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.

Significant differences in gene expression of GABA receptors in peripheral blood leukocytes of migraineurs

Migraine is a debilitating neurovascular disorder, with a substantial genetic component. The exact cause of a migraine attack is unknown; however cortical hyperexcitability is thought to play a role. As Gamma-aminobutyric Acid (GABA) is the major inhibitory neurotransmitter in the brain, malfunctioning of this system may be a cause of the hyperexcitability. To date, there has been limited research examining the gene expression or genetics of GABA receptors in relation to migraine. The aim of our study was to determine if GABA receptors play a role in migraine by investigating their gene expression using profile in migraine affected individuals and non-affected controls by Q-PCR. Gene expression of GABA(A) receptor subunit isoforms (GABRA3, GABRB3, GABRQ) and GABA(B) receptor 2 (GABBR2) was quantified in mRNA obtained from peripheral blood leukocytes from 28 migraine subjects and 22 healthy control subjects. Analysis of results showed that two of the tested genes, GABRA3 and GABBR2, were significantly down regulated in migraineurs (P=0.018; P=0.017), compared to controls. Results from the other tested genes did not show significant gene expression variation. The results indicate that there may be specific GABA receptor gene expression variation in migraine, particularly involving the GABRA3 and GABBR2 genes. This study also identifies GABRA3 and GABBR2 as potential biomarkers to select migraineurs that may be more responsive to GABA agonists with future investigations in this area warranted.

Analysis of the MTHFR C677T variant with migraine phenotypes

Background The methylenetetrahydrofolate reductase (MTHFR) gene variant C677T has been implicated as a genetic risk factor in migraine susceptibility, particularly in Migraine with Aura. Migraine, with and without aura (MA and MO) have many diagnostic characteristics in common. It is postulated that migraine symptomatic characteristics might themselves be influenced by MTHFR. Here we analysed the clinical profile, migraine symptoms, triggers and treatments of 267 migraineurs previously genotyped for the MTHFR C677T variant. The chi-square test was used to analyse all potential relationships between genotype and migraine clinical variables. Regression analyses were performed to assess the association of C677T with all migraine clinical variables after adjusting for gender. Findings The homozygous TT genotype was significantly associated with MA (P < 0.0001) and unilateral head pain (P = 0.002). While the CT genotype was significantly associated with physical activity discomfort (P < 0.001) and stress as a migraine trigger (P = 0.002). Females with the TT genotype were significantly associated with unilateral head pain (P < 0.001) and females with the CT genotype were significantly associated with nausea (P < 0.001), osmophobia (P = 0.002), and the use of natural remedy for migraine treatment (P = 0.003). Conversely, male migraineurs with the TT genotype experienced higher incidences of bilateral head pain (63% vs 34%) and were less likely to use a natural remedy as a migraine treatment compared to female migraineurs (5% vs 20%). Conclusions MTHFR genotype is associated with specific clinical variables of migraine including unilateral head pain, physical activity discomfort and stress.

Heparan sulfate proteoglycans, tumour progression and the cancer stem cell niche

The cancer stem cell hypothesis states that tumours arise from cells with the ability to self-renew and differentiate into multiple cell types, and that these cells persist in tumors as a distinct population that can cause disease relapse and hence metastasis. The crux of this hypothesis is that these cells are the only cells capable of, by themselves, giving rise to new tumours. What proportion of a tumour consists of these stem cells, where are they localised, how are they regulated, and how can we identify them? The stromal cells embedded within the extracellular matrix (ECM) not only provide a scaffold but also produce ECM constituents for use by stem cells. Heparan sulfate proteoglycans (HSPGs) are ubiquitous to this cell niche and interact with a large number of ligands including growth factors, their receptors, and ECM structural components. It is still unclear whether ECM degradation and subsequent metastasis is a result of proteases produced by the tumour cells themselves or by cells within the stromal compartment. The identification of the cellular origin of cancer stem cells along with microenvironmental changes involved in the initiation, progression and the malignant conversion of all cancers is critical to the development of targeted therapeutics. As ubiquitous members of the ECM microenvironment and hence the cancer cell niche, HSPGs are candidates for a central role in these processes.

