The beta isoform of PI3K control of Kras oncogenicity in pancreatic carcinogenesis

La voie de signalisation PI3K/Akt/mTOR est une de plus altérées dans les cancers. Bien que toutes les PI3Ks de classe I (p110α, p110ß, p110δ et p110γ) phosphorylent le PIP2 en PIP3, qui est un important second messager lipidique, chaque isoforme possède des rôles physiologiques et pathologiques spécifiques. Le faible effet des inhibiteurs pan-PI3Ks (ciblant tous les isoformes: p110α, p110ß, p110δ et p110γ), testés en clinique sur différents types de cancer, a mis en évidence la nécessité de comprendre les rôles de chaque isoforme de PI3K dans la cellule cancéreuse, avec des inhibiteurs isoforme-spécifiques de PI3K et des modèles murins qui ciblent conditionnellement leur activité. L'adénocarcinome pancréatique (PDAC) est un des cancers les plus létaux. La voie PI3K est suractivée dans la moitié des PDAC et corrélée à un mauvais pronostic. Différent travaux ont démontré que les deux isoformes ubiquitaires PI3Kα et PI3Kß jouent des rôles différents dans la signalisation cellulaire, la croissance et la transformation oncogénique. Pourtant, comment les différents PI3K contribuent à la formation du PDAC n'est pas entièrement clair. Afin d'élucider ces mécanismes, mon équipe d'accueil, a développé des modèles murins uniques, qui récapitulent la carcinogenèse pancréatique et qui ciblent conditionnellement l'activité de la PI3Kα ou de la PI3Kß spécifiquement dans la cellule épithéliale pancréatique. Alors que l'isoforme a de PI3K est nécessaire à la transformation de cellules acineuses par Kras oncogénique, l'inactivation génétique de p110ß dans les cellules progénitrices pdx1 positives n'empêche pas la formation in vivo de structures canalaires induites par la mutation de Kras (Baer et al., 2014). Cependant, ces lésions sont phénotypiquement différentes et la létalité induite par Kras muté est réduite dans le contexte où PI3Kß est génétiquement inactivé. L'objectif principal de ma thèse était de comprendre le rôle de la PI3Kß au cours de la carcinogenèse pancréatique, induite par Kras oncogénique, avec ou sans p53 muté. Dans un deuxième temps, j'ai aussi analysé l'impact biologique de l'inactivation génétique de la PI3Kß, dans la cellule épithéliale pancréatique, en utilisant un modèle d'accélération de la cancérogenèse pancréatique (induction d'une inflammation dans un contexte génétique Kras muté). En particulier, j'ai étudié les mécanismes d'action de la PI3Kß dans l'oncogénèse et dans la formation du stroma induit par les lésions. Dans nos modèles murins, l'inhibition de PI3Kß protège les souris de la létalité induite par la mutation de Kras et p53 et conduit à la formation de lésions néoplasiques de plus bas grade. De manière intéressante, l'inactivation de la PI3Kß dans les cellules épithéliales du pancréas est responsable de la modification du recrutement des cellules immunitaires et de la réponse stromale à Kras oncogénique ou à l'inflammation. En conclusion, PI3Kß dans la cellule épithéliale pancréatique joue un rôle important dans l'initiation du cancer du pancréas dans la souris, en favorisant la dédifférenciation cellulaire et en reprogrammant le microenvironnement de la tumeur.PI3K/Akt/mTOR pathway is one of the most deregulated signaling pathway in cancers. In particular, PI3Ks are key signal transduction enzymes which are generally considered to be excellent new targets for therapeutic interference. Despite their high similarities, there is strong evidence that class IA PI3K isoforms have non-redundant roles. The modest effect of pan-PI3Ks inhibitors (targeting all p110 isoforms) clinically tested on different types of cancer, highlighted the need to understand the roles of each PI3K isoform in cancer cell, with isoform-specific inhibitors of PI3K and murine models that conditionally target their activity. Class I PI3K signaling is altered (constitutively active) and correlated with a poor prognostic in pancreatic ductal adenocarcinoma. Different works have demonstrated that the two ubiquitously expressed catalytic subunit PI3Kα and PI3Kß play distinct roles in cellular signaling, growth, and oncogenic transformation. Despite that, in pancreatic cancer is still not clear how the different PI3K isoforms contribute to PDAC formation. In order to understand that my team has developed unique mouse models that recapitulate the carcinogenesis of PDAC and that conditionally target the activity of PI3Kα or PI3Kß specifically in pancreatic epithelial cells. While the isoform α of PI3K is necessary for the transformation of acinar cells by oncogenic Kras, the genetic inactivation of the PI3Kß catalytic isoform in the progenitor lineage pdx1 positive does not prevent in vivo the formation of ductal structures induced by the mutation of Kras in the same cell lineage (Baer et al., 2014). However, these lesions are phenotypically different and the lethality induced by such oncogenic mutations is reduced in PI3Kß activity-deficient genetic context. The main aim of my thesis was to understand the role of PI3Kß in PDAC carcinogenesis by first analyzing the biological impact of pancreas-restricted genetic inactivation of PI3Kß during pancreatic cancer progression induced by oncogenic Kras with or without mutated p53 combined with experimentally induced risk factor conditions such as inflammation. In particular, I searched the mechanisms of action of PI3Kß in pancreatic carcinogenesis and in lesion-driven stroma formation. Besides, mice harboring genetic inactivation of PI3Kß in their pancreas are protected from mutated Kras and p53-induced lethality. Interestingly, PI3Kß inactivation in pancreatic epithelial cells is responsible for changing the immune cell recruitment and the stromal response to epithelial oncogenic Kras signal or to inflammation. In conclusion, PI3Kß in pancreatic lineage plays an important role in murine pancreatic cancer initiation, by impacting cell dedifferentiation and reprogramming tumor microenvironment

