Introduction

Lung cancer confers the highest cancer mortality rate worldwide. Two major types of lung cancer, lung squamous cell carcinoma (SCC) and lung adenocarcinoma (ADC), are non-small cell lung cancer with distinct histological features and different genomic alterations [1, 2]. SCC is derived from keratin 5 (KRT5 or K5)-positive basal cells of the pseudostratified airway epithelium in the upper and central lungs, and ADC from the epithelial cells of alveoli [Transgenic mice

A mouse TNFR1 full-length cDNA fragment (1,365 bp) in the CMV-Myc Flag TNFR1 vector (MR226545, OriGene, Rockville, MD) was purified after being digested with KpnI and PmeI. This TNFR1 cDNA fragment was subcloned into the BK5 vector [46] that was digested using the enzyme XhoI. Sequencing was used to confirm the insertion of the TNFR1 cDNA fragment (1,365 bp) into the BK5 vector. The construct was linearized with KpnI digestion. The Transgenic Mouse Model Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research (Frederick, MD), generated the transgenic mice from an FVB background and confirmed the transgenic mice by Southern blotting with a TNFR1 cDNA probe. PCR primers for genoty** the transgenic mice were: 5´-TCA GGG GTG TTG TTT AGA ATG G-3´ and 5´-CAA TAA GAA TAT TTC CAC GCC A-3´ were used to genotype the transgenic mice.

Antibodies

Antibodies used in this study included IKKα (IMG-136A) from IMAGENEX; Lamin B (sc-6216), cytokeratin 5 (K5, sc-17090), p50 (sc-1109), c-myc (sc-764), and IgG (sc-2025) from Santa Cruz Biotechnology; p65 (8242), c-Rel (12707), and E-cadherin (24E10) from Cell Signaling Technology; Sox2 (245610) from R&D Systems; Twist1 (T-6451) and β-actin (A-5441) from Sigma-Aldrich; TNFR1 (ab111119 and ab19139) and histone H3 (acetyl K27, ab4729) from Abcam; UBCH10 (NBP2-20782) from Novus Biologicals; K5 (PRB-160P) from Babco; α-Tublin (ab4074) and histone H3 (acetyl K27, ab4729) from Abcam.

Histopathology, immunoblot analysis, and immunohistochemical and immunofluorescent staining

The Histology and Tissue Core Facility at the Frederick National Laboratory for Cancer Research routinely prepared paraffin sections of mouse organs and performed hematoxylin and eosin staining and immunohistochemical staining for TNFR1, K5, E-cadherin, loricrin, and involucrin. Cell lysates (15 µg) or protein extracts from the tissues (20 µg) were separated on acrylamide gels and proteins detected using immunoblotting with specific antibodies, as previously described [48] and were expressed as a fold change in mRNA expression relative to control values.

Cell proliferation assay

Mouse lung tumor cells (1 × 105 /per well) were seeded into 6 well-plates in DMEM with 5% bovine calf serum. The numbers of the treated cells were counted 2 days after the cells were transfected with a control vector or HA-IKKα plasmid. Data represent the mean ± S.D. of three independent experiments. For the MTT assay to examine cell proliferation, the cells were plated in 96-well culture plate (2000 cells/200 µL/well) and incubated in a CO2 incubator; 20 µL of MTT (5 mg/mL, Sigma-Aldrich, M5655) was added to each well of a single plate and the cells incubated for 4 h at 37 °C. Medium was removed, and the formazan crystals formed by the cells were dissolved using 200 µL of DMSO for 30 min. The absorbance was read at 570 nm.

siRNA transfection and cDNA transfection

All siRNAs were purchased from OriGene (Trilencer-27 siRNA), including Tnfrsf1a (mouse, TG517049), Ube2c (mouse, SR404321), TNFRSF1A (human: SR304883, SR517049; mouse: SR414807), Tnf (mouse, SR406508), and HPRT (positive-control siRNA duplex, SR30003). Transfection with Lipofectamine RNAiMAX was performed in accordance with the manufacturer’s instructions. The target protein expression was analyzed 48 h after transduction. Ube2c cDNA plasmid (MR223758L4V), which encodes UBCH10, was purchased from OriGene Biotechnology Research (Rockville).

