ADAMTS1 alters blood vessel morphology and TSP1 levels in LNCaP and LNCaP-19 prostate tumors

<p>Abstract</p> <p>Background</p> <p>Decreased expression of the angiogenesis inhibitor ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif, 1) has previously been reported during prostate cancer progression. The aim of this study was to investigate the function of ADAMTS1 in prostate tumors.</p> <p>Methods</p> <p>ADAMTS1 was downregulated by shRNA technology in the human prostate cancer cell line LNCaP (androgen-dependent), originally expressing ADAMTS1, and was upregulated by transfection in its subline LNCaP-19 (androgen-independent), expressing low levels of ADAMTS1. Cells were implanted subcutaneously in nude mice and tumor growth, microvessel density (MVD), blood vessel morphology, pericyte coverage and thrombospondin 1 (TSP1) were studied in the tumor xenografts.</p> <p>Results</p> <p>Modified expression of ADAMTS1 resulted in altered blood vessel morphology in the tumors. Low expression levels of ADAMTS1 were associated with small diameter blood vessels both in LNCaP and LNCaP-19 tumors, while high levels of ADAMTS1 were associated with larger vessels. In addition, TSP1 levels in the tumor xenografts were inversely related to ADAMTS1 expression. MVD and pericyte coverage were not affected. Moreover, upregulation of ADAMTS1 inhibited tumor growth of LNCaP-19, as evidenced by delayed tumor establishment. In contrast, downregulation of ADAMTS1 in LNCaP resulted in reduced tumor growth rate.</p> <p>Conclusions</p> <p>The present study demonstrates that ADAMTS1 is an important regulatory factor of angiogenesis and tumor growth in prostate tumors, where modified ADAMTS1 expression resulted in markedly changed blood vessel morphology, possibly related to altered TSP1 levels.</p

Malignant pheochromocytomas/paragangliomas harbor mutations in transport and cell adhesion genes.

One out of ten patients with pheochromocytoma (PCC) and paraganglioma (PGL) develop malignant disease. Today there are no reliable pathological methods to predict malignancy at the time of diagnosis. Tumors harboring mutations in the succinate dehydrogenase subunit B (SDHB) gene often metastasize but the sequential genetic events resulting in malignant progression are not fully understood. The aim of this study was to identify somatic mutations that contribute to the malignant transformation of PCC/PGL. We performed pair-wise (tumor-normal) whole-exome sequencing to analyze the somatic mutational landscape in five malignant and four benign primary PCC/sympathetic PGL (sPGL), including two biological replicates from each specimen. In total, 225 unique somatic mutations were identified in 215 genes, with an average mutation rate of 0.54 mutations/megabase. Malignant tumors had a significantly higher number of mutations compared to benign tumors (

Malignant pheochromocytomas/paragangliomas harbor mutations in transport and cell adhesion genes.

One out of ten patients with pheochromocytoma (PCC) and paraganglioma (PGL) develop malignant disease. Today there are no reliable pathological methods to predict malignancy at the time of diagnosis. Tumors harboring mutations in the succinate dehydrogenase subunit B (SDHB) gene often metastasize but the sequential genetic events resulting in malignant progression are not fully understood. The aim of this study was to identify somatic mutations that contribute to the malignant transformation of PCC/PGL. We performed pair-wise (tumor-normal) whole-exome sequencing to analyze the somatic mutational landscape in five malignant and four benign primary PCC/sympathetic PGL (sPGL), including two biological replicates from each specimen. In total, 225 unique somatic mutations were identified in 215 genes, with an average mutation rate of 0.54 mutations/megabase. Malignant tumors had a significantly higher number of mutations compared to benign tumors (

MYO5B mutations in pheochromocytoma/paraganglioma promote cancer progression

Identification of additional cancer-associated genes and secondary mutations driving the metastatic progression in pheochromocytoma and paraganglioma (PPGL) is important for subtyping, and may provide optimization of therapeutic regimens. We recently reported novel recurrent nonsynonymous mutations in the MYO5B gene in metastatic PPGL. Here, we explored the functional impact of these MYO5B mutations, and analyzed MYO5B expression in primary PPGL tumor cases in relation to mutation status. Immunohistochemistry and mRNA expression analysis in 30 PPGL tumors revealed an increased MYO5B expression in metastatic compared to non-metastatic cases. In addition, subcellular localization of MYO5B protein was altered from cytoplasmic to membranous in some metastatic tumors, and the strongest and most abnormal expression pattern was observed in a paraganglioma harboring a somatic MYO5B:p.G1611S mutation. In addition to five previously discovered MYO5B mutations, the present study of 30 PPGL (8 previous and 22 new samples) also revealed two, and hence recurrent, mutations in the gene paralog MYO5A. The three MYO5B missense mutations with the highest prediction scores (p.L587P, p.G1611S and p.R1641C) were selected and functionally validated using site directed mutagenesis and stable transfection into human neuroblastoma cells (SK-N-AS) and embryonic kidney cells (HEK293). In vitro analysis showed a significant increased proliferation rate in all three MYO5B mutated clones. The two somatically derived mutations, p.L587P and p.G1611S, were also found to increase the migration rate. Expression analysis of MYO5B mutants compared to wild type clones, demonstrated a significant enrichment of genes involved in migration, proliferation, cell adhesion, glucose metabolism, and cellular homeostasis. Our study validates the functional role of novel MYO5B mutations in proliferation and migration, and suggest the MYO5-pathway to be involved in the malignant progression in some PPGL tumors. © 2020 Tomic et al.Export Date: 6 August 2020; Article</p

Clinical data of the PA cases.

