The endosteal niche regulates breast cancer cell dormancy in bone: identification of new molecular determinants

Abstract

Cellular dormancy compromises the long-term survival of breast cancer patients. Bone represents a frequent site for metastasis, where Spindle-shaped N-cadherin⁺CD45⁻ osteoblasts (SNOs) hold dormant metastatic cells in the endosteal niche with a Notch2-dependent mechanism. In this work, we excluded the involvement of Notch1 in SNO-induced breast cancer cellular dormancy by immunofluorescence/immunohistochemistry and molecular approaches, using breast cancer tissues and cell lines. RNAdSeq in human bone metastatic MDA-MB231 breast cancer cells sorted for Notch1^HIGH and Notch2^HIGH expression demonstrated that, compared to their low counterpart, only Notch2^HIGH cells expressed enriched pathways relevant for the metastatic process, including pluripotency and Hematopoietic Stem Cell (HSC) gene signatures. They expressed the HSC-associated genes CXCR4, CD34 and TIE2 and MDA-MB231 cells enriched in the encoded proteins showed lesser proliferation ability. Reduced incidence of osteolytic lesions was induced by CXCR4^HIGH cells intratibially injected in immunocompromised mice, while lower lesion extension was induced by CXCR4^HIGH and TIE2^HIGH injected cells compared to CXCR4^LOW and TIE2^LOW cells. Notch2^HIGH cells were enriched in endoplasmic reticulum stress and unfolded protein response genes and overexpressed the CD177 protein, while the CD177 ligands, Plaur, Itgam and Ceacam 1, were highly expressed in SNOs. Kaplan-Meier plots showed positive correlation between high expression of CD177, ITGAM and CEACAM 1 - but not PLAUR - and overall survival of patients. CD177^HIGH cells were also CXCR4^HIGH, CD34^HIGH and Notch2^HIGH and proliferated less than CD177^LOW cells. These results support the relevance of Notch2 in SNO-mediated cellular dormancy and identified new pathways implicated in bone metastatic breast cancer cell quiescence.

References

[1] Liu R, et al.. Unveiling cancer dormancy: intrinsic mechanisms and extrinsic forces. Cancer Lett., 2024, 591: 216899

[2] Bushnell GG, et al.. Breast cancer dormancy: need for clinically relevant models to address current gaps in knowledge. NPJ Breast Cancer, 2021, 7 Article ID: 66

[3] Yip RKH, et al.. Mammary tumour cells remodel the bone marrow vascular microenvironment to support metastasis. Nat. Commun., 2021, 12 Article ID: 6920

[4] Heidrich I, Deitert B, Werner S, Pantel K. Liquid biopsy for monitoring of tumor dormancy and early detection of disease recurrence in solid tumors. Cancer Metastasis Rev., 2023, 42: 161-182

[5] Kuroda Y, et al.. Developing long bones respond to surrounding tissues by trans-pairing of periosteal osteoclasts and endocortical osteoblasts. Development, 2024, 151: dev202194

[6] Maciel GBM, Maciel RM, Danesi CC. Bone cells and their role in physiological remodeling. Mol. Biol. Rep., 2023, 50: 2857-2863

[7] Arai F, Suda T. Maintenance of quiescent hematopoietic stem cells in the osteoblastic niche. Ann. N. Y. Acad. Sci., 2007, 1106: 41-53

[8] Zhang J, et al.. Identification of the haematopoietic stem cell niche and control of the niche size. Nature, 2003, 425: 836-841

[9] Calvi LM, et al.. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature, 2003, 425: 841-846

[10] Weber JM, et al.. Parathyroid hormone stimulates expression of the Notch ligand Jagged1 in osteoblastic cells. Bone, 2006, 39: 485-493

[11] Capulli M, et al.. Notch2 pathway mediates breast cancer cellular dormancy and mobilisation in bone and contributes to haematopoietic stem cell mimicry. Br. J. Cancer., 2019, 121: 157-171

[12] Maurizi A, Ciocca M, Giuliani C, Di Carlo I, Teti A. Role of neural (N)-cadherin in breast cancer cell stemness and dormancy in the bone microenvironment. Cancers, 2022, 14: 1317

[13] BeLow M, Osipo C. Notch signaling in breast cancer: a role in drug resistance. Cells, 2020, 9: 2204

[14] Barsky L, Cohen-Erez I, Bado I, Zhang XH, Vago R. Review old bone, new tricks. Clin. Exp. Metastasis., 2022, 39: 727-742

[15] Tamizhmani P, et al.. Delineating Notch1 and Notch2: receptor-specific significance and therapeutic importance of pinpoint targeting strategies for hematological malignancies. Eur. J. Haematol., 2025, 114: 213-230

[16] Bui T, et al.. beta-Catenin haploinsufficiency promotes mammary tumorigenesis in an ErbB2-positive basal breast cancer model. Proc. Natl. Acad. Sci. USA, 2017, 114: E707-E716

[17] Sun Y, Sha Y, Cui G, Meng F, Zhong Z. Lysosomal-mediated drug release and activation for cancer therapy and immunotherapy. Adv. Drug Deliv. Rev., 2023, 192: 114624

