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Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium

Abstract

In vitro models of normal mammary epithelium have correlated increased extracellular matrix (ECM) stiffness with malignant phenotypes. However, the role of increased stiffness in this transformation remains unclear because of difficulties in controlling ECM stiffness, composition and architecture independently. Here we demonstrate that interpenetrating networks of reconstituted basement membrane matrix and alginate can be used to modulate ECM stiffness independently of composition and architecture. We find that, in normal mammary epithelial cells, increasing ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. We also find that the combination of stiffness and composition is sensed through β4 integrin, Rac1, and the PI3K pathway, and suggest a mechanism in which an increase in ECM stiffness, without an increase in basement membrane ligands, prevents normal α6β4 integrin clustering into hemidesmosomes.

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Figure 1: The stiffness of interpenetrating networks of alginate and basement-membrane matrix can be modulated independently of cell-adhesion-ligand density for 3D cell culture.
Figure 2: Enhanced stiffness alone leads to the malignant phenotype in MCF10As.
Figure 3: Altered ECM composition can enhance or completely abrogate the effect of increased stiffness on the phenotype of MCF10As.
Figure 4: Mechanotransduction and malignant phenotype of MCF10As in IPNs is mediated through β4 integrin signalling.
Figure 5: Mechanotransduction and malignant phenotype of MCF10As in stiff IPNs is mediated through the PI3K signalling pathway and Rac1 activation.
Figure 6: Proposed mechanism for the impact of ECM stiffness and composition on malignant phenotype.

References

  1. 1

    Bissell, M. J. & Hines, W. C. Why don’t we get more cancer? A proposed role of the microenvironment in restraining cancer progression. Nature Med. 17, 320–329 (2011).

    CAS  Article  Google Scholar 

  2. 2

    Paszek, M. J. et al. Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241–254 (2005).

    CAS  Google Scholar 

  3. 3

    Levental, K. R. et al. Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139, 891–906 (2009).

    CAS  Article  Google Scholar 

  4. 4

    Boyd, N. F., Lockwood, G. A., Byng, J. W., Tritchler, D. L. & Yaffe, M. J. Mammographic densities and breast cancer risk. Cancer Epidemiol. Biomark. Prev. 7, 1133–1144 (1998).

    CAS  Google Scholar 

  5. 5

    Petersen, O., Ronnov-Jessen, L., Howlett, A. & Bissell, M. Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc. Natl Acad. Sci. USA 89, 9064–9068 (1992).

    CAS  Article  Google Scholar 

  6. 6

    Debnath, J. & Brugge, J. S. Modelling glandular epithelial cancers in three-dimensional cultures. Nature Rev. Cancer 5, 675–688 (2005).

    CAS  Article  Google Scholar 

  7. 7

    Weaver, V. M. et al. Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J. Cell Biol. 137, 231–245 (1997).

    CAS  Article  Google Scholar 

  8. 8

    Provenzano, P. P., Inman, D. R., Eliceiri, K. W. & Keely, P. J. Matrix density-induced mechanoregulation of breast cell phenotype, signaling and gene expression through a FAK-ERK linkage. Oncogene 28, 4326–4343 (2009).

    CAS  Article  Google Scholar 

  9. 9

    Giancotti, F. G. Integrin signaling. Science 285, 1028–1033 (1999).

    CAS  Article  Google Scholar 

  10. 10

    Engler, A. et al. Substrate compliance versus ligand density in cell on gel responses. Biophys. J. 86, 617–628 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Brownfield, D. G. et al. Patterned collagen fibers orient branching mammary epithelium through distinct signaling modules. Curr. Biol. 23, 703–709 (2013).

    CAS  Article  Google Scholar 

  12. 12

    Nguyen-Ngoc, K-V. et al. ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium. Proc. Natl Acad. Sci. USA 109, E2595–E2604 (2012).

