Background
Lung cancer, a leading cause of cancer death worldwide, is classified into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). SCLC is characterized by highly aggressive and malignant metastasis. As one of the main features of SCLC is extensive distant metastasis in early phase, it remains one of the most lethal cancers, leading to poor survival with a five-year survival rate of only 3–8% [
1].
Matrix metalloproteinases (MMPs) are the principal enzyme group involved in the degradation of a number of extracellular matrices (ECM). Increased levels of MMPs have been detected in numerous cancers and were correlated with tumor aggressiveness [
2]. For example, MMP-1, −2, −7, −9, −14, and −15 were overexpressed in NSCLC [
3‐
6], and elevated MMP-1, −9, −11, −13, and −14 levels were also shown in SCLC [
7,
8]. Inhibition of MMP transcription prevented invasion
in vitro and decreased the colonization of the lung cancer cells in an
in vivo tail vein metastasis model [
9], indicating that transcriptional regulation is the main regulatory pathway controlling the expression of MMPs. Although interleukin 1 (IL-1), tumor necrosis factor alpha (TNFα), histone acetylation and deacetylation, and DNA methylation affected MMP expression [
10‐
13], clinical trials using MMP inhibitors showed limited benefits to alter the metastatic process [
2,
14]. This data suggests a complex relationship between MMPs and tumor migration. Therefore, investigation of the detailed molecular mechanisms underlying the regulation of MMP expression and the correlation with metastasis in cancer, particularly in SCLC, is warranted.
The E2F1 transcription factor is a well-documented modulator that functions in the regulation of cell cycle, proliferation, and apoptosis. Recent reports have suggested a role for E2F1 in promoting angiogenesis and metastasis through regulation of thrombospondin 1 [
15], platelet-derived growth factor receptor (PDGFR) [
16], vascular endothelial growth factor receptor (VEGFR) [
17], and MMP-9, −14, and −15 [
9]. Additionally, E2F1 could promote lung metastasis of colon cancer [
18] and regulate cellular movement by cell-cell and cell-matrix interactions in yeast [
19,
20]. Although E2F1 is highly expressed in SCLC [
21], the role of E2F1 in the process of invasion and metastasis remains unclear in SCLC.
This study is designed to investigate whether the increased E2F1 participates in the invasion and metastasis through MMP regulation in SCLC. Our results showed that E2F1 was predominantly expressed in SCLC and was an independent and adverse prognosis factor. E2F1 promoted cellular migration through directly modulating the expression of MMP-16 and transcription factors Sp1 and p65 (subunit of NF-kappa B), which in turn regulated MMP-9 expression in SCLC cells.
Methods
Patients
This study consisted of 140 patients (90 SCLC samples, 20 adenocarcinoma samples, 20 squamous and 10 large cell lung cancer samples) between January 2008 and December 2010. Tissue samples were obtained from Qilu Hospital affiliated with Shandong University and Jinan Central Hospital. Among the 90 SCLC tissue samples, 88 cases were biopsy specimens and 2 cases were surgical resections. The clinical data were obtained from the patients’ files (Table
1). This study was approved by the Medical Ethics Committee of Shandong University and all patients provided informed consent when the tissues were donated.
Table 1
The information and clinical characteristics of patients
Aa
| 59.34 | 47-82 | 11 | 9 | 13 | 7 | | | 10 | 6 | 4 |
Sb
| 61.47 | 45-79 | 13 | 7 | 8 | 12 | | | 9 | 6 | 5 |
LCLC | 62.69 | 53-81 | 7 | 3 | 6 | 4 | | | 5 | 3 | 2 |
SCLC | 55.57 | 28-83 | 68 | 22 | 69 | 21 | 22 | 68 | | | |
Cell lines
Human SCLC cell lines (H1688 and H446), a human squamous cell line (SK-MES-1), and a human normal fibroblast epithelial cell line (HFL-1) were purchased from Shanghai Cell Library of Chinese Academy of Science. Human adenocarcinoma cell lines (A549, H292 and H1299) and a human normal bronchial epithelial cell line (HBE) are stored in our lab.
