Background
Lung cancer is the leading cause of cancer-related incidence and mortality throughout the world [
1]. Non-small cell lung cancer (NSCLC) is the major subtype of lung cancer, which is typically divided into two histological subtypes, lung adenocarcinoma (ADC) and lung squamous carcinoma (SCC) [
2]. In addition to differences in morphology, the underlying mechanisms, molecular profiling and therapeutic methods are quite different [
3‐
5]. Therefore, identifying differentially-expressed genes between ADC and SCC is useful to better understand their pathogenesis.
In the process of cancer development, cell junction molecules play a critical role in inhibiting tumor growth and metastasis [
6‐
10]. Previous evidence revealed that desmosomal proteins, as a member of cell junction molecules, are deregulated in various cancers, including lung cancer [
11]. In lung cancer, different members of desmosome genes have different roles in tumor progression. For example, plakophilin 1 (PKP1) can be useful in the diagnosis of patients with SCC [
12,
13]. Plakophilin 3 (PKP3) promotes tumor growth in lung cancer [
14]. Decreased desmocollin 1 (DSC1) expression is associated with poor prognosis in human lung cancer [
15]. Desmoglein 2 (DSG2) acts as an oncogene by accelerating tumor growth of non-small-cell lung carcinoma (NSCLC) [
16]. Desmoglein 3 (DSG3) is underexpressed in lung cancer, and a lower expression of DSG3 is significantly linked to higher tumor grade [
17]. However, the downstream signaling pathway of these desmosomal genes in regulating tumor progression is not entirely clear, except for the three canonical tumor suppressor desmoplakin (DSP), junction plakoglobin (JUP) and desmocollin 3 (DSC3). The expression of DSP and JUP are reduced or absent in lung cancer and re-expression results in reduction of cell growth and migration by suppressing the Wnt signaling pathway in lung cancer [
12‐
14]. DSC3 acts as a tumor suppressor through inhibiting the EGFR/ERK pathway in lung cancer [
17,
18]. In previous studies, higher expression of desmosomal genes were found in SCC tissues compared with ADC tissues [
2,
4,
17,
19,
20]. However, the differentce in regulatory mechanisms of desmosomal genes between ADC and SCC are still not clear.
SOX30 is a member of the Sox family of transcription factors that has been isolated from Fugu, human, mouse and the Nile tilapia [
21]. A high SOX30 expression has a favorable and independent prognostic factor for ADC patients but not for SCC patients [
22]. Our in vitro experiments show that SOX30 overexpression significantly inhibits proliferation by inducing apoptosis in ADC but not in SCC [
19,
22]. These studies indicate that SOX30 may have different functional roles in distinct subtypes of lung cancer. Thus, the different downstream signal mechanism of SOX30 in these two histological subtypes of lung cancer needs to be further investigated.
In this study, our data demonstrates that the expressions of desmosomal genes are regulated by SOX30 in ADC but not in SCC. Mechanistically, SOX30 activates desmosomal gene expression through direct binding to the promoter regions of the desmosomal genes in ADC cells. Our findings elucidate the regulatory mechanisms of desmosomal genes in ADC.
Methods
Cell lines
The lung cancer cell lines (A549, LTEP-a-2, H520 and H226) were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China), cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco, CA). All the cells were maintained at 37 °C with 5% CO2.
Plasmid construction and cell transfection
Construction of SOX30 expression vector was performed as previously described [
19,
22]. For knockdown, two pairs of oligomeric singlestranded oligonucleotides and a pair of negative oligomeric singlestranded oligonucleotides were synthesized, and then inserted into miRNA expression vector pcDNA6.2-GW/EmGFP-miR. Cells were transfected using Lipofectamine2000 Reagent (Invitrogen Preservation, Carlsbad, CA, USA) according to the manufacturer’s instructions. The stably transfected cells were screened under G418 (Calbiochem, La Jolla, CA, USA) or Blasticidin (Sigma). Cell clones were obtained by the limited diluted method.
