Matthew R. Eber

Osteal macrophages support physiologic skeletal remodeling and anabolic actions of parathyroid hormone in bone

Significance Cellular subpopulations in the bone marrow play distinct and unexplored functions in the regulation of the skeleton. A type of blood cell that resides in the bone marrow termed “osteal macrophage” was found to play a role in bone homeostasis by supporting bone formation and mediating parathyroid hormone-dependent bone regeneration. Furthermore, induction of cell death in mature macrophages activated the specialized process of efferocytosis (clearance of dead and dying cells), leading to a marrow microenvironment that supported bone formation. Cellular subpopulations in the bone marrow play distinct and unexplored functions in skeletal homeostasis. This study delineated a unique role of osteal macrophages in bone and parathyroid hormone (PTH)-dependent bone anabolism using murine models of targeted myeloid-lineage cell ablation. Depletion of c-fms+ myeloid lineage cells [via administration of AP20187 in the macrophage Fas-induced apoptosis (MAFIA) mouse model] reduced cortical and trabecular bone mass and attenuated PTH-induced trabecular bone anabolism, supporting the positive function of macrophages in bone homeostasis. Interestingly, using a clodronate liposome model with targeted depletion of mature phagocytic macrophages an opposite effect was found with increased trabecular bone mass and increased PTH-induced anabolism. Apoptotic cells were more numerous in MAFIA versus clodronate-treated mice and flow cytometric analyses of myeloid lineage cells in the bone marrow showed that MAFIA mice had reduced CD68+ cells, whereas clodronate liposome-treated mice had increased CD68+ and CD163+ cells. Clodronate liposomes increased efferocytosis (clearance of apoptotic cells) and gene expression associated with alternatively activated M2 macrophages as well as expression of genes associated with bone formation including Wnt3a, Wnt10b, and Tgfb1. Taken together, depletion of early lineage macrophages resulted in osteopenia with blunted effects of PTH anabolic actions, whereas depletion of differentiated macrophages promoted apoptotic cell clearance and transformed the bone marrow to an osteogenic environment with enhanced PTH anabolism. These data highlight a unique function for osteal macrophages in skeletal homeostasis.

Cyclophosphamide Creates a Receptive Microenvironment for Prostate Cancer Skeletal Metastasis

A number of cancers predominantly metastasize to bone, due to its complex microenvironment and multiple types of constitutive cells. Prostate cancer especially has been shown to localize preferentially to bones with higher marrow cellularity. Using an experimental prostate cancer metastasis model, we investigated the effects of cyclophosphamide, a bone marrow-suppressive chemotherapeutic drug, on the development and growth of metastatic tumors in bone. Priming the murine host with cyclophosphamide before intracardiac tumor cell inoculation was found to significantly promote tumor localization and subsequent growth in bone. Shortly after cyclophosphamide treatment, there was an abrupt expansion of myeloid lineage cells in the bone marrow and the peripheral blood, associated with increases in cytokines with myelogenic potential such as C-C chemokine ligand (CCL)2, interleukin (IL)-6, and VEGF-A. More importantly, neutralizing host-derived murine CCL2, but not IL-6, in the premetastatic murine host significantly reduced the prometastatic effects of cyclophosphamide. Together, our findings suggest that bone marrow perturbation by cytotoxic chemotherapy can contribute to bone metastasis via a transient increase in bone marrow myeloid cells and myelogenic cytokines. These changes can be reversed by inhibition of CCL2.

The Soluble Interleukin-6 Receptor Is a Mediator of Hematopoietic and Skeletal Actions of Parathyroid Hormone

Background: IL-6 signaling plays a role in immune and skeletal systems. Results: sIL-6R mediated PTH-dependent hematopoietic cell expansions and blocking sIL-6R reduced PTH anabolic actions in mice. Conclusion: sIL-6R is a mediator of PTH hematopoietic actions in marrow and anabolic actions in bone. Significance: Novel orphan sIL-6R functions support PTH actions in bone and bone marrow. Both PTH and IL-6 signaling play pivotal roles in hematopoiesis and skeletal biology, but their interdependence is unclear. The purpose of this study was to evaluate the effect of IL-6 and soluble IL-6 receptor (sIL-6R) on hematopoietic and skeletal actions of PTH. In the bone microenvironment, PTH stimulated sIL-6R protein levels in primary osteoblast cultures in vitro and bone marrow in vivo in both IL-6+/+ and IL-6−/− mice. PTH-mediated hematopoietic cell expansion was attenuated in IL-6−/− compared with IL-6+/+ bone marrow, whereas sIL-6R treatment amplified PTH actions in IL-6−/− earlier than IL-6+/+ marrow cultures. Blocking sIL-6R signaling with sgp130 (soluble glycoprotein 130 receptor) inhibited PTH-dependent hematopoietic cell expansion in IL-6−/− marrow. In the skeletal system, although intermittent PTH administration to IL-6+/+ and IL-6−/− mice resulted in similar anabolic actions, blocking sIL-6R significantly attenuated PTH anabolic actions. sIL-6R showed no direct effects on osteoblast proliferation or differentiation in vitro; however, it up-regulated myeloid cell expansion and production of the mesenchymal stem cell recruiting agent, TGF-β1 in the bone marrow microenvironment. Collectively, sIL-6R demonstrated orphan function and mediated PTH anabolic actions in bone in association with support of myeloid lineage cells in the hematopoietic system.

