Explore the vast range of titles available.

Charles Swanton and colleagues used multiregion exome sequencing to study the evolutionary histories of ten clear cell renal cell carcinomas. They observed marked intratumoral heterogeneity in all cases, with extensive evidence of parallel evolution of tumor subclones and only a small number of truncal driver events. Clear cell renal carcinomas (ccRCCs) can display intratumor heterogeneity (ITH). We applied multiregion exome sequencing (M-seq) to resolve the genetic architecture and evolutionary histories of ten ccRCCs. Ultra-deep sequencing identified ITH in all cases. We found that 73–75% of identified ccRCC driver aberrations were subclonal, confounding estimates of driver mutation prevalence. ITH increased with the number of biopsies analyzed, without evidence of saturation in most tumors. Chromosome 3p loss and VHL aberrations were the only ubiquitous events. The proportion of C>T transitions at CpG sites increased during tumor progression. M-seq permits the temporal resolution of ccRCC evolution and refines mutational signatures occurring during tumor development.

Genetic analysis was applied to different regions of renal-cell cancers. The lesions noted in the tumor were not found in every sample, and regions of the tumor had different gene-expression patterns. This suggests that extrapolation from results of a single biopsy may be problematic. Large-scale sequencing analyses of solid cancers have identified extensive heterogeneity between individual tumors. 1 – 6 Genetic intratumor heterogeneity has also been shown 7 – 15 and can contribute to treatment failure and drug resistance. Intratumor heterogeneity may have important consequences for personalized-medicine approaches that commonly rely on single tumor-biopsy samples to portray tumor mutational landscapes. Studies comparing mutational profiles of primary tumors and associated metastatic lesions 16 , 17 or local recurrences 18 have provided evidence of intratumor heterogeneity at nucleotide resolution. Intratumor heterogeneity within primary tumors and associated metastatic sites has not been systematically characterized by next-generation sequencing. We applied exome sequencing, chromosome aberration analysis, . . .

ObjectiveTo use a computational approach to investigate the cellular and extracellular matrix changes that occur with age in the knee joints of mice.MethodsKnee joints from an inbred C57/BL1/6 (ICRFa) mouse colony were harvested at 3–30 months of age. Sections were stained with H&E, Safranin-O, Picro-sirius red and antibodies to matrix metalloproteinase-13 (MMP-13), nitrotyrosine, LC-3B, Bcl-2, and cleaved type II collagen used for immunohistochemistry. Based on this and other data from the literature, a computer simulation model was built using the Systems Biology Markup Language using an iterative approach of data analysis and modelling. Individual parameters were subsequently altered to assess their effect on the model.ResultsA progressive loss of cartilage matrix occurred with age. Nitrotyrosine, MMP-13 and activin receptor-like kinase-1 (ALK1) staining in cartilage increased with age with a concomitant decrease in LC-3B and Bcl-2. Stochastic simulations from the computational model showed a good agreement with these data, once transforming growth factor-β signalling via ALK1/ALK5 receptors was included. Oxidative stress and the interleukin 1 pathway were identified as key factors in driving the cartilage breakdown associated with ageing.ConclusionsA progressive loss of cartilage matrix and cellularity occurs with age. This is accompanied with increased levels of oxidative stress, apoptosis and MMP-13 and a decrease in chondrocyte autophagy. These changes explain the marked predisposition of joints to develop osteoarthritis with age. Computational modelling provides useful insights into the underlying mechanisms involved in age-related changes in musculoskeletal tissues.