    CAS  Article  Google Scholar 

  13. 13

    Wozniak, M. A., Desai, R., Solski, P. A., Der, C. J. & Keely, P. J. ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix. J. Cell Biol. 163, 583–595 (2003).

    CAS  Article  Google Scholar 

  14. 14

    Egeblad, M., Rasch, M. G. & Weaver, V. M. Dynamic interplay between the collagen scaffold and tumor evolution. Curr. Opin. Cell Biol. 22, 697–706 (2010).

    CAS  Article  Google Scholar 

  15. 15

    Lo, C-M., Wang, H-B., Dembo, M. & Wang, Y. Cell movement is guided by the rigidity of the substrate. Biophys. J. 79, 144–152 (2000).

    CAS  Article  Google Scholar 

  16. 16

    Engler, A., Sen, S., Sweeney, H. & Discher, D. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    CAS  Google Scholar 

  17. 17

    Yeung, T. et al. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell. Motil. Cytoskeleton 60, 24–34 (2005).

    Article  Google Scholar 

  18. 18

    Discher, D. E., Janmey, P. & Wang, Y-L. Tissue cells feel and respond to the stiffness of their substrate. Science 310, 1139–1143 (2005).

    CAS  Article  Google Scholar 

  19. 19

    Lee, K. et al. Matrix compliance regulates Rac1b localization, NADPH oxidase assembly, and epithelial-mesenchymal transition. Mol. Biol. Cell 23, 4097–4108 (2012).

    CAS  Article  Google Scholar 

  20. 20

    Peyton, S. R., Raub, C. B., Keschrumrus, V. P. & Putnam, A. J. The use of poly(ethylene glycol) hydrogels to investigate the impact of ECM chemistry and mechanics on smooth muscle cells. Biomaterials 27, 4881–4893 (2006).

    CAS  Article  Google Scholar 

  21. 21

    Huebsch, N. et al. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nature Mater. 9, 518–526 (2010).

    CAS  Google Scholar 

  22. 22

    Ulrich, T. A., Jain, A., Tanner, K., MacKay, J. L. & Kumar, S. Probing cellular mechanobiology in three-dimensional culture with collagen-agarose matrices. Biomaterials 31, 1875–1884 (2010).

    CAS  Article  Google Scholar 

  23. 23

    Khetan, S. et al. Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels. Nature Mater. 12, 458–465 (2013)

    CAS  Google Scholar 

  24. 24

    Mammoto, A. et al. A mechanosensitive transcriptional mechanism that controls angiogenesis. Nature 457, 1103–1108 (2009).

    CAS  Article  Google Scholar 

  25. 25

    Boyd-White, J. & Williams, J. C. Effect of cross-linking on matrix permeability: A model for age-modified basement membranes. Diabetes 45, 348–353 (1996).

    CAS  Article  Google Scholar 

  26. 26

    Drumheller, P. D., Elbert, D. L. & Hubbell, J. A. Multifunctional poly(ethylene glycol) semi-interpenetrating polymer networks as highly selective adhesive substrates for bioadhesive peptide grafting. Biotechnol. Bioeng. 43, 772–780 (1994).

    CAS  Article  Google Scholar 

  27. 27

    Bearinger, J. P. et al. P(AAm-co-EG) interpenetrating polymer networks grafted to oxide surfaces: Surface characterization, protein adsorption, and cell detachment studies. Langmuir 13, 5175–5183 (1997).

    CAS  Article  Google Scholar 

  28. 28

    Elisseeff, J. et al. Photoencapsulation of chondrocytes in poly(ethylene oxide)-based semi-interpenetrating networks. J. Biomed. Mater. Res. 51, 164–171 (2000).

    CAS  Article  Google Scholar 

  29. 29

    Gong, J. P., Katsuyama, Y., Kurokawa, T. & Osada, Y. Double-network hydrogels with extremely high mechanical strength. Adv. Mater. 15, 1155–1158 (2003).

