Background: Notch pathway regulates cell-fate determination in multi-cellular organisms and aberrant activation of Notch pathway has been reported in tumorigenesis of many cancers. We previously reported that Notch3 and its downstream effector, HEY1, were up-regulated by radiation in non-small cell lung cancer (NSCLC) cell lines. Furthermore, we demonstrated that g secretase inhibitor (GSI), when combined with radiation therapy, significantly suppressed the growth of lung cancer cells compared with either GSI or radiation alone. The proposed mechanism involves GSI suppressing radiation-induced Notch activation and the resultant radioresistance. However, little is known about the mechanism of Notch up-regulation induced by radiation. Since the expression of Notch was reported to be activated via HIF under hypoxia, we, in this study, hypothesized that HIF would be involved in radiation-induced Notch activation in NSCLC and thus HIF inhibitor (YC-1) would have an anti-tumor effect when combined with radiation therapy as is the case with GSI.

Materials and Methods: Two Notch-expressing lung cancer cell lines (H460 and HCC2429) were used. We first examined the changes in expressions of HIF pathway and Notch family after radiation under hypoxia using Western blotting (WB) and real time RT-PCR.Notch expression was evaluated after HIF-1α suppression by siRNA targeting HIF-1α. Next, in vitro we investigated an antitumor effect of YC-1, using a MTT proliferation assay and a clonogenic assay, when combined with radiation. And the combined antitumor effect of GSI and YC-1 with radiation was examined using the Fa-CI plot of isobologram analysis. Finally, we utilized a xenograft model and resected some tumors on day 15 for molecular analysis.

Results: Under hypoxia, the expression of HIF-1α was up-regulated when the cells were incubated for 6 h without radiation. Radiation up-regulated the expression of Notch3 and VEGF at 24 h compared with control. Specific suppression of HIF-1α by siRNA down-regulated Notch3 and Hey1 activation associated with radiation. In vitro, IC50 of YC-1 and colony formation were decreased only when radiation treatment was concurrently applied under hypoxia compared with sequential treatment. Moreover, GSI and YC-1 had a synergistic antitumor effect under hypoxia without or with radiation. In vivo, combination of GSI and YC-1 showed the greatest radiosensitivity and reduced radiation-induced Notch activation in molecular analysis, compared with single treatment.

Conclusions: Radiation-induced up-regulation of Notch pathway and HIF-1α might provide therapeutic targets for more effective radiation therapy in NSCLC.

Citation Format: Yasuyuki Ikezawa, Jun Sakakibara-Konishi, Hidenori Mizugaki, Satoshi Oizumi, Masaharu Nishimura. Inhibition of Notch and HIF enhances the antitumor effect of radiation in Notch expressing lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3937. doi:10.1158/1538-7445.AM2014-3937