E-cadherin loss alters cytoskeletal organization and adhesion in non-malignant breast cells but is insufficient to induce an epithelial-mesenchymal transition

BMC Cancer E-cadherin loss alters cytoskeletal organization and adhesion in non-malignant breast cells but is insufficient to induce an epithelial-mesenchymal transition Augustine Chen 0 Henry Beetham 0 Michael A Black 0 Rashmi Priya 1 Bryony J Telford 0 Joanne Guest 0 George A R Wiggins 0 Tanis D Godwin 0 Alpha S Yap 1 Parry J Guilford 0 0 Cancer Genetics Laboratory, Department of Biochemistry, University of Otago , Dunedin 9054 , New Zealand 1 Division of Molecular Cell Biology, Institute for Molecular Bioscience, The University of Queensland , St Lucia, Brisbane 4072 , Australia Background: E-cadherin is an adherens junction protein that forms homophilic intercellular contacts in epithelial cells while also interacting with the intracellular cytoskeletal networks. It has roles including establishment and maintenance of cell polarity, differentiation, migration and signalling in cell proliferation pathways. Its downregulation is commonly observed in epithelial tumours and is a hallmark of the epithelial to mesenchymal transition (EMT). Methods: To improve our understanding of how E-cadherin loss contributes to tumorigenicity, we investigated the impact of its elimination from the non-tumorigenic breast cell line MCF10A. We performed cell-based assays and whole genome RNAseq to characterize an isogenic MCF10A cell line that is devoid of CDH1 expression due to an engineered homozygous 4 bp deletion in CDH1 exon 11. Results: The E-cadherin-deficient line, MCF10A CDH1-/- showed subtle morphological changes, weaker cell-substrate adhesion, delayed migration, but retained cell-cell contact, contact growth inhibition and anchorage-dependent growth. Within the cytoskeleton, the apical microtubule network in the CDH1-deficient cells lacked the radial pattern of organization present in the MCF10A cells and F-actin formed thicker, more numerous stress fibres in the basal part of the cell. Whole genome RNAseq identified compensatory changes in the genes involved in cell-cell adhesion while genes involved in cell-substrate adhesion, notably ITGA1, COL8A1, COL4A2 and COL12A1, were significantly downregulated. Key EMT markers including CDH2, FN1, VIM and VTN were not upregulated although increased expression of proteolytic matrix metalloprotease and kallikrein genes was observed. Conclusions: Overall, our results demonstrated that E-cadherin loss alone was insufficient to induce an EMT or enhance transforming potential in the non-tumorigenic MCF10A cells but was associated with broad transcriptional changes associated with tissue remodelling. CDH1; Cytoskeletal modelling; Adhesion; Migration; EMT - Background E-cadherin, encoded by the tumor suppressor gene CDH1, is a homophilic cell-to-cell adhesion protein localized to the adherens junctions of all epithelial cells [1]. Its cytoplasmic domain effectively creates a bridge between the cytoskeletons of adjacent cells by interacting with both cortical actin filaments and the microtubule network [2]. These and other interactions [3] extend E-cadherins functionality beyond cell-cell adhesion to roles in establishing and maintaining cell polarity, differentiation, stemness, cell migration and the mediation of signalling through various proliferation and survival pathways including WNT and EGFR [1-5]. Abrogation of CDH1 expression by mutation, deletion or promoter hypermethylation is a feature of many epithelial tumours, including prostate, ovarian, lung and hepatocellular carcinomas, and is the hallmark of both the sporadic and familial forms of diffuse gastric cancer (DGC) and lobular breast cancer (LBC) [1,6]. In both LBC and DGC, CDH1 inactivation can be an early initiating event [7,8], whereas in other tumour types including prostate, lung, ovarian and colon, its downregulation is usually considered to be a late event that promotes an increase in invasive capacity [9]. Increased invasiveness following CDH1 downregulation is related, at least in part, to the central role played by E-cadherin in the de-differentiation process known as the epithelialmesenchymal transition (EMT) [10]. During the EMT, epithelial cells lose polarity and normal cell-cell adhesion, acquiring a mesenchymal phenotype with higher motility and an increase in cell-extracellular matrix (ECM) connections [9,11]. The EMT is associated not only with increased tumor invasion and metastasis, but also poor outcome, drug resistance and an increase in the number of cancer stem-like cells [9,12]. E-cadherin downregulation has been shown to be sufficient to induce an EMT in some [4,9,10,13], but not all [14,15], cancer cell lines/models. However, it remains unclear whether its loss can induce an EMT in cells which have not already undergone malignant transformation [16]. Clues to the influence E-cadherin loss has on tumorigenesis and the initiation of the EMT come from study of the multifocal gastric signet ring cell carcinomas (SRCCs) that occur in Hereditary Diffuse Gastric Cancer (HDGC) families. HDGC is a familial cancer syndrome caused by germline mutation of the CDH1 gene and is typified by highly penetrant DGC and an elevated risk of LBC [17]. With few exceptions, mutation carriers develop tens to hundreds of gastric foci of SRCC, sometimes with enrichment in the transition zone between the antrum and body [18]. LBC and lobular carcinoma in situ (LCIS) are also observed to be multifocal in female mutation carriers (V. Blair, pers. comm). The multifocal gastric SRCCs are Ecadherin-negative and almost exclusively stage T1a tumours confined to the lamina propria. Lineage markers suggest that the foci develop from mucous neck cells that have invaded through the basement membrane of the gastric gland [19]. Invasion is likely to be triggered by inactivation of the wild-type CDH1 allele through mechanisms including promoter hypermethylation [6]. In one model [20], E-cadherin loss creates instability in the orientation of the mitotic spindle, leading to a proportion of the cell divisions occurring out of the epithelial plane with subsequent displacement of daughter cells into the lamina propria. The multifocal SRCC foci in the gastric mucosa are known to be relatively indolent, but show unpredictable progression to advanced disease. A small percentage of foci show characteristics of an EMT, and this change is associated with tumour progression [19]. However, the absence of an EMT-like phenotype from the majority of SRCC foci suggests that E-cadherin loss alone is insufficient to induce an EMT in this relatively normal genetic background. MCF10A is a spontaneously immortalized, nontransformed mammary epithelial cell line derived from human fibrocystic tissue. Although it does carry cytogenetic abnormalities associated with in vitro cultured mammary epithelial cells, including p16 and p14ARF deletion and MYC amplification [21], MCF10A is considered a normal breast epithelial cell due to its near diploid, stable karyotype and characteristics of normal breast epithe (...truncated)