RT-PCR and qRT–PCR analysis
The RNA of cells was isolated using the Trizol reagent (Invitrogen, Life Technologies), and conversion of total RNA to cDNA was performed with the Reverse Transcription System (Promega, Madison, WI, USA). All qRT-PCR reactions was performed using the C1000 Real-Time Cycler (Bio-Rad Laboratories, Hercules, CA, USA) and qRT-PCR Master mixes (Promega, Madison, WI, USA). Primers for amplification of the DSC1, DSG1, DSC2, JUP, DSP, PKP1, DSC3, DSG3, PKP3 and ACTIN genes are listed in Additional file
1: Table S1. All experiments were carried out in triplicate, and the 2
-ΔΔt method was used to determine expression of the genes of interest.
MTS
For knockdown, A549 and LTEP-a-2 cells with SOX30 or empty vector stably expression were plated at 4 × 103 cells per well on 96-well plates, and transfected with desmosomal gene miRNA or negative control. Cell proliferation was assessed using MTS Reagent (Promega, Madison, WI, USA) on days 1, 2, 3, 4 and 5 after transfection. The assays were performed in triplicate.
Stable transfected A549 and LTEP-a-2 cells (n = 1500) were seeded into 100 mm cell culture dish and maintained in media for 14 days. Surviving colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Beyotime Biotechnology, China) for 15 min. Then, the cell colonies were eluted by acetic acid and the absorbance of crystal violet were measured at 570 nm. The experiment was carried out in triplicate wells for three times.
Boyden chamber migration/invasion assay
Transwell assays were performed by using transwell plates with 8 μm pore (Corning; 3422). For the migration assay, 1 × 104 Cells were resuspended in serum-free RPMI-1640 medium and plated onto the 24-well upper chamber with the lower chamber filled with complete medium. For the invasion assay, the 24 transwell plate with matrigel (BD biosciences) polymerized at the 24-well upper chamber for 2 h at 37 °C as the intervening invasive barrier. After 24 h incubation at 37 °C, the cells on the upper chamber were removed and the number of cells that migrated or invaded to the lower side were fixed in 4% paraformaldehyde and stained with 1% crystal violet and counted at × 200 magnification in 10 different fields of microscope (Leica; D-35578). The results were determined from three repeated experiments.
In vivo tumorigenicity assay
For the xenograft tumor growth assay, a total of 1 × 106 stable transfected A549 cells suspended in 150 μl PBS were injected subcutaneously into the right flanks of 5-week-old male Balb/c nude mice (n = 4 mice per group), respectively. Tumors size were measured every 3–5 days with calipers after injection, and the tumor volume was calculated based on formula: 0.5 × (length × width2). After 42 days housing, the mice implanted tumors were sacrificed, and liver tissues were dissected and subjected to histological examination. Metastatic nodules were detected by H&E staining and quantified by counting metastatic lesions in each section. The images of the positive areas were taken. All experimental animal procedures were approved by the Institutional Animal Care and Use Committee of Third Military Medical University, China.
WB analysis
WB was performed as previously described [
20]. ACTIN was used as a loading control. The following primary antibodies were used: SOX30 rabbit polyclonal antibody (1:100; Santa Cruz Biotechnology; sc-20,104), DSP rabbit polyclonal antibody (1:100; Santa Cruz Biotechnology; sc-33,586), JUP rabbit polyclonal antibody (1:100; Santa Cruz Biotechnology; sc-7900), DSC3 rabbit polyclonal antibody (1:100; Santa Cruz Biotechnology; sc-48,750), CCND1 rabbit polyclonal antibody (1:100; Santa Cruz Biotechnology; sc-717), MMP7 rabbit polyclonal antibody (1:100; Santa Cruz Biotechnology; sc-30,071), ERK1/2 rabbit polyclonal antibody (1:100; Santa Cruz Biotechnology; sc-93), p-ERK1/2 rabbit polyclonal antibody (1:100; Santa Cruz Biotechnology; sc-101,761) and ACTIN monoclonal antibody (1:2000; Sigma; A5441). ImageJ software was used to quantify the protein expression.
