Cardiac fibroblasts impact myocardial development and remodeling through intercellular contact with cardiomyocytes, but less is known about noncontact, profibrotic signals whereby fibroblasts alter cardiomyocyte behavior. Fibroblasts and cardiomyocytes were harvested from newborn rat ventricles and separated by serial digestion and gradient centrifugation. Cardiomyocytes were cultured in 1) standard medium, 2) standard medium diluted 1:1 with PBS, or 3) standard medium diluted 1:1 with medium conditioned ≥72 h by cardiac fibroblasts. Serum concentrations were held constant under all media conditions, and complete medium exchanges were performed daily. Cardiomyocytes began contracting within 24 h at clonal or mass densities with <5% of cells expressing vimentin. Immunocytochemical analysis revealed progressive expression of α-smooth muscle actin in cardiomyocytes after 24 h in all conditions. Only cardiomyocytes in fibroblast-conditioned medium stopped contracting by 72 h. There was a significant, sustained increase in vimentin expression specific to these cultures (means ± SD: conditioned 46.3 ± 6.0 vs. control 5.3 ± 2.9%, P < 0.00025) typically with cardiac myosin heavy chain coexpression. Proteomics assays revealed 10 cytokines (VEGF, GRO/KC, monocyte chemoattractant protein-1, leptin, macrophage inflammatory protein-1α, IL-6, IL-10, IL-12p70, IL-17, and tumor necrosis factor-α) at or below detection levels in unconditioned medium that were significantly elevated in fibroblast-conditioned medium. Latent transforming growth factor-β and RANTES were present in unconditioned medium but rose to higher levels in conditioned medium. Only granulocyte-macrophage colony-stimulating factor was present above threshold levels in standard medium but decreased with fibroblast conditioning. These data indicated that under the influence of fibroblast-conditioned medium, cardiomyocytes exhibited marked hypertrophy, diminished contractile capacity, and phenotype plasticity distinct from the dedifferentiation program present under standard culture conditions.

Purpose-Recent studies have demonstrated a high frequency of IDH mutations in adult "secondary" malignant gliomas arising from preexisting lower grade lesions, often in young adults, but not in "primary" gliomas. Because pediatric malignant gliomas share some molecular © Springer-Verlag 2010 Correspondence to: Ian F. Pollack. NIH Public Access Author ManuscriptChilds Nerv Syst. Author manuscript; available in PMC 2012 January 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript features with adult secondary gliomas, we questioned whether a subset of these tumors also exhibited IDH mutations.Experimental design-We examined the frequency of IDH mutations, using real-time polymerase chain reaction and sequencing analysis, in a cohort of 43 pediatric primary malignant gliomas treated on the Children's Oncology Group ACNS0423 study. The relationship between IDH mutations and other molecular and clinical factors, and outcome, was evaluated.Results-IDH1 mutations were observed in 7 of 43 (16.3%) tumors; no IDH2 mutations were observed. A striking age association was apparent in that mutations were noted in 7 of 20 tumors (35%) from children ≥14 years, but in 0 of 23 (0%) younger children (p=0.0024). No association was observed with clinical factors other than age. One-year event-free survival was 86±15% in the IDH-mutated group versus 64±8% in the non-mutated group (p=0.03, one-sided logrank test). One-year overall survival was 100% in patients with mutations versus 81±6.7% in those without mutations (p=0.035, one-sided logrank test).Conclusions-IDH1 mutations are common in malignant gliomas in older children, suggesting that a subset of these lesions may be biologically similar to malignant gliomas arising in younger adults and may be associated with a more favorable prognosis.

BACKGROUND:The overall 5-year survival rate of approximately 60% for head and neck cancer patients has remained essentially unchanged over the past 30 years. plays an essential role in cell-cycle control at the G1/S-phase checkpoint. However, the aberrant miR-137 promoter methylation observed in squamous cell carcinoma of the head and neck (SCCHN) suggests a tumor-specific molecular defect that may contribute to disease progression. METHODS: The goal of this study was to assess, in formalin-fixed, paraffin-embedded tumor tissue, the association between miR-137 promoter methylation and survival (both overall and disease free) and with prognostic factors including stage, tumor size, lymph node positivity, tumor grade, and surgical tumor margin positivity. RESULTS: The promoter methylation status of miR-137 was ascertained by methylation-specific polymerase chain reaction and detected in 11 of 67 SCCHN patients (16.4%), with no significant differences according to site (oral cavity, pharynx, larynx). Methylation of the miR-137 promoter was significantly associated with overall survival (hazard ratio, 3.68; 95% confidence interval, 1.01-13.38) but not with disease-free survival or any of the prognostic factors evaluated. CONCLUSIONS: The results of this study indicate that miR-137 is methylated in tumor tissue from pharyngeal and laryngeal squamous cancers, in addition to oral squamous cell carcinoma, and that miR-137 promoter methylation has potential utility as a prognostic marker for SCCHN.

Despite prognostic grading and staging systems, it is a challenge to predict outcomes for patients with pancreatic neuroendocrine tumors (PanNETs). Sequencing studies of PanNETs have identified alterations in death domain-associated protein (DAXX) and ATRX chromatin remodeler (ATRX). In tumors, mutations in DAXX or ATRX and corresponding loss of protein expression correlate with shorter times of disease-free survival and disease-specific survival of patients. However, DAXX or ATRX proteins were lost in only 50% of distant metastases analyzed. We performed whole-exome sequencing analyses of 20 distant metastases from 20 patients with a single non-syndrome, non-functional PanNET. We found distant metastases contained alterations in MEN1 (n=8), ATRX (n=5), DAXX (n=5), TSC2 (n=3), and DEPDC5 (n=3). We found copy number loss of CDKN2A in 15 metastases (75%) and alterations in genes that regulate chromatin remodeling including SETD2 (n=4), ARID1A (n=2), CHD8 (n=2), and DNMT1 (n=2). In a separate analysis of 347 primary PanNETs, we found loss or deletion of DAXX and ATRX, disruption of SETD2 function (based on loss of H3K36me3), loss of ARID1A expression or deletions in CDKN2A in 81% of primary PanNETs with distant metastases. Among patients with loss or deletion of at least 1 of these proteins or genes, 39% survived disease free for 5 years and 44% had disease-specific survival times of 10 years. Among patients without any of these alterations, 98% survived disease free for 5 years and 95% had disease-specific survival times of 10 years. Therefore, primary PanNETs with loss of DAXX, ATRX, H3K36me3, ARID1A, and/or CDKN2A associate with shorter survival times of patients. Our findings indicate that alterations in chromatin remodeling genes and CDKN2A contribute to metastasis of PanNETs.

The tumor microenvironment has been compared with a nonhealing wound involving a complex interaction between multiple cell types. Schwann cells, the key regulators of peripheral nerve repair, have recently been shown to directly affect nonneural wound healing. Their role in cancer progression, however, has been largely limited to neuropathic pain and perineural invasion. In this study, we showed that melanoma activated otherwise dormant functions of Schwann cells aimed at nerve regeneration and wound healing. Such reprogramming of Schwann cells into repair-like cells occurred during the destruction and displacement of neurons as the tumor expanded and via direct signaling from melanoma cells to Schwann cells, resulting in activation of the nerve injury response. Melanoma-activated Schwann cells significantly altered the microenvironment through their modulation of the immune system and the extracellular matrix in a way that promoted melanoma growth in vitro and in vivo. Local inhibition of Schwann cell activity following cutaneous sensory nerve transection in melanoma orthotopic models significantly decreased the rate of tumor growth. Tumor-associated Schwann cells, therefore, can have a significant protumorigenic effect and may present a novel target for cancer therapy.Significance: These findings reveal a role of the nerve injury response, particularly through functions of activated Schwann cells, in promoting melanoma growth. Recently, similar activation of the peripheral glia was described in

Abstract

Dupuytren's contracture (DC) is the most common inherited connective tissue disease of humans and is hypothesized to be associated with aberrant wound healing of the palmar fascia. Fibroblasts and myofibroblasts are believed to play an important role in the genesis of DC and the fibroproliferation and contraction that are hallmarks of this disease. This study compares the gene expression profiles of fibroblasts isolated from DC patients and controls in an attempt to identify key genes whose regulation might be significantly altered in fibroblasts found within the palmar fascia of Dupuytren's patients. Total RNA isolated from diseased palmar fascia (DC) and normal palmar fascia (obtained during carpal tunnel release; 6 samples per group) was subjected to quantitative analyses using two different microarray platforms (GE Code Link™ and Illumina™) to identify and validate differentially expressed genes. The data obtained was analyzed using The Significance Analysis of Microarrays (SAM) software through which we identified 69 and 40 differentially regulated gene transcripts using the CodeLink™ and Illumina™ platforms, respectively. The CodeLink™ platform identified 18 upregulated and 51 downregulated genes. Using the Illumina™ platform, 40 genes were identified as downregulated, eleven of which were identified by both platforms. Quantitative RT-PCR confirmed the downregulation of three high-interest candidate genes which are all components of the extracellular matrix: proteoglycan 4 (PRG4), fibulin-1 (FBLN-1) transcript variant D, and type XV collagen alpha 1 chain. Overall, our study has identified a variety of candidate genes that may be involved in the pathophysiology of Dupuytren's contracture and may ultimately serve as attractive molecular targets for alternative therapies.

No abstract

A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells. Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport. Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein.A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-39 untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.