RANK signaling in mammary spontaneous tumorigenesis and tumor-stromal crosstalk

RESUMEN DE LA TESIS The discovery that RANK signaling mediates the protumorigenic role of progesterone in the mammary gland and its association with poor prognosis in breast cancer has opened the possibility of using RANKL inhibitors for breast cancer prevention but its therapeutic potential remained unexplored. Moreover, RANK expression is enriched in hormone receptor negative adenocarcinomas suggesting additional roles for RANK signaling beyond its hormone-dependent function. This work is mainly focused on exploring the role of RANK signaling on established mammary tumors, which could justify its potential use for breast cancer treatment. With this aim, we have used the mouse mammary tumor virus- Polyoma Middle T (MMTV-PyMT) which mimics RANK and RANKL expression patterns seen in human breast adenocarcinomas. Complementary genetic and pharmacological approaches demonstrate that RANK signaling promotes tumor progression, recurrence and survival of tumor cells by tumor cell intrinsic and tumor cell extrinsic mechanisms. Regarding to the tumor cell intrinsic mechanisms, therapeutic inhibition of RANK signaling drastically reduces the cancer stem cell pool, delays latency, decreases tumor and metastasis initiation and enhances sensitivity to chemotherapy. Mechanistically, genome wide expression analyses showed that anti-RANKL therapy promotes lactogenic differentiation of tumor cells. We further show that RANK signaling in tumor cells negatively regulates Ap2 transcription factors, and enhances the Wnt agonist Rspo1 and the Sca1- population, enriched in tumor initiating cells. Additionally, we found that expression of TFAP2B and the RANK inhibitor, OPG, in human breast cancer correlate and are associated with better relapse free survival. Regarding to the tumor cell extrinsic mechanisms, we found that RANK loss exclusively in tumor cells, drastically changes the tumor infiltrating lymphocytes (TIL)’s landscape. An increase in tumor infiltrating leukocytes and cytotoxic CD8 T lymphocytes, a lower CD4/CD8 ratio and a reduction in the immunosuppressive Ly6G neutrophils, hallmarks associated to anti-tumor response and good outcome, are found in RANK-deficient tumors. Strikingly, a T-cell deficient environment rescues the delayed tumor latency and reduced tumor initiating ability observed upon transplantation of RANK null tumor cells in an immunocompetent environment. Moreover, depletion experiments reveal CD8+ cytotoxic T cells as partially responsible for the induction of RANK null tumor cell death. Importantly, RANKL pharmacological inhibition also results in an increase in the number of infiltrating CD8 T cells and a more active IFNγ response. Mechanistically, we found that RANK depletion in tumor cells leads to decreased survival enhancing antigen presentation which may result into a more efficient anti-tumor immune response. In addition, decreased expression of TGFβ in the tumors, together with the decreased neutrophil infiltration could strengthen further the cytotoxicity of CD8. Despite the initially active immune responses tumors lacking RANK finally evade immune surveillance by activating inhibitory checkpoints. Finally, when anti-RANKL was combined with anti-CTLA4/PD-L1 treatment a synergistic temporary-dependent effect decreasing tumor growth was observed. These results support the use of RANKL inhibitors to reduce recurrence and metastasis and to boost the immune response in breast cancer patients based on its ability to induce tumor cell differentiation and to potentiate the anti-tumor immune response.