Role of the calcium channel Orai3 in chemoresistance in lung cancer : involvement of cancer stem cells

Depuis son approbation par la FDA (Food and Drug Administration) en 1978, la thérapie par les sels de platine reste le traitement chimiothérapeutique standard actuel pour le cancer du poumon non à petites cellules (CPNPC). Cependant, la résistance aux sels de platine évolue rapidement chez les patients atteints de CPNPC et l'une des principales raisons de cette résistance est leur capacité à enrichir la population des cellules souches cancéreuses (CSC). Les canaux calciques de type SOC (Store Operated Channel) représentent la voie d'entrée majeure du calcium dans les cellules non excitables et sont impliqués dans différents processus cancéreux notamment la survie. Parmi ces canaux, le canal calcique Orai3 a été récemment identifié comme un élément important dans la résistance à la chimiothérapie dans les cellules cancéreuses du sein. De plus, ce canal est surexprimé dans les cancers CPNPC, est corrélé au grade tumoral élevé et constitue un marqueur prédictif de métastases et de mauvais pronostic chez les patients atteints de tumeurs CPNPC résécables. In-vitro, Orai3 régule la prolifération des cellules CPNPC d'une façon calcium-dépendante, via la voie Akt. À partir de ces données, mes travaux de thèse ont porté sur l'étude de l'effet potentiel du canal Orai3 dans la résistance au Cisplatine ainsi que les voies de signalisation associées dans le cancer du poumon. Nos résultats, sur lignées cellulaires et sur tissus cancéreux, montrent très clairement que le traitement chimiothérapeutique augmente l'expression d'Orai3. Cliniquement, l'augmentation de l'expression d'Orai3, après un traitement chimiotérapeutique, est associée à la non régression ou à une régression partielle de la tumeur. Dans les lignées cellulaires CPNPC, un traitement au Cisplatine augmente l'expression d'Orai3 d'une façon temps-dépendante. Cet effet s'accompagne par l'augmentation de la population des cellules souches cancéreuses (marquage avec CD133) et aussi des marqueurs des CSC notamment Nanog, SOX2 et Slug. L'invalidation de l'expression d'Orai3 ou la réduction de la concentration du calcium extracellulaire sensibilise les cellules au Cisplatine, et réduit totalement l'expression de Nanog et SOX2 et partiellement l'expression de Slug. Par ailleurs, la surexpression d'Orai3 induit la surexpression de Nanog et SOX2. D'une façon intéressante, nous avons constaté un changement de la fonction d'Orai3 en fonction du traitement au Cisplatine. En effet, Orai3 devient SOC après traitement au Cisplatine, ou dans les cellules surexprimant Orai3, alors qu'il régule l'entrée calcique basal avant le traitement ou dans les cellules transfectées avec le vecteur vide. Cette fonction SOC d'Orai3 est retrouvée aussi dans les cellules souches cancéreuses. Finalement, nous avons montré qu'en inhibant la voie Akt, aucune augmentation des marqueurs de CSC n'a été observée et la réponse au Cisplatine a été favorisée. En conclusion, l'augmentation de l'expression d'Orai3 permet à ce canal de devenir SOC favorisant ainsi l'entrée du calcium. Ce dernier, via la voie Akt, permet l'enrichissement de la population des CSC (augmentation des marqueurs des CSC) insensibles au Cisplatine. L'ensemble de nos résultats montre, pour la première fois, le rôle du canal Orai3 dans la résistance à la chimiothérapie due à l'augmentation de la population des cellules souches. Par conséquent, Orai3 pourrait constituer un biomarqueur prédictif du choix des médicaments chimiothérapeutiquesSince approved by the FDA (Food and Drug Administration) in 1978, platinum doublet therapy remains the current gold standard chemotherapeutic treatment for non-small cell lung cancer (NSCLC). However, resistance to platinum salts evolves rapidly in patients with NSCLC and one of the major reasons behind this resistance is their proved ability to enrich CSC (cancer stem cell) population. Calcium is recognized as an activator of critical signaling pathways, including the ones responsible for resistance to apoptosis. Herein, the calcium channel Orai3 has been recently identified as an important element in the resistance to chemotherapy in breast cancer cells. Furthermore, Orai3 channel is overexpressed in NSCLC tissues, is correlated to a high tumor grade and constitutes a predictive marker of metastasis and survival in resectable NSCLC tumors. In-vitro, Orai3 channel controls cell proliferation in NSCLC cell lines in a calcium dependent manner, via the Akt pathway. In the present work, we investigated the role of Orai3 channel in resistance to Cisplatin in NSCLC and the signaling pathways associated. We found that Orai3 channel becomes overexpressed after treatment with Cisplatin in NSCLC tissues and cell lines. Clinically, Orai3 overexpression after chemotherapy was associated with partial or no tumor regression. In cell lines, Cisplatin treatment increased Orai3 expression in a time-dependent manner and this overexpression was accompanied by an enrichment of CSCs population demonstrated by CD133 staining and an overexpression of the CSCs markers Nanog, SOX-2 and Slug. Moreover, Orai3 silencing or the reduction of extracellular calcium concentration sensitized the cells to Cisplatin and leads to a drastic reduction in the expression of Nanog and SOX-2 and a partial reduction of Slug expression. Furthermore, Orai3 overexpression induced the overexpression of both markers Nanog and SOX-2. Interestingly, we remarked a change in the function of Orai3 upon the treatment with Cisplatin. In basal conditions, Orai3 was found to regulate basal calcium entry in control cells or cells transfected with an empty vector while upon the treatment with Cisplatin or when Orai3 is overexpressed via Orai3 vector transfection, it becomes involved in SOC entry. We also noticed that Orai3 functions as SOC channel in CSCs. Finally, we found that upon the inhibition of the signaling pathway Akt, the expression of the stemness markers didn’t increase and Cisplatin’s efficiency was enhanced. In a conclusion, Orai3 overexpression enables the channel to become a SOC channel and favors the calcium entry. Also, Orai3 channel, via the Akt pathway, promotes the enrichment of CSCs which are insensitive to Cisplatin. Taken together, our results show for the first time that Orai3 channel is able to induce chemoresistance in NSCLC cells by increasing the CSCs population. Our findings provide a new context in the understanding of NSCLC resistance to chemotherapy and present Orai3 as a promising biomarker which could help in the choice of chemotherapeutic drugs for patients with NSCL