Contrôle de l'oncogenicité de Kras et isoforme beta de PI3K dans la cancérogenèse du pancréas

La voie de signalisation PI3K/Akt/mTOR est une de plus altérées dans les cancers. Bien que toutes les PI3Ks de classe I (p110α, p110ß, p110δ et p110γ) phosphorylent le PIP2 en PIP3, qui est un important second messager lipidique, chaque isoforme possède des rôles physiologiques et pathologiques spécifiques. Le faible effet des inhibiteurs pan-PI3Ks (ciblant tous les isoformes: p110α, p110ß, p110δ et p110γ), testés en clinique sur différents types de cancer, a mis en évidence la nécessité de comprendre les rôles de chaque isoforme de PI3K dans la cellule cancéreuse, avec des inhibiteurs isoforme-spécifiques de PI3K et des modèles murins qui ciblent conditionnellement leur activité. L'adénocarcinome pancréatique (PDAC) est un des cancers les plus létaux. La voie PI3K est suractivée dans la moitié des PDAC et corrélée à un mauvais pronostic. Différent travaux ont démontré que les deux isoformes ubiquitaires PI3Kα et PI3Kß jouent des rôles différents dans la signalisation cellulaire, la croissance et la transformation oncogénique. Pourtant, comment les différents PI3K contribuent à la formation du PDAC n'est pas entièrement clair. Afin d'élucider ces mécanismes, mon équipe d'accueil, a développé des modèles murins uniques, qui récapitulent la carcinogenèse pancréatique et qui ciblent conditionnellement l'activité de la PI3Kα ou de la PI3Kß spécifiquement dans la cellule épithéliale pancréatique. Alors que l'isoforme a de PI3K est nécessaire à la transformation de cellules acineuses par Kras oncogénique, l'inactivation génétique de p110ß dans les cellules progénitrices pdx1 positives n'empêche pas la formation in vivo de structures canalaires induites par la mutation de Kras (Baer et al., 2014). Cependant, ces lésions sont phénotypiquement différentes et la létalité induite par Kras muté est réduite dans le contexte où PI3Kß est génétiquement inactivé. L'objectif principal de ma thèse était de comprendre le rôle de la PI3Kß au cours de la carcinogenèse pancréatique, induite par Kras oncogénique, avec ou sans p53 muté. Dans un deuxième temps, j'ai aussi analysé l'impact biologique de l'inactivation génétique de la PI3Kß, dans la cellule épithéliale pancréatique, en utilisant un modèle d'accélération de la cancérogenèse pancréatique (induction d'une inflammation dans un contexte génétique Kras muté). En particulier, j'ai étudié les mécanismes d'action de la PI3Kß dans l'oncogénèse et dans la formation du stroma induit par les lésions. Dans nos modèles murins, l'inhibition de PI3Kß protège les souris de la létalité induite par la mutation de Kras et p53 et conduit à la formation de lésions néoplasiques de plus bas grade. De manière intéressante, l'inactivation de la PI3Kß dans les cellules épithéliales du pancréas est responsable de la modification du recrutement des cellules immunitaires et de la réponse stromale à Kras oncogénique ou à l'inflammation. En conclusion, PI3Kß dans la cellule épithéliale pancréatique joue un rôle