Kristen L. Shogren

MicroRNA-34c inversely couples the biological functions of the runt-related transcription factor RUNX2 and the tumor suppressor p53 in osteosarcoma

Background: Osteosarcoma (OS) is associated with loss of tumor suppressor p53 and increased Runx2. Results: Runx2 and p53 levels are inversely correlated in OS. miR-34c, which targets Runx2, is absent in OS and elevated by p53. Conclusion: p53, miR-34c, and Runx2 form a regulatory loop that is compromised in OS. Significance: RUNX2 could be targeted by miR-34c to prevent OS growth. Osteosarcoma (OS) is a primary bone tumor that is most prevalent during adolescence. RUNX2, which stimulates differentiation and suppresses proliferation of osteoblasts, is deregulated in OS. Here, we define pathological roles of RUNX2 in the etiology of OS and mechanisms by which RUNX2 expression is stimulated. RUNX2 is often highly expressed in human OS biopsies and cell lines. Small interference RNA-mediated depletion of RUNX2 inhibits growth of U2OS OS cells. RUNX2 levels are inversely linked to loss of p53 (which predisposes to OS) in distinct OS cell lines and osteoblasts. RUNX2 protein levels decrease upon stabilization of p53 with the MDM2 inhibitor Nutlin-3. Elevated RUNX2 protein expression is post-transcriptionally regulated and directly linked to diminished expression of several validated RUNX2 targeting microRNAs in human OS cells compared with mesenchymal progenitor cells. The p53-dependent miR-34c is the most significantly down-regulated RUNX2 targeting microRNAs in OS. Exogenous supplementation of miR-34c markedly decreases RUNX2 protein levels, whereas 3′-UTR reporter assays establish RUNX2 as a direct target of miR-34c in OS cells. Importantly, Nutlin-3-mediated stabilization of p53 increases expression of miR-34c and decreases RUNX2. Thus, a novel p53-miR-34c-RUNX2 network controls cell growth of osseous cells and is compromised in OS.

2-methoxyestradiol-induced cell death in osteosarcoma cells is preceded by cell cycle arrest

2‐Methoxyestradiol (2‐ME), a naturally occurring mammalian metabolite of 17β‐Estradiol (E2), induces cell death in osteosarcoma cells. To further understand the molecular mechanisms of action, we have investigated cell cycle progression in 2‐ME‐treated human osteosarcoma (MG63, SaOS‐2 and LM8) cells. At 5 µM, 2‐ME induced growth arrest by inducing a block in cell cycle; 2‐ME‐treatment resulted in 2‐fold increases in G1 phase cells and a decrease in S phase cells in MG63 and SaOS‐2 osteosarcoma cell lines, compared to the appropriate vehicle controls. 2‐ME‐treatment induced a threefold increase in the G2 phase in LM8 osteosarcoma cells. The results demonstrated steroid specificity, as the tumorigenic metabolite, 16α‐hydroxyestradiol (16‐OHE), did not have any effect on cell cycle progression in osteosarcoma cells. The cell cycle arrest coincided with an increase in expression of the cell cycle markers p21, p27 and p53 proteins in 2‐ME‐treated osteosarcoma cells. Also, MG63 cells, transiently transfected with cDNA for a ‘loss of function mutant’ RNA‐dependent protein kinase (PKR) protein, were resistant to 2‐ME‐induced cell cycle arrest. These results suggest that 2‐ME works in concert with factors regulating cell cycle progression, and cell cycle arrest precedes cell death in 2‐ME‐treated osteosarcoma cells. J. Cell. Biochem. 104: 1937–1945, 2008.

Double-Stranded RNA-Dependent Protein Kinase Is Involved in 2-Methoxyestradiol—Mediated Cell Death of Osteosarcoma Cells†

We studied the involvement of interferon‐regulated, PKR on 2‐ME–mediated actions in human osteosarcoma cells. Our results show that PKR is activated by 2‐ME treatment and is necessary for 2‐ME–mediated induction of osteosarcoma cell death.

