New insights into RANK signaling pathway in the mammary gland

Abstract

Receptor activator of nuclear factor kappa-β ligand (RANKL) and its receptor RANK are key mediators of bone resorption1,2. RANKL, its receptor RANK and its natural decoy receptor OPG, which is a competitive inhibitor that binds to RANKL and negatively regulates the pathway3, are members of the tumor necrosis factor (TNF) superfamily. Based on this key role in bone remodeling, a monoclonal antibody against RANKL, called denosumab, has been developed and is used in the clinics for the treatment of osteoporosis and bone metastasis. Moreover, RANK pathway is not only essential for bone remodeling, but also plays important roles in lymph node organogenesis, immune cell activation4,5 and mammary gland development6,7. Disrupted mammary gland development during pregnancy and impaired lactation are observed as a consequence of RANK loss or overexpression (under the MMTV-promoter)7,8. Defective alveologenesis in RANK-null mice has been attributed to decreased proliferation and survival of mammary epithelial cells6.

Overexpression of RANK in mammary epithelial cells enhances proliferation, impairs lactation and disrupts mammary cell fate resulting in the accumulation of MaSCs (mammary stem cells)9. Pharmacological inhibition of RANKL with RANK-Fc, which binds to RANKL and blocks the pathway, completely prevents MPA/DMBA-induced mammary tumor formation in WT mice. Thus, RANKL is the main mediator of the protumorigenic role of progesterone in the mouse mammary gland10. High levels of Rank in both mouse and human mammary epithelial cells (MECs) induce stemness and promote tumorigenesis and metastasis9,11. Conversely, inhibition of RANK pathway using genetic and pharmacological approaches decreases the incidence of spontaneous preneoplasic lesions, tumors and lung metastasis in the oncogene-driven MMTV-PyMT and MMTV-Neu mice10,12. Thus, inhibition of Rank signaling has emerged as a new strategy for breast cancer prevention and treatment.

Unexpectedly, we found a delayed latency in tumor formation and reduced tumor incidence in transgenic mice overexpressing in MECs both Rank and Neu or Rank and PyMT compared to single mutants. Despite the initial attenuation in tumor growth, Rank overexpressing tumors grew faster and seeded more lung metastasis, in agreement with an expansion of the cancer stem cell population. Mechanistically we found that Rank overexpression induced senescence in the mammary epithelia blocking the progression from hyperplasias to mammary intraepithelial neoplasia. High levels of senescence were observed in the mammary glands and cultured MECs of Rank transgenic mice, even in the absence of other oncogenic stimuli. Mice that overexpress Rank in the mammary gland are more susceptible to mammary tumorigenesis driven by carcinogens and spontaneously develop mammary tumors after multiple pregnancies with long latency9,10. These results indicate that eventually Rank-overexpressing MECs evade or scape senescence allowing tumor formation. Moreover, infection of MECs and mouse embryonic fibroblast (MEFs) with Rank overexpressing vectors (but not PyMT or Neu) led to DNA damage and senescence. Like Ras, Rank induces a typical “oncogene-induced senescence (OIS)” through p16/p19. Senescence was also observed in the WT epithelia after Rankl exposure highlighting the physiological relevance of senescence during mammary gland development and tumor initiation. Importantly, Rank-OIS was essential for Rank-driven stemness, as the increased mammosphere forming ability of Rank overexpressing MECs and breast cancer cells is lost after senolytic treatments. We uncovered an unexpected dual role for Rank in the mammary epithelia able to induce stemness as well as OIS, delaying tumor initiation but increasing aggressiveness of established tumors once senescence is overcome. Dougall WC, Glaccum M, Charrier K, et al. RANK is essential for osteoclast and lymph node development. Genes Dev. 1999;13(18):2412-2424. http://www.ncbi.nlm.nih.gov/pubmed/10500098. Accessed January 25, 2017.

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5. Yasuda H, Shima N, Nakagawa N, et al. Osteoclast Differentiation Factor Is a Ligand for Osteoprotegerin Osteoclastogenesis-Inhibitory Factor and Is Identical to TRANCERANKL. Vol 95.; 1998. www.pnas.org. Accessed April 20, 2019.

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8. Cordero A, Pellegrini P, Sanz-Moreno A, et al. Rankl Impairs Lactogenic Differentiation Through Inhibition of the Prolactin/Stat5 Pathway at Midgestation. Stem Cells. 2016;34(4):1027-1039. doi:10.1002/stem.2271

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10. Gonzalez-Suarez E, Jacob AP, Jones J, et al. RANK ligand mediates progestin- induced mammary epithelial proliferation and carcinogenesis. Nature. 2010;468(7320):103-107. doi:10.1038/nature09495

11. Palafox M, Ferrer I, Pellegrini P, et al. RANK Induces Epithelial-Mesenchymal Transition and Stemness in Human Mammary Epithelial Cells and Promotes Tumorigenesis and Metastasis. Cancer Res. 2012;72(11):2879-2888. doi:10.1158/0008-5472.CAN-12-0044

12. Yoldi G, Pellegrini P, Trinidad EM, et al. Tumor and Stem Cell Biology RANK Signaling Blockade Reduces Breast Cancer Recurrence by Inducing Tumor Cell Differentiation. Cancer Res. 2016;76(19):1-13. doi:10.1158/0008-5472.CAN-15- 2745