Human lung cancer cell lines A549, H1792 and H1975 (American Type Culture Collection, Manassas, VA, USA) were cultured in filtered (22 µm vacuum filtration; cat no. 431097; Corning, Inc., Corning, MA, USA) RPMI-1640 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), 1% penicillin-streptomycin and 1% GlutaMAX™ (Thermo Fisher Scientific, Inc.). All cells were maintained at 37°C in 5% CO₂.

A549 lung cancer cell lines were engineered to overexpress MARCKS in a tetracycline-dependent manner as described previously (14). WT-MARCKS and NP-MARCKS sequences (GenScript USA, Inc., Piscataway, NJ, USA) were cloned into a pLenti6.3/TO/V5 (ViraPower HiPerform T-REx Gateway Expression System; cat no. A11141; Invitrogen; Thermo Fisher Scientific, Inc.) lentiviral plasmid as described previously (5). The NP-MARCKS was engineered by substituting the four serine residues in the ED to alanine residues. Exogenous MARCKS was distinguishable from endogenous MARCKS by the addition of a V-5 epitope tag on the C-terminus of WT- and NP-MARCKS.

Lentiviral particles were produced as described previously (14). A total of 4 µg lentiviral packaging plasmid psPAX2 (plasmid no. 12260), lentiviral envelope plasmid PCMV-VSV-G (plasmid no. 8454; both Addgene, Cambridge, Inc., MA, USA) and lentiviral vector plasmid, were mixed with Lipofectamine^® 2000 (cat no. 11668) and Opti-MEM™ media (cat no. 11058; both Invitrogen; Thermo Fisher Scientific, Inc.). The lentiviral mixture was added to the culture media of 293FT cells (Invitrogen; Thermo Fisher Scientific, Inc.) and the media was subsequently replaced the following morning. Lentiviral supernatant was collected after 48 h and filtered through a 0.45 µm filter, aliquoted and stored at −80°C until required. Lentiviral particles were quantified using QuickTiter p24 ELISA (Cell Biolabs, San Diego, CA, USA) (5).

A549 cells were transduced with equal amounts (~30 ng/ml) of p24 quantified tetracycline-repressor (Tet-R) packaged lentiviral particles along with 8 µg/ml polybrene (EMD Millipore, Billerica, MA, USA). A total of 500 µg/ml Geneticin^® (cat no. G418; Thermo Fisher Scientific, Inc.) was used to select for Tet-R positive cells. Tet-R cells were subsequently transduced with equal amounts (~30 ng/ml) of p24 quantified WT- and NP-MARCKS lentiviral particles. A total of 1 µg/ml Blastacidin (Thermo Fisher Scientific, Inc.) was used to select for WT- and NP-MARCKS-expressing cells. MARCKS expression was induced by culturing with 2 µg/ml of the tetracycline homologue doxycycline overnight at 37°C (14).

MARCKS peptide was used as described previously (14). MARCKS-ED tyrosine aminotransferase (TAT) peptide was engineered by conjugating the cell permeable human immunodeficiency virus (HIV) TAT peptide via cysteine bonds to the 25 amino acid ED sequence (KKKKKRFSFKKSFKLSGFSFKKNKK) (15). The control peptide sequence was designed using the ExPAsy random protein sequence generator (www.expasy.org) using the average amino acid composition computed from Swiss-Prot (www.uniprot.org) (CEIEEHAWNTVEMFSSFPGTQLYNDA) to control for peptide size. To eliminate the effect of the positive charges, lysine and arginine residues were changed to glutamates. Peptide was added to cultures 1 h prior to performance of in vivo assays, including immunoblotting, RNA expression analysis, cellular survival and DNA double-strand damage quantification. A dose establishment study identified 6.25 µM as the lowest effective dose (tested, 1–25 µM), which was selected for subsequent studies.

Immunoblotting was performed as previously described by Jarboe et al (5). Mammalian protein extraction reagent lysis buffer supplemented with protease (cat no. P8340; Sigma-Aldrich; Merck Millipore) and phosphatase inhibitors (cat nos., P0044 and P5726; Sigma-Aldrich; Merck Millipore) was used to lyse cells. Protein concentration was determined by using Pierce Bicinchoninic Protein Assay kit (Thermo Fisher Scientific, Inc.) and ~10 µg of protein were separated via electrophoresis on an 8% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Immobilon; Merck Millipore). Blots were blocked in 5% bovine serum albumin (BSA) (Sigma-Aldrich; Merck Millipore) at room temperature for 1 h and probed with the following primary antibodies: Phosphorylated-MARCKS (cat no., ab81295), MARCKS (cat no., ab52616; both Abcam, Cambridge, UK), phosphorylated-Akt (Ser473) (cat no., D9E, 4060), Phospho-Akt (Thr308) (cat no., C31E5E, 2965), Akt (cat no., C67E7, 4691; all Cell Signaling Technology. Inc., Danvers, MA, USA). Antibodies were used at a dilution of 1:1,000 with overnight 4°C incubation. Additionally, actin antibody (cat no. sc-1616; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used at a concentration of 1:5,000 and incubated overnight at 4°C. Blots were washed 3 times for 5 min in TBST. Peroxidase-conjugated Affini Pure Goat Anti-Mouse IgG (cat no., 115-035-166) and Affini-Pure Donkey Anti-Rabbit IgG (cat no. 711-035-152) secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) was added at a dilution of 1:5,000 in 5% BSA-TBST, and incubated for 1 h at room temperature. Enhanced chemiluminescence (ECL) using Western Lighting-Plus ECL substrate (PerkinElmer, Inc., Waltham, MA, USA) was used to visualize the western blots as described previously (14).