[18] Michalczyk M, Humeniuk E, Adamczuk G, Korga-Plewko A. Hyaluronic acid as a modern approach in anticancer therapy-Review. Int. J. Mol. Sci., 2022, 24: 103

[19] Nairuz T, Mahmud Z, Manik RK, Kabir Y. Cancer stem cells: an insight into the development of metastatic tumors and therapy resistance. Stem Cell Rev. Rep., 2023, 19: 1577-1595

[20] Rehman A, Panda SK, Tirino V, Del Vecchio V. Cancer stem cells: detection and characterization from solid tumors. Methods Mol. Biol., 2024, 2835: 215-228

[21] Gondi CS, Rao JS. Cathepsin B as a cancer target. Expert Opin. Ther. Targets, 2013, 17: 281-291

[22] Mukhopadhyay S, Mahapatra KK, Praharaj PP, Patil S, Bhutia SK. Recent progress of autophagy signaling in tumor microenvironment and its targeting for possible cancer therapeutics. Semin, Cancer Biol., 2022, 85: 196-208

[23] Ferraresi A, et al.. How autophagy shapes the tumor microenvironment in ovarian cancer. Front. Oncol., 2020, 10 Article ID: 599915

[24] Hu CT, Wu JR, Wu WS. The role of endosomal signaling triggered by metastatic growth factors in tumor progression. Cell Signal, 2013, 25: 1539-1945

[25] Machado E, et al.. Regulated lysosomal exocytosis mediates cancer progression. Sci. Adv., 2015, 1 Article ID: e1500603

[26] Hwang S-M, Chang S, Rodriguez PC, Cubillos-Ruiz JR. Endoplasmic reticulum stress responses in anticancer immunity. Nat. Rev. Cancer, 2025, 25: 684-702

[27] Chen X, Cubillos-Ruiz JR. Endoplasmic reticulum stress signals in the tumour and its microenvironment. Nat. Rev. Cancer, 2021, 21: 71-88

[28] Lin Y, Jiang M, Chen W, Zhao T, Wei Y. Cancer and ER stress: mutual crosstalk between autophagy, oxidative stress and inflammatory response. Biomed. Pharmacother, 2019, 118: 109249

[29] Wang H, Mi K. Emerging roles of endoplasmic reticulum stress in the cellular plasticity of cancer cells. Front. Oncol., 2023, 13: 1110881

[30] Foote CA, et al.. Endothelial glycocalyx. Compr. Physiol., 2022, 12: 3781-3811

[31] Heldin P, Basu K, Kozlova I, Porsch H. HAS2 and CD44 in breast tumorigenesis. Adv. Cancer Res., 2014, 123: 211-229

[32] Yang Y, et al.. Nuclear transport proteins: structure, function, and disease relevance. Signal Transduct. Target Ther., 2023, 8: 425

[33] Yang H, Beutler B, Zhang D. Emerging roles of spliceosome in cancer and immunity. Protein Cell, 2022, 13: 559-579

[34] Wang Y, Deng B. Hepatocellular carcinoma: molecular mechanism, targeted therapy, and biomarkers. Cancer Metastasis Rev., 2023, 42: 629-652

[35] Dang F, Nie L, Wei W. Ubiquitin signaling in cell cycle control and tumorigenesis. Cell Death Differ., 2021, 28: 427-438

[36] Jamasbi E, Hamelian M, Hossain MA, Varmira K. The cell cycle, cancer development and therapy. Mol. Biol. Rep., 2022, 49: 10875-10883

[37] Yuan H, et al.. Lysine catabolism reprograms tumour immunity through histone crotonylation. Nature, 2023, 617: 818-826

[38] Hashimoto S, Kishimoto T. Roles of RNA-binding proteins in immune diseases and cancer. Semin. Cancer Biol., 2022, 86: 310-324

[39] Mughal MJ, Mahadevappa R, Kwok HF. DNA replication licensing proteins: saints and sinners in cancer. Semin. Cancer Biol., 2019, 58: 11-21

[40] Chatterjee S, Behnam B, Nimmagadda S. The intricate role of CXCR4 in cancer. Adv. Cancer Res., 2014, 124: 31-82

[41] Bianchi ME, Mezzapelle R. The chemokine receptor CXCR4 in cell proliferation and tissue regeneration. Front. Immunol., 2020, 11: 2109

[42] Müller A, et al.. Involvement of chemokine receptors in breast cancer metastasis. Nature, 2001, 410: 50-56

[43] Sugiyama T, Kohara H, Noda M, Nagasawa T. Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches. Immunity, 2006, 25: 977-988

[44] Nakamura Y, et al.. Isolation and characterization of endosteal niche cell populations that regulate hematopoietic stem cells. Blood, 2010, 116: 1422-1432

[45] Wang J, Loberg R, Taichman RS. The pivotal role of CXCL12 (SDF-1)/CXCR4 axis in bone metastasis. Cancer Metastasis Rev., 2006, 25: 573-587

[46] Sidney LE, Branch MJ, Dunphy SE, Dua HS, Hopkinson A. Concise review: evidence for CD34 as a common marker for diverse progenitors. Stem Cells, 2014, 32: 1380-1389

