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Background: Epithelial-mesenchymal transition (EMT) is an early event in tumour invasion and metastasis, and widespread and distant metastasis at early stages is the typical biological behaviour in small cell lung cancer (SCLC). Our previous reports showed that high expression of the transcription factor E2F1 was involved in the invasion and metastasis of SCLC, but the role of E2F1 in the process of EMT in SCLC is unknown., Methods: Immunohistochemistry was performed to evaluate the expressions of EMT related markers. Immunofluorescence was used to detect the expressions of cytoskeletal proteins and EMT related markers when E2F1 was silenced in SCLC cell lines. Adenovirus containing shRNA against E2F1 was used to knock down the E2F1 expression, and the dual luciferase reporter system was employed to clarify the regulatory relationship between E2F1 and ZEB2., Results: In this study, we observed the remodelling of cytoskeletal proteins when E2F1 was silenced in SCLC cell lines, indicating that E2F1 was involved in the EMT in SCLC. Depletion of E2F1 promoted the expression of epithelial markers (CDH1 and CTNNB1) and inhibited the expression of mesenchymal markers (VIM and CDH2) in SCLC cell lines, verifying that E2F1 promotes EMT occurrence. Next, the mechanism by which E2F1 promoted EMT was explored. Among the CDH1 related inhibitory transcriptional regulators ZEB1, ZEB2, SNAI1 and SNAI2, the expression of ZEB2 was the highest in SCLC tissue samples and was highly consistent with E2F1 expression. ChIP-seq data and dual luciferase reporter system analysis confirmed that E2F1 could regulate ZEB2 gene expression., Conclusion: Our data supports that E2F1 promotes EMT by regulating ZEB2 gene expression in SCLC.

Our previous study reported that ADAM12 was highly expressed in small cell lung cancer (SCLC) and could be an effective marker for diagnosis and prognosis. Yet, the reason for the high expression of ADAM12 in SCLC requires further elucidation. Transcription factor E2F1 has been receiving increasing attention due to the complexity and diversity of its function in cancer. In the present study, the expression of ADAM12 was significantly decreased following silencing of E2F1 expression by siRNA, thus indicating that E2F1 may regulate the expression of ADAM12 at the level of transcription. Chromatin immunoprecipitation-to-sequence analysis identified three binding sites for E2F1 in the locus for ADAM12. They were Chr10: 128010444-128011026, located in the intron of ADAM12, named seq0; Chr10: 128076927‑128078127, located in the promoter of ADAM12, named seq1; and Chr10: 128086195‑128086876, located in the upstream 20 kb from the transcription start site of ADAM12, named: seq2. Dual‑luciferase reporter experiments revealed that seq1 not seq0 and seq2 was able to promote the expression of luciferase. Notably, co-transfection of E2F1 significantly increased the activity of seq1 not seq0 and seq2, but quantitative polymerase chain reaction results showed that seq0, seq1 and seq2 could recruit E2F1, indicating that the influence of E2F1 in regulating the expression of ADAM12 was complex. Sequence analysis clarified that seq1 was a part of the ADAM12 promoter, yet the functions of seq0 and seq2 were unknown. Fusion fragments containing seq0-seq1 or seq2-seq1 were analyzed in luciferase constructs. Compared with seq1 alone, the activities of these fusion fragments were non-significantly reduced. The activities of fusion fragments were significantly decreased following co-transfection with E2F1. Thus, the present findings support the conclusion that the E2F1 transcription factor regulates the expression of ADAM12 by binding differential cis-acting elements.

Background: E2F1 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)., Methods: Immunohistochemistry 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., Results: E2F1 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., Conclusions: E2F1 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.