Role of HIF-1α in both resistance to photon and carbon ion irradiations and invasiveness of Head and Neck Squamous Cell Carcinoma (HNSCC) cancer stem cells

Séminaire invité au National Institute of Radiological Sciences (NIRS), Chiba, Japo

Mécanismes moléculaires spécifiques de la réponse aux ions carbone dans les cellules tumorales (souches et non souches) des cancers des Voies Aéro-Digestives Supérieures

Hadrontherapy using carbon ions is an alternative to photon irradiation in the treatment of Head and Neck cancers, because of accurate ballistics and high biological efficiency, including hypoxic tumor areas. These cancers are of poor prognosis because of a high risk of recurrences related to the presence of cancer stem cells (CSCs).The aim of this work was to determine the molecular specificities of the response to carbon ion irradiations compared to photons in two cancer cell lines and their CSCs’ subpopulation, in hypoxic and normoxic conditions. This work focused on the role of the HIF-1α protein in cell survival, since hypoxia promotes its stabilization, but also in the radioresistance; the epithelial-mesenchymal transition (EMT) and the detection and repair of DNA double-strand breaks (DSBs). HIF-1α is stabilized earlier in CSCs compared to non-CSCs. Its activation, as well as the EMT pathways (STAT3, MEK/p38/JNK and Akt/mTOR), are dependent on reactive oxygen species (ROS), whose production is homogeneous in response to photons. At the opposite, the ROS produced in the carbon ion tracks are insufficient to activate HIF-1α and the upstream EMT pathways. Under hypoxic conditions, a relationship has been established between HIF-1α activation and that of the DSBs detection (ATM) and repair (Rad51) pathways (Homologous Recombination). These studies demonstrate that the therapeutic advantage of carbon ions is based on the spatial ROS distribution at the nanoscale and consequently on the non-activation of key pathways involved in tumor cell defenseL’hadronthérapie par ions carbone est une alternative à la radiothérapie photonique dans le traitement des cancers des VADS, en raison d’une balistique précise et d’une efficacité biologique élevée, y compris au sein des zones tumorales hypoxiques. Ces cancers sont de mauvais pronostic en raison d’un risque élevé de récidives liées à la présence de cellules souches cancéreuses (CSCs). L’objectif de ce travail était de déterminer les spécificités moléculaires de la réponse aux ions carbone par rapport aux photons dans deux lignées cellulaires de cancer des VADS et leur sous-population CSCs en hypoxie et normoxie. Il s’est focalisé sur le rôle de la protéine HIF-1α dans la survie cellulaire, dans la mesure où l’hypoxie favorise sa stabilisation mais également la radiorésistance; sur la transition épithélio-mésenchymateuse (EMT) et la détection-réparation des cassures double-brin (CDBs) de l’ADN. HIF-1α est stabilisée plus précocement dans les CSCs par rapport aux non-CSCs. Son activation, tout comme celle des voies de l’EMT (STAT3, MEK/p38/JNK et Akt/mTOR) est dépendante des radicaux libres oxygénés (RLO), dont la production est homogène dans la cellule en réponse aux photons. Par contre, les RLO produits dans la trace des ions carbone ne permettent pas d’activer HIF-1α et les voies de l’EMT. Sous hypoxie, une relation a été établie entre l’activation d’HIF-1α et celles des voies de détection (ATM) et de réparation (Rad51) des CDBs (Recombinaison Homologue). Ces travaux démontrent que l’avantage thérapeutique des ions carbone repose sur la répartition spatiale des RLO à l’échelle nanométrique et consécutivement sur la non-activation de voies clés de la défense cellulaire tumoral

Specific molecular mechanisms of the carbon ion irradiation response in HNSCC (Cancer Stem Cells and non-Cancer Stem Cells)

