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

BMC Cancer

Parry J Guilford²  Alpha S Yap¹  Tanis D Godwin²  George A R Wiggins²  Joanne Guest²  Bryony J Telford²  Rashmi Priya¹  Michael A Black²  Henry Beetham²  Augustine Chen² 

[1]Division of Molecular Cell Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane 4072, Australia

[2]Cancer Genetics Laboratory, Department of Biochemistry, University of Otago, Dunedin 9054, New Zealand

关键词: EMT;    Migration;    Adhesion;    Cytoskeletal modelling;    CDH1;   

Others  :  1125272
DOI  :  10.1186/1471-2407-14-552

 received in 2014-04-04, accepted in 2014-07-15,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Chen et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]van Roy F, Berx G: The cell-cell adhesion molecule E-cadherin. Cell Mol Life Sci: CMLS 2008, 65(23):3756-3788.
• [2]Brieher WM, Yap AS: Cadherin junctions and their cytoskeleton(s). Curr Opin Cell Biol 2013, 25(1):39-46.
• [3]David JM, Rajasekaran AK: Dishonorable discharge: the oncogenic roles of cleaved E-cadherin fragments. Cancer Res 2012, 72(12):2917-2923.
• [4]Mohamet L, Hawkins K, Ward CM: Loss of function of e-cadherin in embryonic stem cells and the relevance to models of tumorigenesis. J Oncol 2011, 2011:352616.
• [5]Jeanes A, Gottardi CJ, Yap AS: Cadherins and cancer: how does cadherin dysfunction promote tumor progression? Oncogene 2008, 27(55):6920-6929.
• [6]Guilford P, Hopkins J, Harraway J, McLeod M, McLeod N, Harawira P, Taite H, Scoular R, Miller A, Reeve AE: E-cadherin germline mutations in familial gastric cancer. Nature 1998, 392:402-405.
• [7]Zou D, Yoon HS, Perez D, Weeks RJ, Guilford P, Humar B: Epigenetic silencing in non-neoplastic epithelia identifies E-cadherin (CDH1) as a target for chemoprevention of lobular neoplasia. J Pathol 2009, 218(2):265-272.
• [8]Humar B, Blair V, Charlton A, More H, Martin I, Guilford P: E-cadherin deficiency initiates gastric signet-ring cell carcinoma in mice and man. Cancer Res 2009, 69(5):2050-2056.
• [9]Thiery JP, Acloque H, Huang RY, Nieto MA: Epithelial-mesenchymal transitions in development and disease. Cell 2009, 139(5):871-890.
• [10]Canel M, Serrels A, Frame MC, Brunton VG: E-cadherin-integrin crosstalk in cancer invasion and metastasis. J Cell Sci 2013, 126(Pt 2):393-401.
• [11]Drasin DJ, Robin TP, Ford HL: Breast cancer epithelial-to-mesenchymal transition: examining the functional consequences of plasticity. Breast Canc Res: BCR 2011, 13(6):226.
• [12]Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, Lander ES: Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 2009, 138(4):645-659.
• [13]Onder TT, Gupta PB, Mani SA, Yang J, Lander ES, Weinberg RA: Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res 2008, 68(10):3645-3654.
• [14]van Horssen R, Hollestelle A, Rens JA, Eggermont AM, Schutte M, Ten Hagen TL: E-cadherin promotor methylation and mutation are inversely related to motility capacity of breast cancer cells. Breast Cancer Res Treat 2012, 136(2):365-377.
• [15]Hollestelle A, Peeters JK, Smid M, Timmermans M, Verhoog LC, Westenend PJ, Heine AA, Chan A, Sieuwerts AM, Wiemer EA, Klijn JG, van der Spek PJ, Foekens JA, Schutte M, den Bakker MA, Martens JW: Loss of E-cadherin is not a necessity for epithelial to mesenchymal transition in human breast cancer. Breast Cancer Res Treat 2013, 138(1):47-57.
• [16]Shamir ER, Pappalardo E, Jorgens DM, Coutinho K, Tsai WT, Aziz K, Auer M, Tran PT, Bader JS, Ewald AJ: Twist1-induced dissemination preserves epithelial identity and requires E-cadherin. J Cell Biol 2014, 204(5):839-856.
• [17]Blair V, Martin I, Shaw D, Winship I, Kerr D, Arnold J, Harawira P, McLeod M, Parry S, Charlton A, Findlay M, Cox B, Humar B, More H, Guilford P: Hereditary diffuse gastric cancer: diagnosis and management. Clin Gastroenterol Hepatol: Offic Clin Pract J Am Gastroenterol Assoc 2006, 4(3):262-275.
• [18]Charlton A, Blair V, Shaw D, Parry S, Guilford P, Martin IG: Hereditary diffuse gastric cancer: predominance of multiple foci of signet ring cell carcinoma in distal stomach and transitional zone. Gut 2004, 53(6):814-820.
• [19]Humar B, Fukuzawa R, Blair V, Dunbier A, More H, Charlton A, Yang HK, Kim WH, Reeve AE, Martin I, Guilford P: Destabilized adhesion in the gastric proliferative zone and c-Src kinase activation mark the development of early diffuse gastric cancer. Cancer Res 2007, 67(6):2480-2489.
• [20]Humar B, Guilford P: Hereditary diffuse gastric cancer and lost cell polarity: a short path to cancer. Future Oncol 2008, 4(2):229-239.
