Pro- and anti-tumour activities of CD146/MCAM in breast cancer result from its heterogeneous expression and association with epithelial to mesenchymal transition.

CD146, also known as melanoma cell adhesion molecule (MCAM), is expressed in numerous cancers and has been implicated in the regulation of metastasis. We show that CD146 negatively regulates transendothelial migration (TEM) in breast cancer. This inhibitory activity is reflected by a reduction in MCAM gene expression and increased promoter methylation in tumour tissue compared to normal breast tissue. However, increased CD146/MCAM expression is associated with poor prognosis in breast cancer, a characteristic that is difficult to reconcile with inhibition of TEM by CD146 and its epigenetic silencing. Single cell transcriptome data revealed MCAM expression in multiple cell types, including the malignant cells, tumour vasculature and normal epithelium. MCAM expressing malignant cells were in the minority and expression was associated with epithelial to mesenchymal transition (EMT). Furthermore, gene expression signatures defining invasiveness and a stem cell-like phenotype were most strongly associated with mesenchymal-like tumour cells with low levels of MCAM mRNA, likely to represent a hybrid epithelial/mesenchymal (E/M) state. Our results show that high levels of MCAM gene expression are associated with poor prognosis in breast cancer because they reflect tumour vascularisation and high levels of EMT. We suggest that high levels of mesenchymal-like malignant cells reflect large populations of hybrid E/M cells and that low CD146 expression on these hybrid cells is permissive for TEM, aiding metastasis.

Tumour cell CD99 regulates transendothelial migration via CDC42 and actin remodelling.

Metastasis requires tumour cells to cross endothelial cell (EC) barriers using pathways similar to those used by leucocytes during inflammation. Cell surface CD99 is expressed by healthy leucocytes and ECs, and participates in inflammatory transendothelial migration (TEM). Tumour cells also express CD99, and we have analysed its role in tumour progression and cancer cell TEM. Tumour cell CD99 was required for adhesion to ECs but inhibited invasion of the endothelial barrier and migratory activity. Furthermore, CD99 depletion in tumour cells caused redistribution of the actin cytoskeleton and increased activity of the Rho GTPase CDC42, known for its role in actin remodelling and cell migration. In a xenograft model of breast cancer, tumour cell CD99 expression inhibited metastatic progression, and patient samples showed reduced expression of the CD99 gene in brain metastases compared to matched primary breast tumours. We conclude that CD99 negatively regulates CDC42 and cell migration. However, CD99 has both pro- and anti-tumour activity, and our data suggest that this results in part from its functional linkage to CDC42 and the diverse signalling pathways downstream of this Rho GTPase. This article has an associated First Person interview with the first author of the paper.

Ultrasound-triggered therapeutic microbubbles enhance the efficacy of cytotoxic drugs by increasing circulation and tumor drug accumulation and limiting bioavailability and toxicity in normal tissues.

Most cancer patients receive chemotherapy at some stage of their treatment which makes improving the efficacy of cytotoxic drugs an ongoing and important goal. Despite large numbers of potent anti-cancer agents being developed, a major obstacle to clinical translation remains the inability to deliver therapeutic doses to a tumor without causing intolerable side effects. To address this problem, there has been intense interest in nanoformulations and targeted delivery to improve cancer outcomes. The aim of this work was to demonstrate how vascular endothelial growth factor receptor 2 (VEGFR2)-targeted, ultrasound-triggered delivery with therapeutic microbubbles (thMBs) could improve the therapeutic range of cytotoxic drugs. Methods: Using a microfluidic microbubble production platform, we generated thMBs comprising VEGFR2-targeted microbubbles with attached liposomal payloads for localised ultrasound-triggered delivery of irinotecan and SN38 in mouse models of colorectal cancer. Intravenous injection into tumor-bearing mice was used to examine targeting efficiency and tumor pharmacodynamics. High-frequency ultrasound and bioluminescent imaging were used to visualise microbubbles in real-time. Tandem mass spectrometry (LC-MS/MS) was used to quantitate intratumoral drug delivery and tissue biodistribution. Finally, 89Zr PET radiotracing was used to compare biodistribution and tumor accumulation of ultrasound-triggered SN38 thMBs with VEGFR2-targeted SN38 liposomes alone. Results: ThMBs specifically bound VEGFR2 in vitro and significantly improved tumor responses to low dose irinotecan and SN38 in human colorectal cancer xenografts. An ultrasound trigger was essential to achieve the selective effects of thMBs as without it, thMBs failed to extend intratumoral drug delivery or demonstrate enhanced tumor responses. Sensitive LC-MS/MS quantification of drugs and their metabolites demonstrated that thMBs extended drug exposure in tumors but limited exposure in healthy tissues, not exposed to ultrasound, by persistent encapsulation of drug prior to elimination. 89Zr PET radiotracing showed that the percentage injected dose in tumors achieved with thMBs was twice that of VEGFR2-targeted SN38 liposomes alone. Conclusions: thMBs provide a generic platform for the targeted, ultrasound-triggered delivery of cytotoxic drugs by enhancing tumor responses to low dose drug delivery via combined effects on circulation, tumor drug accumulation and exposure and altered metabolism in normal tissues.

