Abstract
BackgroundE2F1 transcription factor plays a vital role in the regulation of diverse cellular processes including cell proliferation, apoptosis, invasion and metastasis. E2F1 overexpression has been demonstrated in small cell lung cancer (SCLC), and extensive metastasis in early phase is the most important feature of SCLC. In this study, we investigated the involvement of E2F1 in the process of invasion and metastasis in SCLC by regulating the expression of matrix metalloproteinases (MMPs).MethodsImmunohistochemistry was performed to evaluate the expression of E2F1 and MMPs in SCLC samples in a Chinese Han population. The impact of E2F1 on invasion and metastasis was observed by transwell and wound healing experiments with depletion of E2F1 by specific siRNA. The target genes regulated by E2F1 were identified by chromatin immunoprecipitation (ChIP)-to-sequence, and the expressions of target genes were detected by real time PCR and western blotting. The dual luciferase reporter system was performed to analyze the regulatory relationship between E2F1 and MMPs.ResultsE2F1 is an independent and adverse prognosis factor that is highly expressed in SCLC in a Chinese Han population. Knockdown of E2F1 by specific siRNA resulted in the downregulation of migration and invasion in SCLC. The expressions of MMP-9 and −16 in SCLC were higher than other MMPs, and their expressions were most significantly reduced after silencing E2F1. ChIP-to-sequence and promoter-based luciferase analysis demonstrated that E2F1 directly controlled MMP-16 expression via an E2F1 binding motif in the promoter. Although one E2F1 binding site was predicted in the MMP-9 promoter, luciferase analysis indicated that this binding site was not functionally required. Further study demonstrated that E2F1 transcriptionally controlled the expression of Sp1 and p65, which in turn enhanced the MMP-9 promoter activity in SCLC cells. The associations between E2F1, Sp1, p65, and MMP-9 were validated by immunohistochemistry staining in SCLC tumors.ConclusionsE2F1 acts as a transcriptional activator for MMPs and directly enhances MMP transcription by binding to E2F1 binding sequences in the promoter, or indirectly activates MMPs through enhanced Sp1 and NF-kappa B as a consequence of E2F1 activation in SCLC.
Highlights
E2F1 transcription factor plays a vital role in the regulation of diverse cellular processes including cell proliferation, apoptosis, invasion and metastasis
E2F1 was highly expressed in small cell lung cancer (SCLC) expression of E2F1 had been detected in lung cancer tissue [21,26,27,28,29,30], its expression was inconsistent among different populations, especially in non-small cell lung cancer (NSCLC)
E2F1 expression was positive in 95.56% (86/90) of SCLC, 50% (5/10) of large lung cancer cell (LCLC), and 10% (2/20) of adenocarcinoma samples compared with the normal alveolar sections
Summary
E2F1 transcription factor plays a vital role in the regulation of diverse cellular processes including cell proliferation, apoptosis, invasion and metastasis. We investigated the involvement of E2F1 in the process of invasion and metastasis in SCLC by regulating the expression of matrix metalloproteinases (MMPs). 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. interleukin 1 (IL-1), tumor necrosis factor alpha (TNFα), histone acetylation and deacetylation, and DNA methylation affected MMP expression [10,11,12,13], clinical trials using MMP inhibitors showed limited benefits to alter the metastatic process [2,14]. Investigation of the detailed molecular mechanisms underlying the regulation of MMP expression and the correlation with metastasis in cancer, in SCLC, is warranted
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