L’hadronthérapie par ions carbone est une alternative à la radiothérapie photonique dans le traitement des cancers des VADS, en raison d’une balistique précise et d’une efficacité biologique élevée, y compris au sein des zones tumorales hypoxiques. Ces cancers sont de mauvais pronostic en raison d’un risque élevé de récidives liées à la présence de cellules souches cancéreuses (CSCs). L’objectif de ce travail était de déterminer les spécificités moléculaires de la réponse aux ions carbone par rapport aux photons dans deux lignées cellulaires de cancer des VADS et leur sous-population CSCs en hypoxie et normoxie. Il s’est focalisé sur le rôle de la protéine HIF-1α dans la survie cellulaire, dans la mesure où l’hypoxie favorise sa stabilisation mais également la radiorésistance; sur la transition épithélio-mésenchymateuse (EMT) et la détection-réparation des cassures double-brin (CDBs) de l’ADN. HIF-1α est stabilisée plus précocement dans les CSCs par rapport aux non-CSCs. Son activation, tout comme celle des voies de l’EMT (STAT3, MEK/p38/JNK et Akt/mTOR) est dépendante des radicaux libres oxygénés (RLO), dont la production est homogène dans la cellule en réponse aux photons. Par contre, les RLO produits dans la trace des ions carbone ne permettent pas d’activer HIF-1α et les voies de l’EMT. Sous hypoxie, une relation a été établie entre l’activation d’HIF-1α et celles des voies de détection (ATM) et de réparation (Rad51) des CDBs (Recombinaison Homologue). Ces travaux démontrent que l’avantage thérapeutique des ions carbone repose sur la répartition spatiale des RLO à l’échelle nanométrique et consécutivement sur la non-activation de voies clés de la défense cellulaire tumoraleHadrontherapy using carbon ions is an alternative to photon irradiation in the treatment of Head and Neck cancers, because of accurate ballistics and high biological efficiency, including hypoxic tumor areas. These cancers are of poor prognosis because of a high risk of recurrences related to the presence of cancer stem cells (CSCs).The aim of this work was to determine the molecular specificities of the response to carbon ion irradiations compared to photons in two cancer cell lines and their CSCs’ subpopulation, in hypoxic and normoxic conditions. This work focused on the role of the HIF-1α protein in cell survival, since hypoxia promotes its stabilization, but also in the radioresistance; the epithelial-mesenchymal transition (EMT) and the detection and repair of DNA double-strand breaks (DSBs). HIF-1α is stabilized earlier in CSCs compared to non-CSCs. Its activation, as well as the EMT pathways (STAT3, MEK/p38/JNK and Akt/mTOR), are dependent on reactive oxygen species (ROS), whose production is homogeneous in response to photons. At the opposite, the ROS produced in the carbon ion tracks are insufficient to activate HIF-1α and the upstream EMT pathways. Under hypoxic conditions, a relationship has been established between HIF-1α activation and that of the DSBs detection (ATM) and repair (Rad51) pathways (Homologous Recombination). These studies demonstrate that the therapeutic advantage of carbon ions is based on the spatial ROS distribution at the nanoscale and consequently on the non-activation of key pathways involved in tumor cell defens

Specific molecular mechanisms of the carbon ion irradiation response in HNSCC (Cancer Stem Cells and non-Cancer Stem Cells)

L’hadronthérapie par ions carbone est une alternative à la radiothérapie photonique dans le traitement des cancers des VADS, en raison d’une balistique précise et d’une efficacité biologique élevée, y compris au sein des zones tumorales hypoxiques. Ces cancers sont de mauvais pronostic en raison d’un risque élevé de récidives liées à la présence de cellules souches cancéreuses (CSCs). L’objectif de ce travail était de déterminer les spécificités moléculaires de la réponse aux ions carbone par rapport aux photons dans deux lignées cellulaires de cancer des VADS et leur sous-population CSCs en hypoxie et normoxie. Il s’est focalisé sur le rôle de la protéine HIF-1α dans la survie cellulaire, dans la mesure où l’hypoxie favorise sa stabilisation mais également la radiorésistance; sur la transition épithélio-mésenchymateuse (EMT) et la détection-réparation des cassures double-brin (CDBs) de l’ADN. HIF-1α est stabilisée plus précocement dans les CSCs par rapport aux non-CSCs. Son activation, tout comme celle des voies de l’EMT (STAT3, MEK/p38/JNK et Akt/mTOR) est dépendante des radicaux libres oxygénés (RLO), dont la production est homogène dans la cellule en réponse aux photons. Par contre, les RLO produits dans la trace des ions carbone ne permettent pas d’activer HIF-1α et les voies de l’EMT. Sous hypoxie, une relation a été établie entre l’activation d’HIF-1α et celles des voies de détection (ATM) et de réparation (Rad51) des CDBs (Recombinaison Homologue). Ces travaux démontrent que l’avantage thérapeutique des ions carbone repose sur la répartition spatiale des RLO à l’échelle nanométrique et consécutivement sur la non-activation de voies clés de la défense cellulaire tumoraleHadrontherapy using carbon ions is an alternative to photon irradiation in the treatment of Head and Neck cancers, because of accurate ballistics and high biological efficiency, including hypoxic tumor areas. These cancers are of poor prognosis because of a high risk of recurrences related to the presence of cancer stem cells (CSCs).The aim of this work was to determine the molecular specificities of the response to carbon ion irradiations compared to photons in two cancer cell lines and their CSCs’ subpopulation, in hypoxic and normoxic conditions. This work focused on the role of the HIF-1α protein in cell survival, since hypoxia promotes its stabilization, but also in the radioresistance; the epithelial-mesenchymal transition (EMT) and the detection and repair of DNA double-strand breaks (DSBs). HIF-1α is stabilized earlier in CSCs compared to non-CSCs. Its activation, as well as the EMT pathways (STAT3, MEK/p38/JNK and Akt/mTOR), are dependent on reactive oxygen species (ROS), whose production is homogeneous in response to photons. At the opposite, the ROS produced in the carbon ion tracks are insufficient to activate HIF-1α and the upstream EMT pathways. Under hypoxic conditions, a relationship has been established between HIF-1α activation and that of the DSBs detection (ATM) and repair (Rad51) pathways (Homologous Recombination). These studies demonstrate that the therapeutic advantage of carbon ions is based on the spatial ROS distribution at the nanoscale and consequently on the non-activation of key pathways involved in tumor cell defens

