Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

RANK ligand as a potential target for breast cancer prevention in BRCA1-mutation carriers

Abstract

Individuals who have mutations in the breast-cancer-susceptibility gene BRCA1 (hereafter referred to as BRCA1-mutation carriers) frequently undergo prophylactic mastectomy to minimize their risk of breast cancer. The identification of an effective prevention therapy therefore remains a 'holy grail' for the field. Precancerous BRCA1mut/+ tissue harbors an aberrant population of luminal progenitor cells1, and deregulated progesterone signaling has been implicated in BRCA1-associated oncogenesis2,3,4,5. Coupled with the findings that tumor necrosis factor superfamily member 11 (TNFSF11; also known as RANKL) is a key paracrine effector of progesterone signaling6,7,8,9,10 and that RANKL and its receptor TNFRSF11A (also known as RANK) contribute to mammary tumorigenesis11,12,13, we investigated a role for this pathway in the pre-neoplastic phase of BRCA1-mutation carriers. We identified two subsets of luminal progenitors (RANK+ and RANK) in histologically normal tissue of BRCA1-mutation carriers and showed that RANK+ cells are highly proliferative, have grossly aberrant DNA repair and bear a molecular signature similar to that of basal-like breast cancer. These data suggest that RANK+ and not RANK progenitors are a key target population in these women. Inhibition of RANKL signaling by treatment with denosumab in three-dimensional breast organoids derived from pre-neoplastic BRCA1mut/+ tissue attenuated progesterone-induced proliferation. Notably, proliferation was markedly reduced in breast biopsies from BRCA1-mutation carriers who were treated with denosumab. Furthermore, inhibition of RANKL in a Brca1-deficient mouse model substantially curtailed mammary tumorigenesis. Taken together, these findings identify a targetable pathway in a putative cell-of-origin population in BRCA1-mutation carriers and implicate RANKL blockade as a promising strategy in the prevention of breast cancer.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: RANK expression in pre-neoplastic tissue and tumors from BRCA1-mutation carriers.
Figure 2: RANK+ LP cells are mitotically active and prone to DNA damage.
Figure 3: RANKL is required for progesterone-mediated cell proliferation in BRCA1mut/+ breast tissue.
Figure 4: RANKL inhibition markedly attenuates tumor onset in Brca1-deficient mice.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Lim, E. et al. Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1-mutation carriers. Nat. Med. 15, 907–913 (2009).

    CAS  PubMed  Article  Google Scholar 

  2. King, T.A. et al. Increased progesterone receptor expression in benign epithelium of BRCA1-related breast cancers. Cancer Res. 64, 5051–5053 (2004).

    CAS  PubMed  Article  Google Scholar 

  3. Ma, Y. et al. The breast cancer susceptibility gene BRCA1 regulates progesterone receptor signaling in mammary epithelial cells. Mol. Endocrinol. 20, 14–34 (2006).

    CAS  PubMed  Article  Google Scholar 

  4. Poole, A.J. et al. Prevention of Brca1-mediated mammary tumorigenesis in mice by a progesterone antagonist. Science 314, 1467–1470 (2006).

    CAS  PubMed  Article  Google Scholar 

  5. Widschwendter, M. et al. The sex hormone system in carriers of BRCA1/2 mutations: a case-control study. Lancet Oncol. 14, 1226–1232 (2013).

    CAS  PubMed  Article  Google Scholar 

  6. Asselin-Labat, M.L. et al. Control of mammary stem cell function by steroid hormone signaling. Nature 465, 798–802 (2010).

    CAS  PubMed  Article  Google Scholar 

  7. Beleut, M. et al. Two distinct mechanisms underlie progesterone-induced proliferation in the mammary gland. Proc. Natl. Acad. Sci. USA 107, 2989–2994 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. Fernandez-Valdivia, R. & Lydon, J.P. From the ranks of mammary progesterone mediators, RANKL takes the spotlight. Mol. Cell. Endocrinol. 357, 91–100 (2012).

    CAS  PubMed  Article  Google Scholar 

  9. Joshi, P.A. et al. Progesterone induces adult mammary stem cell expansion. Nature 465, 803–807 (2010).

    CAS  PubMed  Article  Google Scholar 

  10. Tanos, T. et al. Progesterone–RANKL is a major regulatory axis in the human breast. Sci. Transl. Med. 5, 182ra55 (2013).

