LINE-1 ORF-1p functions as a novel HGF/ETS-1 signaling pathway co-activator and promotes the growth of MDA-MB-231 cell Qian Yang a,1 , Fan Feng b,1 , Fan Zhang c , Chunping Wang d , Yinying Lu d , Xudong Gao d , Yunfeng Zhu e,f, ⁎, Yongping Yang a,d, ⁎⁎ a College of Clinical Medicine, Second Military Medical University, Shanghai 200433, PR China b Department of Pharmacy, General Hospital of Shenyang Military Command, 110016 Shenyang, PR China c Department of Medical Oncology, PLA General Hospital, Beijing 100853, PR China d Center of Therapeutic Research for Hepatocellular Carcinoma, 302 Military Hospital, Beijing 100039, PR China e The Institute of Life Sciences and Bio-engineering, Beijing Jiao-tong University, Beijing 100044, PR China f The Key Laboratory of Tumor Center, PLA General Hospital, Beijing 100853, PR China abstract article info Article history: Received 27 July 2013 Received in revised form 25 August 2013 Accepted 27 August 2013 Available online 4 September 2013 Keywords: HGF/ETS-1 signaling LINE-1 ORF-1p Transcriptional activity Protein–protein interaction Triple negative breast cancer Long interspersed nucleotide element (LINE)-1 ORF-1p is encoded by the human pro-oncogene LINE-1. It is involved in the development and progression of several human carcinomas, such as hepatocellular carcinoma and lung and breast cancers. The hepatocyte growth factor (HGF)/ETS-1 signaling pathway is involved in regulation of cancer cell proliferation, metastasis and invasion. The biological function of the interaction between LINE-1 ORF-1p and the HGF/ETS-1 signaling pathway in regulation of human breast cancer proliferation remains largely unknown. Here, we showed that LINE-1 ORF-1p enhanced ETS-1 transcriptional activity and increased expression of downstream genes of ETS-1. Interaction between ETS-1 and LINE-1 ORF-1p was identified by immunoprecipita- tion assays. LINE-1 ORF-1p modulated ETS-1 activity through cytoplasm/nucleus translocation and recruitment to the ETS-1 binding element in the MMP1 gene promoter. We also showed that LINE-1 ORF-1p promoted prolifera- tion and anchorage-independent growth of MDA-MB-231 breast cancer cells. By investigating a novel role of the LINE-1 ORF-1p in the HGF/ETS-1 signaling pathway and MDA-MB-231 cells, we demonstrated that LINE-1 ORF-1p may be a novel ETS-1 coactivator and molecular target for therapy of human triple negative breast cancer. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved. 1. Introduction LINE-1 is characterized by retrotransposon activity and is involved in the development and progression of several cancers via epigenetic regulation [1]. It encodes two proteins: long interspersed nucleotide element (LINE)-1 ORF-1p and LINE-1 ORF-2p [2]. It is reported that LINE-1 ORF-2p is mainly involved in the retrotransposition process of LINE-1. It can be regulated by LINE-1 ORF-1p [2]. In human cancer cells, expression of LINE-1 ORF-1p is more than that of LINE-1 ORF-2p [3]. LINE-1 would be a protein located in nucleus [4]. However, the bio- logical function and detailed molecular mechanisms of LINE-1 ORF-1p are largely unknown. Our previous results indicated that LINE-1 ORF-1p increases growth of several cancer cell lines [5], anchorage-independent growth, and resistance to chemotherapy in hepatocellular carcinoma (HCC) cells. It also increases proliferation of MCF-7 and ZR75-1 breast cancer cells, A549 and h460 lung cancer cells, and PC-3 and LINCaP prostate cancer cells [5–7]. As a protein located in the nucleus, LINE-1 can modulate the activity of transcription factor AR (androgen receptor) through physical interaction [6]. These results suggest that LINE-1 ORF-1p could be a novel co-regulator of nuclear transcription factors. The product of pro-oncogene ETS-1 belongs to the ETS protein fam- ily of transcription factors. It contains the ETS domain (transcription ac- tivation domain) and helix DNA-binding domain [8]. Members of the ETS family are involved in the regulation of proliferation, development, progress, apoptosis, metastasis, invasion and angiogenesis of cancer cells [8]. ETS-1 is present at high levels in breast cancer, ovarian cancer, and cervical carcinoma. A high level of ETS-1 protein is associated with poor prognosis [9]. In the nucleus, ETS-1 binds to the ETS-binding sequences (elements) in the promoter/enhancer regions of their down- stream genes [10], in the presence of hepatocyte growth factor (HGF). The sequence of the ETS binding element is 5′-GGAA/T-3′ [8–10]. In several types of human cancer, the receptor tyrosine kinase c-Met is activated by HGF. The activity of ETS-1 is modulated by c-Met and AKT, and it regulates transcription of several target genes, such as MMP1, MMP9, u-PA and c-Met [7–9]. The transcriptional activity of Cellular Signalling 25 (2013) 2652–2660 ⁎ Correspondence to: Y. Zhu, The College of Life Sciences and Bio-engineering, Beijing Jiaotong University, Beijing 100044, PR China. Tel.: +86 10 51684352, fax.:+86 10 51683887. ⁎⁎ Correspondence to: Y. Yang, Center of Therapeutic Research for Hepatocellular Carcinoma, 302 Military Hospital, Beijing 100039, PR China. Tel./fax: +86 10 63879193. E-mail addresses: zhuyf2004@163.com (Y. Zhu), yongpingyang@hotmail.com (Y. Yang). 1 These authors contributed equally to this work. 0898-6568/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2013.08.029 Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig