Anti-apoptotic role of HIF-1 and AP-1 in paclitaxel exposed breast cancer cells under hypoxia

<p>Abstract</p> <p>Background</p> <p>Hypoxia is a hallmark of solid tumors and is associated with metastases, therapeutic resistance and poor patient survival.</p> <p>Results</p> <p>In this study, we showed that hypoxia protected MDA-MB-231 breast cancer cells against paclitaxel- but not epirubicin-induced apoptosis. The possible implication of HIF-1 and AP-1 in the hypoxia-induced anti-apoptotic pathway was investigated by the use of specific siRNA. Specific inhibition of the expression of these two transcription factors was shown to increase apoptosis induced by chemotherapeutic agents under hypoxia indicating an involvement of HIF-1 and AP-1 in the anti-apoptotic effect of hypoxia. After HIF-1 specific inhibition and using TaqMan Human Apoptosis Array, 8 potential HIF-1 target genes were identified which could take part in this protection. Furthermore, Mcl-1 was shown to be a potential AP-1 target gene which could also participate to the hypoxia-induced chemoresistance.</p> <p>Conclusions</p> <p>Altogether, these data highlight two mechanisms by which hypoxia could mediate its protective role via the activation of two transcription factors and, consecutively, changes in gene expression encoding different anti- and pro-apoptotic proteins.</p

Etude du rôle de l’autophagie et de la réponse UPR dans la résistance des cellules cancéreuses contre l’apoptose induite par le paclitaxel.

