PO-133 Inhibition of HIF-1α enhances radiation sensitivity in tumour cells and mediates tumour growth decrease in mice

Abstract

IntroductionPoor prognosis of many solid tumours is often associated with hypoxic regions and an increased level of hypoxia-inducible-factor-1α (HIF-1α). Previous findings indicate that HIF-1α expression is relevant for radiation resistance. It was shown that HIF-1α is connected to molecules involved in DNA damage repair and checkpoint control. HIF-1α has an enhanced activity in tumours after radiation treatment (IR) and also mediates low radiation sensitivity.Material and methodsIn vitro cultured Lewis Lung Carcinoma (LLC) cells with doxycycline (Dox)-inducible knockdown (KD) of HIF-1α were used. Cells were incubated under hypoxic conditions (Hx, 1%) and irradiated with 5 Gy. Cell cycle distribution analysis was performed by PI staining and FACS. Colony formation assay, caspase-3 assay, PARP-1 cleavage and Annexin/PI staining were used to quantify apoptosis and long term survival. DNA double strand break (DSB) repair was investigated by pulse field gel electrophoresis, Western blot and immunofluorescent staining with γH2AX, 53 BP1, RAD51, BRCA1 antibodies. In vivo the induction of HIF-1α KD was performed by tamoxifen administration to HIF-1αfl/flCre-ERki/+ C57BL/6 mice. The LLC cells with constitutive HIF-1α targeting (shHIF) or scrambled (shscr) shRNA-expressing vectors were inoculated into the flank of the mice. IR was delivered once at day 10 with a dose of 15 Gy using an X-ray source.Results and discussionsWe demonstrated an inhibition of decrement of phosphorylated H2AX after Hx and IR in HIF-1α deficient cells. Fast repair kinetics of the cells remained unchanged, whereas the long term survival of the cells with reduced HIF-1α was decreased. Moreover, these cells displayed an increased rate of apoptosis after IR. We also observed persisted RAD51 foci after 24 hour in HIF-1α deficient cells which indicates an alteration in homologous recombination repair (HRR) of the cells. Furthermore, in vivo experiments with a HIF-1α deficient stromal cells in the mice and injected HIF-1α deficient tumours cells indicate a decrease of tumour growth.ConclusionThese results imply that inactivation of HIF-1α disrupts DSB repair, in particular HRR. Furthermore, they highlight the importance of the interaction of tumour and microenvironment with respect to radiation sensitivity. It was shown that inhibition of HIF-1α enhances radiation sensitivity of tumour cells which is potentially helpful for the development of novel tumour therapies.