• [21]Debnath J, Muthuswamy SK, Brugge JS: Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 2003, 30(3):256-268.
• [22]Soule HD, Maloney TM, Wolman SR, Peterson WD Jr, Brenz R, McGrath CM, Russo J, Pauley RJ, Jones RF, Brooks SC: Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res 1990, 50(18):6075-6086.
• [23]Schneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012, 9(7):671-675.
• [24]Helwani FM, Kovacs EM, Paterson AD, Verma S, Ali RG, Fanning AS, Weed SA, Yap AS: Cortactin is necessary for E-cadherin-mediated contact formation and actin reorganization. J Cell Biol 2004, 164(6):899-910.
• [25]Robinson MD, McCarthy DJ, Smyth GK: edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010, 26(1):139-140.
• [26]Chang JT, Nevins JR: GATHER: a systems approach to interpreting genomic signatures. Bioinformatics 2006, 22(23):2926-2933.
• [27]Huttenlocher A, Horwitz AR: Integrins in cell migration. Cold Spring Harb Perspect Biol 2011, 3(9):a005074.
• [28]Lo SH: Tensin. Int J Biochem Cell Biol 2004, 36(1):31-34.
• [29]Belkin AM, Burridge K: Localization of utrophin and aciculin at sites of cell-matrix and cell-cell adhesion in cultured cells. Exp Cell Res 1995, 221(1):132-140.
• [30]Kumper S, Ridley AJ: p120ctn and P-cadherin but not E-cadherin regulate cell motility and invasion of DU145 prostate cancer cells. PLoS One 2010, 5(7):e11801.
• [31]Huntsman DG, Carneiro F, Lewis FR, MacLeod PM, Hayashi A, Monaghan KG, Maung R, Seruca R, Jackson CE, Caldas C: Early gastric cancer in young, asymptomatic carriers of germ-line E-cadherin mutations. N Engl J Med 2001, 344(25):1904-1909.
• [32]Tinkle CL, Lechler T, Pasolli HA, Fuchs E: Conditional targeting of E-cadherin in skin: insights into hyperproliferative and degenerative responses. Proc Natl Acad Sci U S A 2004, 101(2):552-557.
• [33]Tunggal JA, Helfrich I, Schmitz A, Schwarz H, Gunzel D, Fromm M, Kemler R, Krieg T, Niessen CM: E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions. EMBO J 2005, 24(6):1146-1156.
• [34]Tinkle CL, Pasolli HA, Stokes N, Fuchs E: New insights into cadherin function in epidermal sheet formation and maintenance of tissue integrity. Proc Natl Acad Sci U S A 2008, 105(40):15405-15410.
• [35]Capaldo CT, Macara IG: Depletion of E-cadherin disrupts establishment but not maintenance of cell junctions in Madin-Darby canine kidney epithelial cells. Mol Biol Cell 2007, 18(1):189-200.
• [36]Chausovsky A, Bershadsky AD, Borisy GG: Cadherin-mediated regulation of microtubule dynamics. Nat Cell Biol 2000, 2(11):797-804.
• [37]Meng W, Mushika Y, Ichii T, Takeichi M: Anchorage of microtubule minus ends to adherens junctions regulates epithelial cell-cell contacts. Cell 2008, 135(5):948-959.
• [38]Bulgakova NA, Grigoriev I, Yap AS, Akhmanova A, Brown NH: Dynamic microtubules produce an asymmetric E-cadherin-Bazooka complex to maintain segment boundaries. J Cell Biol 2013, 201(6):887-901.
• [39]Haynes J, Srivastava J, Madson N, Wittmann T, Barber DL: Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression. Mol Biol Cell 2011, 22(24):4750-4764.
• [40]Berrier AL, Yamada KM: Cell-matrix adhesion. J Cell Physiol 2007, 213(3):565-573.
• [41]Weber GF, Bjerke MA, DeSimone DW: Integrins and cadherins join forces to form adhesive networks. J Cell Sci 2011, 124(Pt 8):1183-1193.
• [42]Zhang X, Jiang G, Cai Y, Monkley SJ, Critchley DR, Sheetz MP: Talin depletion reveals independence of initial cell spreading from integrin activation and traction. Nat Cell Biol 2008, 10(9):1062-1068.
• [43]Pankov R, Cukierman E, Katz BZ, Matsumoto K, Lin DC, Lin S, Hahn C, Yamada KM: Integrin dynamics and matrix assembly: tensin-dependent translocation of alpha(5)beta(1) integrins promotes early fibronectin fibrillogenesis. J Cell Biol 2000, 148(5):1075-1090.
• [44]Hazan RB, Qiao R, Keren R, Badano I, Suyama K: Cadherin switch in tumor progression. Ann N Y Acad Sci 2004, 1014:155-163.
• [45]De Craene B, Berx G: Regulatory networks defining EMT during cancer initiation and progression. Nat Rev Cancer 2013, 13(2):97-110.
• [46]Park J, Schwarzbauer JE: Mammary epithelial cell interactions with fibronectin stimulate epithelial-mesenchymal transition. Oncogene 2013, 33(13):1649-1657.
• [47]Radisky ES, Radisky DC: Matrix metalloproteinase-induced epithelial-mesenchymal transition in breast cancer. J Mammary Gland Biol Neoplasia 2010, 15(2):201-212.
• [48]Emberley ED, Murphy LC, Watson PH: S100A7 and the progression of breast cancer. Breast Canc Res: BCR 2004, 6(4):153-159.