Lineage of origin in rhabdomyosarcoma informs pharmacological response.

Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.

Myf5 expression during fetal myogenesis defines the developmental progenitors of adult satellite cells.

Myf5 is a member of the muscle-specific determination genes and plays a critical role in skeletal muscle development. Whereas the expression of Myf5 during embryonic and fetal myogenesis has been extensively studied, its expression in progenitors that will ultimately give rise to adult satellite cells, the stem cells responsible for muscle repair, is still largely unexplored. To investigate this aspect, we have generated a mouse strain carrying a CreER coding sequence in the Myf5 locus. In this strain, Tamoxifen-inducible Cre activity parallels endogenous Myf5 expression. Combining Myf5(CreER) and Cre reporter alleles, we were able to evaluate the contribution of cells expressing Myf5 at distinct developmental stages to the pool of satellite cells in adult hindlimb muscles. Although it was possible to trace back the origin of some rare satellite cells to a subpopulation of Myf5(+ve) progenitors in the limb buds at the late embryonic stage (∼E12), a significant number of satellite cells arise from cells which expressed Myf5 for the first time at the fetal stage (∼E15). These studies provide direct evidence that adult satellite cells derive from progenitors that first express the myogenic determination gene Myf5 during fetal stages of myogenesis.

Rb1 gene inactivation expands satellite cell and postnatal myoblast pools.

Satellite cells are well known as a postnatal skeletal muscle stem cell reservoir that under injury conditions participate in repair. However, mechanisms controlling satellite cell quiescence and activation are the topic of ongoing inquiry by many laboratories. In this study, we investigated whether loss of the cell cycle regulatory factor, pRb, is associated with the re-entry of quiescent satellite cells into replication and subsequent stem cell expansion. By ablation of Rb1 using a Pax7CreER,Rb1 conditional mouse line, satellite cell number was increased 5-fold over 6 months. Furthermore, myoblasts originating from satellite cells lacking Rb1 were also increased 3-fold over 6 months, while terminal differentiation was greatly diminished. Similarly, Pax7CreER,Rb1 mice exhibited muscle fiber hypotrophy in vivo under steady state conditions as well as a delay of muscle regeneration following cardiotoxin-mediated injury. These results suggest that cell cycle re-entry of quiescent satellite cells is accelerated by lack of Rb1, resulting in the expansion of both satellite cells and their progeny in adolescent muscle. Conversely, that sustained Rb1 loss in the satellite cell lineage causes a deficit of muscle fiber formation. However, we also show that pharmacological inhibition of protein phosphatase 1 activity, which will result in pRb inactivation accelerates satellite cell activation and/or expansion in a transient manner. Together, our results raise the possibility that reversible pRb inactivation in satellite cells and inhibition of protein phosphorylation may provide a new therapeutic tool for muscle atrophy by short term expansion of the muscle stem cells and myoblast pool.

Biomarker system for studying muscle, stem cells, and cancer in vivo.

Bioluminescent reporter genes are sensitive in situ tools for following disease progression in preclinical models, albeit they are subject to scattering and absorption in deep tissues. We have generated a bicistronic Cre/LoxP reporter mouse line that pairs the expression of firefly luciferase with quantifiable expression of a human placental alkaline phosphatase that is secreted into the serum (SeAP). With the use of this dual-modality bioreporter with a novel, inducible Pax7-CreER line for tracking muscle satellite cells, we demonstrate the longitudinal kinetics of muscle stem cell turnover, accounting for a doubling of the signal from satellite cell and progeny every 3.93 wk in the transition from adolescence to early adulthood. We also show that this dual-modality bioreporter can be incorporated in preclinical cancer models, whereby SeAP activity is reflective of tumor burden. Thus, this dual bioreporter permits both spatial localization and accurate quantification of biological processes in vivo even when the tissue of interest is deep within the animal.

Involvement of extracellular signal-regulated kinase activation in human osteosarcoma cell resistance to the histone deacetylase inhibitor FK228 [(1S,4S,7Z,10S,16E,21R)-7-ethylidene-4,21-bis(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19,22-pentone].

