Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer

Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA.
Molecular biology of the cell (Impact Factor: 4.47). 05/2011; 22(14):2423-35. DOI: 10.1091/mbc.E11-04-0306
Source: PubMed


Reduced epithelial cadherin (E-cad) is a hallmark of invasive carcinomas that have acquired epithelial-mesenchymal transition (EMT) phenotypes. Here we show that down-regulated E-cad expression induced by transforming growth factor-β (TGF-β) and EMT preceded breast cancer outgrowth in three-dimensional (3D) organotypic assays and in the lungs of mice. Pharmacological inhibitors against focal adhesion kinase prevented metastatic outgrowth of newly seeded organoids, but not that of their fully established counterparts. Interrogating the D2-HAN (hyperplastic alveolar nodule) model of breast cancer dormancy and metastasis showed that dormant D2.OR cells produced branched organoid morphologies in 3D-cultures, and expressed robust quantities of E-cad that was uncoupled from regulation by TGF-β. In contrast, metastatic D2.A1 organoids were spherical and wholly lacked E-cad expression. Interestingly, D2.A1 cells engineered to re-express E-cad formed branched organoids, down-regulated β1 integrin expression, and failed to undergo metastatic outgrowth. The tumor-suppressing function of E-cad was inactivated by increased microenvironmental rigidity, and was not recapitulated by expression of an E-cad mutant lacking its extracellular domain. Twist expression, but not that of Snail, reinitiated metastatic outgrowth in dormant D2.OR cells. Our findings show that EMT and its down-regulated expression of E-cad circumvent breast cancer dormancy in part by facilitating β1 integrin expression necessary for metastatic outgrowth.