Immunohistochemistry
Immunohistochemistry (IHC) was performed according to our previous report [
22,
23]. The dilutions of antibodies were 1:50 for E2F1 (Merk Millipore, USA), MMP-7, MMP-9, MMP-16 (Abgent, China), MMP-2, Sp1, p65 (Santa Cruz Biotechnology, USA) and VEGFR (Cell Signaling Technology, USA). The staining samples were scored by two pathologists without any knowledge of the clinical pathological outcomes. Staining intensity was divided into four grades: 0 as negative; 1 as weak intensity (less than 10% positive); 2 as moderate intensity (more than 10% and less than 60% positive); and 3 as strong intensity (more than 60% positive). Grade 0 was considered as negative expression, and grades 1, 2, and 3 were considered as positive staining.
siRNA transfection
The siRNAs targeting E2F1, Sp1, and p65, and the scramble control siRNA were designed, modified and synthesized by Invitrogen. The siRNA sequences are listed in Table
2. siRNA transfection and experiments were performed using Lipofectamine 2000 as our previous reports [
22,
24,
25].
Table 2
The sequences of siRNA target genes
siRNA1 of E2F1 | 5’-AUGCUACGAAGG UCCUGACACGUCA-3’ |
siRNA2 of E2F1 | 5’-AAAGUUCUCCGAAGAGUCCACGGCU-3’ |
siRNA1 of Sp1 | 5’-AGCCUUG AAGUGUAGCUAU-3’ |
siRNA 2 of Sp1 | 5’-GGUAGCUCUAAGUUUUGAU-3’ |
siRNA1 of p65 | 5’-GATTGAGGAGAAA CGTAAA-3’ |
siRNA2 of p65 | 5’-GATGAGATCTTCCTACTGT-3’ |
Scramble siRNA | 5’-UUCUCCGAACGUGUCACG UTT-3’ |
Real time PCR
Total RNA was extracted by Trizol (Sigma, USA). The reverse transcription was conducted by a cDNA synthesis kit (Ferments, USA) and real time PCR was performed with SYBR Green (TOYOBO, Japan). The primers for target genes are listed in Table
3.
Table 3
The primers of target genes for real time PCR
E2F1 | F: 5’-CATCAGTACCTGGCCGAGAG-3’ |
R: 5’-TGGTGGTCAGATTCAGTGAGG-3’ |
Sp1 | F: 5’-CCACCATGAGCGACCAAGAT-3’ |
R: 5’-TGAAAAGGCACCACCACCAT-3’ |
p65 | F: 5’-CCCACGAGCTTGTAGGAA AGG-3’ |
R: 5’-GGATTCCCAGGTTCTGGAAAC-3’ |
MMP-3 | F: 5’-TGAGGACACC AGCATGAACC-3’ |
R: 5’-CAGGACCACTGTCCTTTCTCC-3’ |
MMP-7 | F: 5’-GAGT GAGCTACAGTGGGAACA-3’ |
R: 5’-CTATGACGCGGGAGTTTAACAT-3’ |
MMP-9 | F: 5’-TTCCAAACCTTTGAGGGCGA-3’ |
R: 5’-GCAAAGGCGTCGTCAATCAC-3’ |
MMP-14 | F: 5’-ATCGCTGCCATGCAGAAGTT-3’ |
R: 5’-TGTCTGGAACACCAC ATCGG-3’ |
MMP-15 | F: 5’-GAGATGCAGCGCTTCTACGG-3’ |
R: 5’-GCTTTCA CTCGTACCCCGAA-3’ |
MMP-16 | F: 5’-TTCGGGGGTGTTTTTCTTGC-3’ |
R: 5’-GGT GGAAGGTAGCCGTACTT-3’ |
VEGFR | F: 5’-AAAGGCACCCAGCACATCAT-3’ |
R: 5’-TCCTTACTCACCATTTCAGGCA-3’ |
Western blotting
Cells were lysed in RIPA lysis buffer. A total of 40 μg protein was separated by SDS-PAGE and samples were electrophoretically transferred onto nitrocellulose membranes. The membranes were blocked with 5% fat-free dry milk and incubated with primary antibodies against E2F1 (1:100, Merk Millipore), Sp1, p65 (1:100, Santa Cruz Biotechnology), MMP-3, −7, −9, −14, −15 and −16 (1:200, Abgent), Vascular endothelial growth factor receptor (VEGFR, 1:1000, Cell Signaling Technology), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:2000, Santa Cruz Biotechnology) at 4°C overnight. The membrane was washed and incubated with HRP-conjugated secondary antibodies for 45 min. The immunoblot bands were detected by an ECL system, and membranes were exposed to X-ray films [
22].
