Angiotensin II-dependent TGF-ß signaling contributes to Loeys-Dietz syndrome vascular pathogenesis.

Loeys-Dietz syndrome (LDS) is a connective tissue disorder that is characterized by a high risk for aneurysm and dissection throughout the arterial tree and phenotypically resembles Marfan syndrome. LDS is caused by heterozygous missense mutations in either TGF-ß receptor gene (TGFBR1 or TGFBR2), which are predicted to result in diminished TGF-ß signaling; however, aortic surgical samples from patients show evidence of paradoxically increased TGF-ß signaling. We generated 2 knockin mouse strains with LDS mutations in either Tgfbr1 or Tgfbr2 and a transgenic mouse overexpressing mutant Tgfbr2. Knockin and transgenic mice, but not haploinsufficient animals, recapitulated the LDS phenotype. While heterozygous mutant cells had diminished signaling in response to exogenous TGF-ß in vitro, they maintained normal levels of Smad2 phosphorylation under steady-state culture conditions, suggesting a chronic compensation. Analysis of TGF-ß signaling in the aortic wall in vivo revealed progressive upregulation of Smad2 phosphorylation and TGF-ß target gene output, which paralleled worsening of aneurysm pathology and coincided with upregulation of TGF-ß1 ligand expression. Importantly, suppression of Smad2 phosphorylation and TGF-ß1 expression correlated with the therapeutic efficacy of the angiotensin II type 1 receptor antagonist losartan. Together, these data suggest that increased TGF-ß signaling contributes to postnatal aneurysm progression in LDS.

Podocalyxin-like protein is expressed in glioblastoma multiforme stem-like cells and is associated with poor outcome.

Glioblastoma multiforme (GBM) is the most common primary malignant adult brain tumor and is associated with poor survival. Recently, stem-like cell populations have been identified in numerous malignancies including GBM. To identify genes whose expression is changed with differentiation, we compared transcript profiles from a GBM oncosphere line before and after differentiation. Bioinformatic analysis of the gene expression profiles identified podocalyxin-like protein (PODXL), a protein highly expressed in human embryonic stem cells, as a potential marker of undifferentiated GBM stem-like cells. The loss of PODXL expression upon differentiation of GBM stem-like cell lines was confirmed by quantitative real-time PCR and flow cytometry. Analytical flow cytometry of numerous GBM oncosphere lines demonstrated PODXL expression in all lines examined. Knockdown studies and flow cytometric cell sorting experiments demonstrated that PODXL is involved in GBM stem-like cell proliferation and oncosphere formation. Compared to PODXL-negative cells, PODXL-positive cells had increased expression of the progenitor/stem cell markers Musashi1, SOX2, and BMI1. Finally, PODXL expression directly correlated with increasing glioma grade and was a marker for poor outcome in patients with GBM. In summary, we have demonstrated that PODXL is expressed in GBM stem-like cells and is involved in cell proliferation and oncosphere formation. Moreover, high PODXL expression correlates with increasing glioma grade and decreased overall survival in patients with GBM.

LMNA-associated cardiocutaneous progeria: an inherited autosomal dominant premature aging syndrome with late onset.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disorder caused by mutations in LMNA, which encodes the nuclear scaffold proteins lamin A and C. In HGPS and related progerias, processing of prelamin A is blocked at a critical step mediated by the zinc metalloprotease ZMPSTE24. LMNA-linked progerias can be grouped into two classes: (1) the processing-deficient, early onset "typical" progerias (e.g., HGPS), and (2) the processing-proficient "atypical" progeria syndromes (APS) that are later in onset. Here we describe a previously unrecognized progeria syndrome with prominent cutaneous and cardiovascular manifestations belonging to the second class. We suggest the name LMNA-associated cardiocutaneous progeria syndrome (LCPS) for this disorder. Affected patients are normal at birth but undergo progressive cutaneous changes in childhood and die in middle age of cardiovascular complications, including accelerated atherosclerosis, calcific valve disease, and cardiomyopathy. In addition, the proband demonstrated cancer susceptibility, a phenotype rarely described for LMNA-based progeria disorders. The LMNA mutation that caused LCPS in this family is a heterozygous c.899A>G (p.D300G) mutation predicted to alter the coiled-coil domain of lamin A/C. In skin fibroblasts isolated from the proband, the processing and levels of lamin A and C are normal. However, nuclear morphology is aberrant and rescued by treatment with farnesyltransferase inhibitors, as is also the case for HGPS and other laminopathies. Our findings advance knowledge of human LMNA progeria syndromes, and raise the possibility that typical and atypical progerias may converge upon a common mechanism to cause premature aging disease.

Regulation of brain tumor dispersal by NKCC1 through a novel role in focal adhesion regulation.

