Abstract
The role that the novel lymphatic-associated adhesion molecule CLEVER-1 plays in breast cancer metastasis has been examined by assessing its expression in human breast tumour specimens and by conducting in vitro experiments to monitor its involvement in regulating cell adhesion to human umbilical vein endothelial cells (HUVEC) and hTERT immortalised lymphatic endothelial cells (LEC). CLEVER-1 expression was examined in tonsil, lymph node and 148 formalin-fixed paraffin-embedded archival breast carcinoma specimens using standard immunohistochemistry protocols. In vitro CLEVER-1 expression was studied, in HUVEC and LEC, via fluorescence-activated cell sorting. Tumour cell (breast MCF-7 and MDA-MB-231, and melanoma SKMEL-30) adhesion and leukocyte adhesion to parental and CLEVER-1 siRNA knockdown endothelial cells was also examined. The results show that, in tissue specimens, CLEVER-1 is present in blood and lymphatic vessels and in certain leukocyte sub-populations (macrophage or dendritic cells). Although expression, in tumours, is higher in blood vessels than in lymphatic vessels (62.4% versus 18.2%), only lymphatic expression is associated with lymph node metastasis (P = 0.027). CLEVER-1 expression in blood vessels and lymphatic vessels correlates with the density of inflammatory infiltrate (P < 0.001 and P = 0.004, respectively) and expression in macrophages (P < 0.001). In vitro results show that although CLEVER-1 is expressed intracellularly in both HUVEC and LEC, only LEC exhibit surface expression. Interestingly, adhesion assays show that tumour cells adhere preferentially to LEC with maximal adhesion exhibited at 30 to 40 minutes. Tumour cells adhere less to CLEVER-1 knockdown LEC than to control LEC. The role of CLEVER-1 in cellular adhesion is being further investigated, using tumour cells and different leukocyte populations, to determine its involvement in adhesion and migration of different cell types across lymphatics.
Highlights
Obesity will soon be the leading preventable risk factor for many cancers
Previous epidemiological studies have investigated the relationship between individual nutrients such as vitamin D and O3 vitamin B12 and mammographic density, a strong marker of breast cancer risk [1], with varied results
We examine prospective data to determine A Bensmail, I Hutcheson, M Giles, J Gee, R Nicholson whether dietary patterns from childhood to adult life affect Tenovus Centre for Cancer Research, Welsh School of Pharmacy, mammographic density
Summary
Obesity will soon be the leading preventable risk factor for many cancers. The insulin-like growth factors (IGFs) have been strongly implicated as important risk factors for many epithelial cancers, including breast cancer, and for mediating the link between nutrition and these cancers. Overexpression of 15-PGDH partially restored sensitivity of TAMr cells to 4-hydroxytamoxifen by the MTT assay, demonstrating that 15-PGDH downregulation plays a functional role in the acquisition of TAMr. Treatment of TAMr MCF-7 cells with a DNA methyltransferase inhibitor (5-azacytidine), and a histone deacetylase inhibitor (trichostatin A), led to re-expression of 15-PGDH mRNA (by quantitative RT-PCR), suggesting that 15-PGDH is silenced via epigenetic mechanisms during the acquisition of TAMr. To address whether 15-PGDH downregulation is involved in clinical TAMr, we assembled a tissue microarray comprising 89 relapsed primary human breast cancers and 234 tamoxifen-sensitive controls. Oestrogen receptor-positive breast cancers develop resistance to anti-oestrogens by utilising alternative growth factor pathways as observed in our tamoxifen-resistant cell line (TAMR) These include EGFR, IGF1-R and Src signalling as well as increased growth and invasion. The tumour size was followed by regular measurement with calipers
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