The histone deacetylase inhibitor depsipeptide [(1S,4S,7Z,10S, 16E,21R)-7-ethylidene-4,21-bis(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19, 22-pentone] (FK228) has attracted a great deal of interest because of its antiproliferative and apoptotic properties in various malignancies. Histone deacetylase inhibitors induce the expression of the multidrug resistance transporter P-glycoprotein (P-gp), and FK228 is a known P-gp substrate. Thus, FK228 seems to induce its own mechanism of drug resistance by up-regulating P-gp. The goal of this study was to establish human FK228-resistant osteosarcoma cell lines and to investigate whether there are mechanisms of FK228 resistance in addition to P-gp up-regulation. After 72 h in culture, the 50% inhibitory concentrations (IC(50)) of FK228 were 4.8 and 991 nM in HOS and HOS/FK8 cells, respectively, and 3.6 and 1420 nM in U2OS and U2OS/FK11 cells, respectively. Increased histone H3 acetylation was observed in FK228-resistant cell lines after a 1-h treatment with 10 nM FK228. Unlike in parental cells, significant P-gp overexpression was detected in FK228-resistant cells, and 10 nM FK228 treatment activated the mitogen-activated protein kinase (MAPK) pathway but did not induce Fas ligand (FasL) up-regulation or c-FLIP down-regulation. However, treatment of FK228-resistant cells with a combination of FK228 and mitogen-activated protein kinase kinase (MEK) inhibitors induced apoptosis, up-regulated FasL, and down-regulated c-FLIP. The expression and function of P-gp were unaltered by treatment with MEK inhibitors. These results indicate that the FK228 resistance of osteosarcoma cells is related to P-gp overexpression and MAPK pathway activation by FK228. MEK or P-gp inhibitors may be useful in overcoming this resistance.

Regeneration of osteonecrosis of canine scapho-lunate using bone marrow stromal cells: possible therapeutic approach for Kienböck disease.

We evaluated the ability of canine bone marrow stromal cells (cBMSCs) to regenerate bone in a cavity of the scapholunate created by curretage and freeze-thawing with liquid nitrogen (LN). Autologous BMSCs were harvested from the iliac crest and expanded in vitro. Their potential to differentiate into osteo-, chondro-, and adipogenic lineages was confirmed using a standard differentiation induction assay. LN-treated scapholunates showed no regeneration of bone tissue when the cavity was left alone, demonstrating severe collapse and deformity as observed in human Kienböck disease. A combination of beta-tri-calcium phosphate and a vascularized bone graft with autologous fibroblasts failed to regenerate bone in the LN-treated cavity. When the same procedure was performed using BMSCs, however, LN-treated scapholunates showed no collapse and deformity, and the cavity was completely filled with normal cancerous bone within 4 weeks. These results suggested the potential of using BMSCs to treat Kienböck disease.

A variant of the SYT-SSX2 fusion gene in a case of synovial sarcoma.

Synovial sarcoma is a malignant soft tissue tumor harboring a tumor-specific fusion gene, SYT-SSX, of which exon 10 of the SYT gene is fused to exon 6 of the SSX gene is the common form. Here we report a case of synovial sarcoma with a novel form of the SYT-SSX2 fusion transcript, in which 75 bases were inserted at the common fusion junction. Computer analyses revealed that 15 bases were from intron 10 of the SYT gene, and 10 from the end of intron 4, and 50 from exon 5 of the SSX2 gene. Precise analyses of genomic breakpoints in SYT and SSX2 loci revealed that the reciprocal translocation creating the fusion gene was associated with a large deletion in both loci. The structure of SYT-SSX2 suggests that the fusion transcript in this case was created using a cryptic splicing acceptor site 15 bases upstream of the genomic fusion point, incorporating intronic sequences in mature mRNA. Reexamination of two variant SYT-SSX2 genes reported previously revealed that unknown sequences inserted at the common junction points were derived from intron sequences, as in the present case.

Disruption of fibroblast growth factor signal pathway inhibits the growth of synovial sarcomas: potential application of signal inhibitors to molecular target therapy.

PURPOSE: Synovial sarcoma is a soft tissue sarcoma, the growth regulatory mechanisms of which are unknown. We investigated the involvement of fibroblast growth factor (FGF) signals in synovial sarcoma and evaluated the therapeutic effect of inhibiting the FGF signal. EXPERIMENTAL DESIGN: The expression of 22 FGF and 4 FGF receptor (FGFR) genes in 18 primary tumors and five cell lines of synovial sarcoma were analyzed by reverse transcription-PCR. Effects of recombinant FGF2, FGF8, and FGF18 for the activation of mitogen-activated protein kinase (MAPK) and the growth of synovial sarcoma cell lines were analyzed. Growth inhibitory effects of FGFR inhibitors on synovial sarcoma cell lines were investigated in vitro and in vivo. RESULTS: Synovial sarcoma cell lines expressed multiple FGF genes especially those expressed in neural tissues, among which FGF8 showed growth stimulatory effects in all synovial sarcoma cell lines. FGF signals in synovial sarcoma induced the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and p38MAPK but not c-Jun NH2-terminal kinase. Disruption of the FGF signaling pathway in synovial sarcoma by specific inhibitors of FGFR caused cell cycle arrest leading to significant growth inhibition both in vitro and in vivo. Growth inhibition by the FGFR inhibitor was associated with a down-regulation of phosphorylated ERK1/2 but not p38MAPK, and an ERK kinase inhibitor also showed growth inhibitory effects for synovial sarcoma, indicating that the growth stimulatory effect of FGF was transmitted through the ERK1/2. CONCLUSIONS: FGF signals have an important role in the growth of synovial sarcoma, and inhibitory molecules will be of potential use for molecular target therapy in synovial sarcoma.

