Caractérisation de nouveaux régulateurs de la sénescence induite par un stress oncogénique dans les cellules épithéliales humaines

Senescence is a stable proliferation arrest triggered by several cellular stresses such as telomeres shortening (replicative senescence) or oncogenic activation (oncogene-induced senescence, OIS). Senescence counteracts proliferation of malignant cells, and as such, constitutes a failsafe program. Senescence was first evidenced in fibroblasts, and most of the following studies were conducted in human or murine fibroblasts, allowing the identification of p53 and pRb pathways as strong regulators of senescence. However the few studies investigating senescence pathways involved in other cell types, such as melanocytes or epithelial cells unveiled new p53/pRB- independent mechanisms. The aim of my thesis was then to characterize new senescence regulators in human epithelial cells. We were first interested in characterizing lysyl oxidase activity (Lox) on OIS escape. LOX enzymes are mainly known to favor metastatic processes. Lox activity inhibition stabilized OIS in vitro, but also senescence in a transgenic murine model of pancreatic ductal adenocarcinoma. This work demonstrated that Lox activity is involved in tumoral initiation by promoting senescence escape. Using a loss-of-function screen, we identified several genes whose down-regulation allowed OIS escape of human mammary epithelial cells. Among them I have characterized ITPR2 and MCU, two calcium-related channels. Loss of ITPR2, known to mediate endoplasmic reticulum (ER) calcium release, as well as loss of MCU, necessary for mitochondrial calcium uptake, enable escape from OIS. During OIS, ITPR2 triggers calcium release from the ER, followed by mitochondrial calcium accumulation through MCU channels. Mitochondrial calcium accumulation leads to a subsequent decrease in mitochondrial membrane potential, reactive oxygen species accumulation and senescence. This ER-mitochondria calcium transport is not restricted to OIS, but is also involved in replicative senescence. Our results show a functional role of calcium release by the ITPR2 channel and its subsequent accumulation in the mitochondriaLa sénescence est un arrêt stable de prolifération mis en place en réponse à différents stress cellulaires, comme le raccourcissement des télomères (sénescence réplicative) ou le stress oncogénique (sénescence induite par un oncogène, OIS) et constitue un processus s'opposant à la prolifération des cellules tumorales. La plupart des études menées dans des fibroblastes ont permis d'identifier p53 et pRb comme acteurs majeurs de la sénescence. Toutefois, l'étude de l'OIS dans les mélanocytes ou les cellules épithéliales a révélé de nouveaux mécanismes, n'impliquant pas obligatoirement ces deux voies canoniques. L'objectif de ma thèse est de caractériser de nouveaux régulateurs de l'OIS dans les cellules épithéliales. Pour cela deux approches différentes ont été utilisées. Dans une première partie, je me suis intéressée aux effets de l'activité portée par les lysyl oxydases (LOX), famille d'enzyme connue pour favoriser le processus métastatique, sur l'échappement à l'OIS. L'inhibition de LOX et LOXL2 stabilise l'OIS dans les cellules épithéliales mammaires humaines et dans un modèle murin d'adénocarcinomes du pancréas. Ce travail a permis de montrer que l'activité Lox est impliquée dans l'initiation tumorale. Dans un deuxième temps, en utilisant un criblage perte de fonction nous avons isolé plusieurs gènes dont l'inhibition stable d'expression permet un échappement à l'OIS dans les cellules épithéliales mammaire humaines. Parmi eux, j'ai caractérisé l'implication de deux canaux calciques, ITPR2 et MCU, dans l'échappement à la sénescence. ITPR2, qui permet la sortie de calcium du réticulum endoplasmique, et MCU, qui permet l'entrée de calcium dans la matrice de la mitochondrie, s'avèrent être deux acteurs nécessaires à la signalisation calcique lors de l'OIS. De manière importante, les mouvements calciques sont associés à une chute du potentiel de membrane mitochondrial, et à la génération d'espèces réactives de l'oxygène. Ce travail montre que les mouvements calciques semblent également être impliqués dans le processus de sénescence réplicativ

Characterization of new oncogene-induced senescence regulators in human epithelial cells

