Secretomic analysis identifies alpha-1 antitrypsin (A1AT) as a required protein in cancer cell migration, invasion, and pericellular fibronectin assembly for facilitating lung colonization of lung adenocarcinoma cells

Abstract

Metastasis is a major obstacle that must be overcome for the successful treatment of lung cancer. Proteins secreted by cancer cells may facilitate the progression of metastasis, particularly within the phases of migration and invasion. To discover metastasis-promoting secretory proteins within cancer cells, we used the label-free quantitative proteomics approach and compared the secretomes from the lung adenocarcinoma cell lines CL1-0 and CL1-5, which exhibit low and high metastatic properties, respectively. By employing quantitative analyses, we identified 660 proteins, 68 of which were considered to be expressed at different levels between the two cell lines. High levels of A1AT were secreted by CL1-5, and the roles of A1AT in the influence of lung adenocarcinoma metastasis were investigated. Molecular and pathological confirmation demonstrated that altered expression of A1AT correlates with the metastatic potential of lung adenocarcinoma. The migration and invasion properties of CL1-5 cells were significantly diminished by reducing the expression and secretion of their A1AT proteins. Conversely, the migration and invasion properties of CL1-0 cells were significantly increased through the overexpression and secretion of A1AT proteins. Furthermore, the assembly levels of the metastasis-promoting pericellular fibronectin (FN1), which facilitates colonization of lung capillary endothelia by adhering to the cell surface receptor dipeptidyl peptidase IV (DPP IV), were higher on the surfaces of suspended CL1-5 cells than on those of the CL1-0 cells. This discovery reflects previous findings in breast cancer. In line with this finding, FN1 assembly and the lung colonization of suspended CL1-5 cells were inhibited when endogenous A1AT protein was knocked down using siRNA. The major thrust of this study is to demonstrate the effects of coupling the label-free proteomics strategy with the secretomes of cancer cells that differentially exhibit invasive and metastatic properties. This provides a new opportunity for the effective identification of metastasis-associated proteins that are secreted by cancer cells and promote experimental metastasis.

Fig. 1.

The influence of cell conditioned media on the migration and invasion of lung adenocarcinoma cells was examined. CL1-0 and CL1-5 cell supernatants were collected after 48 h and then incubated with CL1-5 and CL1-0 cells, respectively, for 48 h. After 48 h, the altered migration and invasion of the cells were investigated using transwell migration and Matrigel invasion assays. Two independent experiments were performed, and each experiment was performed three times. The data are presented as the mean ± S.D., where p < 0.05 was considered statistically significant when analyzed by the Mann-Whitney U test. After conditioning the CL1-5 CM, the migration and invasive ability of CL1-0 cells increased. Conversely, CL1-5 cells were not affected by the CL1-0 CM. The results provide evidence that particular factors produced by the cells could change the migration and invasiveness of lung adenocarcinoma cells. A, Transwell migration assay. A total of 50,000∼100,000 cells were seeded into the upper wells in serum-free medium, and the lower chambers were filled with complete medium, supplemented with 10% FBS, to induce cell migration. After 24 h, the cells that had traversed the filter to the lower chamber were stained with Giemsa stain and counted microscopically (200×) in 10 different fields per filter. B, Matrigel invasion assay. The Matrigel coated the upper wells. Apart from this, the set-up was the same as the migration assay. These results indicated that particular factors produced or secreted by the cells exhibit enhanced or reduced migration or invasion properties.

Fig. 2.

Characterization of secretome samples from CL1-0 and CL1-5 cell lines. A, The distribution of the cytosolic protein tubulin from an exact cell number was evaluated to determine whether the CM contained proteins resulting from cell lysis. The rate of cell lysis was under 0.1% in both CL1 cell lines. B, The schematic representation of the label-free proteomics analysis procedure. The biological triplicates and BSA-spike were performed for the label-free quantitative approach. C, 97.4% (643/660) of the proteins were present in the CM of CL1 cells through multiple routes. These proteins were identified by the following: 1) SignalP and SecretomeP prediction software to predict classical and nonclassical secretion pathways, respectively, as well as TMHMM to predict membrane proteins; 2) the ExoCarta database, which contains results from the exosome human database; and 3) the human plasma proteome database. D, The presence of CD9, CD63, and ALG-2-interacting protein X proteins in the CL1-0 and CL1-5 CM samples proves the existence of exosomes in the CM. The abbreviation, Sup., refers to the supernatant after removing the exosomes and is the negative control. Exo indicates the exosomes were purified from CM samples via the commercial kit. An equal amount of each sample was run on SDS-PAGE and further processed with silver staining in biological triplicate as the loading controls. Each lane was quantified and statistical analyses were performed to confirm if the loading amount was equal.

Fig. 3.

Validation of the differential expressions of the 6 selected proteins. A, Fifty micrograms of protein samples were separated on SDS-PAGE gels, transferred to polyvinylidene difluoride membranes, and probed with the indicated antibodies. Every experiment was performed in triplicate. There were 2 and 4 CM proteins with high expression in CL1-0 and CL1-5, respectively. CALR, GLA, and PGAM1 are shown as CM loading controls. Equal amounts of each cell line with silver staining were used as CM loading controls as well. B, Immunohistochemical staining of A1AT revealed a trend of increasing stain intensity and a positive association with the TNM stage of lung adenocarcinoma tissues. In a well-differentiated case, the A1AT exhibits cytoplasmic staining, especially in the apical surface of the tumor cells (red arrow).

Fig. 4.