Immunofluorescence cell staining (IF)
Lung cancer cell lines with SOX30 or vector control stable expression grown on sterile glass coverslips, washed with PBS, fixed in 4% paraformaldehyde for 15 min and permeabilized by 0.5% Triton for 15 min at room temperature. Following blocking with 1% bovine serum albumin for 30 min, rabbit polyclonal antibody SOX30 (1:100; Santa Cruz Biotechnology; sc-20,104), mouse monoclonal antibody DSP (1:100; Santa Cruz Biotechnology; sc-365,981), mouse monoclonal antibody JUP (1:100; Santa Cruz Biotechnology; sc-33,634) and mouse monoclonal antibody DSC3 (1:100; Santa Cruz Biotechnology; sc-81,806) were incubated overnight at 4 °C. After washing three times, the cells were incubated with an appropriate fluorochrome-conjugated secondary antibody (1:300; Invitrogen; A-11010 and A-11003) for 1 h at 37 °C in the dark. After blue nuclear counterstaining with 4, 6-diamidino-2-phenylindole (Beyotime, Shanghai, China; C1006) for 10 min at room temperature, coverslips were mounted and observed with a fluorescence microscope (Zeiss, Oberkochen, Germany; LSM800). ZEN software was used to quantify the protein expression.
Patient sample and immunohistochemical (IHC) analysis
Tissue microarrays contained a total of 30 lung cancer patient tissue samples including 15 ADCs and 15 SCCs were obtained from the collaboration (Shanghai Biochip Co Ltd., Shanghai, People’s Republic of China). The rabbit polyclonal antibodies used were SOX30 rabbit polyclonal antibody (1:100; Santa Cruz Biotechnology; sc-20,104), DSP rabbit polyclonal antibody (1:100; Santa Cruz Biotechnology; sc-33,586), JUP rabbit polyclonal antibody (1:100; Santa Cruz Biotechnology; sc-7900), DSC3 rabbit polyclonal antibody (1:100; Santa Cruz Biotechnology; sc-48,750). IHC staining was performed as described previously [
19].
Chromatin immunoprecipitation (ChIP) assay
ChIP analysis was performed using a ChIP Assay Kit (Pierce, Rockford, IL, USA). The immunoprecipitated and input DNA was used as a template for RT–PCR analysis using the primers listed in Additional file
1: Table S1.
Chemoncogenic model
C57BL/6 J (SOX30 conditional knockout) mice were obtained from the model animal research center of Nanjing University (Nanjing, China) and maintained in a controlled environment (12-h light/dark cycle, ad libitum access to food and water) at the laboratory animal facility of Third Military Medical University (Chongqing, China). The W/W mice (n = 5) and K/K mice (n = 5) that were used were matched for age (14 weeks) and weight (16–19 g). These mice received a intraperitoneal injection of urethane (1 g/kg in 100 μl saline) once a week for 10 consecutive weeks. Mice were sacrificed after 4 months. Lung neoplastic lesions were observed by H&E staining. All experiments on mice were approved by the Institutional Animal Care and Use Committee of Third Military Medical University, China.
Analysis of publicly available datasets
GSE43580 dataset were used to analyze differential gene expression between ADC and SCC. The protein expression of desmosomal genes in human lung tumor tissues were determined from the human protein atlas (
www.proteinatlas.org). TCGA lung adenocarcinoma RNAseq data (
n = 571) and TCGA lung squamous carcinoma RNAseq data (
n = 553) were used to compare the expression of SOX30 with desmosomal genes.
Statistical analysis
Statistical analyses were performed with the SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Each experiment was performed at least three times. The data were presented as the means ± SD. Results of expression analyses, cell proliferation, colony formation, migration and invasion were analyzed using the two-tailed Student’s t-test. Correlation analysis of gene expression was performed using Spearman’s rank correlation coefficient analysis. A two-sided P-value<0.05 was taken as statistically significant.
