Ian R. Hart

Multiple ways of silencing E‐cadherin gene expression in lobular carcinoma of the breast

The cell‐cell adhesion receptor gene E‐cadherin (CDH1) is expressed by epithelial cells, in which it mediates adhesion and morphogenesis. Invasive lobular carcinoma (ILC) characteristically infiltrates diffusely as single cells; by immunohistochemistry, many of these tumours lack E‐cadherin expression. In the present study we investigated various ways in which loss of function of the E‐cadherin gene could occur in ILCs, namely, promoter methylation, mutation and allelic loss. We analysed 22 ILCs and found 12 (55%) E‐cadherin‐negative samples by immunohistochemical analysis. Methylation‐specific polymerase chain reaction (PCR) showed that 17/22 (77%) of these tumours had methylation of the CDH1 promoter, including 11/12 (91%) of the E‐cadherin‐negative tumours. All 16 exons of E‐cadherin (including intron‐exon boundaries) were amplified from chromosomal DNA and screened for mutations by conformation‐sensitive gel electrophoresis (CSGE). Bands with altered mobility were analysed by direct sequencing. We identified five frameshift mutations, which resulted in downstream stop codons and one splice site mutation in six different tumours (29%). Loss of heterozygosity (LOH) was assessed using microsatellite markers, and 9/18 (50%) informative tumours showed LOH. We conclude that most ILCs show genetic or epigenetic changes affecting the E‐cadherin gene and that many of these tumours lack E‐cadherin expression. In all cases in which there was loss of expression, this was consistent with biallelic inactivation of CDH1 by promoter methylation, mutation or allelic loss in any combination.

αvβ6integrin promotes invasion of squamous carcinoma cells through up-regulation of matrix metalloproteinase-9

The integrin αvβ6 is a fibronectin receptor, which is not detectable on normal epithelium but is neo‐expressed in oral epithelial dysplasia and oral squamous‐cell carcinoma (SCC), suggesting a role in promoting malignant behaviour and tumour progression. We used transfection and retroviral infection to create a panel of SCC cell lines expressing various levels of αvβ6 to examine this possibility. We found that increased expression of αvβ6 in malignant keratinocytes up‐regulates MMP‐9 and MMP‐2 expression and promotes invasion in an MMP‐9‐dependent manner. Our results suggest a possible mechanism for the involvement of αvβ6 in squamous carcinoma in vivo.

Level of expression of E-cadherin mRNA in colorectal cancer correlates with clinical outcome.

A series of colorectal carcinomas (n = 49) resected from patients with known clinical outcomes were analysed for E-cadherin expression using in situ hybridisation to measure mRNA. Patients surviving 5 years or longer (n = 31) exhibited significantly higher levels of E-cadherin mRNA than those surviving less than 5 years (n = 18, P = 0.003). These preliminary results from this small sample suggest that E-cadherin expression may be a useful prognostic marker in colorectal cancer patients.

The Integrins α3β1 and α6β1 Physically and Functionally Associate with CD36 in Human Melanoma Cells REQUIREMENT FOR THE EXTRACELLULAR DOMAIN OF CD36

Lateral association between different transmembrane glycoproteins can serve to modulate integrin function. Here we characterize a physical association between the integrins α3β1 and α6β1 and CD36 on the surface of melanoma cells and show that ectopic expression of CD36 by CD36-negative MV3 melanoma cells increases their haptotactic migration on extracellular matrix components. The association was demonstrated by co-immunoprecipitation, reimmunoprecipitation, and immunoblotting of surface-labeled cells lysed in Brij 96 detergent. Confocal microscopy illustrated the co-association of α3 and CD36 in cell membrane projections and ruffles. A requirement for the extracellular domain of CD36 in this association was shown by co-immunoprecipitation experiments using surface-labeled MV3 melanoma or COS-7 cells that had been transiently transfected with chimeric constructs between CD36 and intercellular adhesion molecule 1 (ICAM-1) or with a truncation mutant of CD36. CD36 is known to engage in signal transduction and to localize to membrane microdomains or rafts in several cell types. Toward a mechanistic explanation for the functional effects of CD36 expression, we demonstrate that in fractionated Triton X-100 lysates of the MV3 cells stably transfected with CD36, CD36 was greatly enriched with the detergent-insoluble fractions that represent plasma membrane rafts. Significantly, when these fractionated lysates were reprobed for endogenous β1 integrin, it was found that a 4-fold increase in the proportion of the mature protein was contained within the detergent-insoluble fractions when extracted from the CD36-transfected cells compared with MV3 cells transfected with vector only. These results suggest that in melanoma cells CD36 expression may induce the sequestration of certain integrins into membrane microdomains and promote cell migration.

