Mutant P53 Upregulates Alpha-1 Antitrypsin (A1AT) Expression and Promotes Invasion In

Mutant p53 upregulates Alpha-1 Antitrypsin (A1AT) expression and promotes invasion in lung cancer

SUPPLEMENTARY MATERIALS AND METHODS

Cell culture and constructs

H1299 (human non small cell lung cancer) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Aldrich, St Louis) supplemented with 10% fetal bovine serum (FBS). HCT116 (human colon cancer) and its TP53-/- derivative were kindly provided by Bert Vogelstein (John Hopkins University, Baltimore) and maintained in DMEM medium supplemented with 10% FBS, HEPES and PSG (Penicillin-streptomycin-glutamine). Lung cancer cells- H2009, SBC-3 and H1466 were a kind gift from Dr. Sandra Hodge (University of Adelaide, Australia) and were maintained as previously described (1). H1299 cells with inducible expression of wild-type or mutant p53 are as described previously (2). EI-H1299 p53 R248Q was engineered to inducibly express either sh.A1AT or a non targeting control through retroviral mediated transduction with viruses generated by TRIPZ-shA1AT or TRIPZ-non targeting (Open Biosystems, Hunstville, AL, USA) vectors, followed by selection in 500 ng/ml puromycin (Sigma-Aldrich, Castle Hill, NSW, Australia). This is called a double inducible cell lines in which (i) treatment with Ponasterone (PonA 2.5 μg/ml) for 24hours induces mutant p53 and (ii) treatment with Doxycycline (Dox 2 μg/ml) for 24 hours induces the expression of sh.A1AT. To transiently silence gene expression in cell lines, cells were transfected with 10μm of various specific siRNA molecules (GenePharma, Shanghai, China; listed in Supplementary table 3) and transfected using Lipofectamine RNAiMax transfection reagent following the manufacturer’s protocol. For constitutive knockdown of A1AT, the pGIPZ lentiviral shRNAmir system (Open Biosystems) expressing two independent A1AT-speific short hairpin RNA (shRNA) oligonucleotides (V3LHS_319251 or V3LHS_319249) was used in accordance with the manufacturer’s protocol (Open Biosystems).

Western Blot analysis and Immunofluorescence

Western blot analysis using whole cell lysates was performed as previously described (3). Antibodies used were: mouse anti-p53 DO-1 (Santa cruz Biotechnology, Santa Cruz, CA), mouse anti β-tubulin (Abcam), mouse anti A1AT (for cell lysates; abcam ab9399), rabbit anti A1AT (for conditioned medium; Spring BioscienceE3442), anti rabbit Fibronectin (abcam ab2413), anti rabbit Vimentin (Abcam ERP3776), anti Rabbit p63 H-129 (Santa Cruz), anti-mouse IgG or anti-rabbit IgG-conjugated (GE Health Care). For western blot analysis using conditioned medium, the medium was concentrated and equal volume of concentrated protein was loaded on 8% polyacrylamide gels. The gels were resolved using SDS PAGE electrophoresis and electrotransferred to Hybond-C Extra nitrocellulose membranes (GE Healthcare). Membranes were probed with rabbit A1AT (1:400; Spring BioscienceE3442) primary antibody. Chemiluminescent detection of protein was done using anti-rabbit secondary antibody conjugated with horseradish peroxidase and the enhanced chemiluminescence kit according to the manufacturer’s instructions (GE Healthcare).

For immunofluorescence, cells were plated at 10% confluence on sterilized glass cover slips, then fixed with 4% paraformaldehyde/PBS and then permeabilized in 0.05% Triton X-100/PBS. Samples were incubated with primary antibody in 10% goat serum for overnight at 4oC and the secondary antibody for 30 minutes at room temperature. Cells were mounted in Vectashield mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Images were acquired using a ZEISS LSM700 confocal microscope.

