Solid tumours are characterised by an altered microenvironment (TME) from the physicochemical point of view, displaying a highly hypoxic and acidic interstitial fluid. Hypoxia results from uncontrolled proliferation, aberrant vascularization and altered cancer cell metabolism. Tumour cellular apparatus adapts to hypoxia by altering its metabolism and behaviour, increasing its migratory and metastatic abilities by the acquisition of a mesenchymal phenotype and selection of aggressive tumour cell clones. Extracellular acidosis is considered a cancer hallmark, acting as a driver of cancer aggressiveness by promoting tumour metastasis and chemoresistance via the selection of more aggressive cell phenotypes, although the underlying mechanism is still not clear. In this context, Ca²⁺ channels represent good target candidates due to their ability to integrate signals from the TME. Ca²⁺ channels are pH and hypoxia sensors and alterations in Ca²⁺ homeostasis in cancer progression and vascularization have been extensively reported. In the present review, we present an up-to-date and critical view on Ca²⁺ permeable ion channels, with a major focus on TRPs, SOCs and PIEZO channels, which are modulated by tumour hypoxia and acidosis, as well as the consequent role of the altered Ca²⁺ signals on cancer progression hallmarks. We believe that a deeper comprehension of the Ca²⁺ signalling and acidic pH/hypoxia interplay will break new ground for the discovery of alternative and attractive therapeutic targets.

Emerging evidence indicates that the TRPM8 channel plays an important role in prostate cancer (PCa) progression, by impairing the motility of these cancer cells. Here, we reveal a novel facet of PCa motility control via direct protein-protein interaction (PPI) of the channel with the small GTPase Rap1A. The functional interaction of the two proteins was assessed by active Rap1 pull-down assays and live-cell imaging experiments. Molecular modeling analysis allowed the identification of four putative residues involved in TRPM8-Rap1A interaction. Point mutations of these sites impaired PPI as shown by GST-pull-down, co-immunoprecipitation, and PLA experiments and revealed their key functional role in the adhesion and migration of PC3 prostate cancer cells. More precisely, TRPM8 inhibits cell migration and adhesion by trapping Rap1A in its GDP-bound inactive form, thus preventing its activation at the plasma membrane. In particular, residues E207 and Y240 in the sequence of TRPM8 and Y32 in that of Rap1A are critical for the interaction between the two proteins not only in PC3 cells but also in cervical (HeLa) and breast (MCF-7) cancer cells. This study deepens our knowledge of the mechanism through which TRPM8 would exert a protective role in cancer progression and provides new insights into the possible use of TRPM8 as a new therapeutic target in cancer treatment.

Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by an acidic microenvironment, which contributes to therapeutic failure. So far there is a lack of knowledge with respect to the role of the acidic microenvironment in the invasive process. This work aimed to study the phenotypic and genetic response of PDAC cells to acidic stress along the different stages of selection. To this end, we subjected the cells to short- and long-term acidic pressure and recovery to pH_e 7.4. This treatment aimed at mimicking PDAC edges and consequent cancer cell escape from the tumor. The impact of acidosis was assessed for cell morphology, proliferation, adhesion, migration, invasion, and epithelial–mesenchymal transition (EMT) via functional in vitro assays and RNA sequencing. Our results indicate that short acidic treatment limits growth, adhesion, invasion, and viability of PDAC cells. As the acid treatment progresses, it selects cancer cells with enhanced migration and invasion abilities induced by EMT, potentiating their metastatic potential when re-exposed to pH_e 7.4. The RNA-seq analysis of PANC-1 cells exposed to short-term acidosis and pH_e-selected recovered to pH_e 7.4 revealed distinct transcriptome rewiring. We describe an enrichment of genes relevant to proliferation, migration, EMT, and invasion in acid-selected cells. Our work clearly demonstrates that upon acidosis stress, PDAC cells acquire more invasive cell phenotypes by promoting EMT and thus paving the way for more aggressive cell phenotypes.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with high mortality due to early metastatic dissemination and high chemoresistance. All these factors are favored by its extracellular matrix (ECM)-rich microenvironment, which is also highly hypoxic and acidic. Gemcitabine (GEM) is still the first-line therapy in PDAC. However, it is quickly deaminated to its inactive metabolite. Several GEM prodrugs have emerged to improve its cytotoxicity. Here, we analyzed how the acidic/hypoxic tumor microenvironment (TME) affects the response of PDAC cell death and invadopodia-mediated ECM proteolysis to both GEM and its C18 prodrug. Methods: For this, two PDAC cell lines, PANC-1 and Mia PaCa-2 were adapted to pH_e 6.6 or not for 1 month, grown as 3D organotypic cultures and exposed to either GEM or C18 in the presence and absence of acidosis and the hypoxia inducer, deferoxamine. Results: We found that C18 has higher cytotoxic and anti-invadopodia activity than GEM in all culture conditions and especially in acid and hypoxic environments. Conclusions: We propose C18 as a more effective approach to conventional GEM in developing new therapeutic strategies overcoming PDAC chemoresistance.