La sénescence est un arrêt stable de prolifération mis en place en réponse à différents stress cellulaires, comme le raccourcissement des télomères (sénescence réplicative) ou le stress oncogénique (sénescence induite par un oncogène, OIS) et constitue un processus s'opposant à la prolifération des cellules tumorales. La plupart des études menées dans des fibroblastes ont permis d'identifier p53 et pRb comme acteurs majeurs de la sénescence. Toutefois, l'étude de l'OIS dans les mélanocytes ou les cellules épithéliales a révélé de nouveaux mécanismes, n'impliquant pas obligatoirement ces deux voies canoniques. L'objectif de ma thèse est de caractériser de nouveaux régulateurs de l'OIS dans les cellules épithéliales. Pour cela deux approches différentes ont été utilisées. Dans une première partie, je me suis intéressée aux effets de l'activité portée par les lysyl oxydases (LOX), famille d'enzyme connue pour favoriser le processus métastatique, sur l'échappement à l'OIS. L'inhibition de LOX et LOXL2 stabilise l'OIS dans les cellules épithéliales mammaires humaines et dans un modèle murin d'adénocarcinomes du pancréas. Ce travail a permis de montrer que l'activité Lox est impliquée dans l'initiation tumorale. Dans un deuxième temps, en utilisant un criblage perte de fonction nous avons isolé plusieurs gènes dont l'inhibition stable d'expression permet un échappement à l'OIS dans les cellules épithéliales mammaire humaines. Parmi eux, j'ai caractérisé l'implication de deux canaux calciques, ITPR2 et MCU, dans l'échappement à la sénescence. ITPR2, qui permet la sortie de calcium du réticulum endoplasmique, et MCU, qui permet l'entrée de calcium dans la matrice de la mitochondrie, s'avèrent être deux acteurs nécessaires à la signalisation calcique lors de l'OIS. De manière importante, les mouvements calciques sont associés à une chute du potentiel de membrane mitochondrial, et à la génération d'espèces réactives de l'oxygène. Ce travail montre que les mouvements calciques semblent également être impliqués dans le processus de sénescence réplicativeSenescence is a stable proliferation arrest triggered by several cellular stresses such as telomeres shortening (replicative senescence) or oncogenic activation (oncogene-induced senescence, OIS). Senescence counteracts proliferation of malignant cells, and as such, constitutes a failsafe program. Senescence was first evidenced in fibroblasts, and most of the following studies were conducted in human or murine fibroblasts, allowing the identification of p53 and pRb pathways as strong regulators of senescence. However the few studies investigating senescence pathways involved in other cell types, such as melanocytes or epithelial cells unveiled new p53/pRB- independent mechanisms. The aim of my thesis was then to characterize new senescence regulators in human epithelial cells. We were first interested in characterizing lysyl oxidase activity (Lox) on OIS escape. LOX enzymes are mainly known to favor metastatic processes. Lox activity inhibition stabilized OIS in vitro, but also senescence in a transgenic murine model of pancreatic ductal adenocarcinoma. This work demonstrated that Lox activity is involved in tumoral initiation by promoting senescence escape. Using a loss-of-function screen, we identified several genes whose down-regulation allowed OIS escape of human mammary epithelial cells. Among them I have characterized ITPR2 and MCU, two calcium-related channels. Loss of ITPR2, known to mediate endoplasmic reticulum (ER) calcium release, as well as loss of MCU, necessary for mitochondrial calcium uptake, enable escape from OIS. During OIS, ITPR2 triggers calcium release from the ER, followed by mitochondrial calcium accumulation through MCU channels. Mitochondrial calcium accumulation leads to a subsequent decrease in mitochondrial membrane potential, reactive oxygen species accumulation and senescence. This ER-mitochondria calcium transport is not restricted to OIS, but is also involved in replicative senescence. Our results show a functional role of calcium release by the ITPR2 channel and its subsequent accumulation in the mitochondri

Tissue specificity of oncogenic BRAF targeted to lung and thyroid through a shared lineage factor

Summary: Cells of origin in cancer determine tumor phenotypes, but whether lineage-defining transcription factors might influence tissue specificity of tumorigenesis among organs with similar developmental traits are unknown. We demonstrate here that tumor development and progression markedly differ in lung and thyroid targeted by Braf mutation in Nkx2.1CreERT2 mice heterozygous for Nkx2-1. In absence of tamoxifen, non-induced Nkx2.1CreERT2;BrafCA/+ mutants developed multiple full-blown lung adenocarcinomas with a latency of 1–3 months whereas thyroid tumors were rare and constrained, although minute BrafCA activation documented by variant allele sequencing was similar in both tissues. Induced oncogene activation accelerated neoplastic growth only in the lungs. By contrast, NKX2-1+ progenitor cells were equally responsive to constitutive expression of mutant Braf during lung and thyroid development. Both lung and thyroid cells transiently downregulated NKX2-1 in early tumor stages. These results indicate that BRAFV600E-induced tumorigenesis obey organ-specific traits that might be differentially modified by a shared lineage factor

<i>KRAS</i> Mutations Impact Clinical Outcome in Metastatic Non-Small Cell Lung Cancer