Glioblastoma (GB) is a highly invasive and lethal brain tumor due to its universal recurrence. Although it has been suggested that the electroneutral Na(+)-K(+)-Cl(-) cotransporter 1 (NKCC1) can play a role in glioma cell migration, the precise mechanism by which this ion transporter contributes to GB aggressiveness remains poorly understood. Here, we focused on the role of NKCC1 in the invasion of human primary glioma cells in vitro and in vivo. NKCC1 expression levels were significantly higher in GB and anaplastic astrocytoma tissues than in grade II glioma and normal cortex. Pharmacological inhibition and shRNA-mediated knockdown of NKCC1 expression led to decreased cell migration and invasion in vitro and in vivo. Surprisingly, knockdown of NKCC1 in glioma cells resulted in the formation of significantly larger focal adhesions and cell traction forces that were approximately 40% lower than control cells. Epidermal growth factor (EGF), which promotes migration of glioma cells, increased the phosphorylation of NKCC1 through a PI3K-dependant mechanism. This finding is potentially related to WNK kinases. Taken together, our findings suggest that NKCC1 modulates migration of glioma cells by two distinct mechanisms: (1) through the regulation of focal adhesion dynamics and cell contractility and (2) through regulation of cell volume through ion transport. Due to the ubiquitous expression of NKCC1 in mammalian tissues, its regulation by WNK kinases may serve as new therapeutic targets for GB aggressiveness and can be exploited by other highly invasive neoplasms.

Generation of chordoma cell line JHC7 and the identification of Brachyury as a novel molecular target.

OBJECT: Chordoma is a malignant bone neoplasm hypothesized to arise from notochordal remnants along the length of the neuraxis. Recent genomic investigation of chordomas has identified T (Brachyury) gene duplication as a major susceptibility mutation in familial chordomas. Brachyury plays a vital role during embryonic development of the notochord and has recently been shown to regulate epithelial-to-mesenchymal transition in epithelial-derived cancers. However, current understanding of the role of this transcription factor in chordoma is limited due to the lack of availability of a fully characterized chordoma cell line expressing Brachyury. Thus, the objective of this study was to establish the first fully characterized primary chordoma cell line expressing gain of the T gene locus that readily recapitulates the original parental tumor phenotype in vitro and in vivo. METHODS: Using an intraoperatively obtained tumor sample from a 61-year-old woman with primary sacral chordoma, a chordoma cell line (JHC7, or Johns Hopkins Chordoma Line 7) was established. Molecular characterization of the primary tumor and cell line was conducted using standard immunostaining and Western blotting. Chromosomal aberrations and genomic amplification of the T gene in this cell line were determined. Using this cell line, a xenograft model was established and the histopathological analysis of the tumor was performed. Silencing of Brachyury and changes in gene expression were assessed. RESULTS: The authors report, for the first time, the successful establishment of a chordoma cell line (JHC7) from a patient with pathologically confirmed sacral chordoma. This cell line readily forms tumors in immunodeficient mice that recapitulate the parental tumor phenotype with conserved histological features consistent with the parental tumor. Furthermore, it is demonstrated for the first time that silencing of Brachyury using short hairpin RNA renders the morphology of chordoma cells to a more differentiated-like state and leads to complete growth arrest and senescence with an inability to be passaged serially in vitro. CONCLUSIONS: This report represents the first xenograft model of a sacral chordoma line described in the literature and the first cell line established with stable Brachyury expression. The authors propose that Brachyury is an attractive therapeutic target in chordoma and that JHC7 will serve as a clinically relevant model for the study of this disease.

Glioblastoma cell growth is suppressed by disruption of Fibroblast Growth Factor pathway signaling.

The Fibroblast Growth Factor (FGF) signaling pathway is reported to stimulate glioblastoma (GBM) growth. In this work we evaluated the effect of FGF2, FGF receptor (FGFR), and small molecule inhibition on GBM cells grown in traditional media, or cultured directly in stem-cell media. These lines each expressed the FGFR1, FGFR3 and FGFR4 receptors. Addition of FGF2 ligand showed significant growth stimulation in 8 of 10 cell lines. Disruption of FGF signaling by a neutralizing FGF2 monoclonal antibody and FGFR1 suppression by RNA interference both partially inhibited cell proliferation. Growth inhibition was temporally correlated with a reduction in MAPK signaling. A receptor tyrosine kinase inhibitor with known FGFR/VEGFR activity, PD173074, showed reproducible growth inhibition. Possible mechanisms of growth suppression by PD173074 were implicated by reduced phosphorylation of AKT and MAPK, known oncogenic signal transducers. Subsequent reduction in the cyclin D1, cyclin D2 and CDK4 cell cycle regulators was also observed. Our results indicate that FGF signaling pathway inhibition as a monotherapy will slow, but not arrest growth of glioblastoma cells.