A1AT has no effect on lung adenocarcinoma cell proliferation. A, Western blot analysis indicated that the expression of A1AT was reduced using siRNA in the cell extract and the CM of the CL1-5 cell line. CALR, GLA, PGAM1, and SDS-PAGE sliver staining were the CM loading controls. (L.C.) indicates loading control. B, Cells (3 × 10³ cells/well) were seeded onto 96-well plates. An MTT assay was performed at specific time points to evaluate cell proliferation after siRNA treatment. The results indicated no difference between the scrambled siRNA control group and the siRNA-treated A1AT group. C, BrdU assay. The results regarding the influences of cell proliferation after reduction of A1AT expression were reassessed. A1AT was overexpressed in CL1-0 cells to discern the influence on proliferation. D, The Western blot indicated that expression and secretion of A1AT was increased in CL1-0 cells via plasmid DNA transfection. E, and F, are the MTT and BrdU assays, respectively. The results reconfirmed the finding that A1AT did not regulate lung adenocarcinoma cell proliferation. N.S. indicates no significance.

Fig. 5.

A1AT prompts migration and invasiveness of lung adenocarcinoma cells. A description of A1AT knockdown and overexpression is provided in Figs. 4A and 4D, respectively. When A1AT was knocked down in CL1-5 cells, the migration and invasiveness of CL1-5 cells was reduced (A, B, and C). A, Cell wound healing assay. The extent of closure was photographed at 0 h and 24 h after treatment at 100× magnification. B, Transwell migration assay at 200× magnification. Migration was quantified by counting cells in six random fields per membrane. C, Matrigel invasion assay at 200× magnification. Invasion was quantified by counting cells in six random fields per membrane. Columns represent the average number of cells per field of at least eighteen fields from three independent experiments. Bars indicate standard deviation. In addition, A1AT was overexpressed in CL1-0 cells to investigate migration and invasiveness. Indeed, the migration and invasiveness of CL1-0 cells was increased in (D) the cell wound healing assay, (E) the transwell migration assay, and (F) the Matrigel invasion assay. All of the data indicate that A1AT protein affects the migration and invasion of lung adenocarcinoma cells. Each assay was performed in independent biological triplicates.

Fig. 6.

A1AT triggers the assembly of pericellular polyFN on lung adenocarcinoma cells to promote lung colonization. A, CL1-0 and CL1-5 cells were trypsinized and placed in a suspension culture for 2 h, stained with rabbit anti-FN antibodies followed by a GFP-conjugated donkey anti-rabbit IgG secondary antibody, observed under a fluorescent microscope, and detected using flow cytometry. Both IF and flow cytometry was used to demonstrate the different levels of FN1 assembly on the cell surfaces of CL1-0 and CL1-5 cells. The assembly levels of pericellular polyFN of CL1-5 cells were higher than those of CL1-0. The Olympus FB1000 microscope was used, and the fluorescent filter set for 488 excitation at 600× magnification. B, Evaluation of polyFN1 assembly on the cell surface after scrambled siRNA control and A1AT siRNA transfection was performed using IF and flow cytometry. The pericellular polyFN assembly of CL1-5 cells was inhibited when the expression of A1AT was reduced by transfecting siRNA. C, In the nude mice model, a tail vein injection was performed using the CL1-5 cells treated with the scrambled siRNA control and A1AT siRNA or CL1-0 cells, using five mice for each treatment. After 8 weeks, the mice were sacrificed and the tumor nodules of their lungs were measured. A tumor size of 0.5 mm in diameter was counted as one nodule. A significant inhibition of lung experimental metastasis was observed in mice that were injected with A1AT siRNA CL1-5 cells. Three of five lungs are shown in (D). E, Represented tumor sections of these three groups were stained with H&E. F, CL1-5 cells were incubated in suspension for 2 h in 20% FBS media. The cells were subsequently treated with myelin basic protein (MBP) and soluble, truncated dipeptidyl peptidase IV (DPP IV) for 20 min. The five mice in each group were sacrificed after 8 weeks, and the lungs were removed and fixed in a 3.7% formalin fixative. On injection of the soluble, truncated DPP IV-treated CL1-5 cells, lung colonization among the nude mice was completely inhibited. There are three representative lungs shown in each group.

Fig. 7.

The proposed mechanisms of A1AT for modulating experimental metastasis in lung adenocarcinoma. A, A1AT influenced polyFN1 assembly but not FN1 synthesis and secretion, as determined by siRNA combined with Western blot analysis. CALR, GLA, PGAM1, and SDS-PAGE silver staining served as the CM loading controls. The abbreviation (L.C.) indicates the loading control. B, The phosphorylation level of PKCε was not affected upon A1AT knockdown in the CL1-5 cells. C, The proposed mechanism of A1AT-modulated polyFN1 assembly on cell surfaces via phosphorylated PKCε regulates A1AT. The Metacore interactome prediction software was used to predict the interaction with PKCε, A1AT, and FN1. The symbols and links shown on the network illustration include the following: transcription factor (), generic kinase (), metalloprotease (), generic protease (), generic binding protein (), lipid phosphatase (), generic receptor (), receptor ligand (), positive effect (), negative effect (), unspecified effect (), the proposed possible pathway (). D, This scheme summarizes the functions in which A1AT is involved when facilitating lung adenocarcinoma experimental metastasis. A1AT mediates migration and invasion abilities but not the proliferation of cancer cells. Additionally, it regulates polyFN assembly on lung adenocarcinoma cell surfaces to colonize on the lung via adhering to DPP IV on lung capillary endothelia.