Semaphorin–plexin signalling genes associated with human breast tumourigenesis

Introduction Gene expression profiling has enabled us to demonstrate the heterogeneity of breast cancers. The potential of a tumour to grow and metastasise is partly dependant on its ability to initiate angiogenesis or growth and remodelling of new blood vessels, usually from a pre-existing vascular network, to ensure delivery of oxygen, nutrients, and growth factors to rapidly dividing transformed cells along with access to the systemic circulation. Cell–cell signalling of semaphorin ligands through interaction with their plexin receptors is important for the homeostasis and morphogenesis of many tissues and has been widely studied for a role in neural connectivity, cancer, cell migration and immune responses. This study investigated the role of four semaphorin/plexin signalling genes in human breast cancers in vivo and in vitro. Materials and methods mRNA was extracted from formalin fixed paraffin embedded archival breast invasive ductal carcinoma tissue samples of progressive grades (grades I–III) and compared to tissue from benign tumours. Gene expression profiles were determined by microarray using the Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays and validated by Q-PCR using a Corbett RotorGene 6000. Following validation, the gene expression profile of the identified targets was correlated with those of the human breast cancer cell lines MCF-7 and MDA-MD-231. Results The array data revealed that 888 genes were found to be significantly (p ≤ 0.05) differentially expressed between grades I and II tumours and 563 genes between grade III and benign tumours. From these genes, we identified four genes involved in semaphorin–plexin signalling including SEMA4D which has previously been identified as being involved in increased angiogenesis in breast cancers, and three other genes, SEMA4F, PLXNA2 and PLXNA3, which in the literature were associated with tumourigenesis, but not directly in breast tumourigenesis. The microarray analysis revealed that SEMA4D was significantly (P = 0.0347) down-regulated in the grade III tumours compared to benign tumours; SEMA4F, was significantly (P = 0.0159) down-regulated between grades I and II tumours; PLXNA2 was significantly (P = 0.036) down-regulated between grade III and benign tumours and PLXNA3 significantly (P = 0.042) up-regulated between grades I and II tumours. Gene expression of SEMA4D was validated using Q-PCR, demonstrating the same expression profile in both data sets. When the sample set was increased to incorporate more cases, SEMA4D continued to follow the same expression profile, including statistical significance for the differences observed and small standard deviations. In vitro the same pattern was present where expression for SEMA4D was significantly higher in MDA-MB-231 cells when compared to MCF-7 cells. The expression of SEMA4F, PLXNA2 and PLXNA3 could not be validated using Q-PCR, however in vitro analysis of these three genes revealed that both SEMA4F and PLXNA3 followed the microarray trend in expression, although they did not reach significance. In contrast, PLXNA2 demonstrated statistical significance and was in concordance with the literature. Discussion We, and others, have proposed SEMA4D to be a gene with a potentially protective effect in benign tumours that contributes to tumour growth and metastatic suppression. Previous data supports a role for SEMA4F as a tumour suppressor in the peripheral nervous system but our data seems to indicate that the gene is involved in tumour progression in breast cancer. Our in vitro analysis of PLXNA2 revealed that the gene has higher expression in more aggressive breast cancer cell types. Finally, our in vitro analysis on PLXNA3 also suggest that this gene may have some form of growth suppressive role in breast cancer, in addition to a similar role for the gene previously reported in ovarian cancer. From the data obtained in this study, SEMA4D may have a role in more aggressive and potentially metastatic breast tumours. Conclusions Semaphorins and their receptors, the plexins, have been implicated in numerous aspects of neural development, however their expression in many other epithelial tissues suggests that the semaphorin–plexin signalling system also contributes to blood vessel growth and development. These findings warrant further investigation of the role of semaphorins and plexins and their role in normal and tumour-induced angiogenesis in vivo and in vitro. This may represent a new front of attack in anti-angiogenic therapies of breast and other cancers.