Tumour cell expression of B7 costimulatory molecules and interleukin-12 or granulocyte-macrophage colony-stimulating factor induces a local antitumour response and may generate systemic protective immunity

Previously, we showed that expression of B7–1 in CMT93 murine colorectal tumour cells inhibited their growth in immunocompetent animals. However, this did not result in any significant increase in systemic protective immunity, relative to that elicited by the parental tumour. To potentiate the effects of B7–1 on systemic immunity, interleukin-12 (IL-12) or granulocyte–macrophage colony-stimulating factor (GM-CSF) was co-expressed with this molecule. These combinations of immunostimulatory molecules were effective in eliciting systemic immunity. We also show that expression of B7–2 led to a local antitumour response as well as significantly raised systemic immunity. In another tumour model, K1735 murine melanoma, which is moderately immunogenic, tumours secreting GM-CSF alone were as effective as the parental tumours in generating protective immunity. Previously, we described the deleterious effect of B7–1 expression on protective immunity. Co-expression of GM-CSF did not counteract this consequence of B7–1 expression. Expression of IL-12 was extremely effective in causing rejection of inoculated tumour cells, but evoked only minimal protective systemic immunity. These results suggest that combining costimulatory molecules and cytokines may be a useful therapeutic approach in some, but not all, tumours.

Rapid solid phase synthesis and biodistribution of 18F-labelled linear peptides

Abstract. A rapid method for radiolabelling short peptides with 18F (t1/2=109.7xa0min) for use in positron emission tomography (PET) was developed. Linear peptides (13mers) were synthesised using solid phase peptide synthesis and 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. The peptides were assembled on a solid-phase polyethylene glycol-polystyrene support using the hyper acid labile linker xanthen-2-oxyvaleric acid and were labelled in situ with 4-[19F]- or 4-[18F]fluorobenzoic acid. Optimum coupling of 4-[19F]fluorobenzoic acid to the peptidyl resin was achieved within 2xa0min using N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-Nmethylmethanaminium hexafluorophosphate N-oxide (HATU/DIPEA), and optimum cleavage was achieved within 7xa0min using trifluoroacetic acid/phenol/water/Triisopropylsilane at 37°C. The linear peptides were rapidly labelled with 4-[18F]fluorobenzoic acid with an overall radiochemical yield of 80%–90% (decay corrected), a radiochemical purity of >95% without HPLC purification and an overall synthesis time of 20xa0min. This novel method was used to label peptides containing the arginine-glycine-aspartic acid (RGD) motif, the binding site of many integrins. In vitro studies showed that the fluorobenzoyl prosthetic group had no deleterious effect on the ability of these peptides to inhibit the binding of human cells via integrins. Biodistribution studies in tumour-bearing mice showed that although the linear peptides were rapidly removed from the circulation by the liver and kidneys, there was a transient and non-RGD-dependent accumulation in the tumour of both the test and the control peptides. The use of more selective peptides with a longer half-life in the circulation combined with this rapid labelling technique will significantly enhance the application of peptides in PET.

Adenovirus type 5 uptake by lung adenocarcinoma cells in culture correlates with Ad5 fibre binding is mediated by alpha(v)beta1 integrin and can be modulated by changes in beta1 integrin function.

Recombinant adenoviruses (Ad) have been employed as vectors for a wide variety of gene therapy applications, but their use has been hindered by problems relating to efficacy and safety. The efficiency of Ad‐mediated gene transfer depends on the interaction of the fibre and penton base proteins with their corresponding cell receptors. Ad infection is initiated by the formation of a high affinity complex between the fibre protein and a host cell protein that for most Ad serotypes is CAR (the coxsackie B virus and Ad receptor). A second molecule, the MHC class I, may also be involved in Ad type 2 and Ad type 5 uptake. Ad internalization results from the interaction of the penton base protein with cell surface integrins αvβ3 and αvβ5. In this study, we addressed the interaction between Ad type 5 (Ad5) and its receptors on lung derived adenocarcinoma cells in culture.