A mechanism to explain chromosomal instability (CIN) in colorectal cancer is demonstrated; three new CIN-suppressor genes ( PIGN , MEX3C and ZNF516 ) encoded on chromosome 18q are identified, the loss of which leads to DNA replication stress, resulting in structural and numerical chromosome segregation errors, which are shown to be identical to phenotypes seen in CIN cells. Cause of chromosome instability in colorectal cancer Chromosomal instability (CIN) occurs in most solid tumours and is associated with poor prognosis and drug resistance. This study demonstrates a link between CIN in colorectal cancer and the loss of a region on chromosome 18q. The authors identify three previously unknown CIN-suppressor genes in this region that, when lost, lead to replication stress resulting in structural and numerical chromosome segregation errors. Supplementing tumour cell lines with nucleosides alleviates replication-associated damage, limits chromosome segregation errors after CIN-suppressor gene silencing and attenuates segregation errors and DNA damage in CIN + cells. These findings point to a genetic mechanism — distinct from mitotic defects — that causes chromosome instability in colorectal tumours and that might be pharmacologically reversible. Cancer chromosomal instability (CIN) results in an increased rate of change of chromosome number and structure and generates intratumour heterogeneity 1 , 2 . CIN is observed in most solid tumours and is associated with both poor prognosis and drug resistance 3 , 4 . Understanding a mechanistic basis for CIN is therefore paramount. Here we find evidence for impaired replication fork progression and increased DNA replication stress in CIN + colorectal cancer (CRC) cells relative to CIN − CRC cells, with structural chromosome abnormalities precipitating chromosome missegregation in mitosis. We identify three new CIN-suppressor genes ( PIGN (also known as MCD4 ), MEX3C ( RKHD2 ) and ZNF516 ( KIAA0222 )) encoded on chromosome 18q that are subject to frequent copy number loss in CIN + CRC. Chromosome 18q loss was temporally associated with aneuploidy onset at the adenoma–carcinoma transition. CIN-suppressor gene silencing leads to DNA replication stress, structural chromosome abnormalities and chromosome missegregation. Supplementing cells with nucleosides, to alleviate replication-associated damage 5 , reduces the frequency of chromosome segregation errors after CIN-suppressor gene silencing, and attenuates segregation errors and DNA damage in CIN + cells. These data implicate a central role for replication stress in the generation of structural and numerical CIN, which may inform new therapeutic approaches to limit intratumour heterogeneity.

The spread of cancer cells from primary tumors to regional lymph nodes is often associated with reduced survival. One prevailing model to explain this association posits that fatal, distant metastases are seeded by lymph node metastases. This view provides a mechanistic basis for the TNM staging system and is the rationale for surgical resection of tumor-draining lymph nodes. Here we examine the evolutionary relationship between primary tumor, lymph node, and distant metastases in human colorectal cancer. Studying 213 archival biopsy samples from 17 patients, we used somatic variants in hypermutable DNA regions to reconstruct high-confidence phylogenetic trees. We found that in 65% of cases, lymphatic and distant metastases arose from independent subclones in the primary tumor, whereas in 35% of cases they shared common subclonal origin. Therefore, two different lineage relationships between lymphatic and distant metastases exist in colorectal cancer.

This study was designed to identify metalloproteinase determinants of macrophage migration and led to the specific hypothesis that matrix metalloproteinase 10 (MMP10/stromelysin-2) facilitates macrophage migration. We first profiled expression of all MMPs in LPS-stimulated primary murine bone marrow-derived macrophages and Raw264.7 cells and found that MMP10 was stimulated early (3 h) and down-regulated later (24 h). Based on this pattern of expression, we speculated that MMP10 plays a role in macrophage responses, such as migration. Indeed, using time lapse microscopy, we found that RNAi silencing of MMP10 in primary macrophages resulted in markedly reduced migration, which was reversed with exogenous active MMP10 protein. Mmp10 (-/-) bone marrow-derived macrophages displayed significantly reduced migration over a two-dimensional fibronectin matrix. Invasion of primary wild-type macrophages into Matrigel supplemented with fibronectin was also markedly impaired in Mmp10 (-/-) cells. MMP10 expression in macrophages thus emerges as an important moderator of cell migration and invasion. These findings support the hypothesis that MMP10 promotes macrophage movement and may have implications in understanding the control of macrophages in several pathologies, including the abnormal wound healing response associated with pro-inflammatory conditions.

Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts. We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells) were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts. We conclude that leptin selectively enhances the expression and secretion of certain matrix metalloproteinases in human gingival fibroblasts, and suggest that gingival fibroblasts may have an ECM-degrading phenotype during conditions of hyperleptinaemia (e.g., obesity, type 2 diabetes mellitus, exogenous leptin therapy).