Osteocyte-induced angiogenesis via VEGF–MAPK-dependent pathways in endothelial cells

Recently, it has been suggested osteocytes control the activities of bone formation (osteoblasts) and resorption (osteoclast), indicating their important regulatory role in bone remodelling. However, to date, the role of osteocytes in controlling bone vascularisation remains unknown. Our aim was to investigate the interaction between endothelial cells and osteocytes and to explore the possible molecular mechanisms during angiogenesis. To model osteocyte/endothelial cell interactions, we co-cultured osteocyte cell line (MLOY4) with endothelial cell line (HUVECs). Co-cultures were performed in 1:1 mixture of osteocytes and endothelial cells or by using the conditioned media (CM) transfer method. Real-time cell migration of HUVECs was measured with the transwell migration assay and xCELLigence system. Expression levels of angiogenesis- related genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of vascular endothelial growth factor (VEGF) and mitogen-activated phosphorylated kinase (MAPK) signaling were monitored by western blotting using relevant antibodies and inhibitors. During the bone formation, it was noted that osteocyte dendritic processes were closely connected to the blood vessels. The CM generated from MLOY4 cells-activated proliferation, migration, tube-like structure formation, and upregulation of angiogenic genes in endothelial cells suggesting that secretory factor(s) from osteocytes could be responsible for angiogenesis. Furthermore, we identified that VEGF secreted from MLOY4-activated VEGFR2–MAPK–ERK-signaling pathways in HUVECs. Inhibiting VEGF and/or MAPK–ERK pathways abrogated osteocyte-mediated angiogenesis in HUVEC cells. Our data suggest an important role of osteocytes in regulating angiogenesis.

Regulation of EP receptors in non-small cell lung cancer by epigenetic modifications

Background: Cyclooxygenase (COX)-2 is frequently overexpressed in non-small cell lung cancer (NSCLC) and results in increased levels of prostaglandin E2 (PGE 2), an important signalling molecule implicated in tumourigenesis. PGE 2 exerts its effects through the E prostanoid (EP) receptors (EPs1-4). Methods: The expression and epigenetic regulation of the EPs were evaluated in a series of resected fresh frozen NSCLC tumours and cell lines. Results: EP expression was dysregulated in NSCLC being up and downregulated compared to matched control samples. For EPs1, 3 and 4 no discernible pattern emerged. EP2 mRNA however was frequently downregulated, with low levels being observed in 13/20 samples as compared to upregulation in 5/20 samples examined. In NSCLC cell lines DNA CpG methylation was found to be important for the regulation of EP3 expression, the demethylating agent decitabine upregulating expression. Histone acetylation was also found to be a critical regulator of EP expression, with the histone deacteylase inhibitors trichostatin A, phenylbutyrate and suberoylanilide hydroxamic acid inducing increased expression of EPs2-4. Direct chromatin remodelling was demonstrated at the promoters for EPs2-4. Conclusions: These results indicate that EP expression is variably altered from tumour to tumour in NSCLC. EP2 expression appears to be predominantly downregulated and may have an important role in the pathogenesis of the disease. Epigenetic regulation of the EPs may be central to the precise role COX-2 may play in the evolution of individual tumours. © 2009 Elsevier Ltd. All rights reserved.

Matrix metalloproteinases during wound healing - a double edged sword

The extracellular matrix (ECM) provides a framework for cells and gives skin its tensile strength and elasticity. Loss of its integrity necessitates the clearing of damaged components and the deposition of firstly a provisional matrix and later remodelling of the ECM to support a functionally intact tissue. Matrix metalloproteinases (MMPs) are an important family of enzymes that function in the breakdown of the ECM and modulate the function of many biologically active molecules housed in the ECM. Through their enzymatic actions MMPs play a role in fundamental processes such as immune cell infiltration and ECM remodelling during wound repair. Their tight control is necessary for timely wound healing and excessive MMP activity participates in the development and persistence of chronic wounds, while reduced activity contributes to fibrosis. A number of inhibitors have been designed to target this activity and improve wound healing with limited success. Novel strategies are currently being investigated to improve wound healing by targeting MMP modulating molecules.