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Available from: Molly A Taylor, Nov 20, 2014
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    • "Pulmonary tumor development was assessed by injection of parental (scrambled shRNA) and Mig6-deficient cells into the lateral tail vein of nu/nu mice (1 × 10 6 cells/mouse). Where indicated tumor growth and metastasis was monitored by in vivo bioluminescent imaging as previously described [1] [2] [18] [20] [22]. Bioluminescent images were captured on a Xenogen IVIS-200 (PerkinElmer). "
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    ABSTRACT: Numerous studies by our lab and others demonstrate that epidermal growth factor receptor (EGFR) plays critical roles in primary breast cancer (BC) initiation, growth and dissemination. However, clinical trials targeting EGFR function in BC have lead to disappointing results. In the current study we sought to identify the mechanisms responsible for this disparity by investigating the function of EGFR across the continuum of the metastatic cascade. We previously established that overexpression of EGFR is sufficient for formation of in situ primary tumors by otherwise nontransformed murine mammary gland cells. Induction of epithelial-mesenchymal transition (EMT) is sufficient to drive the metastasis of these EGFR-transformed tumors. Examining growth factor receptor expression across this and other models revealed a potent downregulation of EGFR through metastatic progression. Consistent with diminution of EGFR following EMT and metastasis EGF stimulation changes from a proliferative to an apoptotic response in in situ versus metastatic tumor cells, respectively. Furthermore, overexpression of EGFR in metastatic MDA-MB-231 BC cells promoted their antitumorigenic response to EGF in three dimensional (3D) metastatic outgrowth assays. In line with the paradoxical function of EGFR through EMT and metastasis we demonstrate that the EGFR inhibitory molecule, Mitogen Induced Gene-6 (Mig6), is tumor suppressive in in situ tumor cells. However, Mig6 expression is absolutely required for prevention of apoptosis and ultimate metastasis of MDA-MB-231 cells. Further understanding of the paradoxical function of EGFR between primary and metastatic tumors will be essential for application of its targeted molecular therapies in BC.
    Preview · Article · Jan 2015 · Neoplasia (New York, N.Y.)
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    • "We recently established that BCs that can downregulate E-cadherin (E-cad) are at a selective advantage to initiate metastatic outgrowth within the pulmonary microenvironment [2,25]. However, these and other studies suggest that, as pulmonary metastases progress from micro- to macroscopic lesions, E-cad expression returns in a manner reminiscent of the differentiation status observed in noninvasive primary tumors [1,2]. Along these lines, the highly metastatic 4T1 model of late-stage BC displays several epithelial characteristics, including the expression of E-cad [2,21]. "
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    ABSTRACT: Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) facilitate breast cancer (BC) metastasis; however, stable molecular changes that result as a consequence of these processes remain poorly defined. Therefore, we sought to identify molecular markers that could identify tumor cells that had completed the EMT:MET cycle in the hopes of targeting unique aspects of metastatic tumor outgrowth. An in vivo reporter system for epithelial-cadherin (E-cad) expression was used to quantify its regulation in metastatic BC cells during primary and metastatic tumor growth. Exogenous addition of Transforming Growth Factor- beta1 (TGF-beta1) was used to induce EMT in an in situ model of BC. Microarray analysis was employed to examine gene expression changes in cells chronically treated with and withdrawn from TGF-beta1, thus completing one full EMT:MET cycle. Changes in fibroblast growth factor receptor 1 (FGFR1) isoform expression were validated using PCR analyses of patient-derived tumor versus matched normal tissues. FGFR1 gene expression was manipulated using shRNA depletion and cDNA rescue. Preclinical pharmacological inhibition of FGFR kinase was employed using the orally available compound BGJ-398. Metastatic BC cells undergo spontaneous downregulation of E-cadherin during primary tumor growth and its expression subsequently returns following initiation of metastatic outgrowth. Exogenous exposure to TGF-beta1 was sufficient to drive the metastasis of an otherwise in situ model of BC, and was similarly associated with a depletion and return of E-cadherin expression during metastatic progression. BC cells treated and withdrawn from TGF-beta stably upregulate a truncated FGFR1-beta splice variant that lacks the outermost extracellular immunoglobulin (Ig) domain. Identification of this FGFR1 splice variant was verified in metastatic human BC cell lines and patient-derived tumor samples. Expression of FGFR1-beta was also dominant in a model of metastatic outgrowth where depletion of FGFR1 and pharmacologic inhibition of FGFR kinase activity both inhibited pulmonary tumor outgrowth. Highlighting the dichotomous nature of FGFR splice variants, recombinant expression of full-length FGFR1-alpha also blocked pulmonary tumor outgrowth. These results strongly suggest that FGFR1-beta is required for the pulmonary outgrowth of metastatic BC. Moreover, FGFR1 isoform expression can be used as a predictive biomarker for therapeutic application of its kinase inhibitors.
    Full-text · Article · Mar 2014 · Breast cancer research: BCR
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    • "Therefore, a more profound understanding of the temporal alterations of cell-cell adhesion during EMT including a quantification of the exerted forces between the cells even down to the single-molecule level is of fundamental importance. Especially, the concerted and time-dependent down-regulation of the adherens junction protein E-cadherin is essential for epithelial-to-mesenchymal transition [52]. Whereas the amount of E-cadherin remains unchanged within the first 2–3 days after cytokine addition, the localization of the protein is completely altered. "
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    ABSTRACT: Structural alterations during epithelial-to-mesenchymal transition (EMT) pose a substantial challenge to the mechanical response of cells and are supposed to be key parameters for an increased malignancy during metastasis. Herein, we report that during EMT, apical tension of the epithelial cell line NMuMG is controlled by cell-cell contacts and the architecture of the underlying actin structures reflecting the mechanistic interplay between cellular structure and mechanics. Using force spectroscopy we find that tension in NMuMG cells slightly increases 24 h after EMT induction, whereas upon reaching the final mesenchymal-like state characterized by a complete loss of intercellular junctions and a concerted down-regulation of the adherens junction protein E-cadherin, the overall tension becomes similar to that of solitary adherent cells and fibroblasts. Interestingly, the contribution of the actin cytoskeleton on apical tension increases significantly upon EMT induction, most likely due to the formation of stable and highly contractile stress fibers which dominate the elastic properties of the cells after the transition. The structural alterations lead to the formation of single, highly motile cells rendering apical tension a good indicator for the cellular state during phenotype switching. In summary, our study paves the way towards a more profound understanding of cellular mechanics governing fundamental morphological programs such as the EMT.
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