Many catabolic stimuli, including interleukin-1 (IL-1) in combination with oncostatin M (OSM), promote cartilage breakdown via the induction of collagen-degrading collagenases such as matrix metalloproteinase 1 (MMP1) and MMP13 in human articular chondrocytes. Indeed, joint diseases with an inflammatory component are characterised by excessive extracellular matrix (ECM) catabolism. Importantly, protein kinase C (PKC) signalling has a primary role in cytokine-induced MMP1/13 expression, and is known to regulate cellular functions associated with pathologies involving ECM remodelling. At present, substrates downstream of PKC remain undefined. Herein, we show that both IL-1- and OSM-induced phosphorylation of protein kinase D (PKD) in human chondrocytes is strongly associated with signalling via the atypical PKCι isoform. Consequently, inhibiting PKD activation with a pan-PKD inhibitor significantly reduced the expression of MMP1/13. Specific gene silencing of the PKD isoforms revealed that only PKD3 (PRKD3) depletion mirrored the observed MMP repression, indicative of the pharmacological inhibitor specifically affecting only this isoform. PRKD3 silencing was also shown to reduce serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphorylation of all three mitogen-activated protein kinase groups. This altered signalling following PRKD3 silencing led to a significant reduction in the expression of the activator protein-1 (AP-1) genes FOS and JUN, critical for the induction of many MMPs including MMP1/13. Furthermore, the AP-1 factor activating transcription factor 3 (ATF3) was also reduced concomitant with the observed reduction in MMP13 expression. Taken together, we highlight an important role for PKD3 in the pro-inflammatory signalling that promotes cartilage destruction.

Objectives To investigate the effect of leptin on cartilage destruction. Methods Collagen release was assessed in bovine cartilage explant cultures, while collagenolytic and gelatinolytic activities in culture supernatants were determined by bioassay and gelatin zymography. The expression of matrix metalloproteinases (MMP) was analysed by real-time RT–PCR. Signalling pathway activation was studied by immunoblotting. Leptin levels in cultured osteoarthritic joint infrapatellar fat pad or peri-enthesal deposit supernatants were measured by immunoassay. Results Leptin, either alone or in synergy with IL-1, significantly induced collagen release from bovine cartilage by upregulating collagenolytic and gelatinolytic activity. In chondrocytes, leptin induced MMP1 and MMP13 expression with a concomitant activation of STAT1, STAT3, STAT5, MAPK (JNK, Erk, p38), Akt and NF-κB signalling pathways. Selective inhibitor blockade of PI3K, p38, Erk and Akt pathways significantly reduced MMP1 and MMP13 expression in chondrocytes, and reduced cartilage collagen release induced by leptin or leptin plus IL-1. JNK inhibition had no effect on leptin-induced MMP13 expression or leptin plus IL-1-induced cartilage collagen release. Conditioned media from cultured white adipose tissue (WAT) from osteoarthritis knee joint fat pads contained leptin, induced cartilage collagen release and increased MMP1 and MMP13 expression in chondrocytes; the latter being partly blocked with an anti-leptin antibody. Conclusions Leptin acts as a pro-inflammatory adipokine with a catabolic role on cartilage metabolism via the upregulation of proteolytic enzymes and acts synergistically with other pro-inflammatory stimuli. This suggests that the infrapatellar fat pad and other WAT in arthritic joints are local producers of leptin, which may contribute to the inflammatory and degenerative processes in cartilage catabolism, providing a mechanistic link between obesity and osteoarthritis.

Osteoarthritis (OA) is a degenerative condition caused by dysregulation of multiple molecular signalling pathways. Such dysregulation results in damage to cartilage, a smooth and protective tissue that enables low friction articulation of synovial joints. Matrix metalloproteinases (MMPs), especially MMP-13, are key enzymes in the cleavage of type II collagen which is a vital component for cartilage integrity. Transforming growth factor beta (TGFβ) can protect against pro-inflammatory cytokine-mediated MMP expression. With age there is a change in the ratio of two TGFβ type I receptors (Alk1/Alk5), a shift that results in TGFβ losing its protective role in cartilage homeostasis. Instead, TGFβ promotes cartilage degradation which correlates with the spontaneous development of OA in murine models. However, the mechanism by which TGFβ protects against pro-inflammatory responses and how this changes with age has not been extensively studied. As TGFβ signalling is complex, we used systems biology to combine experimental and computational outputs to examine how the system changes with age. Experiments showed that the repressive effect of TGFβ on chondrocytes treated with a pro-inflammatory stimulus required Alk5. Computational modelling revealed two independent mechanisms were needed to explain the crosstalk between TGFβ and pro-inflammatory signalling pathways. A novel meta-analysis of microarray data from OA patient tissue was used to create a Cytoscape network representative of human OA and revealed the importance of inflammation. Combining the modelled genes with the microarray network provided a global overview into the crosstalk between the different signalling pathways involved in OA development. Our results provide further insights into the mechanisms that cause TGFβ signalling to change from a protective to a detrimental pathway in cartilage with ageing. Moreover, such a systems biology approach may enable restoration of the protective role of TGFβ as a potential therapy to prevent age-related loss of cartilage and the development of OA.