Loss of epithelial morphology and the acquisition of mesenchymal characteristics are typical for carcinoma cells in tumour progression. In human breast carcinomas, up-regulation of tenascin-C (TN-C) and vimentin (Vim) is frequently observed in cancer cells and correlates with increased malignancy. Thus, it is possible that TN-C is co-expressed with Vim, representing cancer cells that have undergone epithelial-mesenchymal transition (EMT). This study examined 128 breast carcinomas using immunohistochemical techniques to demonstrate that mammary cancer cells are a prominent source of both TN-C and Vim. Statistical analysis revealed a significant association between TN-C and Vim expression in cancer cells. TN-C expression also correlated positively with overexpression of c-erbB-2 oncoprotein and down-regulation of oestrogen receptors (ERs). Eleven human mammary cancer cell lines and two 'normal' cell lines were examined by western blotting and immunohistochemistry. Co-expression of TN-C and Vim was detected in the carcinosarcoma cell line HS 578T, SK-BR-3 (B), fibroblast-like MDA-MB-231 cells, and the myoepithelial cell line HBL 100. These findings suggest that TN-C and Vim, when co-expressed in mammary carcinoma cells, represent regulator genes likely to be involved in EMT during mammary carcinogenesis.

Growth factors are essential for cellular growth and differentiation in both normal and malignant human breast epithelial cells. In the present study we investigated the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGF ) and phorbol myristate acetate (PMA) on chicken ovalbumin upstream promotertranscription factor (COUP-TF) expression in human breast cancer cells. The orphan receptors COUP-TFI and COUP-TFII are members of the nuclear receptor superfamily. The high degree of evolutionary conservation of these proteins strongly argues for an important biological function.COUP-TF expression was highest in SK-BR3 cells (approximately 130 amol/µg total RNA), while the lowest COUP-TF expression was observed in MCF-7 cells (3·5 amol/µg total RNA). While treatment of EGF, TGF and PMA induced expression of COUP-TFII, COUP-TFI did not respond to these agents. Oncostatin M (OSM) is known to exert an antiproliferative effect in breast cancer cells. Treatment of MCF-7 cells with OSM resulted in an approximately 90% reduction of COUP-TFII mRNA expression.In SK-BR3 cells, treatment with the MEK inhibitor UO126 resulted in a profound suppression of endogenous COUP-TFII expression. Furthermore, cotreatment with UO126 prevented induction of COUP-TFII expression by EGF in MCF-7 cells. In conclusion, our data provide evidence, for the first time, that mitogenic substances which activate the MAP kinase pathway, can induce COUP-TFII expression. Our results strongly suggest that an active MAP kinase pathway is essential for COUP-TFII expression in human breast cancer cells.

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Background The presence of hypoxia is a poor prognostic factor in prostate cancer and the hypoxic tumor microenvironment promotes radioresistance. There is potential for drug radiotherapy combinations to improve the therapeutic ratio. We aimed to investigate whether hypoxia-associated genes could be used to identify FDA approved drugs for repurposing for the treatment of hypoxic prostate cancer. Methods Hypoxia associated genes were identified and used in the connectivity mapping software QUADrATIC to identify FDA approved drugs as candidates for repurposing. Drugs identified were tested in vitro in prostate cancer cell lines (DU145, PC3, LNCAP). Cytotoxicity was investigated using the sulforhodamine B assay and radiosensitization using a clonogenic assay in normoxia and hypoxia. Results Menadione and gemcitabine had similar cytotoxicity in normoxia and hypoxia in all three cell lines. In DU145 cells, the radiation sensitizer enhancement ratio (SER) of menadione was 1.02 in normoxia and 1.15 in hypoxia. The SER of gemcitabine was 1.27 in normoxia and 1.09 in hypoxia. No radiosensitization was seen in PC3 cells. Conclusion Connectivity mapping can identify FDA approved drugs for potential repurposing that are linked to a radiobiologically relevant phenotype. Gemcitabine and menadione could be further investigated as potential radiosensitizers in prostate cancer.

6006 Background: Tumour hypoxia is an adverse prognostic factor in head and neck squamous cell carcinoma (HNSCC). We assessed whether patients with hypoxic HNSCC benefited from the addition of nimorazole to definitive radiotherapy (RT). Methods: NIMRAD was a phase III, multi-centre, placebo-controlled, double-blind trial in patients with HNSCC unsuitable for concurrent platinum chemotherapy or cetuximab with definitive RT (NCT01950689). Patients were randomized 1:1 to receive nimorazole (1.2 g/m2 daily, prior to RT) + RT (65 Gy in 30 fractions over 6 weeks) or placebo (taken via the same schedule) + RT. The primary endpoint was loco-regional control (freedom from loco-regional progression, FFLRP) in patients with hypoxic tumours, defined as greater than or equal to the median hypoxia score of the first 50 patients analysed (≥0.079), using a validated 26-gene signature. The planned sample size was 340 patients allowing for signature generation in 85%, assumed HR 0.50 for nimorazole effectiveness in the hypoxic group, and requiring 66 loco-regional failures to have 80% power in a two-tail log-rank test at the 5% significance level. Results: 338 patients were randomised by 19 UK centres from May 2014 to May 2019, with a median follow-up of 3.1 years (95%CI 2.9-3.4). Hypoxia scores were available for 286 (85%). The median patient age was 73 years (range 44-88); and clinical factors were balanced between the arms, both for the whole population and hypoxic group (Table). There were 36 (25.9%) loco-regional failures in the hypoxic group, where nimorazole + RT did not improve FFLRP (adjusted HR 0.72; 95% CI 0.36-1.44; p=0.35), or overall survival (adjusted HR 0.96; 0.53-1.72; p=0.88) compared with placebo + RT. Similarly, nimorazole + RT did not improve FFLRP or OS in the whole population. In total (n=338), 73% of patients allocated nimorazole adhered to the drug for ≥50% of RT fractions. Nimorazole + RT caused more acute nausea compared with placebo + RT (CTCAE v4.0 G1+2: 56.6% vs 42.4%, G3: 10.1% vs 5.3%, respectively; p=0.003), with no differences in other early or late toxicities. Conclusions: Addition of the hypoxia modifier nimorazole to RT for locally advanced HNSCC in older and less fit patients did not improve loco-regional control or survival. Clinical trial information: NCT01950689 . [Table: see text]

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