Stiff stroma increases breast cancer risk by inducing the oncogene ZNF217
Jason J. Northey, Alexander S. Barrett, Irene Acerbi, Mary-Kate Hayward, Stephanie Talamantes, Ivory S. Dean, Janna K. Mouw, Suzanne M. Ponik, Jonathon N. Lakins, Po-Jui Huang, Junmin Wu, Quanming Shi, Susan Samson, Patricia J. Keely, Rita A. Mukhtar, Jan T. Liphardt, John A. Shepherd, E. Shelley Hwang, Yunn-Yi Chen, Kirk C. Hansen, Laurie E. Littlepage, Valerie M. Weaver
Jason J. Northey, Alexander S. Barrett, Irene Acerbi, Mary-Kate Hayward, Stephanie Talamantes, Ivory S. Dean, Janna K. Mouw, Suzanne M. Ponik, Jonathon N. Lakins, Po-Jui Huang, Junmin Wu, Quanming Shi, Susan Samson, Patricia J. Keely, Rita A. Mukhtar, Jan T. Liphardt, John A. Shepherd, E. Shelley Hwang, Yunn-Yi Chen, Kirk C. Hansen, Laurie E. Littlepage, Valerie M. Weaver
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Research Article Oncology

Stiff stroma increases breast cancer risk by inducing the oncogene ZNF217

Abstract

Women with dense breasts have an increased lifetime risk of malignancy that has been attributed to a higher epithelial density. Quantitative proteomics, collagen analysis, and mechanical measurements in normal tissue revealed that stroma in the high-density breast contains more oriented, fibrillar collagen that is stiffer and correlates with higher epithelial cell density. microRNA (miR) profiling of breast tissue identified miR-203 as a matrix stiffness–repressed transcript that is downregulated by collagen density and reduced in the breast epithelium of women with high mammographic density. Culture studies demonstrated that ZNF217 mediates a matrix stiffness– and collagen density–induced increase in Akt activity and mammary epithelial cell proliferation. Manipulation of the epithelium in a mouse model of mammographic density supported a causal relationship between stromal stiffness, reduced miR-203, higher levels of the murine homolog Zfp217, and increased Akt activity and mammary epithelial proliferation. ZNF217 was also increased in the normal breast epithelium of women with high mammographic density, correlated positively with epithelial proliferation and density, and inversely with miR-203. The findings identify ZNF217 as a potential target toward which preexisting therapies, such as the Akt inhibitor triciribine, could be used as a chemopreventive agent to reduce cancer risk in women with high mammographic density.

Authors

Jason J. Northey, Alexander S. Barrett, Irene Acerbi, Mary-Kate Hayward, Stephanie Talamantes, Ivory S. Dean, Janna K. Mouw, Suzanne M. Ponik, Jonathon N. Lakins, Po-Jui Huang, Junmin Wu, Quanming Shi, Susan Samson, Patricia J. Keely, Rita A. Mukhtar, Jan T. Liphardt, John A. Shepherd, E. Shelley Hwang, Yunn-Yi Chen, Kirk C. Hansen, Laurie E. Littlepage, Valerie M. Weaver

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Figure 3

Tissues with high mammographic density are characterized by stiffened collagen fibers, elevated stromal and epithelial density, and expression of collagen cross-linking enzymes.

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Tissues with high mammographic density are characterized by stiffened co...
(A) Representative images from scanning electron microscopy (SEM; n = 5 each for low and high MD). Scale bar: 2 μm. (B) Quantification of levels for COL12A1 from proteomic analysis (MD1–MD4; see Figure 1). (C) Representative images showing immunofluorescence staining of frozen breast tissue sections with antibodies against keratins 8 and 18 (K8+18, green) and COL12A1 (red) (low MD, n = 5, high MD, n = 6 total). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 50 μm. (D–F) Immunohistochemical (IHC) staining of human breast tissues using antibodies against α-smooth muscle actin (α-SMA) (D), lysyl oxidase (LOX) (E), and lysyl hydroxylase 2 (LH2) (F). Scale bar: 100 μm. (G–I) Quantification of positive IHC staining for D–F as indicated (low MD, n = 5; high MD, n = 15 for G; low MD, n = 5; high MD, n = 12 for H; low MD, n = 6; high MD, n = 13 for I). (J) Quantification of atomic force microscopy (AFM) measurements of the mean elastic modulus (ECM stiffness) of human breast tissues (low MD, n = 10; high MD, n = 12). (K) A histogram displaying the top 20% of AFM stiffness values measured in J. (L) Representative images of hematoxylin and eosin–stained (H&E-stained) human breast tissue sections. (M) Quantification of epithelial density expressed as a percentage of epithelial cell area over total area per field of view (low MD, n = 20; high MD, n = 20). (N) Quantification of the average number of acini per terminal ductal lobular unit (TDLU) using the same tissues analyzed in Supplemental Figure 3. (O) A correlation plot of measured epithelial cell density (percentage epithelium in a field of view) versus measured ECM stiffness for a selection of human breast tissues (n = 7). Data are represented as mean ± SEM. #P < 0.1; *P < 0.05; **P < 0.01; ***P < 0.001 by 2-tailed unpaired Student’s t test (H–J and N), 2-tailed Mann-Whitney U test (M and G), or Kruskal-Wallis test followed by Dunn’s multiple-comparison test (B).