Discussion
Desmosomes are intercellular junctions that tether intermediate filaments to the plasma membrane and represent the major adhesive cell junctions of epithelial cells [
30]. It has been reported that desmosome family members play a crucial role in tumor growth and metastasis [
21,
31,
32]. Many studies suggest that the expressions of desmosomal genes can be regulated by transcription factors. For instance, SIP1/ZEB2 induces EMT partly by repressing the expressions of DSP and PKP2 [
33]. DSG4 is repressed by HOXC13, LEF1 and FOXN1 in hair shaft differentiation [
34]. The p53 induced expression of DSC3 implicated in human lung cancer [
28]. Stat3 regulates DSG3 transcription in epithelial keratinocytes [
35]. KLF5 mediates the transcription of DSG2 in mouse colons [
36]. All these data indicated that transcription factors may be important in the regulation of desmosomal genes. Nevertheless, prior to this study, the regulatory mechanisms of desmosomal genes in tumorigenesis remained largely uninvestigated.
In this study, we found differences in expression of desmosmal genes between ADC and SCC. These result indicate that the regulatory mechanisms of desmosomal genes between ADC and SCC are different. Previously, we identified SOX30 as an epigenetically silenced tumor suppressor, inhibiting cell proliferation and inducing cell apoptosis in ADC but not in SCC. Thus, we speculated that the expressions of desmosomal genes might be regulated by SOX30 in ADC. To further explore the regulatory mechanisms of desmosomal genes in ADC and SCC, we analyzed the correlation between desmosomal gene expression and SOX30 expression in ADC and SCC using TCGA data. We found a positive correlation between desmosomal genes and SOX30 in ADC but not in SCC. Further studies demonstrated that SOX30 upregulated the expressions of desmosomal genes by directly binding to the ACAAT motif of their promoter regions in ADC cells, but loss of regulatory function in SCC cells. Because SOX30 acts as a transcription factor, we suspected that there is another molecule necessary for regulatory function of SOX30, and this molecule interferes the function of SOX30 in SCC. Here, we found other desmosomal genes, including DSC1, DSG1, DSC2, PKP1 and DSG3, were also up-regulated by SOX30 (Fig.
2c). Moreover, according to previous reports, PKP3 is dysregulated in lung cancer [
11]. Therefore, we analyzed the correlation between SOX30 and PKP3 and found that there is no correlation between the two (Additional file
4: Figure S3a). The qRT-PCR results confirmed that PKP3 expression is not upregulated by SOX30 overexpression (Additional file
4: Figure S3b). We then analyzed the promoter regions of these desmosomal genes and found all of them contain an ACAAT motif except PKP3 (data not shown), indicating that SOX30 upregulated their expression possibly by binding to their promoter regions and activating transcription. Notably, this hypothesis should be empirically tested, and further studies are required to address this aim.
To define whether desmosomal genes are involved in the antitumor effects of SOX30, we focused on the most differentially expressed desmosomal genes associated with SOX30 and chose the three prominent tumor suppressors DSP, JUP and DSC3. These three desmosomal genes were the main targets through which SOX30 suppresses the tumorigenesis and metastasis of ADC. In vitro and in vivo assays demonstrated that desmosomal genes, DSP, JUP and DSC3 were required for SOX30-medicated cell proliferation and metastasis in ADC. Taken together, these results suggested that the “SOX30-desmosomal genes axis” may act as tumor suppressor in ADC. To obtain further insight into the downstream signaling pathway of SOX30-desmosomal genes axis in inhibiting tumor growth and metastasis of ADC, we detected the change of Wnt and ERK signaling pathway related molecules by WB assays and demonstrated that SOX30 suppressed Wnt and ERK signal in a desmosomal gene dependent manner.
To further evaluate the role of the SOX30-desmosomal gene axis in lung tumor development, the chemical carcinogenesis model of SOX30-knockout mice was employed. We found that knockout of SOX30 promotes lung tumor formation in mice with urethane treatment and is associated with desmosomal gene loss. These results suggested that SOX30-desmosomal gene axis might be involved in the occurrence and development of lung tumor. To evaluate this hypothesis, we plan to establish a SOX30 conditional knockout mice to further clarify the important role of the “SOX30-desmosomal gene axis”.