The pattern of expression of the microtubule‐binding protein RHAMM/IHABP in mammary carcinoma suggests a role in the invasive behaviour of tumour cells

Intracellular hyaluronic acid binding protein (RHAMM/IHABP), which was recently identified as a novel member of the microtubule‐associated protein (MAP) family, has the capacity to interact not only with microtubules but also with microfilaments. The molecule, which is known to be expressed in mammary carcinoma cells, might, through virtue of its intracellular interactions, influence tumour cell morphology and motility. This possibility was examined in a series of 189 mammary carcinomas by immunohistochemistry, using a polyclonal antibody to RHAMM/IHABP. Tumours were selected to include approximately equal numbers of consecutive grade I, II and III ductal carcinomas and invasive lobular carcinomas. Higher grade tumours had significantly lower expression of RHAMM/IHABP in the cytoplasm (p=0.02), but significantly increased expression in trabeculae (p=0.002) and further enhancement at the tumour island edges (p=0.002). Tumours of infiltrating lobular type had stronger expression in the overall cytoplasm (p=0.02) and trabeculae (p=0.08) than carcinomas of ductal type. The presence of strong trabecular expression was associated with a reduced overall survival time (p=0.017). These results suggest that RHAMM/IHABP expression may contribute to the motility and invasiveness of a tumour cell sub‐population in breast cancers. Copyright

BAG-1 expression in human breast cancer: interrelationship between BAG-1 RNA, protein, HSC70 expression and clinico-pathological data

BAG‐1 (BCL‐2 athanogene‐1), a multifunctional protein which associates with steroid hormone receptors (including the oestrogen receptor) and the anti‐apoptotic BCL‐2 protein, regulates steroid hormone‐dependent transcription and apoptosis. Direct interaction with 70u2009kD heat‐shock proteins, HSC70 and HSP70, may mediate the diverse functions of BAG‐1. Immunohistochemistry was used to examine the expression of BAG‐1 and HSC70 in 160 cases of invasive breast cancer. BAG‐1 was expressed in 92% of cases; most tumours exhibited cytoplasmic BAG‐1, while a smaller proportion also had nuclear immunostaining. There was a significant inverse correlation between histological grade and nuclear BAG‐1 expression, with higher‐grade tumours tending to have reduced nuclear BAG‐1 expression, but there was no association with cytoplasmic BAG‐1. There was also no significant correlation between nuclear or cytoplasmic BAG‐1 expression and oestrogen receptor positivity. Since BAG‐1 may be influenced by hormonal background, the relationship between grade and oestrogen receptor was examined separately in pre‐menopausal and post‐menopausal women. The statistically significant correlation between nuclear BAG‐1 expression and low tumour grade was strong in pre‐menopausal, but not apparent in post‐menopausal women. A statistically significant correlation was observed between cytoplasmic, but not nuclear, BAG‐1 expression and oestrogen receptor status in pre‐menopausal, but not post‐menopausal, women. There was no correlation between BAG‐1 protein expression and RNA, suggesting that important post‐transcriptional mechanisms control BAG‐1 expression in vivo. HSC70 was also detected in the majority (97%) of cases, although expression was not correlated with BAG‐1 levels, oestrogen receptor status or tumour grade. Overall survival in cases with high levels of nuclear BAG‐1 expression was improved, though not significantly. These results are consistent with the hypothesis that BAG‐1 plays an important but variable role in breast cancers developing in pre‐menopausal and post‐menopausal women. Copyright

Binding of TGF-β1 latency-associated peptide (LAP) to αvβ6 integrin modulates behaviour of squamous carcinoma cells

The integrin αvβ6 is not detectable on normal keratinocytes in vivo but expression is increased significantly in oral squamous cell carcinoma where this heterodimer has been shown to play a role in cell migration, invasion and protease expression. Although regarded initially as a fibronectin receptor, αvβ6 may bind to arginine-glycine-aspartic acid sequences in other matrix molecules including tenascin and vitronectin. Interestingly, αvβ6 has also been shown to have high affinity for the TGF-β1 latency associated peptide and to participate in the activation of the TGF-β1 latent complex. Since TGF-β1 is present in squamous carcinomas, it is possible that latency associated peptide may modulate malignant keratinocyte behaviour independently from the classical TGF-β signalling pathways through its interaction with integrins. We show here that when latency associated peptide is immobilised onto a surface, it acts as an αvβ6-specific ligand for oral squamous carcinoma cells promoting adhesion and haptotactic migration in addition to αvβ6-dependent increase in pro-MMP-9 expression. In contrast, even very low concentrations of soluble latency associated peptide (0.1u2009μgu2009ml−1) inhibited αvβ6-dependent adhesion, migration and invasion. Thus αvβ6-dependent processes of oral squamous cell carcinoma, is likely to be modulated, not only by the local concentration of latency associated peptide in the stroma, but also whether it is immobilised in the matrix or released as a soluble protein.