Conditioned medium and antibody blocking assay

Cultured cells at 80% confluence were washed with PBS and replaced with fresh Serum free medium and treated (with Ponasterone) as desired. After 72 hours, the conditioned medium was collected and concentrated using Amicon Ultra-Centrifugal filter (Millipore, Billercia, MA, USA) with molecular cutoff of 10 kDa at 12000rpm at 4oC for 30mins. Columns were washed with PBS and concentrated medium was obtained after spinning the column in inverted position. For A1AT blocking assay, EI-H1299 R248Q and H2009 cells were suspended in conditioned medium supplemented with 40μg/ml rabbit monoclonal A1AT antibody (Sigma A0409) as a blocking antibody or rabbit IgG as a control.

iTRAQ LC-MS/MS analysis

Inducible H1299 p53 R248Q and R175H cells were cultured in DMEM complete medium. After about 70-80% confluence, cells were washed 3 times with PBS and cultured in serum free medium in the presence of PonA (2.5μg/ml) or vehicle control (ethanol) for 72 hours. Conditioned medium (secretome) were harvested by centrifugation at 4000 g for 5mins, buffer exchanged using into 0.5M TEAB, 0.02% SDS using 5kDa Vivaspin 6 cartridges and digested with trypsin. The enriched peptides were labeled with iTRAQ reagents (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s protocol. Labeled peptides were mixed together and evaporated before the sample fractionation. The iTRAQ labeled dried peptide mixture were reconstituted and acidified with buffer A (5mM Phosphate 25% Acetonitrile, pH 2.7) for fractionation by SCX HPLC (Agilent 1100 quaternary HPLC system with Polysulfoethyl A 100mmx2.1mm 5μm 200A column). After sample loading and washing with buffer A, addition of buffer B (5mM Phosphate 350mM KCL 25% Acetonitrile, pH 2.7), gradually increased the concentration from 10 to 45% in 70 minutes and then dramatically increased to 100%, after which it remained constant at 100% for 10 mins at a flow of 300 μl/min. The eluent of SCX was collected at every 2mins in the beginning of the gradient and then at 4 mins interval later. The SCX fractions were resuspended in 50μl of loading/desaltation solution (0.1% trifluoroacetic acid and 2% acetonitrile 97.9% water). Sample (40μl) was injected onto a peptide trap (Michrome peptide Captrap) for pre-concentration and desalted with 0.1% formic acid, 2% ACN, at 5μl/min for 10 mins. The peptide trap was then switched into line with the analytical column. Peptides were eluted from the column using a linear solvent gradient, with steps, from mobile phase A: mobile phase B (98:2) to mobile phase A: mobile phase B (65:35) where mobile phase A is 0.1% formic acid and mobile phase B is 90% ACN/0.1% formic acid at 600 nl/min over a 100min period. After peptide elution, the column was cleaned with 95% buffer B for 15mins and then equilibrated with buffer A for 25mins before next sample injection. The reverse phase nanoLC (Eksigent Ultra nanoLC system, Eksigent) eluent was subjected to positive ion nanoflow electrospray analysis in an information dependent acquisition mode (IDA). In IDA mode a TOFMS survey scan was acquired (m/z 400-1500, 0.25 second), with the ten most intense multiply charged ions (counts > 150) in the survey scan sequentially subjected to MS/MS (Triple TOF 5600, AB Sciex) analysis. MS/MS spectra were accumulated for 200 milliseconds in the mass range m/z 100 – 1500 with the total cycle time 2.3 seconds. The analysis of the fractionated proteins resulted in a total of 61249 MS/MS spectra. Peptide and protein quantification was performed on a software tool ProteinPilot V4.2b (AB Sciex) using the Swissprot 2012 Human database. Bias correction was selected. The detected protein threshold (unused ProtScore) was set as larger than 1.3 (better than 95% confidence) to satisfy good quality-scores and high confidences. Altogether 19610 distinct peptides sequences were identified that resulted in identification of 1025 total proteins. To prevent false positive identifications, proteins with P-value < 0.05 (manually validated) were accepted to high confidence protein identification. A total of 85 and 106 proteins from induced EI H1299 R175H and EI H1299 R248Q cell lines fulfilled these criteria. The identified proteins were further selected with the criteria of fold change > 1.6 or less < 1.6, to identify the ‘significantly altered’ (up or down-regulated) proteins. These ‘significantly altered’ proteins were subjected to pathway analysis and correlated with previously published microarray data and selected candidates were tested for functional analysis.