    PubMed  Article  CAS  Google Scholar 

  11. Gonzalez-Suarez, E. et al. RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis. Nature 468, 103–107 (2010).

    CAS  PubMed  Article  Google Scholar 

  12. Schramek, D. et al. Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer. Nature 468, 98–102 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Pellegrini, P. et al. Constitutive activation of RANK disrupts mammary cell fate leading to tumorigenesis. Stem Cells 31, 1954–1965 (2013).

    PubMed  Article  CAS  Google Scholar 

  14. Antoniou, A. et al. Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: a combined analysis of 22 studies. Am. J. Hum. Genet. 72, 1117–1130 (2003).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Perou, C.M. et al. Molecular portraits of human breast tumors. Nature 406, 747–752 (2000).

    CAS  Article  PubMed  Google Scholar 

  16. Turner, N., Tutt, A. & Ashworth, A. Hallmarks of 'BRCAness' in sporadic cancers. Nat. Rev. Cancer 4, 814–819 (2004).

    CAS  PubMed  Article  Google Scholar 

  17. Venkitaraman, A.R. Cancer suppression by the chromosome custodians, BRCA1 and BRCA2. Science 343, 1470–1475 (2014).

    CAS  PubMed  Article  Google Scholar 

  18. Narod, S.A. & Foulkes, W.D. BRCA1 and BRCA2: 1994 and beyond. Nat. Rev. Cancer 4, 665–676 (2004).

    CAS  PubMed  Article  Google Scholar 

  19. Molyneux, G. et al. BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells. Cell Stem Cell 7, 403–417 (2010).

    CAS  PubMed  Article  Google Scholar 

  20. Proia, T.A. et al. Genetic predisposition directs breast cancer phenotype by dictating progenitor cell fate. Cell Stem Cell 8, 149–163 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Joshi, P.A. et al. RANK signaling amplifies WNT-responsive mammary progenitors through R-SPONDIN1. Stem Cell Rep. 5, 31–44 (2015).

    CAS  Article  Google Scholar 

  22. Pal, B. et al. Global changes in the mammary epigenome are induced by hormonal cues and coordinated by Ezh2. Cell Rep. 3, 411–426 (2013).

    CAS  PubMed  Article  Google Scholar 

  23. Eirew, P. et al. A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability. Nat. Med. 14, 1384–1389 (2008).

    CAS  PubMed  Article  Google Scholar 

  24. Eirew, P. et al. Aldehyde dehydrogenase activity is a biomarker of primitive normal human mammary luminal cells. Stem Cells 30, 344–348 (2012).

    CAS  PubMed  Article  Google Scholar 

  25. Liu, S. et al. BRCA1 regulates human mammary stem–progenitor cell fate. Proc. Natl. Acad. Sci. USA 105, 1680–1685 (2008).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  26. Wood, C.E. et al. Progestin effects on cell proliferation pathways in the postmenopausal mammary gland. Breast Cancer Res. 15, R62 (2013).

    PubMed  PubMed Central  Article  Google Scholar 

  27. Pathania, S. et al. BRCA1 haploinsufficiency for replication-stress suppression in primary cells. Nat. Commun. 5, 5496 (2014).

    PubMed  Article  Google Scholar 

  28. Sedic, M. et al. Haploinsufficiency for BRCA1 leads to cell-type-specific genomic instability and premature senescence. Nat. Commun. 6, 7505 (2015).

    CAS  PubMed  Article  Google Scholar 

  29. Kostenuik, P.J. et al. Denosumab, a fully human monoclonal antibody to RANKL, inhibits bone resorption and increases BMD in knock-in mice that express chimeric (murine–human) RANKL. J. Bone Miner. Res. 24, 182–195 (2009).