important dans l'initiation du cancer du pancréas dans la souris, en favorisant la dédifférenciation cellulaire et en reprogrammant le microenvironnement de la tumeur.PI3K/Akt/mTOR pathway is one of the most deregulated signaling pathway in cancers. In particular, PI3Ks are key signal transduction enzymes which are generally considered to be excellent new targets for therapeutic interference. Despite their high similarities, there is strong evidence that class IA PI3K isoforms have non-redundant roles. The modest effect of pan-PI3Ks inhibitors (targeting all p110 isoforms) clinically tested on different types of cancer, highlighted the need to understand the roles of each PI3K isoform in cancer cell, with isoform-specific inhibitors of PI3K and murine models that conditionally target their activity. Class I PI3K signaling is altered (constitutively active) and correlated with a poor prognostic in pancreatic ductal adenocarcinoma. Different works have demonstrated that the two ubiquitously expressed catalytic subunit PI3Kα and PI3Kß play distinct roles in cellular signaling, growth, and oncogenic transformation. Despite that, in pancreatic cancer is still not clear how the different PI3K isoforms contribute to PDAC formation. In order to understand that my team has developed unique mouse models that recapitulate the carcinogenesis of PDAC and that conditionally target the activity of PI3Kα or PI3Kß specifically in pancreatic epithelial cells. While the isoform α of PI3K is necessary for the transformation of acinar cells by oncogenic Kras, the genetic inactivation of the PI3Kß catalytic isoform in the progenitor lineage pdx1 positive does not prevent in vivo the formation of ductal structures induced by the mutation of Kras in the same cell lineage (Baer et al., 2014). However, these lesions are phenotypically different and the lethality induced by such oncogenic mutations is reduced in PI3Kß activity-deficient genetic context. The main aim of my thesis was to understand the role of PI3Kß in PDAC carcinogenesis by first analyzing the biological impact of pancreas-restricted genetic inactivation of PI3Kß during pancreatic cancer progression induced by oncogenic Kras with or without mutated p53 combined with experimentally induced risk factor conditions such as inflammation. In particular, I searched the mechanisms of action of PI3Kß in pancreatic carcinogenesis and in lesion-driven stroma formation. Besides, mice harboring genetic inactivation of PI3Kß in their pancreas are protected from mutated Kras and p53-induced lethality. Interestingly, PI3Kß inactivation in pancreatic epithelial cells is responsible for changing the immune cell recruitment and the stromal response to epithelial oncogenic Kras signal or to inflammation. In conclusion, PI3Kß in pancreatic lineage plays an important role in murine pancreatic cancer initiation, by impacting cell dedifferentiation and reprogramming tumor microenvironment

Signal-Targeted Therapies and Resistance Mechanisms in Pancreatic Cancer: Future Developments Reside in Proteomics