<p>Clinical data of the PA cases.</p

A new <i>GTF2I-BRAF</i> fusion mediating MAPK pathway activation in pilocytic astrocytoma

<div><p>Pilocytic astrocytoma (PA) is the most common pediatric brain tumor. A recurrent feature of PA is deregulation of the mitogen activated protein kinase (MAPK) pathway most often through <i>KIAA1549-BRAF</i> fusion, but also by other <i>BRAF</i>- or <i>RAF1</i>-gene fusions and point mutations (<i>e</i>.<i>g</i>. <i>BRAF</i>V600E). These features may serve as diagnostic and prognostic markers, and also facilitate development of targeted therapy. The aims of this study were to characterize the genetic alterations underlying the development of PA in six tumor cases, and evaluate methods for fusion oncogene detection. Using a combined analysis of RNA sequencing and copy number variation data we identified a new <i>BRAF</i> fusion involving the 5’ gene fusion partner <i>GTF2I</i> (7q11.23), not previously described in PA. The new <i>GTF2I-BRAF</i> 19–10 fusion was found in one case, while the other five cases harbored the frequent <i>KIAA1549-BRAF</i> 16–9 fusion gene. Similar to other <i>BRAF</i> fusions, the <i>GTF2I-BRAF</i> fusion retains an intact <i>BRAF</i> kinase domain while the inhibitory N-terminal domain is lost. Functional studies on <i>GTF2I-BRAF</i> showed elevated MAPK pathway activation compared to <i>BRAF</i>^<i>WT</i>. Comparing fusion detection methods, we found Fluorescence in situ hybridization with <i>BRAF</i> break apart probe as the most sensitive method for detection of different <i>BRAF</i> rearrangements (<i>GTF2I-BRAF</i> and <i>KIAA1549-BRAF</i>). Our finding of a new <i>BRAF</i> fusion in PA further emphasis the important role of B-Raf in tumorigenesis of these tumor types. Moreover, the consistency and growing list of <i>BRAF/RAF</i> gene fusions suggests these rearrangements to be informative tumor markers in molecular diagnostics, which could guide future treatment strategies.</p></div

FISH analysis of <i>BRAF</i> fusions with <i>BRAF</i> break apart assay.

<p>Upper left: Schematic presentation of the <i>BRAF</i> break apart assay, consisting of a 5’ 170 kb green probe and a 178 kb 3’ red probe in 7q34. Upper right (wt BRAF): Metaphase FISH of normal control and Interphase FISH of fusion-negative cell (right corner) showing two wild type <i>BRAF</i> alleles, displayed as a merged (yellow) or two adjacent green (5’)/red (3’) signals. Lower panels: Fusion-positive tumor cells (<i>GTF2I-BRAF</i> in PA3 and <i>KIAA1549-BRAF</i> in PA4) showing the <i>BRAF</i> split pattern; two normal <i>BRAF</i> alleles green /red signals, as well as one additional split <i>BRAF</i> red signal representing the duplicated 3’ region in the fusion gene. The same split signal pattern is seen for different types of <i>BRAF</i> fusions; <i>GTF2I-BRAF</i> in case PA3 and <i>KIAA1549-BRAF</i> in cases PA1-2 and PA4-6 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175638#pone.0175638.s003" target="_blank">S3 Fig</a>). Tissue sections were counterstained with DAPI (blue).</p

Activation of the MAPK pathway.

<p><b>(</b>A<b>)</b> Western Blot of protein lysates from HEK293 cells transiently transfected with pCMV6-Myc-DDK (empty vector), pCMV6-BRAF-Myc-DDK (BRAF^WT) or pCMV6-GTF2I-BRAF-Myc-DDK (GTF2I-BRAF) were probed with antibodies against FLAG-DDK, phosphorylated ERK-Thr202/Tyr204 (pERK), total ERK (tERK) and GAPDH. Bars show relative mean pERK/tERK protein expression for each construct performed in triplicates (mean±SEM) after normalization to GAPDH. (B<b>)</b> Activation of the MAPK pathway in PA tumor tissue. FFPE sections from the six primary PA cases were immunostained with phosphorylated-ERK-Thr202/Tyr204 (pERK) antibody. Tumor tissue (PA1-6) showing perinuclear (arrow), nuclear (arrow head) and to lesser extent cytoplasmic (*) pERK staining. Normal human brain cerebellum reference tissue section from an autopsy specimen showing negative staining for pERK. Negative control with omitted primary antibody showing negative staining for pERK. Some endothelial cells were also positive for pERK. Original magnification x400.</p

Expression analysis of fusion transcripts based on RNA-seq data.

<p>Log2 RNA-seq expression data for three fusion transcripts; <i>GTF2I-BRAF</i> 19–10, <i>DENND2A-GTF2IRD1</i> 14–2, <i>KIAA1549-BRAF</i> 16–9 compared to wild type fusion partner genes in six PA cases. Expression data was calculated as total number of supporting reads normalized to the total number of raw reads in each sample. Exon-exon junction in genes are as follows; <i>GTF2I-BRAF</i> 19–10 (exon 19- exon 10), <i>GTF2I</i> (exon 19- exon 20), <i>BRAF</i> e9-e10 (exon 9- exon 10), <i>DENND2A-GTF2IRD1</i> 14–2 (exon 14- exon 2), <i>KIAA1549-BRAF</i> 16–9 (exon 16- exon 9), <i>DENND2A</i> v1 (transcript version 1, exon 14- exon 15), <i>KIAA1549</i> (exon 16- exon 17), <i>BRAF</i> e8-e9 (exon 8- exon 9).</p