Cell proliferation and cell cycle analysis

To evaluate the direct effect of A1AT on cell proliferation, 1 x 104 cells were seeded in a 96-well plate. Cell growth was determined using the CellTitre-Glo Luminescent Cell Viability assay (Promega) as per the manufacturer’s protocol. Briefly, media was removed from wells, followed by addition of 30μl fresh media and 30μl CellTitre-Glo reagents. Plates were placed on an orbital shaker at ~200rpm for 2mins followed by equilibration at room temperature for 10mins. Plates were read on a luminometer (Lumistar Galaxy). For cell cycle analysis (propidium iodide), cell were harvested and processed (FACS Calibur flow cytometry, Becton Dickinson Immunocytometry Systems) as previously described (3).

Soft Agar Colony formation Assay

To examine anchorage independent growth, 10,000 cells were suspended in 0.32% agarose with medium supplemented with 10% FCS, and seeded in triplicate in 6-well plate pre-coated with 0.72% agar in complete growth medium and cultured for 14 days at 37oC under 5% CO2. Colonies were stained with 0.005% crystal violet in 20% methanol. Colonies were photographed and counted from four microscopic fields per well and expressed as mean of triplicates.

Migration and Inverted Invasion assay

Real time migration assays were performed using Incucyte (Essen, MI, USA). Monolayer cultures were grown on 24 well ImageLock plate. The cultures were scratched using scratch device and washed to remove detached cells. Cells were cultured in a fresh media supplemented with desired treatment. Phase contrast images were captured with a 20X objective every 30mins.

Modified Boyden chamber invasion assays were performed as previously described (4). Briefly 5X104 cells were seeded on the base of matrigel filled transwell chambers (BD). After 6 hours incubation, chambers were washed three times in PBS and incubated in FCS free medium with desired treatment in an upright position for 72 hours. Transwells were placed in cold methanol for 20 min. Cells were then stained with 10μg/ml propidium iodide and invasion toward a gradient of 10% FBS and 25ng/ml Epidermal growth factor (EGF) was measured by Zeiss LSM700 confocal microscopy with serial optical sections being captured at 10μm intervals.

Chicken Chorioallantoic Membrane (CAM) Invasion assay

The CAM invasion assay was performed as previously described (5). Ethical approval for the study was obtained from the University of Adelaide Animal Ethics Review Committee (AEC M-2014-153). Briefly, 5X105 H2009 cells (p53 R273L) stably expressing sh.A1AT or non-targeting control (sh.Ctrl) were mixed with matrigel (8mg/ml, BD Biosciences) and gently inoculated on the upper CAM of day 11 chick embryo. Five days post inoculation, the CAM with developing tumors were excised, paraffin embedded and the invasion of cancer cells into the mesoderm past ectoderm was assessed in the serial sections of CAM stained with haematoxylin, eosin and pan-cytokeratin immunohistochemistry as described previously (5). The antigen retrieval step was performed using EDTA pH8 by heating on high power until boiling (approximately 5min), allowing slides to stand for 5min before microwaving at 50% power for an additional 5min. The area of the tumor in CAM mesoderm layer was quantified using NDP view imaging software (NDP scan software v2.2, Hamamatsu Photonics).