There is an urgent need to identify new predictive biomarkers for treatment response to both platinum doublet chemotherapy (PT) and immune checkpoint blockade (ICB). Here, we evaluated whether treatment outcome could be affected by KRAS mutational status in patients with metastatic (Stage IV) non-small cell lung cancer (NSCLC). All consecutive patients molecularly assessed and diagnosed between 2016–2018 with Stage IV NSCLC in the region of West Sweden were included in this multi-center retrospective study. The primary study outcome was overall survival (OS). Out of 580 Stage IV NSCLC patients, 35.5% harbored an activating mutation in the KRAS gene (KRASMUT). Compared to KRAS wild-type (KRASWT), KRASMUT was a negative factor for OS (p = 0.014). On multivariate analysis, KRASMUT persisted as a negative factor for OS (HR 1.478, 95% CI 1.207–1.709, p n = 195), KRASMUT was a negative factor for survival (p = 0.018), with median OS of 9 months vs. KRASWT at 11 months. On multivariate analysis, KRASMUT persisted as a negative factor for OS (HR 1.564, 95% CI 1.124–2.177, p = 0.008). KRASMUT patients with high PD-L1 expression (PD-L1high) had better OS than PD-L1highKRASWT patients (p = 0.036). In response to first-line ICB, KRASMUT patients had a significantly (p = 0.006) better outcome than KRASWT patients, with a median OS of 23 vs. 6 months. On multivariable Cox analysis, KRASMUT status was an independent prognostic factor for better OS (HR 0.349, 95% CI 0.148–0.822, p = 0.016). kRAS mutations are associated with better response to treatment with immune checkpoint blockade and worse response to platinum doublet chemotherapy as well as shorter general OS in Stage IV NSCLC

The SCN9A channel and plasma membrane depolarization promote cellular senescence through Rb pathway

International audienceOncogenic signals lead to premature senescence in normal human cells causing a proliferation arrest and the elimination of these defective cells by immune cells. Oncogene-induced senescence (OIS) prevents aberrant cell division and tumor initiation. In order to identify new regulators of OIS, we performed a loss-of-function genetic screen and identified that the loss of SCN9A allowed cells to escape from OIS. The expression of this sodium channel increased in senescent cells during OIS. This upregulation was mediated by NF-kappaB transcription factors, which are well-known regulators of senescence. Importantly, the induction of SCN9A by an oncogenic signal or by p53 activation led to plasma membrane depolarization, which in turn, was able to induce premature senescence. Computational and experimental analyses revealed that SCN9A and plasma membrane depolarization mediated the repression of mitotic genes through a calcium/Rb/E2F pathway to promote senescence. Taken together, our work delineates a new pathway, which involves the NF-kappaB transcription factor, SCN9A expression, plasma membrane depolarization, increased calcium, the Rb/E2F pathway and mitotic gene repression in the regulation of senescence. This work thus provides new insight into the involvement of ion channels and plasma membrane potential in the control of senescence

Lysyl oxidase family activity promotes resistance of pancreatic ductal adenocarcinoma to chemotherapy by limiting the intratumoral anticancer drug distribution

Solid tumors often display chemotherapy resistance. Pancreatic ductal adenocarcinoma (PDAC) is the archetype of resistant tumors as current chemotherapies are inefficient. The tumor stroma and extracellular matrix (ECM) are key contributors to PDAC aggressiveness and to limiting the efficacy of chemotherapy. Lysyl oxidase (LOX) family members mediate collagen cross-linking and thus promote ECM stiffening. Our data demonstrate increased LOX, LOXL1, and LOXL2 expression in PDAC, and that the level of fibrillar collagen, which is directly dependent of LOX family activity, is an independent predictive biomarker of adjuvant "Gemcitabinebased chemotherapy" benefit. Experimentally in mice, increased LOX family activity through LOXL2 promotes chemoresistance. This effect of LOX family activity seems to be due to decreased gemcitabine intra-tumoral diffusion. This observation might be explained by increased fibrillar collagen and decreased vessel size observed in tumors with increased LOX family activity. In conclusion, our data support that LOX family activity is both a novel target to improve chemotherapy as well as a novel biomarker to predict gemcitabine benefit in PDAC. Beyond the PDAC, it is possible that targeting LOX family activity might improve efficacy of chemotherapies against different kinds of solid tumors.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Antioxidants Promote Intestinal Tumor Progression in Mice

Dietary antioxidants and supplements are widely used to protect against cancer, even though it is now clear that antioxidants can promote tumor progression by helping cancer cells to overcome barriers of oxidative stress. Although recent studies have, in great detail, explored the role of antioxidants in lung and skin tumors driven by RAS and RAF mutations, little is known about the impact of antioxidant supplementation on other cancers, including Wnt-driven tumors originating from the gut. Here, we show that supplementation with the antioxidants N-acetylcysteine (NAC) and vitamin E promotes intestinal tumor progression in the ApcMin mouse model for familial adenomatous polyposis, a hereditary form of colorectal cancer, driven by Wnt signaling. Both antioxidants increased tumor size in early neoplasias and tumor grades in more advanced lesions without any impact on tumor initiation. Importantly, NAC treatment accelerated tumor progression at plasma concentrations comparable to those obtained in human subjects after prescription doses of the drug. These results demonstrate that antioxidants play an important role in the progression of intestinal tumors, which may have implications for patients with or predisposed to colorectal cancer