    CAS  PubMed  Article  Google Scholar 

  30. Shackleton, M. et al. Generation of a functional mammary gland from a single stem cell. Nature 439, 84–88 (2006).

    CAS  PubMed  Article  Google Scholar 

  31. Shehata, M. et al. Phenotypic and functional characterization of the luminal cell hierarchy of the mammary gland. Breast Cancer Res. 14, R134 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Liu, X. et al. Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer. Proc. Natl. Acad. Sci. USA 104, 12111–12116 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. Fu, N.Y. et al. EGF-mediated induction of Mcl-1 at the switch to lactation is essential for alveolar cell survival. Nat. Cell Biol. 17, 365–375 (2015).

    CAS  PubMed  Article  Google Scholar 

  34. Hartmann, L.C. & Lindor, N.M. The role of risk-reducing surgery in hereditary breast and ovarian cancer. N. Engl. J. Med. 374, 454–468 (2016).

    CAS  PubMed  Article  Google Scholar 

  35. Phillips, K.A. & Lindeman, G.J. Breast cancer prevention for BRCA1- and BRCA-mutation carriers: is there a role for tamoxifen? Future Oncol. 10, 499–502 (2014).

    CAS  PubMed  Article  Google Scholar 

  36. Domchek, S.M. et al. Association of risk-reducing surgery in BRCA1- or BRCA2-mutation carriers with cancer risk and mortality. J. Am. Med. Assoc. 304, 967–975 (2010).

    CAS  Article  Google Scholar 

  37. Rebbeck, T.R. et al. Prophylactic oophorectomy in carriers of BRCA1 or BRCA2 mutations. N. Engl. J. Med. 346, 1616–1622 (2002).

    PubMed  Article  Google Scholar 

  38. Heemskerk-Gerritsen, B.A. et al. Breast cancer risk after salpingo-oophorectomy in healthy BRCA1/2-mutation carriers: revisiting the evidence for risk reduction. J. Natl. Cancer Inst. 107, djv033 (2015).

    PubMed  Article  CAS  Google Scholar 

  39. To, C. et al. The PARP inhibitors veliparib and olaparib are effective chemopreventive agents for delaying mammary tumor development in BRCA1-deficient mice. Cancer Prev. Res. (Phila.) 7, 698–707 (2014).

    CAS  Article  Google Scholar 

  40. Gnant, M. et al. Adjuvant denosumab in breast cancer (ABCSG-18): a multicenter, randomized, double-blind, placebo-controlled trial. Lancet 386, 433–443 (2015).

    CAS  PubMed  Article  Google Scholar 

  41. Mann, G.J. et al. Analysis of cancer risk and BRCA1 and BRCA2 mutation prevalence in the kConFab familial breast cancer resource. Breast Cancer Res. 8, R12 (2006).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  42. Branstetter, D.G. et al. Denosumab induces tumor reduction and bone formation in patients with giant-cell tumor of bone. Clin. Cancer Res. 18, 4415–4424 (2012).

    CAS  PubMed  Article  Google Scholar 

  43. Wagner, K.U. et al. Cre-mediated gene deletion in the mammary gland. Nucleic Acids Res. 25, 4323–4330 (1997).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. Xu, X. et al. Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumor formation. Nat. Genet. 22, 37–43 (1999).

    CAS  PubMed  Article  Google Scholar 

  45. Jacks, T. et al. Tumor spectrum analysis in p53-mutant mice. Curr. Biol. 4, 1–7 (1994).

    CAS  PubMed  Article  Google Scholar 

  46. Oakes, S.R. et al. Sensitization of BCL-2-expressing breast tumors to chemotherapy by the BH3 mimetic ABT-737. Proc. Natl. Acad. Sci. USA 109, 2766–2771 (2012).

    CAS  PubMed  Article  Google Scholar 

  47. Liao, Y., Smyth, G.K. & Shi, W. The Subread aligner: fast, accurate and scalable read-mapping by seed-and-vote. Nucleic Acids Res. 41, e108 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  48. Liao, Y., Smyth, G.K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    CAS  Article  PubMed  Google Scholar 

  49. Robinson, M.D. & Oshlack, A. A scaling-normalization method for differential expression analysis of RNA-seq data. Genome Biol. 11, R25 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  50. Ritchie, M.E. et al. limma powers differential expression analyses for RNA sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  51. Law, C.W., Chen, Y., Shi, W. & Smyth, G.K. voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, R29 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  52. Smyth, G.K. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, e3 (2004).