2‐methoxyestradiol inhibits differentiation and is cytotoxic to osteoclasts

2‐Methoxyestradiol (2‐ME), a naturally occurring metabolite of 17β‐estradiol, is highly cytotoxic to a wide range of tumor cells but is harmless to most normal cells. However, 2‐ME prevented bone loss in ovariectomized rats, suggesting it inhibits bone resorption. These studies were performed to determine the direct effects of 2‐ME on cultured osteoclasts. 2‐ME (2 µM) reduced osteoclast number by more than 95% and induced apoptosis in three cultured osteoclast model systems (RAW 264.7 cells cultured with RANKL, marrow cells co‐cultured with stromal support cells, and spleen cells cultured without support cells in media supplemented with RANKL and macrophage colony stimulating factor (M‐CSF)). The 2‐ME‐mediated effect was ligand specific; 2‐hydroxyestradiol (2‐OHE), the immediate precursor to 2‐ME, exhibited less cytotoxicity; and 2‐methoxyestrone (2‐MEOE1) the estrone analog of 2‐ME, was not cytotoxic. Co‐treatment with ICI 182,780 did not antagonize 2‐ME, suggesting that the cytotoxicity was not estrogen receptor‐dependent. 2‐ME‐induced cell death in RAW 264.7 cells coincided with an increase in gene expression of cytokines implicated in inhibition of differentiation and induction of apoptosis. In addition, the 2‐ME‐mediated decrease in cell survival was partially inhibited by anti‐lymphotoxin(LT)β antibodies, suggesting that 2‐ME‐dependent effects involve LTβ. These results suggest that 2‐ME could be useful for treating skeletal diseases in which bone resorption is increased, such as postmenopausal osteoporosis and cancer metastasis to bone. J. Cell. Biochem. 99: 425–434, 2006.

Osteitis Fibrosa Is Mediated by Platelet-Derived Growth Factor-A Via a Phosphoinositide 3-Kinase-Dependent Signaling Pathway in a Rat Model for Chronic Hyperparathyroidism

Abnormal secretion of PTH by the parathyroid glands contributes to a variety of common skeletal disorders. Prior studies implicate platelet-derived growth factor-A (PDGF-A) as an important mediator of selective PTH actions on bone. The present studies used targeted gene profiling and small-molecule antagonists directed against candidate gene products to elucidate the roles of specific PTH-regulated genes and signaling pathways. A group of 29 genes in rats continuously infused with PTH and cotreated with the PDGF receptor antagonist trapidil were differentially expressed compared with PTH treatment alone. Several of the identified genes were functionally clustered as regulators of fibroblast differentiation and extracellular matrix modeling, including the matrix cross-linking enzyme lysyl oxidase (LOX). Treatment with beta-aminopropionitrile, an irreversible inhibitor of LOX activity, dramatically reduced diffuse mineralization but had no effect on PTH-induced fibrosis. In contrast, the receptor tyrosine kinase inhibitor Gleevec and the phosphoinositide 3-kinase inhibitor wortmannin each reduced bone marrow fibrosis. In summary, the present studies support the hypotheses that PTH-induced bone marrow fibrosis is mediated by PDGF-A via a phosphoinositide 3-kinase-dependent signaling pathway and that increased LOX gene expression plays a key role in abnormal mineralization, a hallmark of chronic hyperparathyroidism.

