237 - HIF-1α plays a key role in the response of HNSCC cancer stem cells to photon and carbon ion exposures

Abstract

Purpose: Head and neck squamous cells carcinomas (HNSCC) have a poor prognosis due to escapement to anti-cancer therapies which leads to locoregional recurrences. The presence of cancer stem cells (CSCs), resistant to chemo- and radio-therapies, could explain these resistances and recurrences. Hadrontherapy has demonstrated favorable results for many cancers, since carbon ions induced more CSC’s cell death than photons. However, for the HNSCC tumor, local control remains low. CSCs are located in a hypoxic microenvironment and hypoxia is well-known for being able to induce the epithelial mesenchymal transition (EMT). The aim of this study is to investigate the response of CSCs to radiotherapy, compared with carbon ions under hypoxic conditions. Many signaling pathways involved in the resistance of CSCs and the invasion/migration process depend on the stabilization of HIF-1α. Reactive Oxygen Species (ROS), which are produced under hypoxia and in response to irradiation, play a key role in this stabilization. However, since few data are available, the mechanisms involving ROS production, HIF-1α stabilization as well as the invasion/migration process that follows, need to be clarified in HNSCC CSCs exposed to carbon or photon radiations in a hypoxic environment. Material and methods: Two HNSCC cells lines, SQ20B and FaDu, and their CSCs were grown in normoxic and hypoxic (1% O2) conditions. CSCs were isolated by flow cytometry cell sorting. Cell survival curves were performed in response to photon (250kV) and carbon ion (75MeV/n, GANIL, France) irradiations in order to define the Oxygen Enhancement Ratio (OER). The expression of HIF-1α was followed by Western-Blots. ROS were quantified with a CM-H2DCFDA dye and Migration/Invasion with Boyden chambers. Results: After photons, HNSCC cells and their CSCs appeared more resistant under hypoxia (OER>1.2) whereas the oxygen effects were cancelled after carbon ions (OER=1). Interestingly, the OER values and the expression of HIF-1α seemed to be linked. HIF-1α was weakly expressed after photon irradiation and fully inhibited after carbon ions. Additionally, under hypoxia, HIF-1α expression appeared earlier in CSCs than in the parental cell lines, confirming their adaptive properties to hypoxia. In CSCs, this expression was correlated with ROS levels. As a consequence, since HIF- 1α is known to promote EMT, under normoxia as well as hypoxia, the invasion/migration abilities of CSCs were more important than in non-CSCs. Finally, after carbon ions, either in normoxic or hypoxic conditions, invasion/migration processes were decreased compared with photons. Conclusion: HIF-1α plays a key role in the radioresistance of CSCs and in their invasiveness abilities. No expression of HIF-1α was found after carbon ion exposure suggesting that carbon ions could be a relevant therapeutic alternative to kill CSCs in their microenvironment.