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The tumor microenvironment modulates tamoxifen resistance in breast cancer: a role for soluble stromal factors and fibronectin through β1 integrin

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Abstract

Tamoxifen resistance has been largely attributed to genetic alterations in the epithelial tumor cells themselves, such as overexpression of HER-2/Neu. However, in the clinic, only about 15–20% of cases of HER-2/Neu amplification has actually been correlated to the acquisition of endocrine resistance, suggesting that other mechanisms must be involved as well. Using the epithelial LM05-E and the fibroblastic LM05-F cell lines, derived from the estrogen dependent spontaneous M05 mouse mammary tumor, as well as MCF-7 cells, we analyzed whether soluble stromal factors or extracellular matrix components protected against tamoxifen induced cell death. Involvement of signaling pathways was determined by using specific inhibitors and western blot, and phosphorylation of the estrogen receptor alpha by western blot and immunofluorescence. Soluble factors produced by the fibroblastic cells protect the epithelial tumor cells from tamoxifen-induced cell death through a mechanism that involves EGFR and matrix metalloproteinases upstream of PI3K/AKT. Exogenous fibronectin by itself confers endocrine resistance through interaction with β1 integrin and activation of PI3K/AKT and MAPK/ERK 1/2 pathways. The conferred resistance is reversed by blocking β1 integrin. We show also that treatment with both conditioned medium and fibronectin leads to the phosphorylation of the estrogen receptor at serine-118, suggesting stromal factors as modulators of ER activity. Our results show that the tumor microenvironment can modulate tamoxifen resistance, providing an alternative explanation for why patients become refractory to hormone-therapy.

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Abbreviations

MMP:

Matrix metalloproteinase

ER:

Estrogen receptor

FN:

Fibronectin

FCM:

LM05-F conditioned medium

NMC:

Non conditioned medium

PI:

Propidium iodide

DAPI:

4′,6-Diamino-2-phenylindole

EGFR:

Epidermal growth factor receptor

pSer-118 ER:

Phosphor-serine-118 ER

ECM:

Extracellular matrix

E2 :

Estradiol

PD:

PD98059

LY:

LY294002

T:

4-OH-tamoxifen

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Acknowledgments

Financial support: MS work is supported by a Grant from the Susan G. Komen for the Cure Foundation (BCTR0600341) and ANPCyT (PICT2008-0325/Préstamo BID); EBKJ by ANPCyT (PICT 00417/Préstamo BID) and UBACyT (M003). MJB’s laboratory is supported by grants from the U.S. Department of Energy, OBER Office of Biological and Environmental Research (DE-AC02-05CH1123), a Distinguished Fellow Award and Low Dose Radiation Program and the Office of Health and Environmental Research, Health Effects Division, (03-76SF00098); by National Cancer Institute awards 5 R01CA064786, R01CA057621, U54CA126552 and U54CA112970; by U.S. Department of Defense (W81XWH0810736).

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The authors declare they have no competing interests.

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Correspondence to Marina Simian.

Additional information

M. Simian and E. B. de Kier Joffé are members of the Research Career, Consejo Nacional de Investigaciones Científicas y Técnicas.

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Supplementary Fig. 1

LM05-E cells were starved for 48 h and then treated for 1 h with either vehicle, the PI3K/AKT inhibitor LY294002 (LY; 10 μM) (a), the MAPK/ERK inhibitor PD98059 (PD; 10 μM) (b) or the EGFR inhibitor AG1478 (AG; 6.4 μM) (c). At that time they were treated or not for 10 min with FCM. Samples were processed for western blot. As shown the three inhibitors were effective at the concentrations used. d LM05-E cells were plated on FN and treated with 10 nM estradiol (E2) or estradiol plus the PI3K/AKT inhibitor LY294002 (LY) or the MAPK/ERK inhibitor PD98059 (PD), both at a 10 μM concentration. Under these experimental conditions none of the inhibitors induced cell death (TIFF 2913 kb)

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Pontiggia, O., Sampayo, R., Raffo, D. et al. The tumor microenvironment modulates tamoxifen resistance in breast cancer: a role for soluble stromal factors and fibronectin through β1 integrin. Breast Cancer Res Treat 133, 459–471 (2012). https://doi.org/10.1007/s10549-011-1766-x

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