RNA-Dependent Protein Kinase Is Essential for 2-Methoxyestradiol-Induced Autophagy in Osteosarcoma Cells

Osteosarcoma is the most common primary malignant bone tumor in children and young adults. Surgical resection and adjunctive chemotherapy are the only widely available options of treatment for this disease. Anti-tumor compound 2-Methoxyestradiol (2-ME) triggers cell death through the induction of apoptosis in osteosarcoma cells, but not in normal osteoblasts. In this report, we have investigated whether autophagy plays a role in 2-ME actions on osteosarcoma cells. Transmission electron microscopy imaging shows that 2-ME treatment leads to the accumulation of autophagosomes in human osteosarcoma cells. 2-ME induces the conversion of the microtubule-associated protein LC3-I to LC3-II, a biochemical marker of autophagy that is correlated with the formation of autophagosomes. Conversion to LC3-II is accompanied by protein degradation in 2-ME-treated cells. 2-ME does not induce autophagosome formation in normal primary human osteoblasts. In addition, 2-ME-dependent autophagosome formation in osteosarcoma cells requires ATG7 expression. Furthermore, 2-ME does not induce accumulation of autophagosomes in osteosarcoma cells that express dominant negative mutant RNA-dependent protein kinase (PKR) and are resistant to anti-proliferative and anti-tumor effects of 2-ME. Taken together, our study shows that 2-ME treatment induces PKR-dependent autophagy in osteosarcoma cells, and that autophagy could play an important role in 2-ME-mediated anti-tumor actions and in the control of osteosarcoma.

Regulation of interferon pathway in 2-methoxyestradiol-treated osteosarcoma cells.

BackgroundOsteosarcoma is a bone tumor that often affects children and young adults. Although a combination of surgery and chemotherapy has improved the survival rate in the past decades, local recurrence and metastases still develop in 40% of patients. A definite therapy is yet to be determined for osteosarcoma. Anti- tumor compound and a metabolite of estrogen, 2-methoxyestradiol (2-ME) induces cell death in osteosarcoma cells. In this report, we have investigated whether interferon (IFN) pathway is involved in 2-ME-induced anti-tumor effects in osteosarcoma cells.Methods2-ME effects on IFN mRNA levels were determined by Real time PCR analysis. Transient transfections followed by reporter assays were used for investigating 2-ME effects on IFN-pathway. Western blot analyses were used to measure protein and phosphorylation levels of IFN-regulated eukaryotic initiation factor-2 alpha (eIF-2α).Results2-ME regulates IFN and IFN-mediated effects in osteosarcoma cells. 2 -ME induces IFN gene activity and expression in osteosarcoma cells. 2-ME treatment induced IFN-stimulated response element (ISRE) sequence-dependent transcription and gamma-activated sequence (GAS)-dependent transcription in several osteosarcoma cells. Whereas, 2-ME did not affect IFN gene and IFN pathways in normal primary human osteoblasts (HOB). 2-ME treatment increased the phosphorylation of eIF-2α in osteosarcoma cells. Furthermore, analysis of osteosarcoma tissues shows that the levels of phosphorylated form of eIF-2α are decreased in tumor compared to normal controls.Conclusions2-ME treatment triggers the induction and activity of IFN and IFN pathway genes in 2-ME-sensitive osteosarcoma tumor cells but not in 2-ME-resistant normal osteoblasts. In addition, IFN-signaling is inhibited in osteosarcoma patients. Thus, IFN pathways play a role in osteosarcoma and in 2-ME-mediated anti-proliferative effects, and therefore targeted induction of IFN signaling could lead to effective treatment strategies in the control of osteosarcoma.

Induction of angiogenesis and osteogenesis in surgically revascularized frozen bone allografts by sustained delivery of FGF-2 and VEGF

Large conventional bone allografts are susceptible to fracture and nonunion due to incomplete revascularization and insufficient bone remodeling. We aim to improve bone blood flow and bone remodeling using surgical angiogenesis combined with delivery of fibroblast growth factor (FGF‐2) and vascular endothelial growth factor (VEGF). Frozen femoral allografts were heterotopically transplanted in a rat model. The saphenous arteriovenous bundle was implanted within the graft medullary canal. Simultaneously, biodegradable microspheres containing phosphate buffered saline (control), FGF‐2, VEGF, or FGF‐2 + VEGF were placed within the graft. Rats were sacrificed at 4 and 18 weeks. Angiogenesis was determined by quantifying bone capillary density and measuring cortical bone blood flow. Bone remodeling was assessed by histology, histomorphometry, and alkaline phosphatase activity. VEGF significantly increased angiogenesis and bone remodeling at 4 and 18 weeks. FGF‐2 did not elicit a strong angiogenic or osteogenic response. No synergistic effect of FGF‐2 + VEGF was observed. VEGF delivered in microspheres had superior long‐term effect on angiogenesis and osteogenesis in surgically revascularized frozen bone structural allografts as compared to FGF‐2 or FGF‐2 + VEGF. Continuous and localized delivery of VEGF by microencapsulation has promising clinical potential by inducing a durable angiogenic and osteogenic response in frozen allografts.