Chromatin immunoprecipitation (ChIP) assay

Cells were crosslinked with 1% formaldehyde and were lysed and sonicated on ice to generate DNA fragments with an average length of 200–800 bp. After preclearing, 1% of each sample was saved as an input fraction. Immunoprecipitation was performed using antibodies specifically recognizing IKKα (M-280, Santa Cruz Biotechnology), p65 and c-Rel. DNA was eluted and purified from complexes, followed by PCR amplification of the target promoters or genomic loci using primers for mouse Ube2c (p-500: 5'-CGG TCC TTG GAC CCT TTA AT-3' and 5'-TGC TAG GTC CTC CCC AGT AA-3'; p-1000: 5'-CGC TGT CTT CAG ACA CTC CA-3' and 5'-GAG ATG GCT CAG CGG TTA AG-3'; p-1500: 5'-ATA CAC CCT GGC TGA CCT TG-3' and 5'-GAA GGT GGT AAC AGG CAG GA-3'), human UBE2C (p-500: 5'-GTG GGC AAA AGG TGA GTG AT-3' and 5'-GAG CTC CTG GTG TGT TCT CC-3'; p-1000: 5'-GAT TAC GGA CGT GAG CCA CT-3' and 5'-GCG AGG GGA AAA ACT AAA GG-3'; p-1500: 5'-TAA GAC CAA CCT GGG AGC AC-3' and 5'-TGG GAG CAA TAA AAG CCA AC-3'), and mouse Tnfrsf1a (p-500: 5'-TGG CGT GAG TGA CTT TAG GT-3' and 5'-CCA CCC CAA GAA CCA ACA AG-3'). Also, the immunoprecipitation was performed using anti-H3K27ac antibody (ab4729, Abcam) in KALLU- and KALLU- cells. Then, DNA was eluted and purified from complexes, followed by PCR amplification of mouse Tnfr1 promoters with its primer including forward: 5'-TGG CGT GAG TGA CTT TAG GT-3' and Reverse: 5'-CCA CCC CAA GAA CCA ACA AG-3'.

Subcutaneous injection for tumor growth and metastasis studies

Approximately 2 × 105 mouse lung SCC cells KALLU+ cells (with or without si-RNA treatment) and KALLU- cells were injected subcutaneously into athymic NCr-nu/nu mice. Following the cell injection, the tumor growth was monitored with caliper measurements. At day 18 after cell injection, animals were euthanized when tumors reached the maximum size allowed by the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the National Institutes of Health for all animal experiments. To study metastasis, the tumors derived from nude mice were digested with 0.05% trypsin solution at 37 °C for 20 min to make a single cell solution. Isolated cells were cultured for 7 days and then subcutaneously injected into nude mice. At day 18 after cell injection, animals were euthanized and the metastases in the liver and lungs were examined.

Analyses for cancer patient survival

OncoLnc and Kaplan–Meier plotter were used to analyze the correlation between gene expression and patient survival rates. OncoLnc contains the survival data of 8,647 patients representing 21 cancer types, along with RNA-seq expression data of numerous genes performed by TCGA. The association of survival with a single gene was directly performed on the OncoLnc database [49]. For double gene analysis, raw data for each gene, including patient ID, survival days, status, and gene expression levels, were downloaded from OncoLnc and combined to compare the survival rate between double-high and double-low gene expression groups. The Kaplan–Meier plotter can be used to evaluate the effect of 54,675 genes on patient survival using 10,461 cancer samples. The “Use multiple gene” function was applied to analyze the correlation between double gene expression and patient survival [50]. The log-rank p-value and hazard ratio with 95% confidence intervals were determined.