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Downregulation of estrogen receptor and modulation of growth of breast cancer cell lines mediated by paracrine stromal cell signals

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Abstract

Purpose

Breast cancers have a poorer prognosis if estrogen receptor expression was lost during recurrence. It is unclear whether this conversion is cell autonomous or whether it can be promoted by the microenvironment during cancer dormancy. We explored the ability of marrow-derived stromal cell lines to arrest co-cultured breast cancer cells and suppress estrogen receptor alpha (ER) expression during arrest, facilitating the emergence of estrogen-independent breast cancer clones.

Methods

Cancer cell growth, ER protein, microRNA, and mRNA levels were measured in breast cancer cell lines exposed to conditioned medium from marrow stromal lines in the presence and absence of estrogen and of signaling pathway modulators.

Results

We demonstrate that paracrine signaling from the stromal cell line HS5 downregulated ER in T47D and MCF7 breast cancer cells. This occurred at the mRNA level and also through decreased ER protein stability. Additionally, conditioned medium (CM) from HS5 arrested the breast cancer cells in G0/G1 in part through interleukin-1 (IL1) and inhibited cancer cell growth despite the activation of proliferative pathways (Erk and AKT) by the CM. Similar findings were observed for CM from the hFOB 1.19 osteoblastic cell line but not from two other fibroblastic marrow lines, HS27A and KM101. HS5-CM inhibition of MCF7 proliferation could not be restored by exogenous ER, but was restored by the IL1-antagonist IL1RA. In the presence of IL1RA, HS5-CM activation of AKT and Erk enabled the outgrowth of breast cancer cells with suppressed ER that were fulvestrant-resistant and estrogen-independent.

Conclusions

We conclude that marrow-derived stromal cells can destabilize estrogen receptor protein to convert the ER status of growth-arrested ER+ breast cancer cell lines. The balance between stromal pro- and anti-proliferative signals controlled the switch from a dormant phenotype to estrogen-independent cancer cell growth.

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Acknowledgements

This project was supported by NIH Grant R01CA154728 (RAS). The project used the UPCI Biostatistics Facility and Cancer Proteomics Facility: Luminex Core that are supported in part by award P30CA047904. We thank Drs. Steffi Oesterreich and Tim Burns (University of Pittsburgh) for helpful comments; Dr. Vera Donnenberg for assistance with flow cytometry and for HFF1 foreskin fibroblasts; and Lucas Santana-Santos for his work on the microRNA analysis algorithm. In addition, we appreciate the generosity of Didier Picard (Universite de Geneve) for the gift of the ER-3′-UTR reporter plasmid; Dr. Sarat Chandarlapaty (Sloan Kettering Cancer Center) for MCF7 cells stably expressing inducible estrogen receptor or vector (mCherry) control; and University of Pittsburgh investigators Steffi Oesterreich for T47D cells, Michael Epperly for KM101 cells, and Adrian Lee for phospho-IGF1R antibody. The 3X ERE TATA luc was a gift from Donald McDonnell (Addgene plasmid # 11354), and pEGFP-C1-ER alpha was a gift from Michael Mancini (Addgene plasmid # 28230). We thank Yingjian Li (University of Pittsburgh) for assistance in exosome preparation. The use of the fluorescence plate reader in the laboratory of Dr. Ben van Houten (University of Pittsburgh) is also appreciated.

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    Authors and Affiliations

    1. Department of Medicine, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Suite 2.26f Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA

      J. Huang, P. Woods, J. P. Goff, C. J. Stehle & R. A. Steinman

    2. School of Medicine, Tsinghua University, Bei**g, China

      J. Huang

    3. Department of Biostatistics, University of Pittsburgh and University of Pittsburgh Cancer Institute, Pittsburgh, USA

      D. Normolle

    4. Department of Computational Biology, University of Pittsburgh and University of Pittsburgh Cancer Institute, Pittsburgh, USA

      P. V. Benos

    5. Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, USA

      R. A. Steinman

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    J. Huang and P. Woods contributed equally to this work.

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    Huang, J., Woods, P., Normolle, D. et al. Downregulation of estrogen receptor and modulation of growth of breast cancer cell lines mediated by paracrine stromal cell signals. Breast Cancer Res Treat 161, 229–243 (2017). https://doi.org/10.1007/s10549-016-4052-0

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