The ‘stealth-bomber’ paradigm for deciphering the tumour response to carbon-ion irradiation

International audienceNumerous studies have demonstrated the higher biological efficacy of carbon-ion irradiation (C-ions) and their ballistic precision compared with photons. At the nanometre scale, the reactive oxygen species (ROS) produced by radiation and responsible for the indirect effects are differentially distributed according to the type of radiation. Photon irradiation induces a homogeneous ROS distribution, whereas ROS remain condensed in clusters in the C-ions tracks. Based on this linear energy transfer-dependent differential nanometric ROS distribution, we propose that the higher biological efficacy and specificities of the molecular response to C-ions rely on a ‘stealth-bomber’ effect. When biological targets are on the trajectories of the particles, the clustered radicals in the tracks are responsible for a ‘bomber’ effect. Furthermore, the low proportion of ROS outside the tracks is not able to trigger the cellular mechanisms of defence and proliferation. The ability of C-ions to deceive the cellular defence of the cancer cells is then categorised as a ‘stealth’ effect. This review aims to classify the biological arguments supporting the paradigm of the ‘stealth-bomber’ as responsible for the biological superiority of C-ions compared with photons. It also explains how and why C-ions will always be more efficient for treating patients with radioresistant cancers than conventional radiotherapy

Role of HIF-1α in the resistance of Cancer Stem Cells to photon and carbon ion irradiations

International audienceHead-and-Neck-Squamous Cell Carcinoma (HNSCC) are resistant to standard treatments, partly due to Cancer Stem Cells (CSCs) localized in hypoxic niches. The protein HIF-1α (Hypoxia-Inducible Factor 1α) is considered as the major transcriptional regulator of the cellular response to oxygen homeostasis. Compared to X-rays, carbon ions relie on better ballistic properties, higher relative biological effectiveness and the absence of oxygen effect. HIF-1α is involved in the resistance to photons whereas its role in response to carbon ions remains unclear. My work aims at clarifying the role of HIF-1 in the response of CSCs to photon (250kV) and carbon ion (75Mev/n and 290Mev/µ) irradiations in normoxic or hypoxic (1% O2) conditions. For two HNSCC cell lines and their CSC subpopulation, in response to photons under hypoxia, an OER upper than 1.2 was associated with HIF-1α expression. This stabilization appears earlier in CSCs than in non-CSCs and is correlated with the variation of ROS levels, confirming the adaptive properties of CSCs to hypoxia. The diffuse ROS production by photons is concomitant with HIF-1α expression and essential to its activation. Compared with photons, the oxygen effect is canceled after carbon ion exposure (OER=1) and no stabilization of HIF-1α was observed in normoxia, probably due to the ROS localization in the track, insufficient to stabilize HIF-1α. Inhibition of HIF-1α with a siRNA leads to the decrease of HNSCC-CSC survival after both radiations in hypoxic conditions (OER<1). Furthermore, radiosensitization is associated with a significant increase of residual DSBs in response to both types of irradiations. Finally, CSCs migrate more and are more invasive than non-CSCs in normoxia and even more under hypoxia. Photon irradiation increases both process under normoxia whereas carbon ions decrease them significantly in normoxic and hypoxic conditions. The inhibition of HIF-1α, in response to both types of irradiations and particularly in hypoxic conditions, is associated with a low proportion of migrating and invasive cells, confirming the role of HIF-1α in the epithelio-mesenchymal transition. These results demonstrate that HIF-1 plays a key role in the response of CSCs and non-CSCs to photon and carbon ion irradiations. It participates to radioresistance by increasing cell survival, DNA repair and invasiveness and contributes to tumor escape. This makes the HIF-1α targeting an attractive therapeutic challenge. Supported by LabEx PRIMES (ANR-11-LABX-0063), France Hadron (ANR-11-INBS-0007), Lyric (INCa-DGOS-4664), Jean-Walter Zellidja Grant (French Academy), Canceropôle Auvergne Rhône-Alpes (CLARA, Mobility Grant