Immunoprecipitation (IP)

Immunoprecipitation was performed as previously described (2). Briefly, subconfluent cultures of inducible p53 mutants were cultures and induced with Ponasterone for 24 hours. Cells were then harvested, lysed in 500 μl of lysis buffer (20mM Tris-HCL at pH8, 1mM EDTA, 0.5% NP-40, 150mM NaCl, 10% glycerol and protease inhibitors, sonicated and centrifuged at 13,200 rpm for 10min at 4oC. Following clarification, Inputs were taken and lysates were incubated with 500ng of either p53 antibody (DO-1, Santa Cruz) or mouse IgG for 1 hour at 4oC with rotation followed by the precipitation with Protein-G-Sepharose beads (GE Healthcare) and incubation for an additional 1hour at 4oC. Beads were washed three times with 400 μl of lysis buffer, protein complexes were eluted with SDS loading buffer and heated for 5 min at 95oC. Western blot analysis of inputs and immunoprecipitated protein complexes was performed as previously described (3). Protein samples were loaded on 8% polyacrylamide gels. The gels were resolved using SDS PAGE electrophoresis and electrotransferred to Hybond-C Extra nitrocellulose membranes (GE Healthcare) 8% SDS-gel.

Lung tissue microarrays and immunohistochemistry

Ethical approval was obtained from the Royal Adelaide Hospital Human Research Ethics Committee (140301b). Primary lung adenocarcinoma (ADC) samples were obtained from 107 patients with stage I-III tumors resected at Royal Prince Alfred Hospital,Sydney, NSW, Australia. Representative tumor samples and matching normal alveolar lung parenchyma and bronchial epithelium were constructed in tissue microarrays as previously described (6). Briefly, each core was 1mm in diameter and consisted of 3-6 tumor core replicates with its matching normal control. ADC tumors were staged using the AJCC tumor, node and metastasis (TNM) classification. The cohort consisted of 55 (52%) stage I, 39 (37%) stage II and 11 (11%) stage III tumors. Immunohistochemistry was performed to determine A1AT and p53 expression. Slides were heated at 50οC for 2 hours followed by dewaxing 3X5min in xylene, rehydration 3X5min with 100% ethanol and 2X5min with PBS. Endogenous peroxide activity was quenched by treating the slides in H2O2 in PBS at room temperature followed by 2X5min wash in PBS. Next, antigen retrieval was performed in 600ml of 1mM EDTA (pH 8) by heating on high power until boiling (approximately 5min), allowing slides to stand for 5min before microwaving at 50% power for an additional 5min. Slides were allowed to cool for 60minutes and washed 2 X 3min in PBS. Sections were encircled with a wax pen and primary antibody diluted in PBS with 10% normal goat serum was applied overnight at 4οC (mouse anti-p53 DO-1 1:100 (Santa cruz Biotechnology, Santa Cruz, CA); or mouse anti-A1AT 1:50 (Abcam ab9399). Slides were washed twice for 5min in PBS, followed by incubation with secondary antibody diluted in PBS (1:400) with 10% normal goat serum for 60min at room temperature (goat anti-mouse biotinylated, Dako). Next, Streptavidin (1:500 dilution in PBS; Dako) was added and incubated for 60min in a humid chamber. 3,3-Diaminobenzidine (DAB) and H2O2 solutions were added and incubated at room temperature for 6mins. Sections were counterstained with Lillie-Mayer haematoxylin for 15-30sec, washed in a running tap water and dehydrated using increasing concentration of ethanol (70%, 90% and 100%) and 3X5mins washed with xylene. Slides were mounted with DPX.

Statistical Analysis

Statistical analysis of data was done in GraphPad prism using two-sided Student’s t-test. The correlation between A1AT expression and clinicopathological parameters were assessed using Chi-square test. Kaplan-Meier plots and the log rank test were used for survival analysis. P-values less than 0.05 were considered as statistically significant.

Supplementary References

1. Dehle FC, Mukaro VR, Jurisevic C, Moffat D, Ahern J, Hodge G, et al. Defective Lung Macrophage Function in Lung Cancer±Chronic Obstructive Pulmonary Disease (COPD/Emphysema)-Mediated by Cancer Cell Production of PGE2? PLoS ONE. 2013;8(4):e61573.