RNA expression analysis was performed at the UAB Nanostring Laboratory (Birmingham, AL, USA) (www.uab.edu/medicine/radonc/en/nanostring). The nCounter^® GX Human Cancer Reference kit (NanoString Technologies, Inc., Seattle, WA, USA) was used to analyze RNA expression of 230 human cancer-associated genes. MARCKS expression in A549 NP-MARCKS and A549 WT-MARCKS cells was induced by overnight incubation at 37°C with doxycycline, and total RNA was collected the following day. A total of 100 ng RNA was prepared in a 30 µl reaction volume and run on the automated nCounter system. Quality control and normalization were performed on the raw data using positive and negative control spots on the included GX Human Cancer Reference Panel chip and five housekeeping reference genes (16,17).

Cells were plated in black 96-well plates (cat no. 3603; Costar; Thermo Fisher Scientific, Inc.) and allowed to adhere overnight. The following morning the control or ED peptides were added. A total of 1 h following peptide treatment, cells in the irradiation group were subjected to 0, 5 or 8 Gy irradiation using a 320 kV X-ray irradiator (Kimtron Inc., Woodbury, CT, USA). A total of 4 days following peptide addition or irradiation, ATP levels were measured via the ATPlite™ Luminescence Assay System (PerkinElmer, Inc., Waltham, MA, USA) using a Synergy H1 Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA).

The animal protocol was provided by the University of Alabama at Birmingham's Institutional Animal Care and Use Committee. Mice were housed at ~24°C and given free access to food and water. A 12 h light/dark schedule was provided for the mice. A total of 1 million WT- or NP-MARCKS-expressing A549 lung cancer cells, were injected into the flanks of ~7 week old female athymic nude mice (~17 g) (Charles River Laboratories, Hartford, CT, USA). Cells were mixed in a 1:1 mixture of PBS:Matrigel^® (BD Biosciences, San Jose, CA, USA) to a final volume of 50 µl and loaded into a 1 ml syringe with a 25 gx 5/8” needle (Monoject™ Tuberculin Syringe; cat no. #8881501640; Covidien, Dublin, Ireland) (18). When tumors were palpable, mouse food was subsequently supplemented with doxycycline (cat no. TD.05125; Harlan Laboratories, Madison, WI, USA) (19). WT- and NP-MARCKS expression was induced with doxycycline-supplemented mouse food for 7 days prior to tumor volume calculation. Tumor volume was measured using Vernier calipers using the formula: (Length × width²)/2 (20,21). Values are presented as the mean fold change in tumor volume ± standard error of the mean (SEM). Mice were euthanized with CO₂ inhalation as set forth by institutional guidelines.

Following overnight induction of MARCKS expression, lung cancer cells were treated with the control or ED peptides 1 h prior to 8 Gy irradiation. As described previously, cells were fixed and stained with an anti-phosphorylated-γH2AX-S139 antibody and counterstained with DAPI (14). γH2AX staining foci marking DNA double-strand breaks were analyzed using an EVOS FL digital inverted fluorescence microscope (Thermo Fisher Scientific, Inc.). Positive events were defined as ≥10 foci per cell, and positive and negative controls were included in all experiments (14,22–24).

Statistics calculations and data graphing was performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). Analysis of variance followed by the Bonferroni correction post hoc test was used to quantify DNA damage, and a Student's t-test was performed to analyze survival assays. All statistics were two-sided and P<0.05 was considered to indicate a statistically significant difference. Values are presented as the mean ± SEM.

