Conception and design: Y.P. Sher, M.C. Hung
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.Y. Lin, H.J. Chen, G.C. Tseng
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.C. Huang, L.C. Liang, T.P. Lu, T.T. Kuo, Q.Y. Kuok
Writing, review, and/or revision of the manuscript: C.Y. Lin, J.L. Hsu, M.C. Hung, Y.P. Sher Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.Y. Sung
Study supervision: Y.P. Sher
Grant Support
This work was supported by grants from National Science Council (2314-B-039-029-MY3, NSC101-2325-B-039-008 and NSC102-2628-B-039-010-MY3 to Y.-P.S.; NSC99-2632-B-039-001-MY3 to M.-C.H), International Research-Intensive Centers of Excellence in Taiwan (NSC102-2911-I-002-303 to M.-C.H.), Taiwan Department of Health, China Medical University Hospital Cancer Research Center of Excellence (CRC; DOH102-TD-C-111-005), CMU102-BC-5, and CMU99-NTU-08.
Acknowledgment
The authors thank Diane Hackett from The Department of Scientific Publications at MD Anderson Cancer Center for editing our manuscript.
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Figure Legends
Figure 1. Knockdown of ADAM9 decreases lung cancer metastatic to the brain. A, flowchart for establishing cell sublines of lung cancer metastatic to the brain. F4-luc cells were injected intracardially into SCID mice and cultured from dissected brain tissue. The cycle was repeated
expression is shown. ADAM9 proteins were detected in non-reducing gels using the antibody from R&D (MAB939, R&D Systems). B, migration ability of ADAM9 knockdown (Bm7-shADAM9) and control (Bm7-shGFP) cells was evaluated by wound-healing assay. Western blot analysis of ADAM9 protein expression is shown at the top. EF1α served as protein-loading control. C, trans-BBB migration assay of ADAM9 knockdown and control cells in vitro. Top, the images of trans-migrated cells. Bottom, quantitation of the number of trans-migrated cells from three independent experiments. *p < 0.05 and ** p < 0.01. D, relative binding activity of each indicated cell line to human brain endothelial cells (HBMEC) at 0.5 hr and 2 hr. The binding activity of Bm7-shGFP at 2 hr was set as 100% for comparison between cell lines. ** p < 0.01.
E, Bm7-shGFP, Bm7-shADAM9-C, or Bm7-shADAM9-E cells were injected intracardially into SCID mice and monitored under IVIS detection. The number of mice in which the result was detected out of the total mice in each group is shown in parenthesis. F, the metastatic lung tumor from the brain of mice bearing Bm7-shGFP cells was confirmed by pathological hematoxylin and eosin staining.
Figure 2. ADAM9-regulated genes and associated networks in lung cancer cells metastatic to brain identified from transcriptome microarray analysis. A, hierarchical clustering of the 182 differentially expressed genes by comparing control versus ADAM9 knockdown and brain-metastatic cell sublines (Bm2 and Bm7) versus parental lung cancer cells. Red indicates genes that were upregulated; green indicates downregulated genes. B, Ingenuity Pathway Analysis (IPA) showing the gene network in cellular movement, molecular transport, and neurological disease. C, Western blot analysis of ADAM9 and CDCP1 in parental cells and brain-metastatic cell sublines (left, lung cancer cells; right, breast cancer cells). The numbers below the middle
panel represent the levels of full-length and cleaved CDCP1 in F4 and Bm7 cells relative to CL1-0 cells. D, Western blot analysis of ADAM9 and CDCP1 in Bm7 cells treated with BB-94, a broad-spectrum inhibitor of metalloproteinases. The numbers below the top panel represent the levels of ADAM9 expression in BB-94-treated relative to DMSO-treated Bm7 cells. The numbers below the middle panel represent the levels of full-length and cleaved CDCP1 in BB-94-treated relative to DMSO-treated Bm7 cells. E, Western blot analysis of ADAM9 and CDCP1 in A549 cells treated with BB-94. The numbers below the top panel represent the levels of ADAM9 expression in BB-94-treated relative to DMSO-treated A549 cells. The numbers below the middle panel represent the levels of full-length and cleaved CDCP1 in BB-94-treated relative to DMSO-treated A549 cells. Western blots shown in panels C, D, and E were probed with anti-ADAM9 antibody (#2099, Cell Signaling) under reducing conditions. EF1α served as protein-loading control.
