Tumor necrosis factor-alpha (TNF-α) is known to mediate bone resorption; however, its role in osteogenesis has not been fully elucidated. In order to investigate the direct role of TNF-α signaling in the recruitment and differentiation of osteoblasts, two separate models of bone repair were used, marrow ablation and simple transverse fractures. These models were carried out in the tibiae of both wild-type and knock-out mice in which both TNF-α receptors (p55^–/–/p75^–/–) had been ablated. Marrow ablation is a unique model in which robust intramembranous bone formation is induced without an endochondral component, followed by remodeling and restoration of the original trabecular architecture of the bone marrow. In contrast, fracture repair proceeds concurrently through both endochondral and intramembranous processes of new bone tissue formation. In both models of bone repair, healing was delayed in the TNF-α receptor (p55^–/–/p75^–/–) deficient mice. In the marrow ablation model, young osteoblasts were recruited into the marrow space by day three in the wild-type mice, while the TNF-α (p55^–/–/p75^–/–) mice had only granulation tissue in the marrow cavity. Type I collagen and osteocalcin mRNA expressions were reduced ∼30 and ∼50%, respectively, of the control values in the TNF-α receptor ablated mice. In the fracture repair model there was almost a complete absence of the initial intramembranous bone formation on the periosteal surface in the TNF-α (p55^–/–/p75^–/–) mice. As healing progressed however, the callus tissues were greatly enlarged, and there was a delay in hypertrophy of the chondrocytes and the resorption of cartilage tissue. While during the initial period of fracture repair there was a marked reduction in the expression of both type I collagen and osteocalcin mRNAs in the TNF-α (p55^–/–/p75^–/–) mice, levels of these mRNAs were elevated by ∼10–20% over the wild type at the later time points in the absence of endochondral resorption of the callus. The lack of inhibition of osteogenesis during endochondral resorption suggests that a different set of signals are involved in the recruitment of osteogenic cells during endochondral repair then during intramembranous bone formation. Co-culture of chondrocytes with a mesenchymal stem cell line was carried out to examine if chondrocytes themselves produced paracrine factors that promote osteogenic differentiation. These experiments demonstrated that chondrocytes do indeed produce factors that promoted osteogenic differentiation. In summary, the results presented here suggest that TNF-α plays a crucial role in promoting postnatal bone repair through the induction of osteoprogenitor cell recruitment or osteogenic cell activation in the context of intramembranous bone formation. These results further suggest that the signals that promote osteogenesis during endochondral bone formation are different from those involved in intramembranous bone formation.

Medulloblastoma, the most common pediatric brain tumor, is thought to arise from deregulated proliferation of cerebellar granule precursor (CGP) cells. Sonic hedgehog (Shh) is the primary mitogen that regulates proliferation of CGP cells during the early stages of postnatal cerebellum development. Aberrant activation of Shh signaling during this time has been associated with hyperplasia of CGP cells and eventually may lead to the development of medulloblastoma. The molecular targets of Shh signaling involved in medulloblastoma formation are still not well-understood. Here, we show that Shh regulates sustained activation of histone deacetylases (HDACs) and that this activity is required for continued proliferation of CGP cells. Suppression of HDAC activity not only blocked the Shh-induced CGP proliferation in primary cell cultures, but also ameliorated aberrant CGP proliferation at the external germinal layer (EGL) in a medulloblastoma mouse model. Increased levels of mRNA and protein of several HDAC family members were found in medulloblastoma compared to wild type cerebellum suggesting that HDAC activity is required for the survival/progression of tumor cells. The identification of a role of HDACs in the early steps of medulloblastoma formation suggests there may be a therapeutic potential for HDAC inhibitors in this disease.

