Molecular and Cellular Pathobiology SND1 Acts Downstream of TGFb1 and Upstreamof Smurf1 to Promote Breast Cancer Metastasis

SND1 is an AEG-1/MTDH/LYRIC-binding protein that is upregulated in numerous human cancers, where it has been assigned multiple functional roles. In this study, we report its association with the TGFb1 signaling pathway, which promotes epithelial–mesenchymal transition (EMT) in breast cancer. SND1 was upregulated in breast cancer tissues, in particular in primary invasive ductal carcinomas. Transcriptional activation of the SND1 gene was controlled by the TGFb1/Smad pathway, specifically by activation of the Smad2/Smad3 complex. The SND1 promoter region contained several Smad-specific recognition domains (RD motifs), which were recognized and bound by the Smad complex that enhanced the transcriptional activation of SND1. We found that SND1 promoted expression of the E3 ubiquitin ligase Smurf1, leading to RhoA ubiquitination and degradation. RhoA degradation in breast cancer cells disrupted F-actin cytoskeletal organization, reduced cell adhesion, increased cell migration and invasion, and promoted metastasis. Overall, our results define a novel role for SND1 in regulating breast tumorigenesis and metastasis. Cancer Res; 75(7); 1275–86. 2015 AACR. Introduction Breast cancer is the most commonly diagnosed cancer in women and the leading cause of female cancer death worldwide (1). Distant metastasis and invasion of advanced cancer are responsible for more than 90% of cancer-related deaths (2). However, themolecularmechanisms underlying the breast cancer tumorigenesis and metastasis are largely unknown. The TGFb signaling pathway is a key player in the epithelial– mesenchymal transition (EMT) in breast cancer. The misregulation of TGFb signaling may promote tumor invasion and migration, leading to detrimental consequences for prognosis in patients with breast cancer (3–5). The TGFb signaling pathway contains different R-Smads (Smad1, Smad2, Smad3, and Smad5) and co-Smad (Smad4) transcription factors (6–8). Smad1, Smad5, and Smad8 are specifically activated by the bone morphogenetic protein (BMP) receptors, whereas Smad2 and Smad3 are specifically activated by TGFb-related receptors (9). In the activated pathway, the phosphorylated R-Smads enter the nucleus to form complex with co-Smad Smad4 and then combine with specific Smad recognition domains (RD) in the promoter region of target genes (10). Staphylococcal nuclease domain-containing 1 (SND1), also known as Tudor staphylococcal nuclease (Tudor-SN) or p100 protein, ubiquitously exists in human and many other species. Proteomic analysis of clinical breast cancer samples shows a close correlation of elevated SND1 expression with cancer metastasis (11). It was also reported that SND1 could interact with astrocyte elevated gene-1 (AEG-1) in breast cancer and strongly promote lung metastasis (12). In our earlier work using chromatin immunoprecipitation guided ligation and selection (ChIP-GLAS) assays, we have demonstrated that SND1 is potentially involved in the TGFb signaling pathway in breast cancer cells (13). However, it remains unclear how SND1 is upregulated in breast cancer and how SND1 participates in breast cancer metastasis. In the present study, we demonstrate for the first time that SND1 is a novel target of the Smad complex. The gene transcriptional activation of SND1 could be regulated by the TGFb/ Smad pathway. In addition, SND1 mediates the gene transcriptional activation of Smurf1, which is an E3 ubiquitin ligase, promoting RhoA ubiquitination, causing F-actin rearrangement, and leading to increased invasiveness and metastasis of breast cancer. Materials and Methods Cell lines, siRNA, and cell transfection The cell lines MCF-7, T47D, SK-BR-3, MDA-MB-231, and BT549 were obtained from the ATCC. MDA-MB-231 was Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China. Departmentof Immunology,SchoolofBasicMedicalSciences,TianjinMedical University, Tianjin, China. Laboratory of Molecular Immunology, Research Center of Basic Medical Science, Tianjin Medical University, Tianjin,China. TianjinKeyLaboratoryofCellular andMolecular Immunology and Key Laboratory of the Educational Ministry of China, TianjinMedical University,Tianjin, China. Department of Immunology, University of Manitoba, Winnipeg, Canada. Department of Cardiovascular Surgery, Tianjin Medical University General Hospital, Tianjin, China. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). L. Yu and X. Liu contributed equally to this article. Corresponding Authors: Jie Yang, Tianjin Medical University, Heping District, Qixiangtai Road, No. 22, Tianjin 300070, China. Phone: 86-22-23542520; Fax: 86-22-23542581; E-mail: [email protected]; and Zhi Yao, Phone: 86-2283332817; Fax: 86-22-83332817; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-14-2387 2015 American Association for Cancer Research. Cancer Research www.aacrjournals.org 1275 on August 6, 2017. © 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst January 16, 2015; DOI: 10.1158/0008-5472.CAN-14-2387