Abstract 1480: Reactive oxygen generated by shh signaling contributes to lymphangiogenesis in the non-small cell lung cancer

Abstract

Approximately 90% of all cancer deaths arise from the metastatic spread of primary tumours. Recent reports have demonstrated that the Sonic hedgehog (shh) signaling pathway plays an important role in the metastasis of various organs, including the prostate, pancreas, gastrointestinal tract, esophageal and other tumors. Recently, we found that increased shh producing cells correlate with high migration, invasiveness and increased lymphangiogenesis in Non-small-cell lung cancer (NSCLC), but the mechanism is unknown. Furthermore, blockade of shh pathway by target antibodies inhibited shh-induced tumor growth, metastasis and lymphangiogenesis. In this study, we investigated the hypothesis that expression and function of the shh pathway in human Non-small cell lung cancer. We performed immunohistochemistry on lung tumor biopsies showed that expressions of shh, Gli-1, VEGF-D and LYVE-1 were enhanced in lymph node metastasis specimens compared with lymph node negative lung cancer specimens. To investigate the role of shh signaling as it relates to invasive phenotypes and the motility of NSCLC. We found that recombinant human shh (N-shh) enhanced cell motility and invasiveness in gastric cancer cells, whereas no increase was observed in cells that were treated with KAAD-cyclopamine(SMO inhibitor). The overall cell growth was not affected by treatment with N-Shh when cells were treated with the same concentration and for the same length of time, which indicates that the difference in motility was not due to an effect on proliferation. Reverse transcription-PCR analysis was performed to find lymphangiogenesis factor of mRNA levels. The expressions of shh, Gli-1, VEGF-D and LYVE-1 was also enhanced by treatment with N-shh, but not KAAD-cyclopamine. We further analysed the affects of shh signaling in vitro by stimulating a normal human lymphatic endothelial cell line (HMVEC) with N-shh. In vitro lymphangiogenesis assay using the HMVEC cell line. Stimulation with N-shh enhanced tube formation on matrigel. Stimulation with shh significantly increased in vitro lymphangiogenesis, but not KAAD-cyclopamine. To investigate the role and mechanism for shh signaling in regulating the lymphangiogenetic progression of NSCLC. Tumour hypoxia not only induces tumour angiogenesis, but also modulates the expression of several genes that have been implicated in tumour metastasis. We next explored how the shh signaling and resultant expression of LYVE-1 and VEGF-D regulate metastasis. Because expression of LYVE-1 and VEGF-D may lead to overproduction of reactive oxygen species (ROS), we estimated the amounts of ROS, and found that the N-shh in NSCLC showed enhanced ROS production, whereas the KAAD-cyclopamine did not. These results indicate that shh signaling pathway can contribute to ROS gerenates by enhancing the metastatic and lymphangiogenetic potential of tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1480. doi:10.1158/1538-7445.AM2011-1480