Figure 3. ADAM9 enhances cleaved CDCP1 protein level and the function of CDCP1 in anoikis resistance. A, Western blot analysis of ADAM9 and CDCP1 in 293 cells transfected with ADAM9 expression or control vector. B, Western blot analysis of expression of CDCP1 and its downstream proteins in control and ADAM9 knockdown cells. P/T indicates the quantified ratio of phosphorylated proteins to total proteins. ADAM9 proteins were detected in non-reducing gels using the antibody from R&D (MAB939, R&D Systems). C, protein stability of ADAM9 and CDCP1 in ADAM9 knockdown cells treated with cycloheximide at different incubation times was analyzed by Western blot (top) and quantified (bottom). The intensity of the band at 0 hr was used as a control (100%). D, detection of CDCP1 phosphotyrosine levels. Total CDCP1 proteins were immunoprecipitated from control and ADAM9 knockdown cells and detected
using anti-phosphotyrosine antibody. E, colony formation of ADAM9 knockdown cells by soft agar assay. Representative results showing the number of colonies (each one > 200 m in diameter) from stained soft agar plates after 4 weeks’ growth. F, the percentage of apoptosis in Bm7-shGFP, Bm7-shADAM9E, Bm7-shCDCP1-A1, and Bm7-shCDCP1-B2 cells was determined by flow cytometry using Annexin-V (high) and PI staining (low) under anchorage-free culture conditions at different time points. Each experiment was repeated independently and showed a similar trend. Raw data are shown in Fig. S3A. G, plasmid mixture of CDCP1 and GFP (molar ratios = 5:1) was transiently transfected into Bm7-shADAM9-E cells. After 24 hr, the 16 hr-migration distance (motile activity) of Bm7-shADAM9-E cells was measured by time-lapse video microscopy in each group (left) and quantified (right). Transfected Bm7-shADAM9-E cells with high GFP signal represents CDCP1-transfected group whereas low GFP signal represents control cells. Error bars, SD; **, p < 0.01. Western blot analysis of CDCP1 is shown at the top.
Figure 4. The tPA activity correlates with conversion of full-length CDCP1 to the cleavage form.
A, RT-qPCR of tPA (top) and PAI-1 (bottom) in control and ADAM9 knockdown cells. B, Western blot of ADAM9, CDCP1, tPA, and PAI-1 expression in control and ADAM9 knockdown cells. C, activity of tPA in different conditioned media from the indicated cell cultures. D, activity of tPA versus the ratio of CDCP1 cleavage over the full-length form; R = 0.89. E, the percentage of apoptotic cells was determined by flow cytometry under anchorage-free culture conditions at different time points. Raw data are shown in Supplementary Fig. S3A.
F, Bm7-shADAM9-E cells were cultured with or without serum and treated with different concentrations of tPA (Boehringer Ingelheim, Ingelheim, Germany). The numbers below the
CDCP1 blot represent the levels of CDCP1 cleavage in cells treated with tPA treatment relative to cells without tPA treatment in serum-containing or -free culture condition. G, Western blot analysis of immunoprecipitated CDCP1 proteins in ADAM9 knockdown stable cells treated with indicated proteases (top). The numbers below the panel represent the ratio of CDCP1 cleavage over full-length and were plotted against various concentrations of protease treatment (bottom).
Stable clones of ADAM9 knockdown cells were used instead of the pooled population.