Wound healing analysis and transwell experiments
The wound healing experiment was performed according to a previous report [
9]. The cells were scratched by a 10 μl pipette tip and photographed by microscopy at 0, 12, and 24 h. The transwell experiment was conducted according to the manufacturer’s instruction (BD Company). A total of 60 μl of matrigel was placed into the upper chamber and plates were incubated for 3 h at 37°C. After the matrigel solidified, 1 × 10
4 cells were plated into the upper chamber with media containing 1% fetal bovine serum. Media containing 10% fetal bovine serum was placed into the lower well. After 72 h, the matrigel was cleaned and the cells were stained by Gimsa Dye. The cells that invaded through the chamber were quantified by counting three fields.
ChIP-to-sequence
Chromatin immunoprecipitation (ChIP) was conducted according to the manual supplied by Merck Millipore Company (ChIP Assay kit, Cat. No.: 17–295). Cells (5 × 10
7) were prepared and cross-linked by 1% final concentration of formaldehyde at 37°C for 10 min. Cells were centrifuged at 2,000 rpm for 4 min at 4°C, and then collected and incubated in SDS Lysis Buffer on ice for 10 min. The genomic DNA was sheared with Sonicate (36% strengthen, 25 sec and 30 cycle) and the average length of the fragments generated was 200 bp. Protein A agarose beads were added to the samples for 30 min at 4°C with agitation. Next, E2F1 antibody (4 μg) or equal amount of normal mouse IgG was added into the samples, and samples were incubated at 4°C with rotation overnight. The agarose beads were collected by gentle centrifugation (800 rpm) for 5 min and washed five times. Reverse cross-linking was performed with high salt solution (5 M NaCl) and the DNA fragments were obtained. The cyclin D1 primer was used as a positive control in real-time PCR. The DNA fragments were sequenced by BGI Company (
http://www.genomics.cn). The Hiseq2000 50SE sequencing platform was used and the data analysis algorithm included SOAP2.20 comparison and MACS peak calling. Clean data was obtained by filtering the low quality data according to a certain criteria: the sequences not containing adapter, N less than 10%, quality values less than 20 and ratio less than 50%. For the peak value, the filtering was conducted according to the
p value obtained by MACS analysis. The data was discarded when the
p value was higher than 1
e-5, which ensured the fidelity of the data and exclusion of false positives.
Construction of the MMP-9, MMP-16, Sp1 and p65 luciferase reporter constructs
Genomic DNA was extracted from H1688 cells, and MMP-9, MMP-16, Sp1 and p65 were amplified by PCR using primer sequences shown in Additional file
1: Table S1. The PCR DNA fragments were extracted by a Gel Extraction kit (Invitrogen, USA). The PCR fragments and pGL3-basic luciferase reporter vector (Promega, USA) were digested with FastDigest SacI, NheI or XhoI (Thermo, USA), extracted and ligated with T4 DNA Ligase (TakaRa, Japan) to generate the four luciferase reporter constructs. The binding site mutants were constructed by overlap PCR and nested PCR, and the primers were listed in Additional file
1: Table S1. The constructs were confirmed through sequencing by BioSune Company.
Transient transfections and luciferase assays
Cells were transiently transfected with 0.5 μg of luciferase reporters and 0.3 μg of E2F1, Sp1, or p65 expression vector with Lipofectamine 2000 (Invitrogen). Cotransfection with 0.02 μg of the pRL-TK Renilla reniformis luciferase served as a normalizing control. Luciferase assays were performed using the Dual Luciferase Assay System (Promega).
Statistical analysis
SPSS 17.0 was used as the statistical software. The immunohistochemistry samples were treated with Chi Square test. The association and statistical difference between E2F1 lower, moderate, and higher and clinicopathological variables was analyzed by Spearman’s analysis and χ2 test. Univariate survival rate was analyzed by the Kaplan-Meier method, and the significant were tested by Log-Rank test. Multivariate survival analysis was performed by using Cox’s regression. The expression differences among target genes were analyzed using paired t test. P < 0.05 was considered to be statistically significant.
Discussion
Transcription factor E2F1 gains more attention due to its predominant functions in controlling cell cycle, tumorigenesis, apoptosis, and aggressiveness [
21,
26‐
29]. Our studies revealed that E2F1 was highly expressed in SCLC of Chinese Han population, associated with high expressions of MMP-7, −9, and −16, but not MMP-2. Overexpression of E2F1 facilitated the expressions of MMP-9 and −16 genes in SCLC. We showed for the first time that MMP-9 expression was transcriptional regulated by Sp1 and NF-kappa B as a consequence of activation of E2F1 in SCLC.