International audienceFor patients with metastatic pancreatic cancer that are not eligible for surgery, signal-targeted therapies have so far failed to significantly improve survival. These therapeutic options have been tested in phase II/III clinical trials mostly in combination with the reference treatment gemcitabine. Innovative therapies aim to annihilate oncogenic dependency, or to normalize the tumoural stroma to allow immune cells to function and/or re-vascularisation to occur. Large scale transcriptomic and genomic analysis revealed that pancreatic cancers display great heterogeneity but failed to clearly delineate specific oncogene dependency, besides oncogenic Kras. Beyond these approaches, proteomics appears to be an appropriate approach to classify signal dependency and to identify specific alterations at the targetable level. However, due to difficulties in sampling, proteomic data for this pathology are scarce. In this review, we will discuss the current state of clinical trials for targeted therapies against pancreatic cancer. We will then highlight the most recent proteomic data for pancreatic tumours and their metastasis, which could help to identify major oncogenic signalling dependencies, as well as provide future leads to explain why pancreatic tumours are intrinsically resistant to signal-targeted therapies. We will finally discuss how studies on phosphatidylinositol-3-kinase (PI3K) signalling, as the paradigmatic pro-tumoural signal downstream of oncogenic Kras in pancreatic cancer, would benefit from exploratory proteomics to increase the efficiency of targeted therapies

Atomic force microscopy-single-molecule force spectroscopy unveils GPCR cell surface architecture

International audienceG protein-coupled receptors (GPCRs) form the largest family of cell surface receptors. Despite considerable insights into their pharmacology, the GPCR architecture at the cell surface still remains largely unexplored. Herein, we present the specific unfolding of different GPCRs at the surface of living mammalian cells by atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS). Mathematical analysis of the GPCR unfolding distances at resting state revealed the presence of different receptor populations relying on distinct oligomeric states which are receptor-specific and receptor expression-dependent. Moreover, we show that the oligomer size dictates the receptor spatial organization with nanoclusters of high-order oligomers while lower-order complexes spread over the whole cell surface. Finally, the receptor activity reshapes both the oligomeric populations and their spatial arrangement. These results add an additional level of complexity to the GPCR pharmacology until now considered to arise from a single receptor population at the cell surface.Atomic force microscopy-based single molecule force spectroscopy reveals the unfolding of G-protein coupled receptors on the surface of living mammalian cells

Mechanical Control of Cell Proliferation Increases Resistance to Chemotherapeutic Agents

International audienceWhile many cellular mechanisms leading to chemotherapeutic resistance have been identified, there is an increasing realization that tumor-stroma interactions also play an important role. In particular, mechanical alterations are inherent to solid cancer progression and profoundly impact cell physiology. Here, we explore the influence of compressive stress on the efficacy of chemotherapeutics in pancreatic cancer spheroids. We find that increased compressive stress leads to decreased drug efficacy. Theoretical modeling and experiments suggest that mechanical stress decreases cell proliferation which in turn reduces the efficacy of chemotherapeutics that target proliferating cells. Our work highlights a mechanical form of drug resistance and suggests new strategies for therapy

Targeting the Sphingosine 1-Phosphate Axis Exerts Potent Antitumor Activity in BRAFi-Resistant Melanomas

Abstract BRAF inhibitors (BRAFi) are used to treat patients with melanoma harboring the V600E mutation. However, resistance to BRAFi is inevitable. Here, we identified sphingosine 1-phosphate (S1P) receptors as regulators of BRAFV600E-mutant melanoma cell-autonomous resistance to BRAFi. Moreover, our results reveal a distinct sphingolipid profile, that is, a tendency for increased very long-chain ceramide species, in the plasma of patients with melanoma who achieve a response to BRAFi therapy as compared with patients with progressive disease. Treatment with BRAFi resulted in a strong decrease in S1PR1/3 expression in sensitive but not in resistant cells. Genetic and pharmacologic interventions, that increase ceramide/S1P ratio, downregulated S1PR expression and blocked BRAFi-resistant melanoma cell growth. This effect was associated with a decreased expression of MITF and Bcl-2. Moreover, the BH3 mimetic ABT-737 improved the antitumor activity of approaches targeting S1P-metabolizing enzymes in BRAFi-resistant melanoma cells. Collectively, our findings indicate that targeting the S1P/S1PR axis could provide effective therapeutic options for patients with melanoma who relapse after BRAFi therapy