Solid tumours are characterized by an altered microenvironment (TME) from the physi-cochemical point of view, displaying a highly hypoxic and acidic interstitial fluid. Hy-poxia results from uncontrolled proliferation, aberrant vascularization and altered can-cer cell metabolism. Tumour cellular apparatus adapt to hypoxia by altering its metab-olism and behaviour, increasing its migratory and metastatic abilities by acquisition of a mesenchymal phenotype and selection of aggressive tumour cell clones. Extracellular acidosis is considered a cancer hallmark, acting as a driver of cancer aggressiveness by promoting tumour metastasis and chemoresistance by selecting for more aggressive cell phenotypes, although the underlying mechanism is still not clear. In this context, Ca2+ channels represent good target candidates due to their ability to integrate signals from the TME. Ca2+ channels are pH and hypoxia sensors and alterations in Ca2+ homeostasis in cancer progression and vascularization have been extensively reported. The present review will focus on Ca2+ permeable ion channels, with a major focus on TRP, SOC and PIEZO channels, that are modulated by tumour hypoxia and acidosis as well as the role of the resulted altered Ca2+ signals on cancer progression hallmarks. A deeper compre-hension of the Ca2+ signaling and acidic pH/hypoxia interplay will break new ground for the discovery of alternative and attractive therapeutics targets.

Interaction du microenvironnement tumoral acide et de la signalisation calcique dans la progression de l'adénocarcinome du canal pancréatique (PDAC): L'adénocarcinome canalaire pancréatique (PDAC) est une maladie mortelle caractérisée par un micro-environnement tumoral (TME) extrêmement acide (˂pHe 6,5) qui joue un rôle important dans son début et sa progression. Dans ce contexte, les canaux ioniques perméables au Ca2+ représentent de bons candidats cibles en raison de leur capacité à intégrer des signaux provenant de la TME. Les canaux Ca2+ sont en effet des capteurs de pH capables d'intégrer les signaux de la TME pour activer les voies intracellulaires en aval liées à la progression du PDAC. Bien que les rôles de l'acidose tumorale et de la signalisation du Ca2+ dans la progression du cancer soient bien établis, l'hypothèse d'une TME acide utilisant la signalisation du Ca2+ comme voie préférentielle pour soutenir la progression tumorale n'a pas encore été suffisamment explorée.Mon travail de doctorat visait à étudier les changements phénotypiques et génétiques des cellules PDAC lors d'un stress acide au cours des différentes étapes de sélection et à évaluer comment l'acidose tumorale module les signaux Ca2+ et les phénotypes dans les lignées cellulaires PDAC, avec un accent particulier sur les oscillations Ca2+ et Store-Operated Ca2+ entry (SOCE). À cette fin, les cellules PANC-1 et Mia PaCa-2 ont été soumises à une pression acide à court et à long terme et à une récupération à pHe 7,4. Ce dernier traitement visait à imiter les bords du PDAC et l'évasion consécutif des cellules cancéreuses de la tumeur. L'impact de l'acidose a été évalué sur la morphologie cellulaire, la prolifération, l'adhésion, la migration, l'invasion, l'activité des invadopodes et la transition épithélio-mésenchymateuse (TEM) par des tests fonctionnels in vitro et le séquençage de l'ARN, ainsi que sur les signaux Ca2+ intracellulaires avec Fura-2. Nos résultats indiquent qu'un court traitement acide limite la croissance, l'adhésion, l'invasion et la viabilité des cellules PDAC. Au fur et à mesure que le traitement acide progresse, il sélectionne les cellules cancéreuses ayant des capacités de migration et d'invasion accrues induites par l'EMT, ce qui augmente encore leur potentiel métastatique lorsqu'elles sont réexposées à un pHe 7,4. L'analyse RNA-seq des cellules PANC-1 exposées à une acidose de courte durée et récupérées à pHe 7,4 a révélé un recâblage transcriptomique distinct. Il est intéressant de noter que les cellules PANC-1 sont caractérisées par des oscillations Ca2+ plus lentes pendant une exposition à l'acide à court terme par rapport aux cellules de contrôle et par une tendance à la régulation négative d'ORAI1 au niveau de l'ARNm, tandis que l'acidose à long terme et le rétablissement à un pH neutre déterminent le rétablissement d'oscillations Ca2+ rapides et la régulation positive d'ORAI1. Dans tous nos modèles cellulaires, les oscillations du Ca2+ sont dépendantes de SOCE, car le blocage d'ORAI1 par Synta66 et siORAI1 entraîne une altération de l'initiation et du maintien des oscillations du Ca2+. Ces données sont en corrélation avec le SOCE dans les cellules PANC-1, qui est diminuée pendant le traitement acide à court terme, et augmentée dans les cellules sélectionnées pour l'acide avec et sans récupération à pHe 7,4. Enfin, l'entrée de Ca2+ médiée par ORAI1 pourrait être impliquée dans l'activation des cascades de signalisation qui conduisent à l'augmentation de la migration et de l'invasion de tous les modèles cellulaires exposés à un pHe acide, car le traitement par Synta66 et siORAI1 n'ont pas affecté l'invasion et la migration des cellules de contrôle.En conclusion, nos résultats montrent que la sélection induite par l'acide contribue à l'acquisition d'un phénotype plus agressif dans les cellules de PDAC, caractérisé par une augmentation du SOCE, nécessaire à la génération d'oscillations rapides de Ca2+ qui peuvent activer des voies de signalisation Ca2+-dépendantes impliquées dans la progression du PDAC.