Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Jun 22:12:266.
doi: 10.1186/1471-2407-12-266.

Hornerin, an S100 family protein, is functional in breast cells and aberrantly expressed in breast cancer

Jodie M Fleming  1 , Erika Ginsburg, Shannon D Oliver, Paul Goldsmith, Barbara K Vonderhaar
Affiliations

Hornerin, an S100 family protein, is functional in breast cells and aberrantly expressed in breast cancer

Jodie M Fleming et al. BMC Cancer. .

Abstract

Background: Recent evidence suggests an emerging role for S100 protein in breast cancer and tumor progression. These ubiquitous proteins are involved in numerous normal and pathological cell functions including inflammatory and immune responses, Ca(2+) homeostasis, the dynamics of cytoskeleton constituents, as well as cell proliferation, differentiation, and death. Our previous proteomic analysis demonstrated the presence of hornerin, an S100 family member, in breast tissue and extracellular matrix. Hornerin has been reported in healthy skin as well as psoriatic and regenerating skin after wound healing, suggesting a role in inflammatory/immune response or proliferation. In the present study we investigated hornerin's potential role in normal breast cells and breast cancer.

Methods: The expression levels and localization of hornerin in human breast tissue, breast tumor biopsies, primary breast cells and breast cancer cell lines, as well as murine mammary tissue were measured via immunohistochemistry, western blot analysis and PCR. Antibodies were developed against the N- and C-terminus of the protein for detection of proteolytic fragments and their specific subcellular localization via fluorescent immunocytochemisty. Lastly, cells were treated with H(2)O(2) to detect changes in hornerin expression during induction of apoptosis/necrosis.

Results: Breast epithelial cells and stromal fibroblasts and macrophages express hornerin and show unique regulation of expression during distinct phases of mammary development. Furthermore, hornerin expression is decreased in invasive ductal carcinomas compared to invasive lobular carcinomas and less aggressive breast carcinoma phenotypes, and cellular expression of hornerin is altered during induction of apoptosis. Finally, we demonstrate the presence of post-translational fragments that display differential subcellular localization.

Conclusions: Our data opens new possibilities for hornerin and its proteolytic fragments in the control of mammary cell function and breast cancer.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Immunolocalization of hornerin expression in breast tissue. (A) Reduction mammoplasty tissue sections were subjected to immunohistochemical analysis using a hornerin specific antibody or corresponding negative control. A minimum of 10 patient samples were analyzed. Yellow bar = 200 μM, red bar = 50 μM. (B) Western blot analysis of hornerin expression in primary breast fibroblast and epithelial cell whole cell lysates isolated from reduction mammoplasty tissue samples. A minimum of five patient samples were analyzed per cell type. Representative data from five breast fibroblasts and three breast epithelial samples are shown. (C) Western blot analysis of exosomes isolated from primary breast fibroblast and epithelial cell cultures and representative TEM image of purified exosomes 30,000X. FB = fibroblasts, Epi = epithelial cells.
Figure 2
Figure 2
Hornerin expression throughout the stages of mammary development. (A) Representative images of mammary glands isolated from the indicated stages of development that were subjected to immunohistochemical analysis using a hornerin-specific antibody or corresponding negative control. (B) Data represent the mean +/− SD of a minimum of three glands quantitated using Image J64. *P < 0.05 compared to nulliparous and pregnant glands. (C) Data represent the mean +/− SD of number of hornerin positive macrophages counted per 40x field; a minimum of three glands was counted at each developmental stage and three fields were counted per slide. * *P < 0.05 compared to nulliparous glands. (D) Representative images of immune cells in mammary glands that were subjected to immunohistochemical analysis using a hornerin-specific antibody and the corresponding PCR analysis of hornerin in human monocytes/macrophages isolated from peripheral blood; cells were treated +/− 20 ng/ml of LPS/IFNγ for five days. N = nulliparous week 5, Pg = pregnancy day 9, L D1 = lactation day 1, L D15 = lactation day 15, I D1 = involution day 1, I D7 = involution day 7. Yellow bar = 200 μm, Red bar = 50 μm.
Figure 3
Figure 3
Hornerin expression in an in vitro model of breast cancer progression. (A) Quantitative real time PCR analysis of hornerin transcript abundance in proliferating MCF10A cell line series. Quantified data were normalized to the housekeeping gene GAPDH. (B) Western blot analysis of hornerin expression in proliferating MCF10A cell line series; α-tubulin was used as a loading control. (C) Data represent mean +/− SD of three independent experiments. Quantified values were expressed relative to corresponding abundance of tubulin protein expression *P < 0.05.
Figure 4
Figure 4
Correlation of hornerin expression with breast cancer subtype and TNM staging. (A) Representative images of breast cancer biopsies that were subjected to immunohistochemical analysis using a hornerin-specific antibody or corresponding negative control (219 individual patient samples analyzed). Yellow bar = 200 μM. Hornerin expression in correlation with (B) breast cancer subtype IDC = invasive ductal carcinoma. ILC = invasive lobular carcinoma, (C) tumor grade, and (D) lymph node metastasis. Data represent mean relative intensity +/− SD. *P < 0.05.
Figure 5
Figure 5
Localization of hornerin fragments in breast cancer cells. (A) Representative western blot of hornerin products in breast cancer cell lines. ER = estrogen receptor, PR = progesterone receptor; α-tubulin was used as a loading control. (B) Representative western blot of MCF10AI subcellular fractionated products. N = nuclear, C = cytoplasmic, M = membrane fraction. Lamin A/C indicates purity of subcellular fractions. (C) Confocal microscopy images of hornerin localization using antibodies directed against the proteins N-terminus and C-terminus in the indicated breast cancer cell lines (63x magnification). For each cell line, the top panel shows hornerin staining (green), while the bottom panel shows both the nuclear (DNA stained with DAPI, blue) and hornerin staining (green).
Figure 6
Figure 6
Induction of cell death events increases hornerin expression and fragmentation. (A) Quantitative real time PCR analysis of hornerin transcript abundance in MCF10AI cells treated overnight with increasing concentrations of H2O2. Data are the mean +/− SEM of three independent experiments; * P < 0.05. (B) Representative western blot of hornerin fragments, caspase 3 and cleaved caspase 3 in MCF10AI cells treated with the indicated concentrations of H2O2. α-Tubulin was used as a loading control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Carafoli E. Calcium signaling: a tale for all seasons. Proc Natl Acad Sci USA. 2002;99(3):1115–1122. - PMC - PubMed
    1. Leclerc E, Heizmann CW. The importance of Ca2+/Zn2+ signaling S100 proteins and RAGE in translational medicine. Front Biosci (Schol Ed) 2011;3:1232–1262. - PubMed
    1. Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 2003;4(7):517–529. - PubMed
    1. Heizmann CW, Ackermann GE, Galichet A. Pathologies involving the S100 proteins and RAGE. Subcell Biochem. 2007;45:93–138. - PubMed
    1. McKiernan E, McDermott EW, Evoy D, Crown J, Duffy MJ. The role of S100 genes in breast cancer progression. Tumour Biol. 2011;32(3):441–450. - PubMed

Publication types

MeSH terms