A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis.

During angiogenesis, Rho-GTPases influence endothelial cell migration and cell-cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell-cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4 (dedicator of cytokinesis 4). DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4. VEGF-dependent Rac activation via DOCK4 is necessary for CDC42 activation to signal filopodia formation and depends on the activation of RHOG through the RHOG GEF, SGEF. VEGF promotes interaction of DOCK4 with the CDC42 GEF DOCK9. These studies identify a novel Rho-family GTPase activation cascade for the formation of endothelial cell filopodial protrusions necessary for tubule remodelling, thereby influencing subsequent stages of lumen morphogenesis.

VORFFIP-driven dock: V-D2OCK, a fast and accurate protein docking strategy.

The experimental determination of the structure of protein complexes cannot keep pace with the generation of interactomic data, hence resulting in an ever-expanding gap. As the structural details of protein complexes are central to a full understanding of the function and dynamics of the cell machinery, alternative strategies are needed to circumvent the bottleneck in structure determination. Computational protein docking is a valid and valuable approach to model the structure of protein complexes. In this work, we describe a novel computational strategy to predict the structure of protein complexes based on data-driven docking: VORFFIP-driven dock (V-D2OCK). This new approach makes use of our newly described method to predict functional sites in protein structures, VORFFIP, to define the region to be sampled during docking and structural clustering to reduce the number of models to be examined by users. V-D2OCK has been benchmarked using a validated and diverse set of protein complexes and compared to a state-of-art docking method. The speed and accuracy compared to contemporary tools justifies the potential use of VD2OCK for high-throughput, genome-wide, protein docking. Finally, we have developed a web interface that allows users to browser and visualize V-D2OCK predictions from the convenience of their web-browsers.

Regional differences in prostaglandin E2 metabolism in human colorectal cancer liver metastases.

BACKGROUND: Prostaglandin (PG) E2 plays a critical role in colorectal cancer (CRC) progression, including epithelial-mesenchymal transition (EMT). Activity of the rate-limiting enzyme for PGE2 catabolism (15-hydroxyprostaglandin dehydrogenase [15-PGDH]) is dependent on availability of NAD+. We tested the hypothesis that there is intra-tumoral variability in PGE2 content, as well as in levels and activity of 15-PGDH, in human CRC liver metastases (CRCLM). To understand possible underlying mechanisms, we investigated the relationship between hypoxia, 15-PGDH and PGE2 in human CRC cells in vitro. METHODS: Tissue from the periphery and centre of 20 human CRCLM was analysed for PGE2 levels, 15-PGDH and cyclooxygenase (COX)-2 expression, 15-PGDH activity, and NAD+/NADH levels. EMT of LIM1863 human CRC cells was induced by transforming growth factor (TGF) ß. RESULTS: PGE2 levels were significantly higher in the centre of CRCLM compared with peripheral tissue (P = 0.04). There were increased levels of 15-PGDH protein in the centre of CRCLM associated with reduced 15-PGDH activity and low NAD+/NADH levels. There was no significant heterogeneity in COX-2 protein expression. NAD+ availability controlled 15-PGDH activity in human CRC cells in vitro. Hypoxia induced 15-PGDH expression in human CRC cells and promoted EMT, in a similar manner to PGE2. Combined 15-PGDH expression and loss of membranous E-cadherin (EMT biomarker) were present in the centre of human CRCLM in vivo. CONCLUSIONS: There is significant intra-tumoral heterogeneity in PGE2 content, 15-PGDH activity and NAD+ availability in human CRCLM. Tumour micro-environment (including hypoxia)-driven differences in PGE2 metabolism should be targeted for novel treatment of advanced CRC.

A holistic in silico approach to predict functional sites in protein structures.

MOTIVATION: Proteins execute and coordinate cellular functions by interacting with other biomolecules. Among these interactions, protein-protein (including peptide-mediated), protein-DNA and protein-RNA interactions cover a wide range of critical processes and cellular functions. The functional characterization of proteins requires the description and mapping of functional biomolecular interactions and the identification and characterization of functional sites is an important step towards this end. RESULTS: We have developed a novel computational method, Multi-VORFFIP (MV), a tool to predicts protein-, peptide-, DNA- and RNA-binding sites in proteins. MV utilizes a wide range of structural, evolutionary, experimental and energy-based information that is integrated into a common probabilistic framework by means of a Random Forest ensemble classifier. While remaining competitive when compared with current methods, MV is a centralized resource for the prediction of functional sites and is interfaced by a powerful web application tailored to facilitate the use of the method and analysis of predictions to non-expert end-users. AVAILABILITY: http://www.bioinsilico.org/MVORFFIP

High-frequency ultrasound for in vivo measurement of colon wall thickness in mice.