Toll-like receptor and pro-inflammatory cytokine expression during prolonged hyperinsulinaemia in horses : implications for laminitis

Equine laminitis, a disease of the lamellar structure of the horse’s hoof, can be incited by numerous factors that include inflammatory and metabolic aetiologies. However, the role of inflammation in hyperinsulinaemic laminitis has not been adequately defined. Tolllike receptor (TLR) activation results in up-regulation of inflammatory pathways and the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-�), and may be a pathogenic factor in laminitis. The aim of this study was to determine whether TLR4 expression and subsequent pro-inflammatory cytokine production is increased in lamellae and skeletal muscle during equine hyperinsulinaemia. Standardbred horses were treated with either a prolonged, euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged, glucose infusion (p-GI), which induced marked and moderate hyperinsulinaemia, respectively. Age-matched control horses were treated simultaneously with a balanced electrolyte solution. Treated horses developed clinical (p-EHC) or subclinical (p-GI) laminitis, whereas controls did not. Skeletal muscle and lamellar protein extracts were analysed by Western blotting for TLR4, IL-6, TNF-� and suppressor of cytokine signalling 3 (SOCS3) expression. Lamellar protein expression of TLR4 and TNF-�, but not IL-6, was increased by the p-EHC, compared to control horses. A significant positive correlation was found between lamellar TLR4 and SOCS3. Skeletal muscle protein expression of TLR4 signalling parameters did not differ between control and p-EHC-treated horses. Similarly, the p-GI did not result in up-regulation of lamellar protein expression of any parameter. The results suggest that insulin-sensitive tissues may not accurately reflect lamellar pathology during hyperinsulinaemia. While TLR4 is present in the lamellae, its activation appears unlikely to contribute significantly to the developmental pathogenesis of hyperinsulinaemic laminitis. However, inflammation may have a role to play in the later stages (e.g., repair or remodelling) of the disease.

Chlamydia pneumoniae infection of Dentritic cells and its role in asthma exacerbations

Chlamydia infections are associated with exacerbations of asthma however the mechanisms are poorly understood. In this thesis we infected dendritic cells from healthy controls and asthmatic patients to determine if the immune response to chlamydial infection by these key immune cells could explain this association of chlamydial infection with asthma attacks. Infected dendritic cells from asthmatic patients showed increased expression of multiple inflammatory cytokine genes and genes for several tissue remodelling proteins, suggesting that infected dendritic cells play a central role in driving the airways damage associated with asthma. The findings provide a greater understanding of the role of infections in asthma and may provide a basis for new therapies to treat this important disease.

Sharpening our tools – translating research into practice

Providing culturally appropriate health communication tools at a community level, whilst meeting funding objectives set by Government led initiatives, can be challenging. Literature states that a translational research framework fostering community communication can encourage the development of appropriate communication tools to facilitate transfer of health information between community and researchers. Reflections from initial Need for Feed cooking and nutrition education program trials in remote Indigenous communities across Cape York indicated program resources were neither meeting community nor researchers needs. In response, a translational research framework was modelled with collaborative partnerships formed between researchers and community with the aim of modifying current resources. Local working groups were established to facilitate communication and guide continual remodelling and retrial of resources for successive programs. Feedback from working groups indicated community members wanted resources with more pictures and less words. Partnership with Chronic Disease Resources Online (CDRO) led to the development of pictorial resources including 3 evaluation tools, 27 recipe sets and 10 education support materials. Between June to December 2012 resources were trialled across 4 Cape York communities with 69 school aged children and 4 community elders. Qualitative data has indicated high satisfaction with modified pictorial resources, proving pictorial resources to be an effective and culturally appropriate method to both communicate health messages to community and facilitate flow of evaluation data to researchers. A translational research framework fostering communication between community and researchers can potentially enhance the quality of health communication tools.

Doxycycline-inducible expression of SPARC/Osteonectin/BM40 in MDA-MB-231 human breast cancer cells results in growth inhibition

SPARC (secreted protein acidic and rich in cysteine)/BM40/Osteonectin is a matricellular protein with multiple effects on cell behaviour. In vitro, its major known functions are anti-adhesive and anti-proliferative, and it is associated with tissue remodelling and cancer in vivo. SPARC is overexpressed in many cancers, including breast cancer, and the effects of SPARC seem to be cell type-specific. To study the effects of SPARC on breast cancer, we transfected SPARC into the MDA-MB-231 BAG, human breast cancer cell line using the Tet-On inducible system. By western analysis, we found low background levels in the MDA-MB-231 BAG and clone X parental cells, and prominent induction of SPARC protein expression after doxycycline treatment in SPARC transfected clones X5, X21, X24 and X75. Induction of SPARC expression did not affect cell morphology or adhesiveness to collagens type I and IV, but it slowed the rate of proliferation in adherent cultures. Cell cycle analysis showed that SPARC slowed the progression to S phase. Doxycycline induction of SPARC also slowed the rate of monolayer wound closure in the cultured wound healing assay. Thymidine inhibition of proliferation abrogated this effect, confirming that it was due to anti-proliferation rather than inhibition of migration. Consistent with this, we were unable to detect any differences in migration and Matrigel outgrowth analysis of doxycycline-stimulated cells. We conclude that SPARC is inhibitory to human breast cancer cell proliferation, and does not stimulate migration, in contrast to its stimulatory effects reported for melanoma (proliferation and migration) and glioma (migration) cells. Similar growth repression by SPARC has been reported for ovarian cancer cells, and this may be a common feature among carcinomas.

