Etude du réticulum endoplasmique et de la mitochondrie en tant que cibles pour la chimiothérapie anti cancéreuse

A travers les travaux que j ai menés au cours de ma thèse, je me suis intéressée au rôle de la mitochondrie et du réticulum endoplasmique (RE) en tant que cibles pour la chimiothérapie anticancéreuse. Ainsi, j ai (i) étudié le rôle de l isoforme 4 de l ANT (ANT4) dans l apoptose des cellules cancéreuses et démontré sa fonction anti-apoptotique, (ii) recherché, parmi les différents membres du PTP, lesquels seraient des cibles permettant de sensibiliser les cellules cancéreuses à l apoptose induite par des agents pontants de l ADN (cisplatine et melphalan) et montré que la surexpression génétique ou pharmacologique de VDAC1 sensibilise les cellules cancéreuses à l apoptose induite par ces agents génotoxiques, (iii) testé le potentiel chimiothérapeutique de la withaférine A (WFA) sur différentes lignées de mélanomes humains et montré que WFA induit l apoptose dans ces cellules via une production d EAO et une diminution de l expression de Bcl-2, et (iv) étudié les mécanismes moléculaires et cellulaires de la mort provoquée par l inhibiteur de Hsp90 PU-H71 sur des lignées cancéreuses et mis en évidence la capacité de cette molécule à induire un stress RE, la voie mitochondriale de l apoptose et à contourner la protection conférée par Bcl-2. J ai également montré que les cellules déficientes pour Bax sont résistantes à l apoptose induite par PU-H71 mais peuvent être sensibilisées à la mort cellulaire en utilisant un traitement combinant PU-H71 et le melphalan ou le cisplatine. Dans ce travail, nous avons donc identifié différentes cibles cellulaires et molécules prometteuses pour le traitement des cancers, notamment dans le cas de tumeurs résistantes aux traitements actuels.During my Ph.D thesis, I studied the endoplasmic reticulum (ER) and the mitochondria as potential targets for anticancer chemotherapy. The role of the isoforme 4 of the ANT (ANT4) in the apoptosis of cancer cells was studied and we demonstrated the anti-apoptotic function of this protein. Besides, we investigated which members of PTP were able to sensitize cancer cells to apoptosis induced by alkylating agents such as cisplatin or melphalan, and showed that genetic or pharmacologic overexpression of VDAC1 sensitizes cancer cells to apoptosis induced by these genotoxic compounds. Furthermore, the chemotherapeutic potential of withaferin A (WFA) against different human melanoma cell lines was tested and we demonstrated that WFA triggers apoptosis through ROS production and decrease of Bcl-2 expression. Finally, the molecular and cellular mechanisms of cell death induced by PU-H71, an Hsp90 inhibitor, were evaluated on cancer cell lines, and we showed that PU-H71 induces ER stress and mitochondrial pathway of apoptosis, and overcomes the protection conferred by Bcl-2 overexpression in cancer cells. Moreover, Bax deficient cells, which are resistant to PU-H71-induced cell death, were sensitized to apoptosis by a combined treatment using PU-H71 and melphalan or cisplatin. Consequently, through this work we identified different cellular targets and promising molecules for cancer treatment, particularly in the case of tumors resistant to conventional chemotherapy.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF

Role of the permeability transition pore complex in lethal crosstalk between endoplasmic reticulum and mitochondria

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Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions

Intrarenal interleukin-15 (IL-15) participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15) isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC) to peritumoral (ptumTEC), tumoral (RCC), and cancer stem cells (CSC/CD105+). RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα) chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15) isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα). This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105+, where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105+ from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa) displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, “apparently normal” ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral “preneoplastic” environment committed to favor tumor progression

The TLR3 L412F polymorphism prevents TLR3-mediated tumor cell death induction in pediatric sarcomas

Abstract Toll-like receptor 3 (TLR3) is a pattern recognition receptor mainly known for its role in innate immune response to infection. Indeed, binding of double-stranded RNA (dsRNA) to TLR3 triggers a pro-inflammatory cascade leading to cytokine release and immune cell activation. Its anti-tumoral potential has emerged progressively, associated with a direct impact on tumor cell death induction and with an indirect action on immune system reactivation. Accordingly, TLR3 agonists are currently being tested in clinical trials for several adult cancers. Meanwhile, TLR3 variants have been linked to auto-immune disorders, and as risk factors of viral infection and cancers. However, aside from neuroblastoma, TLR3 role in childhood cancers has not been evaluated. Here, by integrating public transcriptomic data of pediatric tumors, we unveil that high TLR3 expression is largely associated with a better prognosis in childhood sarcomas. Using osteosarcomas and rhabdomyosarcomas as models, we show that TLR3 efficiently drives tumor cell death in vitro and induces tumor regression in vivo. Interestingly, this anti-tumoral effect was lost in cells expressing the homozygous TLR3 L412F polymorphism, which is enriched in a rhabdomyosarcomas cohort. Thus, our results demonstrate the therapeutic potential associated with the targeting of TLR3 in pediatric sarcomas, but also the need to stratify patients eligible for this clinical approach with respect to the TLR3 variants expressed

Cardiolipin content controls mitochondrial coupling and energetic efficiency in muscle

International audienceUnbalanced energy partitioning participates in the rise of obesity, a major public health concern in many countries. Increasing basal energy expenditure has been proposed as a strategy to fight obesity yet raises efficiency and safety concerns. Here, we show that mice deficient for a muscle-specific enzyme of very-long-chain fatty acid synthesis display increased basal energy expenditure and protection against high-fat diet–induced obesity. Mechanistically, muscle-specific modulation of the very-long-chain fatty acid pathway was associated with a reduced content of the inner mitochondrial membrane phospholipid cardiolipin and a blunted coupling efficiency between the respiratory chain and adenosine 5′-triphosphate (ATP) synthase, which was restored by cardiolipin enrichment. Our study reveals that selective increase of lipid oxidative capacities in skeletal muscle, through the cardiolipin-dependent lowering of mitochondrial ATP production, provides an effective option against obesity at the whole-body level

SIRT1 protects the heart from ER stress-induced cell death through eIF2α deacetylation

Over the past decade, endoplasmic reticulum (ER) stress has emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases including heart failure. Cardiac therapy based on ER stress modulation is viewed as a promising avenue toward effective therapies for the diseased heart. Here, we tested whether sirtuin-1 (SIRT1), a NAD(+)-dependent deacetylase, participates in modulating ER stress response in the heart. Using cardiomyocytes and adult-inducible SIRT1 knockout mice, we demonstrate that SIRT1 inhibition or deficiency increases ER stress-induced cardiac injury, whereas activation of SIRT1 by the SIRT1-activating compound STAC-3 is protective. Analysis of the expression of markers of the three main branches of the unfolded protein response (i.e., PERK/eIF2α, ATF6 and IRE1) showed that SIRT1 protects cardiomyocytes from ER stress-induced apoptosis by attenuating PERK/eIF2α pathway activation. We also present evidence that SIRT1 physically interacts with and deacetylates eIF2α. Mass spectrometry analysis identified lysines K141 and K143 as the acetylation sites on eIF2α targeted by SIRT1. Furthermore, mutation of K143 to arginine to mimic eIF2α deacetylation confers protection against ER stress-induced apoptosis. Collectively, our findings indicate that eIF2α deacetylation on lysine K143 by SIRT1 is a novel regulatory mechanism for protecting cardiac cells from ER stress and suggest that activation of SIRT1 has potential as a therapeutic approach to protect the heart against ER stress-induced injury