Loss of CSMD 1 expression disrupts mammary duct formation while enhancing proliferation , migration and invasion

The CUB and sushi multiple domains 1 (CSMD1) gene maps to chromosome 8p23, a region deleted in many cancers. Loss of CSMD1 expression is associated with poor prognosis in breast cancer suggesting that it acts as a tumour suppressor in this cancer. However, the function of CSMD1 is largely unknown. Herein, we investigated CSMD1 functions in cell line models. CSMD1 expression was suppressed in MCF10A and LNCaP cells using short hairpin RNA. Functional assays were performed focusing on the ‘normal’ MCF10A cell line. Suppression of CSMD1 significantly increased the proliferation, cell migration and invasiveness of MCF10A cells compared to shcontrols. shCSMD1 cells also showed significantly reduced adhesion to Matrigel and fibronectin. In a three-dimensional Matrigel model of MCF10A cells, reduced CSMD1 expression resulted in the development of larger and more poorly differentiated breast acini-like structures that displayed impaired lumen formation. Loss of CSMD1 expression disrupts a model of mammary duct formation while enhancing proliferation, migration and invasion. Our data suggest that CSMD1 is involved in the suppression of a transformed phenotype. Introduction CUB and sushi multiple domains-1 (CSMD1) is a very large gene which contains 71 exons that span over 2 Mb of genomic DNA on chromosome 8p23 (1). Multiple splice variants exist for CSMD1 and these encode proteins of varying length. The largest transcript is 14.3 kb long and this encodes a 3,565 amino acid protein (1). The full-length protein is a membrane protein with an extracellular region containing 14 CUB and 28 sushi domains, a single transmembrane domain and a short cytoplasmic domain that contains a putative tyrosine phosphorylation site. CSMD1 belongs to the CSMD gene family whose members also include the structurally similar proteins CSMD2 and CSMD3 (1-3). The function of CSMD1 is largely unknown. Although CSMD1 has been shown to inhibit C3 deposition onto the surface of cells leading to the inhibition of the classical complement pathway (4,5). The structure of CSMD1 predicts that CSMD1 is a receptor for unknown ligand(s) and is involved in signal transduction (1). CSMD1 is believed to act as a tumour suppressor based on a number of observations. CSMD1 is located on chromosome 8p23, a region that is frequently deleted in different types of cancer (1,6-10). In addition, reduced CSMD1 mRNA and protein expression has been observed in different cancers (11-17). An array comparative genomic hybridization study revealed a high rate of loss of 8p23.2 containing the CSMD1 gene in advanced prostrate cancer samples. A further small real-time PCR study identified a decrease in CSMD1 mRNA levels in higher stage prostrate cancer samples (11). Similarly reduced CSMD1 expression at the mRNA level has been identified in colorectal cancer associated with reduced patient survival (12). Furthermore, somatic mutations were detected in CSMD1 in 11% (6/54) of colorectal cancer patients along side DNA methylation and allele loss, predominantly in early onset patients (13). Previously, we demonstrated in a large series of breast cancers that 79/275 (28.7%) had reduced CSMD1 protein expression. Low CSMD1 expression was significantly associated with high tumour grade (p=0.003) and decreased overall survival (p=0.018). Importantly multivariate analysis showed that CSMD1 was also an independent predictor of overall survival (p=0.03) (14). Loss of CSMD1 expression disrupts mammary duct formation while enhancing proliferation, migration and invasion MOHAMED KAMAL1,2, DeboRah L. hoLLIDay3, ewan e. MoRRISon1, VaLeRIe SPeIRS3, CARMEL TOOMES1 and SANDRA M. BELL1 1Leeds Institute of biomedical and Clinical Sciences, University of Leeds, wellcome Trust brenner building, St. James's University Hospital, Leeds LS9 7TF, UK; 2Department of Zoology, Faculty of Science, University of Benha, Benha 13518, Egypt; 3Leeds Institute of Cancer and Pathology, University of Leeds, Wellcome Trust Brenner Building, St. James's University Hospital, Leeds LS9 7TF, UK Received August 17, 2016; Accepted May 11, 2017 DoI: 10.3892/or.2017. 5656 Correspondence to: Dr Sandra M. bell, Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Wellcome Trust Brenner Building, St. James's University Hospital, Beckett Street, Leeds LS9 7TF, UK E-mail: medsmb@leeds.ac. uk Abbreviations: CDCP1, CUB domain containing protein 1; CSMD1, CUB and sushi multiple domains 1; CSMD2, CUB and sushi multiple domains 2; CSMD3, CUB and sushi multiple domains 3; ECM, extracellular matrix; FCS, foetal calf serum; IhC, immunohistochemistry; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; qRT-PCR, quantitative real-time polymerase chain reaction; RPLP0, 60S acidic ribosomal protein P0; RT-PCR, reverse transcriptase polymerase chain reaction; SFM, serum-free medium; shRNA, short hairpin RNA