PTHrP(12-48) Modulates the Bone Marrow Microenvironment and Suppresses Human Osteoclast Differentiation and Lifespan

Abstract

Bone is a common site for metastasis in breast cancer patients and is associated with a series of complications that significantly compromise patient survival, partially due to the advanced stage of disease at the time of detection. Currently, no clinically-approved biomarkers can identify or predict the development of bone metastasis. We recently identified a unique peptide fragment of parathyroid hormone-related protein (PTHrP), PTHrP(12-48), as a validated serum biomarker in breast cancer patients that correlates with and predicts the presence of bone metastases. In this study, the biological activity and mode of action of PTHrP(12-48) was investigated. Sequence-based and structure-based bioinformatics techniques predicted that the PTHrP(12-48) fragment formed an alpha helical core followed by an unstructured region after residue 40 or 42. Thereafter, detailed structure alignment and molecular docking simulations predicted a lack of interaction between PTHrP(12-48) and the cognate PTH1 receptor (PTHR1). The in silico prediction was confirmed by the lack of PTHrP(12-48)-stimulated cAMP accumulation in PTHR1-expressing human SaOS2 cells. Using a specific human PTHrP(12-48) antibody that we developed, PTHrP(12-48) was immunolocalized in primary and bone metastatic human breast cancer cells, as well as within human osteoclasts (OCLs) in bone metastasis biopsies, with little or no localization in other resident bone or bone marrow cells. In vitro, PTHrP(12-48) was internalized into cultured primary human OCLs and their precursors within 60 min. Interestingly, PTHrP(12-48) treatment dose-dependently suppressed osteoclastogenesis, via the induction of apoptosis in both OCL precursors as well as in mature OCLs, as measured by the activation of cleaved caspase 3. Collectively, these data suggest that PTHrP(12-48) is a bioactive breast cancer–derived peptide that locally regulates the differentiation of hematopoietic cells and the activity of osteoclasts within the tumor–bone marrow microenvironment, perhaps to facilitate tumor control of bone.

Targeting the RANKL/RANK/OPG Axis for Cancer Therapy

“…RANK (TNFRSF11A, OFE, ODFR, TRANCE-R, ODAR, CD265) ( 4 , 12 , 13 ) and RANKL (TNFSF11, TRANCE, ODF, and OPGL) ( 14 – 17 ) are a receptor-ligand pair of the TNF receptor superfamily discovered at the end of the last millennium and were identified as key regulators of osteoclast development and bone metabolism ( 12 , 18 ) ( Figure 1 ). Factors that can induce bone resorption, such as the sex hormone progesterone, vitamin D3, PTHrP, IL-1, IL-11, IL-17, or TNF-α ( 19 – 23 ) act on osteoblasts to induce RANKL expression, which then binds to its receptor RANK on the surface of osteoclast progenitor cells, inducing pre-osteoclast differentiation into multinucleated, fully-functional osteoclasts. RANKL also plays an important role in the continued survival and function of osteoclasts ( 24 – 27 ) ( Figure 1 ).…”

“…In bone tissue, the tumor microenvironment includes immune and tumor cells, as well as osteoblasts and osteoclasts, all of which participate in a “vicious cycle” that accelerates osteolysis and cancer cell proliferation through, in part, the RANK/RANKL/OPG axis ( 2 , 108 ). For instance, cancer cells can increase the expression of RANKL in osteoclasts by secreting parathyroid hormone-related peptide (PTH-rP) ( 23 , 109 ). Tumor cells can also directly express RANKL and secrete cytokines such as interleukin (IL)-1α, 6, 8, 11; TNF-α; macrophage colony-stimulating factor (M-CSF); or prostaglandin E2 (PGE2), all of which promote osteoclast differentiation and survival, resulting in local osteolysis which supports metastatic growth ( 110 – 117 ).…”

Integration of Molecular Docking and In Vitro Studies: A Powerful Approach for Drug Discovery in Breast Cancer

“…In vitro analysis supported this model: PTHrP(12-48) treatment does not promote an increase in cAMP in PTHR1-expressing SaOS2 cells. In conclusion, these data indicate that PTHrP acts in the regulation of the differentiation of hematopoietic cells and regulates the osteoclasts within the tumor-bone marrow microenvironment, possibly to induce bone metastasis [55].…”

“…A first circulating fragment of parathyroid hormone-related protein (PTHrP), PTHrP , has been proposed as a biomarker associated with the presence of bone metastases. Kamalakar et al [55] investigated the biological processes and mechanisms of action of PTHrP . First of all, they predicted the tertiary structure of PTHrP(12-48) through bioinformatics analyses, and the molecular modeling found that PTHrP interacts via a weak binding with the PTH1 receptor (PTHR1).…”

Chitosan-Collagen 3D Matrix Mimics Trabecular Bone and Regulates RANKL-Mediated Paracrine Cues of Differentiated Osteoblast and Mesenchymal Stem Cells for Bone Marrow Macrophage-Derived Osteoclastogenesis

“…The bone marrow of the femur and tibia of wild and OVX-mice was flushed out with ice-cold PBS and was cultured in a T75 cell culture flask (Costar) (20 mL final volume) containing 50 ng/mL recombinant Macrophage colony-stimulating factor (mCSF) (R&D Industries, Minneapolis, MN, USA) in endotoxin-free RPMI-10% heat-inactivated fetal bovine serum. Then, mouse bone marrow macrophage (mBMM) (10 5 cells/well/48 well plates) were treated with RANKL (50 ng/mL), mCSF (30 ng/mL), and rPTHr11 (50 nM/mL) as previously described [21,26,27].…”