell lines infected with NCI-H290/sALCAM or NCI-H290/GFP (1 × 106) virus into the right thoracic cavity. NCI-H290/sALCAM group showed a significantly prolonged survival (p = 0.01 and log-rank test). MM, malignant mesothelioma; sALCAM, soluble activated leukocyte cell-adhesion molecule. Journal of Thoracic Oncology 2012 7, DOI: ( /JTO.0b013e31824af2db) Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

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