Matrix metalloproteinase localisation by in situ-RT-PCR in archival human breast biopsy material

Utilising archival human breast cancer biopsy material we examined the stromal/epithelial interactions of several matrix metalloproteinases (MMPs) using in situ-RT-PCR (IS-RT-PCR). In breast cancer, the stromal/epithelial interactions that occur, and the site of production of these proteases, are central to understanding their role in invasive and metastatic processes. We examined MT1-MMP (MMP-14, membrane type-1-MMP), MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) for their localisation profile in progressive breast cancer biopsy material (poorly differentiated invasive breast carcinoma (PDIBC), invasive breast carcinomas (IBC) and lymph node metastases (LNM)). Expression of MT1-MMP, MMP-1 and MMP-3 was observed in both the tumour epithelial and surrounding stromal cells in most tissue sections examined. MT1-MMP expression was predominantly localised to the tumour component in the pre-invasive lesions. MMP-1 gene expression was relatively well distributed between both tissue compartments, while MMP-3 demonstrated highest expression levels in the stromal tissue surrounding the epithelial tumour cells. The results demonstrate the ability to distinguish compartmental gene expression profiles using IS-RT-PCR. Further, we suggest a role for MT1-MMP in early tumour progression, expression of MMP-1 during metastasis and focal expression pattern of MMP-3 in areas of expansion. These expression profiles may provide markers for early breast cancer diagnoses and present potential therapeutic targets.

Genetic investigation of methylenetetrahydrofolate reductase (MTHFR) and catechol-O-methyl transferase (COMT) in multiple sclerosis

Multiple sclerosis (MS) is a chronic neurological disease characterized by central nervous system (CNS) inflammation and demyelination. The C677T substitution variant in the methylenetetrahydrofolate reductase (MTHFR) gene has been associated with increased levels of circulating homocysteine and is a mild risk factor for vascular disease. Higher blood levels of homocysteine have also been reported in MS. Thus, the C677T mutation of the MTHFR gene may influence MS susceptibility. Noradrenaline, a neurotransmitter believed to play an immunosupressive role in neuroinflammatory disorders, is catabolized by catechol-O-methyl transferase (COMT). The COMT G158A substitution results in a three- to four-fold decreased activity of the COMT enzyme, which may influence CNS synaptic catecholamine breakdown and could also play a role in MS inflammation. We tested DNA from Australian MS patients and unaffected control subjects, matched for gender, age and ethnicity. Specifically, we genotyped the MTHFR C677T and the COMT G158A mutations. Genotype distributions showed that the homozygous mutant MTHFR genotype (T/T) and the COMT (H/H) genotype were slightly over-represented in the MS group (16% versus 11% and 24% versus 19%, respectively), but both variations failed to reach statistical significance (P=0.15 and P=0.32, respectively). Hence, results from the present study do not support a major role for either functional gene mutation in MS susceptibility.

In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts

Background Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult. Methods We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state. Results In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma. Conclusion We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in vivo. Induction of MMP gene expression in both the epithelial tumour cells and surrounding stromal cells is associated with increased metastatic potential. Our data demonstrate the contribution of the stroma to epithelial MMP gene expression, and highlight the complexity of the role of MMPs in the stromal-epithelial interactions within breast carcinoma.

Analysis of chromosome 1 microsatellite markers and the FHM2-ATP1A2 gene mutations in migraine pedigrees