2‐methoxyestradiol‐mediated anti‐tumor effect increases osteoprotegrin expression in osteosarcoma cells

Osteosarcoma is a bone tumor that frequently develops during adolescence. 2‐Methoxyestradiol (2‐ME), a naturally occurring metabolite of 17β‐estradiol, induces cell cycle arrest and cell death in human osteosarcoma cells. To investigate whether the osteoprotegrin (OPG) protein plays a role in 2‐ME actions, we studied the effect of 2‐ME treatment on OPG gene expression in human osteosarcoma cells. 2‐ME treatment induced OPG gene promoter activity and mRNA levels. Also, Western blot analysis showed that 2‐ME treatment increased OPG protein levels in MG63, KHOS, 143B and LM7 osteosarcoma cells by 3‐, 1.9‐, 2.8‐, and 2.5‐fold, respectively, but did not affect OPG expression in normal bone cells. In addition, increases in OPG protein levels were observed in osteosarcoma cell culture media after 3 days of 2‐ME treatment. The effect of 2‐ME on osteosarcoma cells was ligand‐specific as parent estrogen, 17β‐estradiol and a tumorigenic estrogen metabolite, 16α‐hydroxyestradiol, which do not affect osteosarcoma cell cycle and cell death, had no effect on OPG protein expression. Furthermore, co‐treating osteosarcoma cells with OPG protein did not further enhance 2‐ME‐mediated anti‐tumor effects. OPG‐released in 2‐ME‐treated cultures led to an increase in osteoblastic activity and a decrease in osteoclast number, respectively. These findings suggest that OPG is not directly involved in 2‐ME‐mediated anti‐proliferative effects in osteosarcoma cells, but rather participates in anti‐resorptive functions of 2‐ME in bone tumor environment. J. Cell. Biochem. 109: 950–956, 2010.

2‐Methoxyestradiol‐Mediated Induction of Frzb Contributes to Cell Death and Autophagy in MG63 Osteosarcoma Cells

Osteosarcoma is a bone tumor that mainly affects children and adolescents. Although its pathogenesis is still not fully understood, activation of Wnt signaling has been implicated in the development and metastasis of osteosarcoma. In this report, we have investigated the effect of the anti‐tumor compound, 2‐methoxyestradiol (2‐ME) on Wnt antagonist frizzled‐related protein b (Frzb), also known as secreted frizzled‐related protein (sFRP)3 in human osteosarcoma (MG63) cells. Our results show that 2‐ME treatment induces Frzb gene promoter activity, and increases Frzb mRNA and protein levels in osteosarcoma cells. In addition, 2‐ME treatment regulates downstream Wnt signaling, increasing the cytoplasmic levels of β‐catenin, and blocking β‐catenin‐mediated Wnt activation in osteosarcoma cells. 2‐ME‐mediated induction of Frzb protein expression is specific to osteosarcoma cells, as it does not affect Frzb expression in normal primary human osteoblasts. Furthermore, 2‐ME‐induced apoptosis and autophagy are blocked in osteosarcoma cells transfected with Frzb siRNAs. Taken together, these studies demonstrate that Frzb protein plays an important role in 2‐ME‐mediated anti‐tumor mechanisms in osteosarcoma cells. J. Cell. Biochem. 118: 1497–1504, 2017.