Induction of E-Cadherin in Lung Cancer and Interaction with Growth Suppression by Histone Deacetylase Inhibition Masatoshi Kakihana, MD, Tatsuo Ohira,

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Induction of E-Cadherin in Lung Cancer and Interaction with Growth Suppression by Histone Deacetylase Inhibition Masatoshi Kakihana, MD, Tatsuo Ohira, MD, Daniel Chan, PhD, Robin B. Webster, PhD, Harubumi Kato, MD, Harry A. Drabkin, MD, Robert M. Gemmill, PhD Journal of Thoracic Oncology Volume 4, Issue 12, Pages 1455-1465 (December 2009) DOI: 10.1097/JTO.0b013e3181bc9419 Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 Induction of E-cadherin in non-small cell lung cancer (NSCLC) cell lines by histone deacetylase (HDAC) inhibition. A, Time course of E-cadherin induction in H661 and H460 cells with trichostatin A (TSA), valproic acid (VPA), MS-275, and vorinostat (VOR). The indicated concentrations of each agent were added to cultures at time zero and samples taken over 4 days; agents were present throughout the time course. Equal aliquots of protein lysates were immunoblotted for E-cadherin. Equivalent loading was verified using anti-β-actin (H661) or Coomassie blue staining (H460, Coom.). B, Dose-response to MS-275 and vorinostat. Cultures of H661 and H460 cells were treated with the indicated concentrations of MS-275 or vorinostat for 24 hours and analyzed for E-cadherin levels and actin. C, Persistence of E-cadherin induction. H661 cells were treated with TSA or MS-275 for 12 hours followed by wash out with fresh medium at zero time. Samples were then harvested at the indicated time points and analyzed for E-cadherin and actin. D, Time course of acetylated histone H4 accumulation by HDAC inhibitors. H661 and H460 cells were treated with the indicated concentrations of TSA and VPA and samples analyzed for acetylated Histone H4 levels over time. Journal of Thoracic Oncology 2009 4, 1455-1465DOI: (10.1097/JTO.0b013e3181bc9419) Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 Growth inhibition after MS-275 treatment in non-small cell lung cancer (NSCLC) lines. Dimethylthiazol tetrazolium bromide (MTT) growth assays for three representative cell lines show the extreme responses among the panel of 12 NSCLCs tested. Cells were seeded into 96-well microtitre plates at between 2000 and 5000/well. MS-275 was added from 25 nM to 25 μM and treatment continued for 3 days. IC50 values for the complete cell line panel are provided in Table 1. Journal of Thoracic Oncology 2009 4, 1455-1465DOI: (10.1097/JTO.0b013e3181bc9419) Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 E-cadherin induction contributes to antiproliferative effects of histone deacetylase (HDAC) inhibition. A, National Cancer Institute-H661 cells were stably transfected with a shRNA construct targeting cadherin (CDH) 1 mRNA or a nontargeting control. Transfected cells were tested for E-cadherin protein levels by Western blot after 24 hours MS-275 treatment using the indicated doses. B, E-cadherin knockdown increased resistance to MS-275. Dimethylthiazol tetrazolium bromide (MTT) assays were performed on control and CDH1 knockdown H661 cells. Values are the average of two independent experiments, each performed in triplicate. Growth was significantly restored in CDH1 knockdown cells compared with controls when treated with 4 μM MS-275 (p = 0.001, Tukey's honestly significant difference test). Journal of Thoracic Oncology 2009 4, 1455-1465DOI: (10.1097/JTO.0b013e3181bc9419) Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 4 Zinc finger E box-binding homeobox (ZEB)-1 and -2 inhibit E-cadherin in H157 cells in a histone deacetylase (HDAC) class I/II independent manner. A, Six non-small cell lung cancer (NSCLC) cell lines with negative or low-E-cadherin expression were treated for 24 hours with trichostatin A (TSA, 0.33 μM) or MS-275 (5 μM). Protein lysates (10 μg) were analyzed for induced E-cadherin levels on Western blots. E-cadherin remained undetectable