OBJECTIVES: The aims of the study were: (i) to extend our linkage analysis of chromosome 1q microsatellite markers in predominantly migraine with aura pedigrees and (ii) to test the novel FHM-2 ATP1A2 gene for involvement in these migraine affected pedigrees and a previous pedigree (MF14) showing evidence of linkage of markers to C1q31. METHODS: A chromosome 1 scan (31 markers) was performed in 21 multiplex pedigrees affected predominantly with migraine with aura (MA). The known FHM-2 ATP1A2 gene mutations were tested, by sequencing, for the involvement in MA and migraine without aura (MO) in these pedigrees. Sequencing was performed in the coding areas of the ATP1A2 gene through three MA individuals from MF14. RESULTS: Evidence for linkage was obtained at C1q23 to markers spanning the ATP1A2 gene. However, testing of the known ATP1A2 gene mutations (for FHM) in common migraine probands of pedigrees showing excess allele sharing was negative. Sequencing of the entire coding areas of the gene through all the three MA affected from MF14 was also negative for mutations. DISCUSSION: Microsatellite markers on chromosome 1q23 show evidence of excess allele sharing in MA and some MO pedigrees, suggesting linkage to the common forms of migraine and the presence of a susceptibility gene in this region. The FHM-2 (ATP1A2 gene) does not seem to be involved in the common types of migraine. Despite certain clinical characteristics, the genetic correlation between FHM and familial typical migraine remains unclear. Several candidate genes lie within the C1q23 and C1q31 cytogenetic regions; therefore, further studies are needed.

Association of the NuMA region on chromosome 11q13 with breast cancer susceptibility

The development of breast cancer is a complex process that involves multiple genes at many stages, from initial cell cycle dysregulation to disease progression. To identify genetic variations that influence this process, we conducted a large-scale association study using a collection of German cases and controls and >25,000 SNPs located within 16,000 genes. One of the loci identified was located on chromosome 11q13 [odds ratio (OR)=1.85, P=0.017]. The initial association was subsequently tested in two independent breast cancer collections. In both sample sets, the frequency of the susceptibility allele was increased in the cases (OR=1.6, P=0.01). The susceptibility allele was also associated with an increase in cancer family history (P=0.1). Fine mapping showed that the region of association extends approximately 300 kb and spans several genes, including the gene encoding the nuclear mitotic apparatus protein (NuMA). A nonsynonymous SNP (A794G) in NuMA was identified that showed a stronger association with breast cancer risk than the initial marker SNP (OR=2.8, P=0.005 initial sample; OR=2.1, P=0.002 combined). NuMA is a cell cycle-related protein essential for normal mitosis that is degraded in early apoptosis. NuMA-retinoic acid receptor alpha fusion proteins have been described in acute promyelocytic leukemia. Although the potential functional relevance of the A794G variation requires further biological validation, we conclude that variations in the NuMA gene are likely responsible for the observed increased breast cancer risk.

Genetic polymorphisms in DPF3 associated with risk of breast cancer and lymph node metastases

Background Several studies have identified rare genetic variations responsible for many cases of familial breast cancer but their contribution to total breast cancer incidence is relatively small. More common genetic variations with low penetrance have been postulated to account for a higher proportion of the population risk of breast cancer. Methods and Results In an effort to identify genes that influence non-familial breast cancer risk, we tested over 25,000 single nucleotide polymorphisms (SNPs) located within approximately 14,000 genes in a large-scale case-control study in 254 German women with breast cancer and 268 age-matched women without malignant disease. We identified a marker on chromosome 14q24.3-q31.1 that was marginally associated with breast cancer status (OR = 1.5, P = 0.07). Genotypes for this SNP were also significantly associated with indicators of breast cancer severity, including presence of lymph node metastases ( P = 0.006) and earlier age of onset ( P = 0.01). The association with breast cancer status was replicated in two independent samples (OR = 1.35, P = 0.05). High-density association fine mapping showed that the association spanned about 80 kb of the zinc-finger gene DPF3 (also known as CERD4 ). One SNP in intron 1 was found to be more strongly associated with breast cancer status in all three sample collections (OR = 1.6, P = 0.003) as well as with increased lymph node metastases ( P = 0.01) and tumor size ( P = 0.01). Conclusion Polymorphisms in the 5' region of DPF3 were associated with increased risk of breast cancer development, lymph node metastases, age of onset, and tumor size in women of European ancestry. This large-scale association study suggests that genetic variation in DPF3 contributes to breast cancer susceptibility and severity.