    CAS  Article  Google Scholar 

  30. 30

    Park, Y. D., Tirelli, N. & Hubbell, J. A. Photopolymerized hyaluronic acid-based hydrogels and interpenetrating networks. Biomaterials 24, 893–900 (2003).

    CAS  Article  Google Scholar 

  31. 31

    Li, Y. J., Chung, E. H., Rodriguez, R. T., Firpo, M. T. & Healy, K. E. Hydrogels as artificial matrices for human embryonic stem cell self-renewal. J. Biomed. Mater. Res. A 79, 1–5 (2006).

    Article  Google Scholar 

  32. 32

    Buxton, A. N. et al. Design and characterization of poly(ethylene glycol) photopolymerizable semi-interpenetrating networks for chondrogenesis of human mesenchymal stem cells. Tissue Eng. 13, 2549–2560 (2007).

    CAS  Article  Google Scholar 

  33. 33

    Rowley, J. A., Madlambayan, G. & Mooney, D. J. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20, 45–53 (1999).

    CAS  Article  Google Scholar 

  34. 34

    Kong, H. J., Kim, C. J., Huebsch, N., Weitz, D. & Mooney, D. J. Noninvasive probing of the spatial organization of polymer chains in hydrogels using fluorescence resonance energy transfer (FRET). J. Am. Chem. Soc. 129, 4518–4519 (2007).

    CAS  Article  Google Scholar 

  35. 35

    Kleinman, H. K. et al. Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma . Biochemistry 21, 6188–6193 (1982).

    CAS  Article  Google Scholar 

  36. 36

    Tanner, K., Mori, H., Mroue, R., Bruni-Cardoso, A. & Bissell, M. J. Coherent angular motion in the establishment of multicellular architecture of glandular tissues. Proc. Natl Acad. Sci. USA 109, 1973–1978 (2012).

    CAS  Article  Google Scholar 

  37. 37

    Tse, J. M. et al. Mechanical compression drives cancer cells toward invasive phenotype. Proc. Natl Acad. Sci. USA 109, 911–916 (2012).

    CAS  Article  Google Scholar 

  38. 38

    Yuan, T. L. & Cantley, L. C. PI3K pathway alterations in cancer: variations on a theme. Oncogene 27, 5497–5510 (2008).

    CAS  Article  Google Scholar 

  39. 39

    Ali, S. & Coombes, R. C. Estrogen receptor α in human breast cancer: occurrence and significance. J. Mammary Gland Biol. Neoplasia 5, 271–281 (2000).

    CAS  Article  Google Scholar 

  40. 40

    Sun, M. et al. Phosphatidylinositol-3-OH Kinase (PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen receptor α (ERα) via interaction between ERα and PI3K. Cancer Res. 61, 5985–5991 (2001).

    CAS  Google Scholar 

  41. 41

    Humphries, J. D., Byron, A. & Humphries, M. J. Integrin ligands at a glance. J. Cell Sci. 119, 3901–3903 (2006).

    CAS  Article  Google Scholar 

  42. 42

    Niessen, C. M. et al. The α6β4 integrin is a receptor for both laminin and kalinin. Exp. Cell Res. 211, 360–367 (1994).

    CAS  Article  Google Scholar 

  43. 43

    Lee, E. C., Lotz, M. M., Steele, G. D. Jr & Mercurio, A. M. The integrin α6β4 is a laminin receptor. J. Cell Biol. 117, 671–678 (1992).

    CAS  Article  Google Scholar 

  44. 44

    Spinardi, L., Einheber, S., Cullen, T., Milner, T. A. & Giancotti, F. G. A recombinant tail-less integrin β 4 subunit disrupts hemidesmosomes, but does not suppress α6β4-mediated cell adhesion to laminins. J. Cell Biol. 129, 473–487 (1995).

    CAS  Article  Google Scholar 

  45. 45

    Litjens, S. H. M., de Pereda, J. M. & Sonnenberg, A. Current insights into the formation and breakdown of hemidesmosomes. Trends Cell Biol. 16, 376–383 (2006).

