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RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis

Abstract

RANK ligand (RANKL), a TNF-related molecule, is essential for osteoclast formation, function and survival through interaction with its receptor RANK1,2. Mammary glands of RANK- and RANKL-deficient mice develop normally during sexual maturation, but fail to form lobuloalveolar structures during pregnancy because of defective proliferation and increased apoptosis of mammary epithelium3. It has been shown that RANKL is responsible for the major proliferative response of mouse mammary epithelium to progesterone during mammary lactational morphogenesis4, and in mouse models, manipulated to induce activation of the RANK/RANKL pathway in the absence of strict hormonal control, inappropriate mammary proliferation is observed5,6. However, there is no evidence so far of a functional contribution of RANKL to tumorigenesis. Here we show that RANK and RANKL are expressed within normal, pre-malignant and neoplastic mammary epithelium, and using complementary gain-of-function (mouse mammary tumour virus (MMTV)-RANK transgenic mice) and loss-of function (pharmacological inhibition of RANKL) approaches, define a direct contribution of this pathway in mammary tumorigenesis. Accelerated pre-neoplasias and increased mammary tumour formation were observed in MMTV-RANK transgenic mice after multiparity or treatment with carcinogen and hormone (progesterone). Reciprocally, selective pharmacological inhibition of RANKL attenuated mammary tumour development not only in hormone- and carcinogen-treated MMTV-RANK and wild-type mice, but also in the MMTV-neu transgenic spontaneous tumour model. The reduction in tumorigenesis upon RANKL inhibition was preceded by a reduction in pre-neoplasias as well as rapid and sustained reductions in hormone- and carcinogen-induced mammary epithelial proliferation and cyclin D1 levels. Collectively, our results indicate that RANKL inhibition is acting directly on hormone-induced mammary epithelium at early stages in tumorigenesis, and the permissive contribution of progesterone to increased mammary cancer incidence is due to RANKL-dependent proliferative changes in the mammary epithelium. The current study highlights a potential role for RANKL inhibition in the management of proliferative breast disease.

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Figure 1: Decreased latency of mammary tumour formation and accelerated pre-neoplastic changes in mammary glands of MMTV-RANK mice treated with hormone and carcinogen.
Figure 2: RANKL is localized in PR + cells at multiple stages during mouse tumorigenesis, and RANKL and RANK are expressed in invasive carcinoma of the human breast.
Figure 3: RANK-Fc inhibits MPA/DMBA-induced mammary tumour formation which is preceded by a protective effect on early pre-neoplastic changes in the mammary gland.
Figure 4: RANK and RANKL are expressed in normal mammary epithelium of MMTV- neu mice, and RANK-Fc decreases spontaneous mammary tumorigenesis and lung metastasis in this model.

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Acknowledgements

We thank G. Begley, C. Queva and L. Kirsch for critical reading of the manuscript. The authors would like to thank W. Weng for statistical analysis; H. Li-Ya, L. Woody, K. Stocking, B. Saxbe, D. Hill, L. Cherepow, S. Allred, A. Winters and R. Soriano for technical assistance; M. Blake and M. Tometsko for critical discussions and technical assistance; I. Sarosi, C. Johnson and J. Hawkins for providing tissue specimens; and T. Chang, A. Foreman-Wykert and G. Smith for editorial support.

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E.G.-S. and A.P.J. conducted the largest portion of experiments, analysed data, designed experiments and helped to write the manuscript; J.J., R.M., R.E. and M.P.R.-M. conducted experiments and analysed data; J.P., D.B. and W.C.D. designed and supervised the study, analysed data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to William C. Dougall.

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All authors are Amgen employees and stockholders who contributed extensively to the work presented in this paper.

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Gonzalez-Suarez, E., Jacob, A., Jones, J. et al. RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis. Nature 468, 103–107 (2010). https://doi.org/10.1038/nature09495

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