Large-scale association study identifies ICAM gene region as breast and prostate cancer susceptibility locus

We conducted a large-scale association study to identify genes that influence nonfamilial breast cancer risk using a collection of German cases and matched controls and >25,000 single nucleotide polymorphisms located within 16,000 genes. One of the candidate loci identified was located on chromosome 19p13.2 [odds ratio (OR) = 1.5, P = 0.001]. The effect was substantially stronger in the subset of cases with reported family history of breast cancer (OR = 3.4, P = 0.001). The finding was subsequently replicated in two independent collections (combined OR = 1.4, P < 0.001) and was also associated with predisposition to prostate cancer in an independent sample set of prostate cancer cases and matched controls (OR = 1.4, P = 0.002). High-density single nucleotide polymorphism mapping showed that the extent of association spans 20 kb and includes the intercellular adhesion molecule genes ICAM1, ICAM4, and ICAM5. Although genetic variants in ICAM5 showed the strongest association with disease status, ICAM1 is expressed at highest levels in normal and tumor breast tissue. A variant in ICAM5 was also associated with disease progression and prognosis. Because ICAMs are suitable targets for antibodies and small molecules, these findings may not only provide diagnostic and prognostic markers but also new therapeutic opportunities in breast and prostate cancer.

Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

Background Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

IS-RT-PCR assay detection of MT-MMP in a human breast cancer cell line

The in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) is a method that allows the direct localisation of gene expression. The method utilises the dual buffer mediated activity of the enzyme rTth DNA polymerase enabling both reverse transcription and DNA amplification. Labelled nucleoside triphosphates allow the site of expression to be labelled, rather than the PCR primers themselves, giving a more accurate localisation of transcript expression and decreased background than standard in situ hybridisation (ISH) assays. The MDA-MB-231 human breast carcinoma (HBC) cell line was assayed via the IS-RT-PCR technique, using primers encoding MT-MMP (membrane-type matrix metalloproteinase) and human β-actin. Our results clearly indicate baseline expression of MT-MMP in the relatively invasive MDA-MB-231 cell line at a signal intensity similar to the housekeeping gene β-actin, and results following induction with Concanavalin A (Con A) are consistent with our previous results obtained via Northern blotting.

Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR

With an increased emphasis on genotyping of single nucleotide polymorphisms (SNPs) in disease association studies, the genotyping platform of choice is constantly evolving. In addition, the development of more specific SNP assays and appropriate genotype validation applications is becoming increasingly critical to elucidate ambiguous genotypes. In this study, we have used SNP specific Locked Nucleic Acid (LNA) hybridization probes on a real-time PCR platform to genotype an association cohort and propose three criteria to address ambiguous genotypes. Based on the kinetic properties of PCR amplification, the three criteria address PCR amplification efficiency, the net fluorescent difference between maximal and minimal fluorescent signals and the beginning of the exponential growth phase of the reaction. Initially observed SNP allelic discrimination curves were confirmed by DNA sequencing (n = 50) and application of our three genotype criteria corroborated both sequencing and observed real-time PCR results. In addition, the tested Caucasian association cohort was in Hardy-Weinberg equilibrium and observed allele frequencies were very similar to two independently tested Caucasian association cohorts for the same tested SNP. We present here a novel approach to effectively determine ambiguous genotypes generated from a real-time PCR platform. Application of our three novel criteria provides an easy to use semi-automated genotype confirmation protocol.

A genetic variant located in miR-423 is associated with reduced breast cancer risk

Background/Aim: Since microRNAs (miRNAs) act as translational regulators of multiple genes, single nucleotide polymorphisms (SNP) in them can have potentially wide-ranging effects. Using an association approach, this research examined the effects of the rs6505162 SNP, an A>C polymorphism located in the premiRNA region of miR-423, on breast cancer development. Materials and Methods: Caucasian Australian women with breast cancer and controls matched for age and ethnicity were genotyped for rs6505162 and their genotypic and allelic frequencies analysed for significant differences. Results: Analysis indicated that there were significant differences between the case and control populations (χ 2=6.70, p=0.035), with the CC genotype conferring reduced risk of breast cancer development (odds ratio=0.50 95% confidence interval=0.27-0.92, p=0.03). Conclusion: Further functional research is required to determine the mechanism of action of this SNP on miRNA function.