    CAS  Article  Google Scholar 

  46. 46

    Underwood, J. M. et al. The ultrastructure of MCF-10A acini. J. Cell. Physiol. 208, 141–148 (2006).

    CAS  Article  Google Scholar 

  47. 47

    Marinkovich, M. P. Tumour microenvironment: Laminin 332 in squamous-cell carcinoma. Nat. Rev. Cancer 7, 370–380 (2007).

    CAS  Article  Google Scholar 

  48. 48

    Giancotti, F. G. Targeting integrin β4 for cancer and anti-angiogenic therapy. Trends Pharmacol. Sci. 28, 506–511 (2007).

    CAS  Article  Google Scholar 

  49. 49

    Shaw, L. M., Rabinovitz, I., Wang, H. H., Toker, A. & Mercurio, A. M. Activation of phosphoinositide 3-OH kinase by the α6β4 integrin promotes carcinoma invasion. Cell 91, 949–960 (1997).

    CAS  Article  Google Scholar 

  50. 50

    Liu, H., Radisky, D. C., Wang, F. & Bissell, M. J. Polarity and proliferation are controlled by distinct signaling pathways downstream of PI3-kinase in breast epithelial tumor cells. J. Cell Biol. 164, 603–612 (2004).

    CAS  Article  Google Scholar 

  51. 51

    Geuijen, C. A. W. & Sonnenberg, A. Dynamics of the α6β4 integrin in keratinocytes. Mol. Biol. Cell 13, 3845–3858 (2002).

    CAS  Article  Google Scholar 

  52. 52

    Hakkinen, K. M., Harunaga, J. S., Doyle, A. D. & Yamada, K. M. Direct comparisons of the morphology, migration, cell adhesions, and actin cytoskeleton of fibroblasts in four different three-dimensional extracellular matrices. Tissue Eng. Part A 17, 713–724 (2011).

    CAS  Article  Google Scholar 

  53. 53

    Leung, C. T. & Brugge, J. S. Outgrowth of single oncogene-expressing cells from suppressive epithelial environments. Nature 482, 410–413 (2012).

    CAS  Article  Google Scholar 

  54. 54

    Bilodeau, G. G. Regular pyramid punch problem. J. Appl. Mech. 59, 519–523 (1992).

    Article  Google Scholar 

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Acknowledgements

The authors acknowledge the help of A. Li, D. Klumpers, A. Mao and other members of the Mooney lab. The authors also thank J. Brugge (Harvard Medical School) for providing the β4-integrin and Rac1 mutant plasmids, L. Lichten (Qiagen) for help with RNA arrays, Louise Jawerth/Weitz lab for help/use of the rheometer, M. Ericsson and L. Trakimas of the Harvard Medical School EM facility for help with transmission electron microscopy, P. Mali (Harvard Medical School) for discussions, and the Bauer Core for flow sorting. This work was supported by an NIH F32 grant to O.C. (CA153802), fellowships from NSERC and HHMI for S.T.K., fellowships from FCT, FCG and FLAD for C.B.d.C., and NIH (R01EB015498) and MRSEC (DMR-0820484) grants to D.J.M. This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Infrastructure Network (NNIN).

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O.C. and D.J.M. designed the IPNs. O.C., S.T.K. and D.J.M. designed the experiments. O.C., S.T.K., C.B.d.C., J-W.S. and C.S.V. conducted experiments and analysed data. C.B.d.C. designed and conducted the RNA expression arrays. K.H.A. conducted the comparison of in vitro results to human-breast-cancer samples. O.C., S.T.K., C.B.d.C. and D.J.M. wrote the manuscript.

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Correspondence to David J. Mooney.

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Chaudhuri, O., Koshy, S., Branco da Cunha, C. et al. Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium. Nature Mater 13, 970–978 (2014). https://doi.org/10.1038/nmat4009

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