It has been reported that E2F1 was highly expressed in SCLC and promoted SCLC cell proliferation [
21]. However, its expression level in NSCLC showed inconsistent. Eymin’s and Kuhn’s results showed that E2F1 expression was lower in NSCLC [
21,
29], but the studies by Hung, Huang and Gorgoulis displayed that E2F1 was highly expressed in NSCLC [
27,
28,
30]. Here we detected the expression of E2F1 in lung cancer among a Chinese Han population. Our results were consistent with Eymin’s results that E2F1 was highly expressed in SCLC, but not NSCLC [
21]. Further investigation is required to examine the level in populations with large numbers of samples, and to clarify the relationship between E2F1 and lung cancer.
Overexpressions of MMPs were considered to play an important role in metastatic spread of SCLC. Michael
et al. detected the expressions of MMPs and reported a deficiency of MMP-2 in SCLC [
8]. Our results were consistent with their discovery. This study was the first to report the expression of MMP-16 in all SCLC samples (90 of 90). Together this indicated that MMP-16 played an important role in the process of invasion and metastasis of SCLC, and high expression of E2F1 may be the main driver to promote MMP-16 expression.
Some investigators reported that Sp1 or NF-kappa B could regulate the expression of MMP-13, −9 and −2 [
33‐
35]. In our study, E2F1 regulated the expression of MMP-9 mediated by Sp1 and NF-kappa B, indicating the importance of E2F1 in facilitating the expressions of MMP genes. The role of E2F1 in metastatic process was recently investigated in different cancer types. Klein-Szanto’s study showed that E2F1 gene transfer enhanced the invasion of head and neck carcinoma cells [
37]. Chellappan’s group demonstrated that E2F1 influenced metastasis by targeting MMP family members, FLT-1, KDR, and angiopoietin 2 [
9,
17]. In agreement with these observations, we provided additional evidence that Sp1 and NF-kappa B, transcriptional activated by E2F1, promoted aggressive phenotype via upregulation of MMP-9 that was highly expressed in SCLC.
Because genes usually contain multiple binding sites for many transcription factors, it is essential to explore the detailed interactions between transcription factors and DNA or other proteins. E2F1 and Sp1 bind through specific domains in each protein, and their physical interaction and functional synergism appears to be required for the regulation of many genes, including DHFR, MYCN, murine thymidine kinase, and transglutaminase type 1 [
38‐
41]. Several investigators reported a positive interaction between E2F1 and p65. E2F1 firmly bound IκB (Inhibitor of NF-kappa B) to NF-kappa B and inhibited cell adhesion in human aortic endothelial cells [
42]. E2F1 also cooperated with NF-kappa B to regulate BNIP3 to control cell survival [
43,
44]. In our study, we found that E2F1 upregulated the expression of Sp1 and p65 in SCLC, which in turn activated the expression of MMP-9. It remains unclear whether high level of E2F1 cooperates with Sp1 or p65 to regulate other genes involved in malignant phenotype of SCLC. Although E2F1 expression varies in different types of lung cancer, ours together with other’s finding demonstrated that overexpression of E2F1, at least partially, contributed to invasion and metastasis in both SCLC and NSCLC [
9,
21]. Further investigation is required to test a possibility whether E2F1 acts as a target for SCLC therapy.
Acknowledgements
We would like to thank prof. Srikumar P. Chellappan (University of South Florida, Tampa, Florida) for kindly providing the plasmid expressing E2F1. This work was supported by the National Natural Science Foundation of China (No. 81273533 to ZL, YG, HJ, TZ and HY, No. 81302017 to ZL, CJ and HY).
Competing interests
All authors declare that there are no conflicts of interest.
Authors’ contributions
ZL carried out the design of the experiment, participated in immunohistochemistry, rela-time PCR, western blotting, dual luciferase, statistical analysis, and drafted the manuscript. YG carried out the immunohistochemistry, plasmid construction and luciferase analysis. HJ carried out the Chromatin immunoprecipitation. TZ and CJ carried out the collection the clinical samples and the scores of IHC staining. CY polished and modified the language. HY conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.