BackgroundThe Sonic hedgehog (Shh) signaling pathway plays an important role in cerebellar development, and mutations leading to hyperactive Shh signaling have been associated with certain forms of medulloblastoma, a common form of pediatric brain cancer. While the fundamentals of this pathway are known, the molecular targets contributing to Shh-mediated proliferation and transformation are still poorly understood. Na,K-ATPase is a ubiquitous enzyme that maintains intracellular ion homeostasis and functions as a signaling scaffold and a cell adhesion molecule. Changes in Na,K-ATPase function and subunit expression have been reported in several cancers and loss of the β1-subunit has been associated with a poorly differentiated phenotype in carcinoma but its role in medulloblastoma progression is not known.MethodsHuman medulloblastoma cell lines and primary cultures of cerebellar granule cell precursors (CGP) were used to determine whether Shh regulates Na,K-ATPase expression. Smo/Smo medulloblastoma were used to assess the Na,K-ATPase levels in vivo. Na,K-ATPase β1-subunit was knocked down in DAOY cells to test its role in medulloblastoma cell proliferation and tumorigenicity.ResultsNa,K-ATPase β1-subunit levels increased with differentiation in normal CGP cells. Activation of Shh signaling resulted in reduced β1-subunit mRNA and protein levels and was mimicked by overexpression of Gli1and Bmi1, both members of the Shh signaling cascade; overexpression of Bmi1 reduced β1-subunit promoter activity. In human medulloblastoma cells, low β1-subunit levels were associated with increased cell proliferation and in vivo tumorigenesis.ConclusionsNa,K-ATPase β1-subunit is a target of the Shh signaling pathway and loss of β1-subunit expression may contribute to tumor development and progression not only in carcinoma but also in medulloblastoma, a tumor of neuronal origin.

Medulloblastoma, which usually arises in the cerebellum, is the most common malignant brain cancer in children and is still associated with substantial mortality and survivors often suffer from serious life-long therapy-related side effects. In recent years several medulloblastoma subtypes with distinct developmental origins, genetic profiles, pathway signatures, and clinicopathological features have been identified. The current consensus divides medulloblastoma tumors into four subgroups, WNT, sonic hedgehog (Shh), Group 3 and Group 4 allowing for different targeted therapeutic approaches. Not specifically restricted to a defined subgroup, aberrant activation of the epidermal growth factor receptor family (EGFR/ErbB) has been reported in a large percentage of medulloblastoma and has been associated with poor outcome. The Shh pathway is an important signaling pathway involved in cerebellar development. Shh is secreted from the Purkinje cells, and acts as a mitogen for cerebellar granule precursor (CGP) cells. Mutations leading to hyperactive Shh signaling have been associated with improper migration and differentiation of CGP cells and medulloblastoma formation. Still, the molecular targets contributing to Shh-mediated proliferation in these cells are poorly understood. Na,K-ATPase is a membrane protein that maintains intracellular ion homeostasis, and is responsible for generating ion gradients across cell membranes. Consisting of a catalytic alpha subunit and a beta subunit, the enzyme not only pumps sodium ions out and potassium ions into the cell using ATP but also functions a signaling scaffold and a cell adhesion molecule. Changes in Na,K-ATPase function and expression have been reported in various cancers and may contribute to tumor development and progression. We now show that the Na,K-ATPase beta1-subunit is drastically reduced in medulloblastoma tumors of mice with aberrant activation of Shh signaling. In addition, Shh activation prevented the upregulation of the beta1-subunit in primary cultures of normal cerebellar granule cells and the polycomb transcription factor Bmi1 that is induced upon activation of Shh signaling repressed beta1-subunit levels. Furthermore, shRNA-mediated knockdown of the beta1-subunit increased cell proliferation and tumorigenicity of medulloblastoma cells. Nevertheless, cardiac glycosides that inhibit the pump function of Na,K-ATPase inhibited EGF-induced signaling and cell motility suggesting that Na,K-ATPase alpha and beta subunit may have dual functions in CGP cells and medulloblastoma. Citation Format: Zhiqin Li, Alisa Litan, Seung Joon Lee, Bruce Graves, Sonali P. Barwe, Sigrid A. Langhans. Distinct roles of Na,K-ATPase function and expression in medulloblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 707. doi:10.1158/1538-7445.AM2015-707