MARCKS ED is known to have multiple biological functions; however, limited information is available regarding the involvement of MARCKS in lung cancer biology (25). Reversible phosphorylation of MARCKS ED is important in regulating the function and subcellular localization of MARCKS (10,26). In the present study, A549 lung cancer cells were engineered to express WT- or NP-MARCKS under the regulation of a tetracycline inducible promoter. Western blotting (Fig. 1A) confirmed that A549 cells were able to overexpress WT- and NP-MARCKS in a tetracycline/doxycycline-inducible manner. Due to the ability of MARCKS to regulate multiple signaling cascades (25), the effect of MARCKS phosphorylation on gene expression was analyzed. Using the nCounter^® GX Human Cancer Reference kit, differences in gene expression between A549 cells overexpressing WT- and NP-MARCKS were investigated. A total of 19 genes exhibited a >1.5 fold increase in expression in the WT-MARCKS cell line compared to the NP-MARCKS cell line, including siah E3 ubiquitin protein ligase 1, androgen receptor, matrix metalloproteinase (MMP) 1, platelet derived growth factor subunit A, protein tyrosine kinase 7, notch 1, MYB proto-oncogene, transcription factor, caspase 10, Wnt family member 10B, MMP9, retinoic acid receptor alpha, WD repeat domain 87, v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog, B-cell CLL/lymphoma 6, leukemia inhibitory factor, fibroblast growth factor receptor 4, S100 calcium binding protein A4, cytochrome P450 family 1 subfamily A member 1 and Fos proto-oncogene, AP-1 transcription factor subunit (Fig. 1B). Several of these genes are associated with tumorigenesis and cancer progression (27–31). Cell viability assays were performed to investigate if the ED phosphorylation status was important in lung cancer proliferation. The ATPlite assay indicated significantly increased levels of ATP in the WT-MARCKS A549 cells compared with the NP-MARCKS cell line (Fig. 1C; P=0.002), suggesting that non-phosphorylated MARCKS impedes cell viability. In addition, in vivo mouse studies were used to assess the ability of MARCKS ED phosphorylation status to influence lung cancer growth. WT- and NP- MARCKS A549 cells were injected into the flanks of athymic nude mice. Mouse food was supplemented with doxycycline to induce the expression of WT- and NP- MARCKS (19). The change in tumor volume was compared between the WT- and NP-MARCKS A549 cells over a period of 40 days. WT-MARCKS transfected mice exhibited a greater increase in tumor volume compared with NP-MARCKS transfected mice (Fig. 1D). By modeling the data to a nonlinear regression fit for exponential growth, a significant difference between the WT- and NP-MARCKS tumors (P<0.0001) was observed. The results of the present study suggest that the phosphorylation status of MARCKS ED influences gene expression, viability and tumor growth.

Previously, a 25-amino acid peptide mimetic of MARCKS ED was used to alter lung cancer biology (14). The 25-amino acid ED peptide was conjugated to the cell permeable TAT-peptide, which allowed the ED mimic to enter the cell. Swiss-Prot software calculated a control peptide comprised of 25 random amino acids to control for peptide length. The previous investigation observed changes in cell physiology due to the ED peptide; however, the ability of the peptide to influence MARCKS phosphorylation or proliferation was not investigated (14). Dose titration determined that 6.25 µM was the ideal concentration (data not shown). A549, H1792 and H1975 lung cancer cell lines were cultured without peptide, with 6.25 µM control peptide or with 6.25 µM ED mimetic peptide. Western blot analysis revealed that the ED peptide mimetic was able to decrease the level of phosphorylated MARCKS in the three lung cancer cell lines (Fig. 2A). Decreasing the level of phosphorylated MARCKS may lead to increased plasma membrane-associated MARCKS and decreased Akt activation. Treatment of A549, and to a lesser extent, H1975, cells with the ED-peptide was able to decrease levels of Akt phosphorylated at the serine 473 and threonine 308 residues (Fig. 2A). In addition, the ED-peptide was able to reduce the total level of Akt, which was most pronounced in the H1792 cells (Fig. 2A). Notably, minimal phosphorylated T308 Akt was observed in the H1792 cells in all conditions, though phosphorylated serine 473 Akt was detectable and was mildly suppressed with the ED-peptide. The effect of MARCKS ED peptide on lung tumor growth was investigated. ATP levels in A549, H1972 and H1975 cell lines treated with the ED peptide mimetic were normalized to ATP levels in lung cancer cells with no peptide intervention. A significant decrease in ATP levels in the A549 (P=0.045), H1792 (P=0.000002) and H1975 (P=0.000003) lung cancer cells following 4 days of treatment with the ED peptide was observed compared with the control cells (Fig. 2B).

Previously, lentiviral manipulation was used to overexpress NP-MARCKS in A549 cells and their sensitivity to radiation increased (14). In the present study, the effect of treatment with the MARCKS ED peptide mimetic on the radiation sensitivity of lung cancer cells was analyzed. A549, H1792 and H1975 cells were cultured with either the control or ED peptides 1 h prior to treatment with ionizing radiation. The three cell lines exhibited significantly decreased survival following ED peptide treatment compared with the control group at irradiation levels 0, 5 and 8 Gy (Fig. 3A; P<0.05). DNA double-strand breaks in A549 and H1975 cells were examined by measuring γH2AX foci (22). γH2AX foci increased in all groups 1 h following irradiation; however, 24 h following irradiation A549 and H1975 cells treated with the control peptide exhibited basal level γH2AX foci staining. Conversely, A549 and H1975 cells treated with the ED peptide demonstrated persistent γH2AX foci staining (Fig. 3B; P<0.01 compared with the control). The results of the present study suggest that altering the phosphorylation status of MARCKS ED increases lung cell sensitivity to radiation and prolongs DNA damage following irradiation.