Crest maturation at the cardiomyocyte surface contributes to a new late postnatal development stage that controls the diastolic function of the adult heart

Abstract RATIONALE In addition to its typical rod-shape, the mammalian adult cardiomyocyte (CM) harbors a unique lateral membrane surface architecture with periodic crests, relying on the presence of subsarcolemmal mitochondria (SSM) the role of which is still unknown. OBJECTIVE To investigate the development and functional role of CM crests during the postnatal period. METHODS AND RESULTS Electron/confocal microscopy and western-blot of left ventricular tissues from rat hearts indicated a late CM surface crest maturation, between postnatal day 20 (P20) and P60, as shown by substantial SSM swelling and increased claudin-5 cell surface expression. The P20-P60 postnatal stage also correlates with an ultimate maturation of the T-Tubules and the intercalated disk. At the cellular level, we identified an atypical CM hypertrophy characterized by an increase in long- and short-axes without myofibril addition and with sarcomere lateral stretching, indicative of lateral stretch-based CM hypertrophy. We confirmed the P20-P60 hypertrophy at the organ level by echocardiography but also demonstrated a transcriptomic program after P20 targeting all the cardiac cell populations. At the functional level, using Doppler echocardiography, we found that the P20-P60 period is specifically dedicated to the improvement of relaxation. Mechanistically, using CM-specific knock-out mice, we identified ephrin-B1 as a determinant of CM crest maturation after P20 controlling lateral CM stretch-hypertrophy and relaxation. Interestingly, while young adult Efnb1 CMspe−/− mice essentially show a relaxation impairment with exercise intolerance, they progressively switch toward heart failure with 100% KO mice dying after 13 months. CONCLUSIONS This study highlights a new late P20-P60 postnatal developmental stage of the heart in rodents during which the CM surface crests mature through an ephrin-B1-dependant mechanism and regulate the diastolic function. Moreover, we demonstrate for the first time that the CM crest architecture is cardioprotective

Ephrin-B1 regulates the adult diastolic function through a late postnatal maturation of cardiomyocyte surface crests

The rod-shaped adult cardiomyocyte (CM) harbors a unique architecture of its lateral surface with periodic crests, relying on the presence of subsarcolemmal mitochondria (SSM) with unknown role. Here, we investigated the development and functional role of CM crests during the postnatal period. We found in rodents that CM crest maturation occurs late between postnatal day 20 (P20) and P60 through both SSM biogenesis, swelling and crest-crest lateral interactions between adjacent CM, promoting tissue compaction. At the functional level, we showed that the P20-P60 period is dedicated to the improvement of relaxation. Interestingly, crest maturation specifically contributes to an atypical CM hypertrophy of its short axis, without myofibril addition, but relying on CM lateral stretching. Mechanistically, using constitutive and conditional CM-specific knock-out mice, we identified ephrin-B1, a lateral membrane stabilizer, as a molecular determinant of P20-P60 crest maturation, governing both the CM lateral stretch and the diastolic function, thus highly suggesting a link between crest maturity and diastole. Remarkably, while young adult CM-specific Efnb1 KO mice essentially exhibit an impairment of the ventricular diastole with preserved ejection fraction and exercise intolerance, they progressively switch toward systolic heart failure with 100% KO mice dying after 13 months, indicative of a critical role of CM-ephrin-B1 in the adult heart function. This study highlights the molecular determinants and the biological implication of a new late P20-P60 postnatal developmental stage of the heart in rodents during which, in part, ephrin-B1 specifically regulates the maturation of the CM surface crests and of the diastolic function