Rôle du canal calcique Orai3 dans la résistance à la chimiothérapie dans le cancer du poumon : implication des cellules souches cancéreuses

Since approved by the FDA (Food and Drug Administration) in 1978, platinum doublet therapy remains the current gold standard chemotherapeutic treatment for non-small cell lung cancer (NSCLC). However, resistance to platinum salts evolves rapidly in patients with NSCLC and one of the major reasons behind this resistance is their proved ability to enrich CSC (cancer stem cell) population. Calcium is recognized as an activator of critical signaling pathways, including the ones responsible for resistance to apoptosis. Herein, the calcium channel Orai3 has been recently identified as an important element in the resistance to chemotherapy in breast cancer cells. Furthermore, Orai3 channel is overexpressed in NSCLC tissues, is correlated to a high tumor grade and constitutes a predictive marker of metastasis and survival in resectable NSCLC tumors. In-vitro, Orai3 channel controls cell proliferation in NSCLC cell lines in a calcium dependent manner, via the Akt pathway. In the present work, we investigated the role of Orai3 channel in resistance to Cisplatin in NSCLC and the signaling pathways associated. We found that Orai3 channel becomes overexpressed after treatment with Cisplatin in NSCLC tissues and cell lines. Clinically, Orai3 overexpression after chemotherapy was associated with partial or no tumor regression. In cell lines, Cisplatin treatment increased Orai3 expression in a time-dependent manner and this overexpression was accompanied by an enrichment of CSCs population demonstrated by CD133 staining and an overexpression of the CSCs markers Nanog, SOX-2 and Slug. Moreover, Orai3 silencing or the reduction of extracellular calcium concentration sensitized the cells to Cisplatin and leads to a drastic reduction in the expression of Nanog and SOX-2 and a partial reduction of Slug expression. Furthermore, Orai3 overexpression induced the overexpression of both markers Nanog and SOX-2. Interestingly, we remarked a change in the function of Orai3 upon the treatment with Cisplatin. In basal conditions, Orai3 was found to regulate basal calcium entry in control cells or cells transfected with an empty vector while upon the treatment with Cisplatin or when Orai3 is overexpressed via Orai3 vector transfection, it becomes involved in SOC entry. We also noticed that Orai3 functions as SOC channel in CSCs. Finally, we found that upon the inhibition of the signaling pathway Akt, the expression of the stemness markers didn’t increase and Cisplatin’s efficiency was enhanced. In a conclusion, Orai3 overexpression enables the channel to become a SOC channel and favors the calcium entry. Also, Orai3 channel, via the Akt pathway, promotes the enrichment of CSCs which are insensitive to Cisplatin. Taken together, our results show for the first time that Orai3 channel is able to induce chemoresistance in NSCLC cells by increasing the CSCs population. Our findings provide a new context in the understanding of NSCLC resistance to chemotherapy and present Orai3 as a promising biomarker which could help in the choice of chemotherapeutic drugs for patients with NSCLCDepuis son approbation par la FDA (Food and Drug Administration) en 1978, la thérapie par les sels de platine reste le