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Chandra Goparaju, PhD, Jessica S

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1 Overexpression of EPH Receptor B2 in Malignant Mesothelioma Correlates with Oncogenic Behavior 
Chandra Goparaju, PhD, Jessica S. Donington, MD, Tsungda Hsu, PhD, Ryan Harrington, BS, Nathalie Hirsch, BS, Harvey I. Pass, MD  Journal of Thoracic Oncology  Volume 8, Issue 9, Pages (September 2013) DOI: /JTO.0b013e31829ceb6a Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

2 FIGURE 1 EPHB2 is overexpressed in MM tumors and cell lines. A, Affymetrix HG 1.0ST microarrays reveals a 3.75-fold (p = 2.39E-05) increase in EPHB2 gene-expression levels in tumors compared with matched normal peritoneum. B, Western blot analysis of tissue lysates also demonstrates overexpression of EPHB2 in clinical MM tumor specimens compared with matched normal peritoneum. C, Semiquantitative RT-PCR analysis reveals elevated levels of EPHB2 in MM cell lines compared with benign cell lines. PPIA, peptidylproplyl isomerase; EPHB2, ephrin B2 receptor; MM, malignant mesothelioma; NYU, New York University. Journal of Thoracic Oncology 2013 8, DOI: ( /JTO.0b013e31829ceb6a) Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

3 FIGURE 2 Targeted knockdown of EPHB2 with shRNA results in a significant decrease in EPHB2 protein expression (A) in H2595 and HP-1 cells. B, Immunofluorescence studies demonstrate decreased EPHB2 expression in H2595 and HP-1 MM cells after expression of its specific shRNA. EPHB2, ephrin B2 receptor. Journal of Thoracic Oncology 2013 8, DOI: ( /JTO.0b013e31829ceb6a) Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

4 FIGURE 3 A, qRT-PCR and Western blot analysis of EPHB2 downstream genes: Targeted knockdown of EPHB2 expression by its shRNA causes an increased expression of caspase 1 and caspase 8 mRNA (A) and protein (B). A decreased expression of BCL2L1, BIRC5, matrix metalloproteinase, and vascular endothelial growth factor mRNA (A) and protein (B) is noticed in H2595 and HP-1 cells. C, Caspase-8 activity: A significant increase in the caspase-8 activity in apoptotic cells is seen after forced expression of EPHB2-shRNA in H2595 and HP-1 cells compared with scrambled control. Peptidylproplyl isomerase A gene was used as a normalization control in both semi and qRT-PCR analysis. EPHB2, ephrin B2 receptor; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; EPHB2, ephrin B2 receptor. Journal of Thoracic Oncology 2013 8, DOI: ( /JTO.0b013e31829ceb6a) Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

5 FIGURE 4 Biological significance of EPHB2 in MM is demonstrated in in vitro functional assays after suppression of EPHB2 expressions by its shRNA. A, Cell-proliferation assay: a significant decrease in cell proliferation with a fold change of 1.4-fold to 2.0-fold (p < 0.001) was noticed in both H2595 and HP-1 cells. B, Scratch closure assay: a reduction in horizontal migration by 29% (p < ) in H2595 and 25% (p < ) in HP-1 is noticed. C, Cell invasion is significantly decreased in H2595 by 2.3-fold to 2.7-fold (p < 2.00E-05) and in HP-1 by 1.5-fold to 1.7 fold (p < 2.00E-05) compared with scrambled controls. D, Colony formation: significant decrease in the size and number of colonies was noted in both H2595 and HP-1 MM cells. However, no significant changes were noticed in LP9 cells. EPHB2, ephrin B2 receptor, MM, malignant mesothelioma. Journal of Thoracic Oncology 2013 8, DOI: ( /JTO.0b013e31829ceb6a) Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

6 FIGURE 5 Blocking peptide and antibody experiments in H2595 and HP-1 cells show a significant decrease in cell proliferation (A) and invasion (B). EPHB2, ephrin B2 receptor. Journal of Thoracic Oncology 2013 8, DOI: ( /JTO.0b013e31829ceb6a) Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

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