Mouse models are becoming increasingly important in the study of molecular mechanisms of colorectal disease and in the development of novel therapeutics. To enhance this phase of preclinical research, cost-effective, easy to use noninvasive imaging is required to detect and monitor changes in the colon wall associated with disease pathology. This study investigated the feasibility of using 40-MHz (high frequency) B-mode ultrasound (HF-US) to image the normal mouse colon and measure its thickness in vivo by establishing a robust imaging protocol and conducting a blinded comparison of colon wall thickness (CWT) measurement between and within operators. The in vivo and ex vivo appearance of mouse colon under HF-US revealed distinct patterns. Colon wall thickness was reproducibly and accurately measured using HF-US compared with histology measurement. The technique was more sensitive in detecting changes in CWT in distal than proximal colon as it showed the highest level of inter- and intraoperator reproducibility. Using the protocol described, it is possible to detect changes in thickness of 0.09 mm and 0.25 mm in distal and proximal colon, respectively. In conclusion, HF-US provides an easy to use and noninvasive method to perform anatomical investigations of mouse colon and to monitor changes in CWT.

Improving the prediction of protein binding sites by combining heterogeneous data and Voronoi diagrams.

BACKGROUND: Protein binding site prediction by computational means can yield valuable information that complements and guides experimental approaches to determine the structure of protein complexes. Predictions become even more relevant and timely given the current resolution of protein interaction maps, where there is a very large and still expanding gap between the available information on: (i) which proteins interact and (ii) how proteins interact. Proteins interact through exposed residues that present differential physicochemical properties, and these can be exploited to identify protein interfaces. RESULTS: Here we present VORFFIP, a novel method for protein binding site prediction. The method makes use of broad set of heterogeneous data and defined of residue environment, by means of Voronoi Diagrams that are integrated by a two-steps Random Forest ensemble classifier. Four sets of residue features (structural, energy terms, sequence conservation, and crystallographic B-factors) used in different combinations together with three definitions of residue environment (Voronoi Diagrams, sequence sliding window, and Euclidian distance) have been analyzed in order to maximize the performance of the method. CONCLUSIONS: The integration of different forms information such as structural features, energy term, evolutionary conservation and crystallographic B-factors, improves the performance of binding site prediction. Including the information of neighbouring residues also improves the prediction of protein interfaces. Among the different approaches that can be used to define the environment of exposed residues, Voronoi Diagrams provide the most accurate description. Finally, VORFFIP compares favourably to other methods reported in the recent literature.

Lymphatic vessel distribution in the mucosa and submucosa and potential implications for T1 colorectal tumors.

PURPOSE: Colorectal cancer spreads to lymph nodes via surrounding lymphatic vasculature. Once this spread has occurred, the prognosis of the patient is significantly worse. Lymphatics are difficult to identify on hematoxylin and eosin stains and lack of specific markers has meant that little is known about their distribution in colorectal tissue. The national bowel cancer screening program has resulted in an increase in the diagnosis of T1 colorectal cancers. Patients with suitable T1 tumors can avoid bowel resections and their associated morbidity with the advances in local resection techniques. This means, however, that formal staging and lymph node assessment cannot be performed. Prognostic tools are required to predict risk of lymph node metastases. Studies assessing risk of lymph node spread in T1 tumors have found that invasion of the tumor into the deepest third of the submusosa affords a much greater risk. We hypothesized that this might be due to the quantity or characteristics of lymphatic vasculature in this third. METHODS: A specific lymphatic marker, D2-40 was applied to 5-µm sections of normal colorectal tissue from 45 patients. Slides were scanned and analyzed using Aperio's ImageScope software for PC. Analysis boxes of fixed area were placed within the mucosal layer and within each third of the submucosal layer allowing characteristics of the lymphatics in each third to be quantified individually. RESULTS: Lymphatic vessels were found in the mucosal layer of all samples although these were significantly smaller than the submucosal vessels (P = .0005). Lymphatics were significantly more numerous in the superficial third of the submucosa (P = .0005); however, vessel size was similar in Sm1, Sm2, and Sm3. CONCLUSION: The deepest third of the submucosa contains the smallest number of lymphatic vessels despite invasion into this layer being associated with a higher risk of lymph node spread.

Angiogenesis - understanding the mathematical challenge.