A major contribution of the HIF-1α pathway in the oxygen effect

International audienceOxygen plays an essential role in the response to ionizing radiation through the production of ROS induced by water radiolysis while hypoxia may lead to radioresistance. However, compared to photons, carbon ions have the advantage of a high relative biological efficiency (EBR) independently of the oxygen concentration. The protein HIF-1α (Hypoxia-Inducible Factor 1α) is considered as the major transcriptional regulator of the cellular response to oxygen homeostasis. HIF-1α is thus involved in the resistance to photons whereas its role in the response to carbon ions remains unclear. This work aims at clarifying the role of HIF-1 in the oxygen effect. Cancer Stem Cells (CSCs) isolated from Head-and-Neck-Squamous Cell Carcinoma (HNSCC) were irradiated with photons (250kV) and carbon ions (75Mev/n) in normoxic or hypoxic (1% O2) conditions. For two HNSCC cell lines and their CSC subpopulation, in response to photons under hypoxia, an OER (Oxygen Enhancement Ratio calculated as the ratio of dose inducing 10% survival after photon or carbon ion irradiation in hypoxic versus normoxic conditions) upper than 1.2 was associated with HIF-1α expression. This stabilization appears earlier in CSCs than in non-CSCs and is correlated with the variation of ROS levels, confirming the adaptive properties of CSCs (located in tumor hypoxic niches) to hypoxia. The diffuse ROS production by photons (measured by flow cytometry) is concomitant with HIF-1α expression and essential to its activation. Compared with photons, the oxygen effect is canceled after carbon ion exposure (OER=1) and no expression of HIF-1α was observed in normoxia, probably due to the ROS localization in the track, insufficient to stabilize HIF-1α. Inhibition of HIF-1α with a siRNA leads to the decrease of HNSCC-CSC survival, and subsequently radiosensitization, after both types of radiations in hypoxic conditions (OER<1). The signaling pathways leading to HIF-1α expression are currently investigated.These results demonstrate that HIF-1 plays a key role in the response of CSCs and non-CSCs to photon and carbon ion irradiations, participating to radioresistance by increasing cell survival. This makes the HIF-1α targeting an attractive therapeutic challenge. Supported by LabEx PRIMES (ANR11LABX0063), France Hadron (ANR11INBS0007) and Lyric (INCaDGOS4664

Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

International audienceDespite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local recurrence and distant metastasis. One hypothesis to explain this recurrence is the presence of cancer stem-like cells (CSCs) that present inherent chemo- and radio-resistance. In order to develop new therapeutic strategies, it is necessary to have experimental models that validate the effectiveness of targeted treatments and therefore to have reliable methods for the identification and isolation of CSCs. To this end, we present a protocol for the isolation of CSCs from human HNSCC cell lines that relies on the combination of two successive cell sortings performed by fluorescence activated cell sorting (FACS). The first one is based on the property of CSCs to overexpress ATP-Binding Cassette (ABC) transporter proteins and thus exclude, among others, vital DNA dyes such as Hoechst 33342. The cells sorted with this method are identified as a "side population" (SP). As the SP cells represent a low percentage (<5%) of parental cells, a growing phase is necessary in order to increase their number before the second cell sorting. The next step allows for the selection of cells that possess two other HNSCC stem cell characteristics i.e. high expression level of the cell surface marker CD44 (CD44high) and the over-expression of aldehyde dehydrogenase (ALDHhigh). Since the use of a single marker has numerous limitations and pitfalls for the isolation of CSCs, the combination of SP, CD44 and ALDH markers will provide a useful tool to isolate CSCs for further analytical and functional assays requiring viable cells. The stem-like characteristics of CSCs was finally validated in vitro by the formation of tumorispheres and the expression of β-catenin

Role of HIF-1α in the migration/invasion processes in response to photon and C-ion irradiations

International audiencePurpose: The survival rate of patients with HNSCC remains low due to recurrences. Metastasis, but also cancer stem cells (CSC), located in hypoxic niches, play a key role in these relapses. HIF-1α protein (Hypoxia Inducible Factor 1α), stabilized by hypoxia and radical species induced by ionizing radiations, is involved in the radio-resistance to photons but also in the epithelial mesenchymal transition (EMT). The understanding of the differential molecular mechanisms HIF-1α-dependent involved in the EMT and induced by photons and C-ion radiations must be deepened under normoxic conditions and particularly under hypoxia in CSCs, especially since hypoxia constitutes a tumor microenvironment promoting metastases and CSCs

Ciblage des cellules souches cancéreuses dans le cancer ORL: effet synergique du cetuximab à l’ABT-199 en association à l’irradiation photonique

National audienceLes cellules souches cancéreuses (CSCs) de cancer ORL, sous-population hautement migratoire, semblent être à l’origine de la résistance aux traitements. L’objectif du travail était d’étudier l’effet synergique d’un inhibiteur spécifique de Bcl-2, l’ABT-199, avec un anticorps monoclonal anti-EGFR, le cetuximab, en association avec l’irradiation photonique sur une lignée de cancer ORL et sa sous-population de CSCs