Neuropilin-1 expression in adenocarcinoma and squamous cell carcinoma of the lung is differentially regulated by hypoxia.

17152 Background: Neuropilin-1 (NRP-1) is an isoform-specific receptor for VEGF165 and semaphorin3A, initially discovered on migrating neurons. NRP-1 expression has been reported on a number of tumour cell lines in the absence of the other VEGF receptors, where it mediates survival signals. In this study we examined the regulation of NRP-1 by hypoxia, its effect on survival in a panel of lung cancer cell lines and its potential as a biomarker in retrospective human lung tumours. METHODS A549, SK-MES1, H460 and H647 cells were grown in serum depleted media (0.5%) in normoxic or hypoxic (0.1% O2) conditions and screened for NRP-1 expression by western and immunocytochemistry analysis. Cell survival and apoptosis was determined using BrdU and Annexin-V/PI staining respectively following treatment with an antibody to the extracellular NRP-1 domain. A panel of 100 retrospective resected lung tumours and matched normal samples were stained for NRP-1 expression by immunhistochemistry. RESULTS A549, SKMES-1 and H647 cell lines all expressed NRP-1 and displayed reduced survival following treatment with NRP-1 antibody (1ug/ml) compared to controls (A549 46%, SKMES-1 61%, H647 53%). H460 did not express NRP-1 and no survival inhibition was seen in the cell line (104%). Reduced survival was accompanied by increased apoptosis in all NRP-1 positive cell lines. Hypoxia strongly increased NRP-1 expression in the A549 adenocarcinoma (AC) cell line, while NRP-1 was decreased in SKMES-1 squamous cell carcinoma (SCC) following hypoxia. Neutralisation of NRP-1 had a greater effect in A549 cells under hypoxia (37%), with a lesser effect in SKMES-1 cells (82%). Western analysis of matched frozen normal and lung cancer biopsies showed NRP-1 overexpression in AC and decreased expression in SCC relative to normal. High NRP-1 expression was confirmed in AC and large cell carcinoma by immunohistochemistry, relative to normal. However, SCCs had a lower level of NRP-1 staining, supporting the results by western analysis and following hypoxia in vitro. CONCLUSIONS These results implicate NRP-1 as an important survival pathway in lung cancer. Hypoxia differentially regulated NRP-1 mediated survival implicating this pathway as a potential therapeutic strategy in AC. No significant financial relationships to disclose.