Biological mathematics is based on the development of mathematical descriptions of biological systems and behaviour. We are interested in developing mathematical models of capillary sprouting, and have adopted a novel approach to our modelling, in that the mathematics is based on the biochemistry underpinning cell behaviour. By considering the crucial steps of the angiogenic process, and through an understanding of the biochemistry involved, we successfully developed a preliminary model of angiogenesis. More importantly, our approach is applicable to many other areas of biological research. As mathematics remains a mystery to the majority of life scientists, we have aimed to describe our mathematical modelling strategy in biological terms. The assumptions and simplifications that form the basis of the modelling are explained, pinpointing the manner in which the different biological processes are linked via the mathematics. Examples of simulations using the mathematical model are shown, highlighting the success of our approach.

Angiopoietins in tumours: the angiogenic switch.

On first view, the literature pertaining to the expression of the angiopoietins in tumours is confusing and does not readily offer a consensus pattern. Apparently conflicting publications report increased, decreased or unchanged expression levels of both angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in a wide range of tumours. However, closer scrutiny of the literature, taking into account relative increases or decreases of each factor, reveals a consensus pattern, seen in almost all instances of expression profiling of the angiopoietins in tumours. What becomes apparent is that although absolute levels of either angiopoietin may increase or decrease, the ratio of Ang-1:Ang-2 shifts in favour of Ang-2. Given that Ang-2 is a destabilization factor, rendering vasculature in a more plastic state amenable to sprouting (under the influence of vascular endothelial growth factor, VEGF) or regression, this analysis suggests that tumours shift the angiogenic balance towards a pro-angiogenic state through altering the balance between the angiopoietins. This in turn implicates Ang-2 as a candidate for the angiogenic switch and also as an important potential therapeutic target.

Not just angiogenesis--wider roles for the angiopoietins.

Since the discovery of the angiopoietins, much interest has been focused on their biological actions and their potential use as therapeutic targets. It is generally accepted that the angiopoietins play an important role in angiogenesis and hence are described as angiogenic factors. However, it is becoming increasingly clear that this is not their only role and it is likely that the angiopoietins have important roles in a wider range of biological and pathological functions.

Lymphatics and vascular channels; the differences behind similarity.

Although sharing many features and functions, there is a mounting body of evidence to suggest that there are subtle differences between lymphatics and vascular channels. New data suggest that Ets-1 and Ets-1-target MMP genes are differentially expressed in lymphatic and vascular channels. Since it is generally accepted that the Ets family of transcription factors play a significant role in the regulation of the expression of angiogenic factors, the finding that Ets-1 is apparently not involved in lymphangiogenesis may highlight one aspect where angiogenesis and lymphangiogenesis are differently regulated.

Angiopoietins, growth factors, and vascular morphology in early arthritis.

OBJECTIVE: To examine angiogenic growth factors in patients with early, untreated inflammatory arthritides and controls. METHODS: Synovial membrane (SM) infiltrate and Ang1, Ang2, and vascular endothelial growth factor (VEGF) mRNA and protein expression were examined using immunohistochemistry and in situ hybridization. Synovial fluid (SF) VEGF, transforming growth factor-beta (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) protein were measured by ELISA. Vascular morphology was assessed at arthroscopy. RESULTS: Ang2 mRNA and protein expression was observed in early psoriatic arthritis (PsA) and rheumatoid arthritis (RA) SM. Expression of Ang2 and VEGF was significantly greater in early PsA SM and correlated strongly. SF VEGF and TGF-beta 1 concentrations were also significantly higher in early PsA compared to RA. Distinct vascular morphology, with tortuous vessels in PsA, correlated with microscopic vascular scores (r = 0.54, p = 0.005) and VEGF levels (r = 0.51, p = 0.01). Ang1 mRNA and protein expression was observed, but concentrations were markedly lower than for Ang2 and VEGF. Clinical disease activity, SM infiltration, and SF TNF-alpha concentrations were similar in both groups. CONCLUSION: This is the first report of angiopoietin expression in early inflammatory arthritis. There is a close relationship between angiopoietins, VEGF, TGF-beta, and vascular morphology. There is differential angiogenesis at an early stage of inflammation, with major pathogenic and therapeutic implications.

cDNA cloning and molecular characterization of mink SMAD4.

The Smad family of proteins have been implicated as major components of the TGF beta signalling pathway and are important mediators of its pleiotrophic effects. Here we describe the cloning and characterization of the mink (Mustela vison) ortholog of Smad4. Mink Smad4 has a high level of conservation to its human counterpart showing 96% homology at the DNA level and 99% at the amino acid level. This is in agreement with the close homologies seen for the rat and mouse orthologs. In vitro transcription and translation shows the expression of a protein of predicted molecular weight, of identical size to its human counterpart.