Molecular Pathways: Niches in Metastatic Dormancy

Despite the best available treatments for primary tumors, cancer can return, even after a long disease-free interval. During this period, cancer cells are believed to lie dormant in either primary sites, metastatic sites, or independent sites like bone marrow, effectively escaping adjuvant cytotoxic treatments. To date, little is known about how these cells transition to dormancy, or how they are reactivated if cancer recurs. Recent studies have revealed the effects of tumor microenvironment or niche on the regulation of tumor dormancy via the signaling pathways of growth arrest–specific 6, bone morphogenetic protein 7, and TGFβ1, and that the balance between activation of p38 MAPK and ERK MAPK plays a pivotal role in tumor dormancy. In this review, we discuss tumor dormancy from the perspective of the niche and consider potential therapeutic targets. Greater understanding of the mechanisms involved will help guide innovation in the care of patients with advanced cancer. Clin Cancer Res; 20(13); 3384–9. ©2014 AACR.

Proteoglycan 4: A Dynamic Regulator of Skeletogenesis and Parathyroid Hormone Skeletal Anabolism

Proteoglycan 4 (Prg4), known for its lubricating and protective actions in joints, is a strong candidate regulator of skeletal homeostasis and parathyroid hormone (PTH) anabolism. Prg4 is a PTH‐responsive gene in bone and liver. Prg4 null mutant mice were used to investigate the impact of proteoglycan 4 on skeletal development, remodeling, and PTH anabolic actions. Young Prg4 mutant and wild‐type mice were administered intermittent PTH(1–34) or vehicle daily from 4 to 21 days. Young Prg4 mutant mice had decreased growth plate hypertrophic zones, trabecular bone, and serum bone formation markers versus wild‐type mice, but responded with a similar anabolic response to PTH. Adult Prg4 mutant and wild‐type mice were administered intermittent PTH(1–34) or vehicle daily from 16 to 22 weeks. Adult Prg4 mutant mice had decreased trabecular and cortical bone, and blunted PTH‐mediated increases in bone mass. Joint range of motion and animal mobility were lower in adult Prg4 mutant versus wild‐type mice. Adult Prg4 mutant mice had decreased marrow and liver fibroblast growth factor 2 (FGF‐2) mRNA and reduced serum FGF‐2, which were normalized by PTH. A single dose of PTH decreased the PTH/PTHrP receptor (PPR), and increased Prg4 and FGF‐2 to a similar extent in liver and bone. Proteoglycan 4 supports endochondral bone formation and the attainment of peak trabecular bone mass, and appears to support skeletal homeostasis indirectly by protecting joint function. Bone‐ and liver‐derived FGF‐2 likely regulate proteoglycan 4 actions supporting trabeculae formation. Blunted PTH anabolic responses in adult Prg4 mutant mice are associated with altered biomechanical impact secondary to joint failure.

The marrow niche controls the cancer stem cell phenotype of disseminated prostate cancer.

Dissemination of cancer stem cells (CSCs) serves as the basis of metastasis. Recently, we demonstrated that circulating prostate cancer targets the hematopoietic stem cell (HSCs) ‘niche’ in marrow during dissemination. Once in the niche, disseminated tumor cells (DTCs) may remain dormant for extended periods. As the major function of the HSC niche is to maintain stem cell functions, we hypothesized that the niche regulates CSC activities of DTCs. Here we show that DTCs recovered from marrow were significantly enriched for a CSC phenotype. Critically, the conversion of DTCs to CSCs is regulated by niche-derived GAS6 through the Mer/mTOR; molecules previously shown to regulate dormancy. The data demonstrate that the niche plays a significant role in maintaining tumor-initiating prostate cancer in marrow and suggests a functional relationship between CSCs and dormancy. Understanding how the marrow niche regulates the conversion of DTCs to CSCs is critical for the development of therapeutics specifically targeting skeletal bone metastasis and dormancy.

Axl is required for TGF-β2-induced dormancy of prostate cancer cells in the bone marrow.