Cells Tissues Organs : preface

This volume stems from the 1st International Conference on Epithelial-Mesenchymal Transitions (EMT), which was convened by the editors on October 5–8, 2003 in the beautiful setting of Port Douglas, Queensland, Australia. EMT, the name given to the transformation of cells arranged in a coherent layer – epithelial cells – to more individualistic and potential motile cells – mesenchymal cells – was recognized decades ago by Prof. Elisabeth (Betty) Hay (Harvard Medical School, Boston, Mass., USA) as a primary mechanism in embryogenesis for remodelling tissues. More recently EMT has been seen as crucial to the spread and invasion of carcinoma, and more recently still, EMT-like changes have been detected in various pathologies marked by fi brosis. Despite the basic and clinical importance of EMT, this extremely rapidly growing fi eld had never previously had a conference devoted to it, and indeed the disciplines of developmental biology, cancer and pathology rarely interact although they have much to share. The chapters assembled for this volume encompass these three major themes of the meeting, development, pathology and cancer, and further highlight the commonality in terms of mechanisms and outcomes among them...

Characterization and novel activation of 72-kDa metalloproteinase in retinal interphotoreceptor matrix and Y-79 cell culture medium

Analysis of bovine interphotoreceptor matrix and conditioned medium from human Y-79 retinoblastoma cells by gelatin SDS-PAGE zymography reveals abundant activity of a 72-kDa M(r) gelatinase. The 72-kDa gelatinase from either source is inhibited by EDTA but not aprotinin or NEM, indicating that it is a metalloproteinase (MMP). The 72-kDa MMP is converted to a 62-kDa species with APMA treatment after gelatin sepharose affinity purification typical of previously described gelatinase MMP-2. The latent 72-kDa gelatinase from either bovine IPM or Y-79 media autoactivates without APMA in the presence of calcium and zinc after 72 hr at 37°C, producing a fully active mixture of proteinase species, 50 (48 in Y-79 medium), 38 and 35 kDa in size. The presence of inhibitory activity was examined in both whole bovine IPM and IPM fractions separated by SDS-PAGE. Whole IPM inhibited gelatinolytic activity of autoactivated Y-79-derived MMP in a dose-dependent manner. Inhibitory activities are observed in two protein fractions of 27-42 and 20-25 kDa. Western blots using antibodies to human tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) reveal the presence of two TIMP-1-like proteins at 21 and 29 kDa in inhibitory fractions of the bovine IPM. TIMP-2 was not detected in the inhibitory IPM fractions, consistent with the observed autoactivation of bovine IPM 72-kDa gelatinase. Potential roles for this IPM MMP-TIMP system include physiologic remodelling of the neural retina-RPE cell interface and digestion of shed rod outer segment as well as pathological processes such as retinal detachment, PE cell migration, neovascularization and tumor progression. Cultured Y-79 cells appear to be a good model for studying the production and regulation of this proteinase system.

The orphan nuclear receptor LRH-1 promotes breast cancer motility and invasion

The orphan nuclear receptor liver receptor homologue-1 (LRH-1) has roles in the development, cholesterol and bile acid homeostasis, and steroidogenesis. It also enhances proliferation and cell cycle progression of cancer cells. In breast cancer, LRH-1 expression is associated with invasive breast cancer; positively correlates with ERα status and aromatase activity; and promotes oestrogen-dependent cell proliferation. However, the mechanism of action of LRH-1 in breast cancer epithelial cells is still not clear. By silencing or over-expressing LRH-1 in ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells, we have demonstrated that LRH-1 promotes motility and cell invasiveness. Similar effects were observed in the non-tumourigenic mammary epithelial cell line, MCF-10A. Remodelling of the actin cytoskeleton and E-cadherin cleavage was observed with LRH-1 over-expression, contributing to increased migratory and invasive properties. Additionally, in LRH-1 over-expressing cells, the truncation of the 120 kDa E-cadherin to the inactive 97 kDa form was observed. These post-translational modifications in E-cadherin may be associated with LRH-1-dependent changes to matrix metalloproteinase 9 expression. These findings suggest a new role of LRH-1 in promoting migration and invasion in breast cancer, independent of oestrogen sensitivity. Therefore, LRH-1 may represent a new target for breast cancer therapeutics.