PGE2 signal through EP2 promotes the growth of articular chondrocytes.

UNLABELLED: EP2 was identified as the major PGE2 receptor expressed in articular cartilage. An EP2 agonist increased intracellular cAMP in articular chondrocytes, stimulating DNA synthesis in both monolayer and 3D cultures. Hence, the EP2 agonist may be a potent therapeutic agent for degenerative cartilage diseases. INTRODUCTION: Prostaglandin E2 (PGE2) exhibits pleiotropic effects in various types of tissue through four types of receptors, EP1-4. We examined the expression of EPs and effects of agonists for each EP on articular chondrocytes. MATERIALS AND METHODS: The expression of each EP in articular chondrocytes was examined by immunohistochemistry and RT-PCR. A chondrocyte cell line, MMA2, was established from articular cartilage of p53(-/-) mice and used to analyze the effects of agonists for each EP. A search for molecules downstream of the PGE2 signal through the EP2 agonist was made by cDNA microarray analysis. The growth-promoting effect of the EP2 agonist on chondrocytes surrounded by cartilage matrix was examined in an organ culture of rat femora. RESULTS AND CONCLUSION: EP2 was identified as the major EP expressed in articular cartilage. Treatment of MMA2 cells with specific agonists for each EP showed that only the EP2 agonist significantly increased intracellular cAMP levels in a dose-dependent manner. Gene expression profiling of MMA2 revealed a set of genes upregulated by the EP2 agonist, including several growth-promoting and apoptosis-protecting genes such as the cyclin D1, fibronectin, integrin alpha5, AP2alpha, and 14-3-3gamma genes. The upregulation of these genes by the EP2 agonist was confirmed in human articular chondrocytes by quantitative mRNA analysis. On treatment with the EP2 agonist, human articular chondrocytes showed an increase in the incorporation of 5-bromo-2-deoxyuracil (BrdU), and the organ culture of rat femora showed an increase of proliferating cell nuclear antigen (PCNA) staining in articular chondrocytes surrounded by cartilage matrix, suggesting growth-promoting effects of the PGE2 signal through EP2 in articular cartilage. These results suggested that the PGE2 signal through EP2 enhances the growth of articular chondrocytes, and the EP2 agonist is a candidate for a new therapeutic compound for the treatment of degenerative cartilage diseases.

FR901228 induces tumor regression associated with induction of Fas ligand and activation of Fas signaling in human osteosarcoma cells.

We investigated the antitumor effects of FR901228, a HDAC inhibitor, on human osteosarcoma cells, in vitro and in vivo to explore its possible utility in the treatment of pediatric bone cancers. FR901228 caused marked growth inhibition with a 50% inhibitory concentration of 1.2-7.3 nM and induction of apoptosis in all eight osteosarcoma cell lines tested. These effects of FR901228 were also observed in vivo xenograft models on BALB/c nude mice, and treatment with 5.6 mg/kg/day resulting in a >70% reduction in the mean final tumor volume compared with the mean initial tumor volume. TUNEL assays demonstrated extensive apoptosis in tumor sections of mice treated with FR901228. Induction of apoptosis was preceded by increased expression of Fas ligand (FasL) mRNA, resulting in expression of membrane-bound FasL, which was followed by sequential activation of caspase-8 and -3. The level of apoptosis induction was reduced using a neutralizing anti-FasL antibody and overexpression of either the dominant-negative FADD or the viral FLICE inhibitory protein. Furthermore, treatment with a suboptimal dose of FR901228 greatly sensitized osteosarcoma cells to agonistic anti-Fas antibody-mediated apoptosis. These findings suggest that FR901228 is a highly promising antitumor agent against osteosarcoma, inducing apoptosis by the activation of the Fas/FasL system.