Disseminated prostate cancer (PCa) cells in the marrow survive for years without evidence of proliferation, while maintaining the capacity to develop into metastatic lesions. These dormant disseminated tumor cells (DTCs) may reside in close proximity to osteoblasts, while expressing high levels of Axl, one of the tyrosine kinase receptors for growth arrest specific 6 (Gas6). Yet how Axl regulates DTC proliferation in marrow remains undefined. Here, we explored the impact of the loss of Axl in PCa cells (PC3 and DU145) on the induction of their dormancy when they are co-cultured with a pre-osteoblastic cell line, MC3T3-E1. MC3T3-E1 cells dramatically decrease the proliferation of PCa cells, however this suppressive effect of osteoblasts is significantly reduced by the reduction of Axl expression in PCa cells. Interestingly, expression of both TGF-β and its receptors were regulated by Axl expression in PCa cells, while specific blockade of TGF-β signaling limited the ability of the osteoblasts to induce dormancy of PCa cells. Finally, we found that both Gas6 and Axl are required for TGF-β2-mediated cell growth suppression. Taken together, these data suggest that a loop between the Gas6/Axl axis and TGF-β2 signaling plays a significant role in the induction of PCa cell dormancy.

Proteoglycan 4, a Novel Immunomodulatory Factor, Regulates Parathyroid Hormone Actions on Hematopoietic Cells

Proteoglycan 4 (PRG4), a critical protective factor in articular joints, is implicated in hematopoietic progenitor cell expansion and megakaryopoiesis. PRG4 loss-of-function mutations result in camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome, which is characterized primarily by precocious joint failure. PRG4 was identified as a novel parathyroid hormone (PTH) responsiveness gene in osteoblastic cells in bone, and was investigated as a potential mediator of PTH actions on hematopoiesis. Sixteen-week-old Prg4(-/-) mutant and Prg4(+/+) wild-type mice were treated daily with intermittent PTH (residues 1-34) or vehicle for 6 weeks. At 22 weeks of age, Prg4 mutant mice had increased peripheral blood neutrophils and decreased marrow B220(+) (B-lymphocytic) cells, which were normalized by PTH. The PTH-induced increase in marrow Lin(-)Sca-1(+)c-Kit(+) (hematopoietic progenitor) cells was blunted in mutant mice. Basal and PTH-stimulated stromal cell-derived factor-1 (SDF-1) was decreased in mutant mice, suggesting SDF-1 as a candidate regulator of proteoglycan 4 actions on hematopoiesis in vivo. PTH stimulation of IL-6 mRNA was greater in mutant than in wild-type calvaria and bone marrow, suggesting a compensatory mechanism in the PTH-induced increase in marrow hematopoietic progenitor cells. In summary, proteoglycan 4 is a novel PTH-responsive factor regulating immune cells and PTH actions on marrow hematopoietic progenitor cells.

Mer Tyrosine Kinase Regulates Disseminated Prostate Cancer Cellular Dormancy

Many prostate cancer (PCa) recurrences are thought to be due to reactivation of disseminated tumor cells (DTCs). We previously found a role of the TAM family of receptor tyrosine kinases TYRO3, AXL, and MERTK in PCa dormancy regulation. However, the mechanism and contributions of the individual TAM receptors is largely unknown. Knockdown of MERTK, but not AXL or TYRO3 by shRNA in PCa cells induced a decreased ratio of P‐Erk1/2 to P‐p38, increased expression of p27, NR2F1, SOX2, and NANOG, induced higher levels of histone H3K9me3 and H3K27me3, and induced a G1/G0 arrest, all of which are associated with dormancy. Similar effects were also observed with siRNA. Most importantly, knockdown of MERTK in PCa cells increased metastasis free survival in an intra‐cardiac injection mouse xenograft model. MERTK knockdown also failed to inhibit PCa growth in vitro and subcutaneous growth in vivo, which suggests that MERTK has specificity for dormancy regulation or requires a signal from the PCa microenvironment. The effects of MERTK on the cell cycle and histone methylation were reversed by p38 inhibitor SB203580, which indicates the importance of MAP kinases for MERTK dormancy regulation. Overall, this study shows that MERTK stimulates PCa dormancy escape through a MAP kinase dependent mechanism, also involving p27, pluripotency transcription factors, and histone methylation. J. Cell. Biochem. 118: 891–902, 2017.

Skeletal complications in cancer patients with bone metastases.

As a result of significant improvements in current therapies, the life expectancy of cancer patients with bone metastases has dramatically improved. Unfortunately, these patients often experience skeletal complications that significantly impair their quality of life. The major skeletal complications associated with bone metastases include: cancer‐induced bone pain, hypercalcemia, pathological bone fractures, metastatic epidural spinal cord compression and cancer cachexia. Once cancer cells invade the bone, they perturb the normal physiology of the marrow microenvironment, resulting in bone destruction, which is believed to be a direct cause of skeletal complications. However, full understanding of the mechanisms responsible for these complications remains unknown. In the present review, we discuss the complications associated with bone metastases along with matched conventional therapeutic strategies. A better understanding of this topic is crucial, as targeting skeletal complications can improve both the morbidity and mortality of patients suffering from bone metastases.