    Article  Google Scholar 

  53. Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    CAS  PubMed  Article  Google Scholar 

  54. Wu, D. et al. ROAST: rotation gene set tests for complex microarray experiments. Bioinformatics 26, 2176–2182 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to the women who generously donated breast tissue for our studies and to the surgical, pathology and tissue bank colleagues for their substantial assistance and support. We thank L. Taylor, K. Shackleton, S. Nightingale and H. Liu for expert assistance; the Animal, FACS, Imaging and Histology facilities at WEHI; C. Perou and K. Hoadley for kind provision of the PAM50 subtypes; and A.Y. Rhee (Amgen Inc.) for assistance with the manuscript submission. We are grateful to H. Thorne and all kConFab staff, members and families for their contributions to this resource and to the Victorian Cancer Biobank (supported by the Victorian Government), which also provided coded breast tissue and data. We thank H. Yasuda for advice on the antibody to mouse RANKL and K.U. Wagner (Eppley Institute, Nebraska) for MMTV-Cre A mice. This work was supported by the Australian National Health and Medical Research Council (NHMRC) (grant numbers 1016701 (J.E.V. and G.J.L.), 1040978 (G.B.M., J.E.V. and G.J.L.) and 1054618 (G.K.S.)), NHMRC Independent Research Institute Infrastructure Support Scheme (IRIISS) (to WEHI; E.N., F.V., B.P., G.G., L.W., S.W.L., G.K.S., J.E.V. and G.J.L.), the Victorian State Government through the Victorian Cancer Agency and Operational Infrastructure Support, the National Breast Cancer Foundation (Australia) (grant numbers NC-13-32 and PS-15-042; to J.E.V. and G.J.L.); Amgen Inc. (J.E.V. and G.J.L.), the Qualtrough Cancer Research Fund (J.E.V. and G.J.L.), the Joan Marshall Breast Cancer Research Fund (J.E.V. and G.J.L.) and the Australian Cancer Research Foundation (to WEHI; E.N., F.V., B.P., G.G., L.W., S.W.L., G.K.S., J.E.V. and G.J.L.). E.N. is supported by a Cancer Council Victoria Scholarship and a Cancer Therapeutics CRC Top-Up Scholarship; B.P. is supported by a VCA Early Career Seed Grant 13035; G.K.S. and G.J.L. are supported by NHMRC Fellowships 1058892 (G.K.S.), 637307 (G.J.L.) and 1078730 (G.J.L.); and J.E.V. is supported by NHMRC Australia Fellowship 1037230.

Author information

Authors and Affiliations

Authors

Consortia

Contributions

E.N. designed and performed experiments, carried out data analysis and contributed to manuscript writing; B.P. performed RNA-seq and mRNA studies; D.B. performed pathology scoring and analysis of tumor and normal tissue samples; F.V. performed in vivo studies and analysis; G.G. and G.K.S. carried out bioinformatics analysis; L.W. developed a new scoring assay; and G.J.L. and S.W.L. developed and implemented the investigator-sponsored (ISS) BRCA-D study protocol. No Amgen authors were directly or indirectly involved in the ISS design, implementation, analysis or ISS-derived reported results (Fig. 3d and Supplementary Fig. 4b); G.B.M. provided material and discussions; kConFab provided annotated samples; K.R., L.-Y.H. and R.S. assisted with histological analysis of tumor and normal tissue samples; and J.E.V. and G.J.L. conceived the study, designed experiments and wrote the paper in collaboration with W.C.D.

Corresponding authors

Correspondence to Jane E Visvader or Geoffrey J Lindeman.

Ethics declarations

Competing interests

D.B., K.R., L.-Y.H., R.S. and W.C.D. were former Amgen employees. D.B., K.R. and L.-Y.H. currently hold Amgen stock. Amgen Inc. contributed reagents and some financial support for this study, including financial support for the investigator-sponsored study, BRCA-D.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1–5 (PDF 7177 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nolan, E., Vaillant, F., Branstetter, D. et al. RANK ligand as a potential target for breast cancer prevention in BRCA1-mutation carriers. Nat Med 22, 933–939 (2016). https://doi.org/10.1038/nm.4118

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.4118

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing