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DNA Methylation Mediated Control of Gene Expression Is Critical for Development of Crown Gall Tumors
(2013)
Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA–encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA–mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene expression, physiological processes, and the development of crown gall tumors.
Background: Measles virus (MV) causes T cell suppression by interference with phosphatidylinositol-3-kinase (PI3K) activation. We previously found that this interference affected the activity of splice regulatory proteins and a T cell inhibitory protein isoform was produced from an alternatively spliced pre-mRNA. Hypothesis: Differentially regulated and alternatively splice variant transcripts accumulating in response to PI3K abrogation in T cells potentially encode proteins involved in T cell silencing. Methods: To test this hypothesis at the cellular level, we performed a Human Exon 1.0 ST Array on RNAs isolated from T cells stimulated only or stimulated after PI3K inhibition. We developed a simple algorithm based on a splicing index to detect genes that undergo alternative splicing (AS) or are differentially regulated (RG) upon T cell suppression. Results: Applying our algorithm to the data, 9% of the genes were assigned as AS, while only 3% were attributed to RG. Though there are overlaps, AS and RG genes differed with regard to functional regulation, and were found to be enriched in different functional groups. AS genes targeted extracellular matrix (ECM)-receptor interaction and focal adhesion pathways, while RG genes were mainly enriched in cytokine-receptor interaction and Jak-STAT. When combined, AS/RG dependent alterations targeted pathways essential for T cell receptor signaling, cytoskeletal dynamics and cell cycle entry. Conclusions: PI3K abrogation interferes with key T cell activation processes through both differential expression and alternative splicing, which together actively contribute to T cell suppression.
Clear cell renal cell carcinoma (ccRCC) characterized by a tumor thrombus (TT) extending into the inferior vena cava (IVC) generally indicates poor prognosis. Nevertheless, the risk for tumor recurrence after nephrectomy and thrombectomy varies. An applicable and accurate prediction system to select ccRCC patients with TT of the IVC (ccRCC/TT) at high risk after nephrectomy is urgently needed, but has not been established up to now. To our knowledge, a possible role of microRNAs (miRs) for the development of ccRCC/TT or their impact as prognostic markers in ccRCC/TT has not been explored yet. Therefore, we analyzed the expression of the previously described onco-miRs miR-200c, miR-210, miR-126, miR-221, let-7b, miR-21, miR-143 and miR-141 in a study collective of 74 ccRCC patients. Using the expression profiles of these eight miRs we developed classification systems that accurately differentiate ccRCC from non-cancerous renal tissue and ccRCC/TT from tumors without TT. In the subgroup of 37 ccRCC/TT cases we found that miR-21, miR-126, and miR-221 predicted cancer related death (CRD) accurately and independently from other clinico-pathological features. Furthermore, a combined risk score based on the expression of miR-21, miR-126 and miR-221 was developed and showed high sensitivity and specificity to predict cancer specific survival (CSS) in ccRCC/TT. Using the combined risk score we were able to classify ccRCC/TT patients correctly into high and low risk cases. The risk stratification by the combined risk score (CRS) will benefit from further cohort validation and might have potential for clinical application as a molecular prediction system to identify high- risk ccRCC/TT patients.
The initial stages of the interaction between the host and Aspergillus fumigatus at the alveolar surface of the human lung are critical in the establishment of aspergillosis. Using an in vitro bilayer model of the alveolus, including both the epithelium (human lung adenocarcinoma epithelial cell line, A549) and endothelium (human pulmonary artery epithelial cells, HPAEC) on transwell membranes, it was possible to closely replicate the in vivo conditions. Two distinct sub-groups of dendritic cells (DC), monocyte-derived DC (moDC) and myeloid DC (mDC), were included in the model to examine immune responses to fungal infection at the alveolar surface. RNA in high quantity and quality was extracted from the cell layers on the transwell membrane to allow gene expression analysis using tailored custom-made microarrays, containing probes for 117 immune-relevant genes. This microarray data indicated minimal induction of immune gene expression in A549 alveolar epithelial cells in response to germ tubes of A. fumigatus. In contrast, the addition of DC to the system greatly increased the number of differentially expressed immune genes. moDC exhibited increased expression of genes including CLEC7A, CD209 and CCL18 in the absence of A. fumigatus compared to mDC. In the presence of A. fumigatus, both DC subgroups exhibited up-regulation of genes identified in previous studies as being associated with the exposure of DC to A. fumigatus and exhibiting chemotactic properties for neutrophils, including CXCL2, CXCL5, CCL20, and IL1B. This model closely approximated the human alveolus allowing for an analysis of the host pathogen interface that complements existing animal models of IA.
Background
Renal cell carcinoma (RCC) is marked by high mortality rate. To date, no robust risk stratification by clinical or molecular prognosticators of cancer-specific survival (CSS) has been established for early stages. Transcriptional profiling of small non-coding RNA gene products (miRNAs) seems promising for prognostic stratification. The expression of miR-21 and miR-126 was analysed in a large cohort of RCC patients; a combined risk score (CRS)-model was constructed based on expression levels of both miRNAs.
Methods
Expression of miR-21 and miR-126 was evaluated by qRT-PCR in tumour and adjacent non-neoplastic tissue in n = 139 clear cell RCC patients. Relation of miR-21 and miR-126 expression with various clinical parameters was assessed. Parameters were analysed by uni- and multivariate COX regression. A factor derived from the z-score resulting from the COX model was determined for both miRs separately and a combined risk score (CRS) was calculated multiplying the relative expression of miR-21 and miR-126 by this factor. The best fitting COX model was selected by relative goodness-of-fit with the Akaike information criterion (AIC).
Results
RCC with and without miR-21 up- and miR-126 downregulation differed significantly in synchronous metastatic status and CSS. Upregulation of miR-21 and downregulation of miR-126 were independently prognostic. A combined risk score (CRS) based on the expression of both miRs showed high sensitivity and specificity in predicting CSS and prediction was independent from any other clinico-pathological parameter. Association of CRS with CSS was successfully validated in a testing cohort containing patients with high and low risk for progressive disease.
Conclusions
A combined expression level of miR-21 and miR-126 accurately predicted CSS in two independent RCC cohorts and seems feasible for clinical application in assessing prognosis.
Swords are exaggerated male ornaments of swordtail fishes that have been of great interest to evolutionary biologists ever since Darwin described them in the Descent of Man (1871). They are a novel sexually selected trait derived from modified ventral caudal fin rays and are only found in the genus Xiphophorus. Another phylogenetically more widespread and older male trait is the gonopodium, an intromittent organ found in all poeciliid fishes, that is derived from a modified anal fin. Despite many evolutionary and behavioral studies on both traits, little is known so far about the molecular mechanisms underlying their development. By investigating transcriptomic changes (utilizing a RNA-Seq approach) in response to testosterone treatment in the swordtail fish, Xiphophorus hellerii, we aimed to better understand the architecture of the gene regulatory networks underpinning the development of these two evolutionary novelties. Large numbers of genes with tissue-specific expression patterns were identified. Among the sword genes those involved in embryonic organ development, sexual character development and coloration were highly expressed, while in the gonopodium rather more morphogenesis-related genes were found. Interestingly, many genes and genetic pathways are shared between both developing novel traits derived from median fins: the sword and the gonopodium. Our analyses show that a larger set of gene networks was co-opted during the development and evolution of the older gonopodium than in the younger, and morphologically less complex trait, the sword. We provide a catalog of candidate genes for future efforts to dissect the development of those sexually selected exaggerated male traits in swordtails.
Dysregulated IGFBP5 expression causes axon degeneration and motoneuron loss in diabetic neuropathy
(2015)
Diabetic neuropathy (DNP), afflicting sensory and motor nerve fibers, is a major complication in diabetes.The underlying cellular mechanisms of axon degeneration are poorly understood. IGFBP5, an inhibitory binding protein for insulin-like growth factor 1 (IGF1) is highly up-regulated in nerve biopsies of patients with DNP. We investigated the pathogenic relevance of this finding in transgenic mice overexpressing IGFBP5 in motor axons and sensory nerve fibers. These mice develop motor axonopathy and sensory deficits similar to those seen in DNP. Motor axon degeneration was also observed in mice in which the IGF1 receptor(IGF1R) was conditionally depleted in motoneurons, indicating that reduced activity of IGF1 on IGF1R in motoneurons is responsible for the observed effect. These data provide evidence that elevated expression of IGFBP5 in diabetic nerves reduces the availability of IGF1 for IGF1R on motor axons, thus leading to progressive neurodegeneration. Inhibition of IGFBP5 could thus offer novel treatment strategies for DNP.