Figure 5. Blocking the ADAM9-CDCP1-tPA axis in lung cancer cells prolongs animal survival time. A, the migration distance (motile activity) for ADAM9, CDCP1, tPA and PAI-1 knockdown cells was measured by time-lapse video microscopy (top), and quantified (bottom).
Error bars, SD from three independent experiments; *, p < 0.05; **, p < 0.01. B, Bm7 cells with indicated shRNA knockdown were intracardially injected into SCID mice and mice survival time was monitored for 50 days. The number (n) of mice shown in each group was from two independent experiments. C, time-course analysis of sub-G1 fraction from the cell cycle.
Knockdown cells as indicated were cultured in anchorage-free conditions and subjected to cell cycle analysis. Sub-G1 fraction values at 0, 24, and 48 h are shown. D, Western blot analysis of indicated proteins in Bm7 cells after dexamethasone (Dexa) or dasatinib (Dasa) treatment for 24 hr. Media-alone treatment served as negative control. E, cytotoxic effects of dexamethasone and/or dasatinib after 72-hr treatment. Dose-dependent cytotoxic fraction (top); combination index (CI) summary (bottom) at 50, 75, and 90% inhibition (ED50, ED75, and ED90) for two cell lines treatments with a combination of dexamethasone and dasatinib are shown. A CI value
< 1 indicates synergism.
Figure 6. High expression of ADAM9 and CDCP1 in primary tumors predicts mortality in lung adenocarcinoma patients. A, Kaplan-Meier survival analyses of lung adenocarcinoma patients in the indicated groups calculated by dividing the lung adenocarcinoma patients into different groups via the gene expression level in three independent datasets. H indicates high expression and L indicates low expression above or below the median, respectively. DH, SH, and DL indicate dual-high, single-high, and dual-low expression of ADAM9 and CDCP1, respectively.
The number of patients (n) in each group is shown. B, Cox proportional hazard regression analyses of overall survival of lung adenocarcinoma patients with ADAM9 or CDCP1 expression. C, ranking of the p value of ADAM9 plus CDCP1 among the null p-value baseline obtained by combining a fixed predictor with the other genes randomly selected from the original gene pool. Fixed predictor: ADAM9 in the GSE11969 dataset and CDCP1 in the GSE8894 and Shedden datasets. p value of ADAM9High/CDCP1High versus ADAM9Low/CDCP1Low is marked by the red line. D, comparison of tPA expression in DH, SH, and DL patient groups in combined GSE8894 and GSE11969 datasets using Fisher’s exact test. E, comparison of tPA expression in DH, SH, and DL patient groups in the Shedden dataset using Fisher’s exact test.
Figure 7. Detection of ADAM9, CDCP1, and tPA protein expression in human lung cancer specimens. A, IHC analysis of ADAM9 in lung cancer tissue array scored by staining intensity from 0 to 3+ (0, negative; 1, weak; 2, moderate; 3, strong) by histologists. Matched lung adenocarcinoma tissue and adjacent normal lung tissue from the same patients were analyzed for the distribution of ADAM9 staining by the McNemar method. Representative staining for ADAM9 is shown below the table. B, RT-qPCR analysis of ADAM9 mRNA expression in lung cancer cells and metastatic lung cancer specimens from different patients. HPRT was served as
internal control. Abbreviations: N, normal part; mets, metastatic tumor. C, IHC analysis of ADAM9/CDCP1/tPA expression in 84 clinical paraffin block specimens from four groups of lung adenocarcinoma patients. A score > 1+ indicates positive detection. D, correlation of ADAM9 and tPA expression in all 84 clinical paraffin block specimens. E, Trend-change plots of staining scores in paired primary lung (Group III) and metastatic lung tumors (Group IV).
Each line indicates the trend change between a primary and metastatic lung tumor. Red:
increasing trend; blue: decreasing trend; black: no changes in the trend; p value was calculated by Wilcoxon Signed Ranks Test. F, the indicated detection rate of ADAM9/CDCP1/tPA in all four groups as defined in (C).