Angiotensin II type 1 receptor blockade attenuates TGF-beta-induced failure of muscle regeneration in multiple myopathic states.

Skeletal muscle has the ability to achieve rapid repair in response to injury or disease. Many individuals with Marfan syndrome (MFS), caused by a deficiency of extracellular fibrillin-1, exhibit myopathy and often are unable to increase muscle mass despite physical exercise. Evidence suggests that selected manifestations of MFS reflect excessive signaling by transforming growth factor (TGF)-beta (refs. 2,3). TGF-beta is a known inhibitor of terminal differentiation of cultured myoblasts; however, the functional contribution of TGF-beta signaling to disease pathogenesis in various inherited myopathic states in vivo remains unknown. Here we show that increased TGF-beta activity leads to failed muscle regeneration in fibrillin-1-deficient mice. Systemic antagonism of TGF-beta through administration of TGF-beta-neutralizing antibody or the angiotensin II type 1 receptor blocker losartan normalizes muscle architecture, repair and function in vivo. Moreover, we show TGF-beta-induced failure of muscle regeneration and a similar therapeutic response in a dystrophin-deficient mouse model of Duchenne muscular dystrophy.

Separable roles for rent1/hUpf1 in altered splicing and decay of nonsense transcripts.

The mechanism by which disruption of reading frame can influence pre-messenger RNA (pre-mRNA) processing is poorly understood. We assessed the role of factors essential for nonsense-mediated mRNA decay (NMD) in nonsense-mediated altered splicing (NAS) with the use of RNA interference (RNAi) in mammalian cells. Inhibition of rent1/hUpf1 expression abrogated both NMD and NAS of nonsense T cell receptor beta transcripts. In contrast, inhibition of rent2/hUpf2 expression did not disrupt NAS despite achieving comparable stabilization of nonsense transcripts. We also demonstrate that NAS and NMD are genetically separable functions of rent1/hUpf1. Additionally, rent1/hUpf1 enters the nucleus where it may directly influence early events in mRNA biogenesis. This provides compelling evidence that NAS relies on a component of the nonsense surveillance machinery but is not an indirect consequence of NMD.

Patterns of gene expression and a transactivation function exhibited by the vGCR (ORF74) chemokine receptor protein of Kaposi's sarcoma-associated herpesvirus.

The ORF74 or vGCR gene encoded by Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8) has properties of a ligand-independent membrane receptor signaling protein with angiogenic properties that is predicted to play a key role in the biology of the virus. We have examined the expression of vGCR mRNA and protein in primary effusion lymphoma (PEL) cell lines, PEL and multicentric Castleman's disease (MCD) tumors, Kaposi's sarcoma lesions and infected endothelial cell cultures. The vGCR gene proved to be expressed in PEL cell lines as a large spliced bicistronic mRNA of 3.2 kb that also encompasses the upstream vOX2 (K14) gene. This mRNA species was induced strongly by phorbol ester (TPA) and sodium butyrate treatment in the BCBL-1 cell line, but only weakly in the HBL6 cell line, and was classified as a relatively late and low-abundance delayed early class lytic cycle gene product. A complex bipartite upstream lytic cycle promoter for this mRNA was nestled within the intron of the 5'-overlapping but oppositely oriented latent-state transcription unit for LANA1/vCYC-D/vFLIP and responded strongly to both TPA induction and cotransfection with the KSHV RNA transactivator protein (RTA or ORF50) in transient reporter gene assays. A vGCR protein product of 45 kDa that readily dimerized was detected by Western blotting and in vitro translation and was localized in a cytoplasmic and membrane pattern in DNA-transfected Vero and 293T cells or adenovirus vGCR-transduced dermal microvascular endothelial cells (DMVEC) as detected by indirect immunofluorescence assay (IFA) and immunohistochemistry with a specific rabbit anti-vGCR antibody. Similarly, a subfraction of KSHV-positive cultured PEL cells and of KSHV (JSC-1) persistently infected DMVEC cells displayed cytoplasmic vGCR protein expression, but only after TPA or spontaneous lytic cycle induction, respectively. The vGCR protein was also detectable by immunohistochemical staining in a small fraction (0.5 to 3%) of the cells in PEL and MCD tumor and nodular Kaposi's sarcoma lesion specimens that were apparently undergoing lytic cycle expression. These properties are difficult to reconcile with the vGCR protein's playing a direct role in spindle cell proliferation, transformation, or latency, but could be compatible with proposed contributions to angiogenesis via downstream paracrine effects. The ability of vGCR to transactivate expression of both several KSHV promoter-driven luciferase (LUC) reporter genes and an NFkappaB motif containing the chloramphenicol acetyltransferase (CAT) reporter gene may also suggest an unexpected regulatory role in viral gene expression.