traitement chimiothérapeutique standard actuel pour le cancer du poumon non à petites cellules (CPNPC). Cependant, la résistance aux sels de platine évolue rapidement chez les patients atteints de CPNPC et l'une des principales raisons de cette résistance est leur capacité à enrichir la population des cellules souches cancéreuses (CSC). Les canaux calciques de type SOC (Store Operated Channel) représentent la voie d'entrée majeure du calcium dans les cellules non excitables et sont impliqués dans différents processus cancéreux notamment la survie. Parmi ces canaux, le canal calcique Orai3 a été récemment identifié comme un élément important dans la résistance à la chimiothérapie dans les cellules cancéreuses du sein. De plus, ce canal est surexprimé dans les cancers CPNPC, est corrélé au grade tumoral élevé et constitue un marqueur prédictif de métastases et de mauvais pronostic chez les patients atteints de tumeurs CPNPC résécables. In-vitro, Orai3 régule la prolifération des cellules CPNPC d'une façon calcium-dépendante, via la voie Akt. À partir de ces données, mes travaux de thèse ont porté sur l'étude de l'effet potentiel du canal Orai3 dans la résistance au Cisplatine ainsi que les voies de signalisation associées dans le cancer du poumon. Nos résultats, sur lignées cellulaires et sur tissus cancéreux, montrent très clairement que le traitement chimiothérapeutique augmente l'expression d'Orai3. Cliniquement, l'augmentation de l'expression d'Orai3, après un traitement chimiotérapeutique, est associée à la non régression ou à une régression partielle de la tumeur. Dans les lignées cellulaires CPNPC, un traitement au Cisplatine augmente l'expression d'Orai3 d'une façon temps-dépendante. Cet effet s'accompagne par l'augmentation de la population des cellules souches cancéreuses (marquage avec CD133) et aussi des marqueurs des CSC notamment Nanog, SOX2 et Slug. L'invalidation de l'expression d'Orai3 ou la réduction de la concentration du calcium extracellulaire sensibilise les cellules au Cisplatine, et réduit totalement l'expression de Nanog et SOX2 et partiellement l'expression de Slug. Par ailleurs, la surexpression d'Orai3 induit la surexpression de Nanog et SOX2. D'une façon intéressante, nous avons constaté un changement de la fonction d'Orai3 en fonction du traitement au Cisplatine. En effet, Orai3 devient SOC après traitement au Cisplatine, ou dans les cellules surexprimant Orai3, alors qu'il régule l'entrée calcique basal avant le traitement ou dans les cellules transfectées avec le vecteur vide. Cette fonction SOC d'Orai3 est retrouvée aussi dans les cellules souches cancéreuses. Finalement, nous avons montré qu'en inhibant la voie Akt, aucune augmentation des marqueurs de CSC n'a été observée et la réponse au Cisplatine a été favorisée. En conclusion, l'augmentation de l'expression d'Orai3 permet à ce canal de devenir SOC favorisant ainsi l'entrée du calcium. Ce dernier, via la voie Akt, permet l'enrichissement de la population des CSC (augmentation des marqueurs des CSC) insensibles au Cisplatine. L'ensemble de nos résultats montre, pour la première fois, le rôle du canal Orai3 dans la résistance à la chimiothérapie due à l'augmentation de la population des cellules souches. Par conséquent, Orai3 pourrait constituer un biomarqueur prédictif du choix des médicaments chimiothérapeutique