in H157, H1299, and H1703 cells after HDACi treatment. E-cadherin precursor (135 kDa) was observed only in H2122 (mature E-cadherin migrates at 120 kDa). B, Synergistic induction of E-cadherin message by joint knockdown of ZEB-1 and -2. H157 cells were mock transfected (mock) or transfected with control, ZEB-1, or ZEB-2-specific siRNAs, alone and in combination. Cells were harvested 48 hours posttransfection and processed for RNA and protein. ZEB-1, ZEB-2 and CDH1 were analyzed by real-time reverse-transcriptase polymerase chain reaction (RT-PCR); bars are plotted as log10 of expression relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). C, Protein samples from similarly treated cells were analyzed for ZEB-1 and E-cadherin on Western blots. ZEB-1-specific siRNA achieved substantial knockdown of protein (B, lane 3), but no further knockdown was observed on addition of a second anti-ZEB-1 siRNA (B and C, lane 4). Knockdown of ZEB-2 was verified by real-time PCR because available anti-ZEB-2 antibodies were nonspecific. E-cadherin was detectably induced only in cells knocked down for both ZEB-1 and -2 (lane 6). Journal of Thoracic Oncology 2009 4, 1455-1465DOI: (10.1097/JTO.0b013e3181bc9419) Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 5 E-cadherin expression blocks tumorigenicity of NCI-H157 cells. A, NCI-H157 cells were stably cotransfected with pEGFP-C1 and an E-cadherin expression construct (independent clones in lanes 3 and 4, pBATEM) or pEGFP-C1 alone (lane 2). Western blot for E-cadherin verified expression (top), whereas tubulin provided a loading control (bottom). B, Orthotopic tumorigenicity assay. Ten million cells (H157/GFP controls or H157/E-cadherin transfectants) were introduced intratracheally into the left lung of irradiated athymic nude rats (six/group) and tumor growth monitored for 60 days. H157 cells stably transfected with EGFP-C1 formed rapidly growing, metastatic tumors by 42 days postinoculation (dark arrowhead). The E6 transfectant at 123 days had formed a typically small tumor (white arrowhead) in the left lung without metastasis. C, Kaplan-Meier survival plots are shown for animals injected with the indicated transfectants (six animals/group). GFP, green fluorescence protein; NCI, National Cancer Institute. Journal of Thoracic Oncology 2009 4, 1455-1465DOI: (10.1097/JTO.0b013e3181bc9419) Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 6 MS-275 induces apoptosis in non-small cell lung cancer (NSCLC) cell lines. A–D, The indicated NSCLC cell lines were incubated with medium (controls), MS-275 (10 μM), or trichostatin A (TSA, 0.33 μM), as indicated, for 48 hours. The percentage of apoptotic cells was determined by flow cytometry using dual staining with YO-PRO1 and propidium iodide. A, C, Flow analysis of untreated control cultures for H661 (A) and H290 (C). B, D, Flow analysis of H661 cells after treatment with TSA (B) and H290 cells after treatment with MS-275 (D). E,F, MS-275 activates caspase-3 in sensitive NSCLC cell lines. E, F, The moderately sensitive line H647 and the resistant line A549 were treated with MS-275 at different doses (E) or times (F) and examined for biochemical changes indicative of apoptosis. In (E), cultures were treated for 36 hours with the indicated doses of MS-275 (μM); in (F), cultures were treated with 5 μM MS-275 for the indicated times. Lysates were analyzed by Western blot for activated caspase 3 and poly ADP ribose polymerase (PARP) cleavage. Tubulin provided loading controls. (G) Bcl-2 protein levels were measured in 14 non-small cell lung cancer (NSCLC) lines, as indicated. Signals from a shorter exposure (not shown) were densitized and normalized to the level obtained in A549 to yield relative expression (Rel Exp). A Coomassie stained gel (Coom.) provided a loading control. Journal of Thoracic Oncology 2009 4, 1455-1465DOI: (10.1097/JTO.0b013e3181bc9419) Copyright © 2009 International Association for the Study of Lung Cancer Terms and Conditions