Background
A growing number of studies report an abnormal expression of Piwi-interacting RNAs (piRNAs) and the piRNA processing enzyme Piwi in many cancers. Whether this finding is an epiphenomenon of the chaotic molecular biology of the fast dividing, neoplastically transformed cells or is functionally relevant to tumorigenesisis is difficult to discern at present. To better understand the role of piRNAs in cancer development small laboratory fish models can make a valuable contribution. However, little is known about piRNAs in somatic and neoplastic tissues of fish.
Results
To identify piRNA clusters that might be involved in melanoma pathogenesis, we use several transgenic lines of medaka, and platyfish/swordtail hybrids, which develop various types of melanoma. In these tumors Piwi, is expressed at different levels, depending on tumor type. To quantify piRNA levels, whole piRNA populations of testes and melanomas of different histotypes were sequenced. Because no reference piRNA cluster set for medaka or Xiphophorus was yet available we developed a software pipeline to detect piRNA clusters in our samples and clusters were selected that were enriched in one or more samples. We found several loci to be overexpressed or down-regulated in different melanoma subtypes as compared to hyperpigmented skin. Furthermore, cluster analysis revealed a clear distinction between testes, low-grade and high-grade malignant melanoma in medaka.
Conclusions
Our data imply that dysregulation of piRNA expression may be associated with development of melanoma. Our results also reinforce the importance of fish as a suitable model system to study the role of piRNAs in tumorigenesis.
The process of tumor invasion requires degradation of extracellular matrix by proteolytic enzymes. Cancer cells form protrusive invadopodia, which produce and release matrix metalloproteinases (MMPs) to degrade the basement membrane thereby enabling metastasis. We investigated the effect of LASP1, a newly identified protein in invadopodia, on expression, secretion and activation of MMPs in invasive breast tumor cell lines.
By analyzing microarray data of in-house generated control and LASP1-depleted MDA-MB-231 breast cancer cells, we observed downregulation of MMP1, -3 and -9 upon LASP1 depletion. This was confirmed by Western blot analysis. Conversely, rescue experiments restored in part MMP expression and secretion. The regulatory effect of LASP1 on MMP expression was also observed in BT-20 breast cancer cells as well as in prostate and bladder cancer cell lines.
In line with bioinformatic FunRich analysis of our data, which mapped a high regulation of transcription factors by LASP1, public microarray data analysis detected a correlation between high LASP1 expression and enhanced c-Fos levels, a protein that is part of the transcription factor AP-1 and known to regulate MMP expression. Compatibly, in luciferase reporter assays, AP-1 showed a decreased transcriptional activity after LASP1 knockdown.
Zymography assays and Western blot analysis revealed an additional promotion of MMP secretion into the extracellular matrix by LASP1, thus, most likely, altering the microenvironment during cancer progression.
The newly identified role of LASP1 in regulating matrix degradation by affecting MMP transcription and secretion elucidated the migratory potential of LASP1 overexpressing aggressive tumor cells in earlier studies.
Immature or semi-mature dendritic cells (DCs) represent tolerogenic maturation stages that can convert naive T cells into Foxp3\(^{+}\) induced regulatory T cells (iTreg). Here we found that murine bone marrow-derived DCs (BM-DCs) treated with cholera toxin (CT) matured by up-regulating MHC-II and costimulatory molecules using either high or low doses of CT (CT\(^{hi}\), CT\(^{lo}\)) or with cAMP, a known mediator CT signals. However, all three conditions also induced mRNA of both isoforms of the tolerogenic molecule cytotoxic T lymphocyte antigen 2 (CTLA-2α and CTLA-2β). Only DCs matured under CT\(^{hi}\) conditions secreted IL-1β, IL-6 and IL-23 leading to the instruction of Th17 cell polarization. In contrast, CT\(^{lo}\)- or cAMP-DCs resembled semi-mature DCs and enhanced TGF-β-dependent Foxp3\(^{+}\) iTreg conversion. iTreg conversion could be reduced using siRNA blocking of CTLA-2 and reversely, addition of recombinant CTLA-2α increased iTreg conversion in vitro. Injection of CT\(^{lo}\)- or cAMP-DCs exerted MOG peptide-specific protective effects in experimental autoimmune encephalomyelitis (EAE) by inducing Foxp3\(^{+}\) Tregs and reducing Th17 responses. Together, we identified CTLA-2 production by DCs as a novel tolerogenic mediator of TGF-β-mediated iTreg induction in vitro and in vivo. The CT-induced and cAMP-mediated up-regulation of CTLA-2 also may point to a novel immune evasion mechanism of Vibrio cholerae.