[47] Anjos-Afonso F, et al.. Single cell analyses identify a highly regenerative and homogenous human CD34+ hematopoietic stem cell population. Nat. Commun., 2022, 13 Article ID: 2048

[48] Gomei Y, et al.. Functional differences between two Tie2 ligands, angiopoietin-1 and -2, in regulation of adult bone marrow hematopoietic stem cells. Exp. Hematol., 2010, 38: 82-89

[49] Nie Y, Han Y-C, Zou Y-R. CXCR4 is required for the quiescence of primitive hematopoietic cells. J. Exp. Med., 2008, 205: 777-783

[50] Wang N, et al.. Mitotic quiescence, but not unique “stemness,” marks the phenotype of bone metastasis-initiating cells in prostate cancer. FASEB J., 2015, 19: 3141-3150

[51] Smith JT, Chai RC. Bone niches in the regulation of tumour cell dormancy. J. Bone Oncol., 2024, 47: 100621

[52] Mayhew V, Omokehinde T, Johnson RW. Tumor dormancy in bone. Cancer Rep., 2020, 3: e1156

[53] Azab AK, et al.. CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myeloma cells with the bone marrow microenvironment and enhances their sensitivity to therapy. Blood, 2009, 113: 4341-4351

[54] Drescher F, et al.. TIE2 induces breast cancer cell dormancy and inhibits the development of osteolytic bone metastases. Cancers, 2020, 12: 868

[55] Ranganathan AC, Adam AP, Zhang L, Aguirre-Ghiso JA. Tumor cell dormancy induced by p38SAPK and ER-stress signaling: an adaptive advantage for metastatic cells?. Cancer Biol. Ther., 2006, 5: 729-735

[56] Calvo V, et al.. A PERK-specific inhibitor blocks metastatic progression by limiting integrated stress response-dependent survival of quiescent cancer cells. Clin. Cancer Res., 2023, 29: 5155-5172

[57] Hsu SK, et al.. Unfolded protein response (UPR) in survival, dormancy, immunosuppression, metastasis, and treatments of cancer cells. Int. J. Mol. Sci., 2019, 20: 2518

[58] Yamazaki H, et al.. Ribosome binding protein GCN1 regulates the cell cycle and cell proliferation and is essential for the embryonic development of mice. PLoS Genet, 2020, 16 Article ID: e1008693

[59] Yin Y, et al.. Overactivation of XBP1 in plasma cells implies worse survival through innate immunity in esophageal squamous cell carcinoma. Cancer Lett., 2024, 597: 217045

[60] Bai M, et al.. CD177 modulates human neutrophil migration through activation-mediated integrin and chemoreceptor regulation. Blood, 2017, 130: 2092-2100

[61] Jerke U, et al.. Complement receptor Mac-1 is an adaptor for NB1 (CD177)-mediated PR3-ANCA neutrophil activation. J. Biol. Chem., 2011, 286: 7070-7081

[62] Jerke U, Marino SF, Daumke O, Kettritz R. Characterization of the CD177 interaction with the ANCA antigen proteinase 3. Sci. Rep., 2017, 7 Article ID: 43328

[63] Kluz PN, et al.. Cancer cell-intrinsic function of CD177 in attenuating beta-catenin signaling. Oncogene, 2020, 39: 2877-2889

[64] Kim MC, et al.. CD177 modulates the function and homeostasis of tumor-infiltrating regulatory T cells. Nat. Commun., 2021, 12 Article ID: 5764

[65] Zhou G, et al.. CD177⁺ neutrophils suppress epithelial cell tumourigenesis in colitis-associated cancer and predict good prognosis in colorectal cancer. Carcinogenesis, 2018, 39: 272-282

[66] Liang T, et al.. TYROBP, TLR4 and ITGAM regulated macrophages polarization and immune checkpoints expression in osteosarcoma. Sci. Rep., 2021, 11 Article ID: 19315

[67] Zhu J, Min N, Gong W, Chen Y, Li X. Identification of hub genes and biological mechanisms associated with non-alcoholic fatty liver disease and triple-negative breast cancer. Life, 2023, 13: 998

[68] Zhang B, Liu R, Huang H, Wang C, Yang C. Identifying CEACAM1 as a potential prognostic biomarker for basal-like breast cancer by bioinformatics analysis and in vitro experiments. J. Cancer, 2024, 15: 6468-6478

[69] Andres S, Edwards AB, Wittliff JL. Expression of urokinase-type plasminogen activator (uPA), its receptor (uPAR), and inhibitor (PAI-1) in human breast carcinomas and their clinical relevance. J. Clin. Lab. Anal., 2012, 26: 93-103

[70] Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol., 2010, 11 Article ID: R14

[71] Supek F, Bošnjak M, Škunca N, Šmuc T. Revigo summarizes and visualizes long lists of gene ontology terms. PLoS One, 2011, 6: e21800

[72] Bouxsein ML, et al.. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J. Bone Miner. Res., 2010, 25: 1468-1486

Funding

Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)(20116)

RIGHTS & PERMISSIONS

The Author(s)