Role of dentin matrix protein 1 in cartilage redifferentiation and osteoarthritis

Objective The aim of this study was to test the possible involvement, relevance and significance of dentin matrix protein 1 (DMP1) in chondrocyte redifferentiation and OA. Methods To examine the function of DMP1 in vitro, bone marrow stromal cells (BMSCs) and articular chondrocytes (ACs) were isolated and differentiated in micromasses in the presence or absence of DMP1 small interfering RNA and analysed for chondrogenic phenotype. The association of DMP1 expression with OA progression was analysed time dependently in the OA menisectomy rat model and in grade-specific OA human samples. Results It was found that DMP1 was strongly related to chondrogenesis, which was evidenced by the strong expression of DMP1 in the 14.5-day mouse embryonic cartilage development stage and in femoral heads of post-natal days 0 and 4. In vitro chondrogenesis in BMSCs and ACs was accompanied by a gradual increase in DMP1 expression at both the gene and protein levels. In addition, knockdown of DMP1 expression led to decreased chondrocyte marker genes, such as COL2A1, ACAN and SOX9, and an increase in the expression of COL10A and MMP13 in ACs. Moreover, treatment with IL-1β, a well-known catabolic culprit of proteoglycan matrix loss, significantly reduced the expression of DMP1. Furthermore, we also observed the suppression of DMP1 protein in a grade-specific manner in knee joint samples from patients with OA. In the menisectomy-induced OA model, an increase in the Mankin score was accompanied by the gradual loss of DMP1 expression. Conclusion Observations from this study suggest that DMP1 may play an important role in maintaining the chondrogenic phenotype and its possible involvement in altered cartilage matrix remodelling and degradation in disease conditions like OA.

Physical mechanism of the compressive response of F-actin networks : significance of crosslinker unbinding events

A model of crosslinker unbinding is implemented in a highly coarsegrained granular model of F-actin cytoskeleton. We employ this specific granular model to study the mechanisms of the compressive responses of F-actin networks. It is found that the compressive response of F-actin cytoskeleton has dependency on the strain rate. The evolution of deformation energy in the network indicates that crosslinker unbinding events can induce the remodelling of F-actin cytoskeleton in response to external loadings. The internal stress in F-actin cytoskeleton can efficiently dissipate with the help of crosslinker unbinding, which could lead to the spontaneous relaxation of living cells.

Tetraspanins as regulators of the tumour microenvironment : implications for metastasis and therapeutic strategies

One of the hallmarks of cancer is the ability to activate invasion and metastasis (Hanahan et al., 2011). Cancer morbidity and mortality are largely related to the spread of the primary, localised tumour to adjacent and distant sites (Pantel et al., 2004). Appropriate management and treatment decisions of predicting metastatic disease at the time of diagnosis is thus crucial, which supports better understanding of the metastatic process. There are common events that occur during metastasis: dissociation from the primary tumour mass, reorganisation/remodelling of extracellular matrix, cell migration, recognition and transversal of endothelial cells and the vascular circulation and lodgement and proliferation within ectopic stroma (Wells, 2006). One of the key and initial events is the increased capability of cancer cells to move, escaping the regulation of normal physiological control. The cellular cytoskeleton plays an important role in cancer cell motility and active cytoskeletal rearrangement can result in metastatic disease. This active change in cytoskeletal dynamics results in manipulation of plasma membrane and cellular balance between cellular adhesion and motility which in turn determines cancer cell movement. Members of the tetraspanins play important roles in regulation of cancer migration and cancer-endothelial cell interactions, which are critical for cancer invasion and metastasis. Their involvements in active cytoskeletal dynamics, cancer metastasis and potential clinical application will be discussed in this review. In particular, tetraspanin member, CD151, is highlighted for its major role in cancer invasion and metastasis