Orai3-Mediates Cisplatin-Resistance in Non-Small Cell Lung Cancer Cells by Enriching Cancer Stem Cell Population through PI3K/AKT Pathway

International audienceSimple Summary Lung cancer is recognized for having a very poor prognosis with an overall survival rate of 5-years not exceeding 15%. Platinum-doublet therapy is the most current chemotherapeutic treatment used to treat lung tumors. However, resistance to such drugs evolves rapidly in patients with non-small cell lung cancer (NSCLC) and is one of the major reasons behind therapy failure. Tumor recurrence due to chemoresistance is mainly attributed to the presence of cancer stem cells (CSCs) subpopulations. Thus, the identification of resistance actors and markers is necessary. The Orai3 channel has been recently identified as a predictive marker of metastasis and survival in resectable NSCLC tumors. Our results show, for the first time, that the Orai3 channel is able to induce chemoresistance by enriching CSCs population. Our findings present Orai3 as a promising predictive biomarker which could help with selecting chemotherapeutic drugs. The development of the resistance to platinum salts is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). Among the reasons underlying this resistance is the enrichment of cancer stem cells (CSCs) populations. Several studies have reported the involvement of calcium channels in chemoresistance. The Orai3 channel is overexpressed and constitutes a predictive marker of metastasis in NSCLC tumors. Here, we investigated its role in CSCs populations induced by Cisplatin (CDDP) in two NSCLC cell lines. We found that CDDP treatment increased Orai3 expression, but not Orai1 or STIM1 expression, as well as an enhancement of CSCs markers. Moreover, Orai3 silencing or the reduction of extracellular calcium concentration sensitized the cells to CDDP and led to a reduction in the expression of Nanog and SOX-2. Orai3 contributed to SOCE (Store-operated Calcium entry) in both CDDP-treated and CD133(+) subpopulation cells that overexpress Nanog and SOX-2. Interestingly, the ectopic overexpression of Orai3, in the two NSCLC cell lines, lead to an increase of SOCE and expression of CSCs markers. Furthermore, CD133(+) cells were unable to overexpress neither Nanog nor SOX-2 when incubated with PI3K inhibitor. Finally, Orai3 silencing reduced Akt phosphorylation. Our work reveals a link between Orai3, CSCs and resistance to CDDP in NSCLC cells

Ion Channels: New Actors Playing in Chemotherapeutic Resistance

In the battle against cancer cells, therapeutic modalities are drastically limited by intrinsic or acquired drug resistance. Resistance to therapy is not only common, but expected: if systemic agents used for cancer treatment are usually active at the beginning of therapy (i.e., 90% of primary breast cancers and 50% of metastases), about 30% of patients with early-stage breast cancer will have recurrent disease. Altered expression of ion channels is now considered as one of the hallmarks of cancer, and several ion channels have been linked to cancer cell resistance. While ion channels have been associated with cell death, apoptosis and even chemoresistance since the late 80s, the molecular mechanisms linking ion channel expression and/or function with chemotherapy have mostly emerged in the last ten years. In this review, we will highlight the relationships between ion channels and resistance to chemotherapy, with a special emphasis on the underlying molecular mechanisms

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licenseBackground: Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide. Methods: A multimethods analysis was performed as part of the GlobalSurg 3 study—a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital. Findings: Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3·85 [95% CI 2·58–5·75]; p<0·0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63·0% vs 82·7%; OR 0·35 [0·23–0·53]; p<0·0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer. Interpretation: Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised. Funding: National Institute for Health and Care Research