Cancer cell resistance against chemotherapy is still a heavy burden to improve anticancer treatments. Understanding the mechanisms responsible for the resistance against chemotherapy-induced cell death is of interest since the number of patients with cancer increases and relapse is commonly observed. Development of hypoxic regions is known to promote cancer cell adaptation to the stressful tumoral microenvironment and resistance against anti-cancer therapies. The HIF-1 signaling pathway, activated under hypoxia, promotes cancer cell adaptation and resistance against chemotherapy. Other biological processes are also activated under hypoxia and/or after chemotherapy treatment and are known to promote cancer cell survival. Autophagy and UPR (unfolded protein response) are part of these processes. Therefore, during the first part of this work, we sought to evaluate the role of autophagy and hypoxia on the taxol-induced apoptosis in MDA-MB-231 breast cancer cells. Results showed that taxol induced apoptosis after 16 h of incubation, and that hypoxia protected MDA-MB- 231 cells from taxol-induced apoptosis. In parallel, taxol induced autophagy activation already after 2 h of incubation both under normoxia and hypoxia. Autophagy activation during taxol exposure was shown to be a protective mechanism against taxol-induced cell death both under normoxia and hypoxia. However, at longer incubation times, the autophagic process reached a saturation point under normoxia leading to cell death, whereas under hypoxia, autophagy flow still correctly took place allowing the cells to survive. Autophagy induction was initiated via mechanistic target of rapamycin (mTOR) inhibition, which is more important in cells exposed to hypoxia. Taxol also induced c-Jun N-terminal kinase (JNK) activation and phosphorylation of its substrates B-cell CLL/lymphoma 2 (Bcl2) and Bcl2-like 1 (BclXL) under normoxia and hypoxia very early after taxol exposure. Bcl2 and BclXL phosphorylation was decreased more importantly under hypoxia after long incubation times. The role of JNK in autophagy and apoptosis induction was studied using siRNAs. The results showed that JNK activation promoted resistance against taxol-induced apoptosis under normoxia and hypoxia without being involved in induction of autophagy. In conclusion, the resistance against taxol-induced cell death observed under hypoxia can be explained by a more effective autophagic flow activated via the classical mTOR pathway and by a mechanism involving JNK, which could be dependent on Bcl2 and BclXL phosphorylation but independent of JNK-induced autophagy activation. During the second part of this work, the impact of UPR combined to hypoxia on autophagy and apoptosis activation during taxol exposure was investigated in MDA-MB-231 breast cancer cells. Results showed that taxol rapidly induced endoplasmic reticulum (ER) stress and UPR activation and that hypoxia modulated taxol-induced UPR activation. The putative involvement of PERK (protein kinase double-stranded RNA-dependent (PRK)-like ER Kinase), ATF6 (activating transcription factor 6) and IRE1 (inositol-requiring enzyme-1) signaling pathways in autophagy or in apoptosis regulation after taxol exposure was investigated. No link between the activation of these three ER stress sensors and autophagy or apoptosis regulation was evidenced. However, UPR played a role in the induction of VEGF (vascular Endothelial Growth Factor) secretion triggered by taxol exposure specifically in cells incubated under hypoxia. Results also showed that ATF4 (activating transcription factor 4) activation was involved in taxol-induced autophagy completion and in the mechanisms leading to cancer cell adaptation and resistance against taxol-induced cell death. Finally, our results demonstrate that the expression of ATF4, in association with hypoxia-induced genes, could be used as a biomarker of a poor prognosis in human breast cancer patients supporting the conclusion that ATF4 might play a role in adaptation and resistance of breast cancer cells to chemotherapy in hypoxic tumors.La résistance des cellules cancéreuses à la chimiothérapie est un problème crucial pour améliorer les traitements anticancéreux. La compréhension des mécanismes responsables de la résistance à la mort cellulaire induite par la chimiothérapie est toujours d’actualité car le nombre de patients atteints d’un cancer ne cesse d’augmenter et des rechutes sont souvent observées. Le développement de régions hypoxiques au sein des tumeurs est connu pour favoriser l’adaptation des cellules cancéreuses à leur environnement stressant et la résistance aux thérapies anticancéreuses. Les voies de signalisation dépendantes de HIF-1 (hypoxiainducible factor-1), qui sont activées en hypoxie, favorisent l’adaptation des cellules cancéreuses et la résistance à la chimiothérapie. D’autres processus biologiques, activés en hypoxie et/ou suite à un traitement chimiothérapeutique, sont également connus pour favoriser la survie des cellules cancéreuses. L’autophagie et l’UPR font partie de ces processus biologiques. Pour cette raison, durant la première partie de ce travail, nous avons étudié le rôle de l’autophagie et de l’hypoxie dans l’apoptose induite par le taxol dans les cellules MDA-MB- 231 issues d’un cancer du sein triple négatif. Les résultats montrent que le taxol induit l’apoptose après 16 heures d’incubation et que l’hypoxie protège les cellules MDA-MB-231 de l’apoptose induite par le taxol. En parallèle, le taxol induit l’activation de l’autophagie après 2 heures d’incubation en normoxie et en hypoxie. L’activation de l’autophagie dans les cellules incubées avec du taxol est un mécanisme protecteur contre l’apoptose induite par le taxol en normoxie et en hypoxie. Cependant, après des temps d’incubation plus longs, le processus autophagique atteint un seuil de saturation en normoxie, conduisant à la mort cellulaire. En hypoxie, le flux autophagique se déroule correctement même après des temps d’incubation plus longs, ce qui permet aux cellules de survivre. L’autophagie est activée via l’inhibition de mTOR (mechanistic target of rapamycin), qui est plus importante dans les cellules incubées en hypoxie. Le taxol induit également l’activation de la JNK (c-Jun Nterminal kinase) et la phosphorylation de ses substrats Bcl2 (B-cell CLL/lymphoma 2) et BclXL (Bcl2-like 1) en normoxie et en hypoxie, très tôt au cours de l’exposition au taxol. Après des temps d’incubation plus longs, la phosphorylation de Bcl2 et BclXL diminue de manière plus importante dans les cellules incubées en hypoxie. Le rôle de la JNK dans l’activation de l’autophagie et de l’apoptose a été étudié à l’aide de siRNAs. Les résultats montrent que l’activation de la JNK favorise la résistance à l’apoptose induite par le taxol en normoxie et en hypoxie sans être impliquée dans l’activation de l’autophagie. En conclusion, la résistance à la mort cellulaire induite par le taxol observée en hypoxie peut être expliquée par un flux autophagique plus efficace activé par la voie classique mTOR et également pas un mécanisme impliquant la JNK, qui pourrait dépendre de la phosphorylation de Bcl2 et BclXL mais qui ne dépend pas de l’activation de l’autophagie par la JNK. Durant la seconde partie de ce travail, l’impact de l’UPR, combinée à l’hypoxie, sur l’activation de l’autophagie et de l’apoptose, durant une exposition au taxol, fut étudié dans les cellules MDA-MB-231. Les résultats montrent que le taxol induit rapidement un stress au réticulum endoplasmique et l’activation de l’UPR et que l’hypoxie module l’activation de l’UPR induite par le taxol. L’implication des voies de signalisation en aval de PERK (protein kinase double-stranded RNA-dependent (PRK)-like ER Kinase), d’ATF6 (activating transcription factor 6) et d’IRE1 (inositol-requiring enzyme-1) dans la régulation de l’autophagie ou de l’apoptose lors d’une incubation en présence de taxol fut investiguée. Aucun lien entre l’activation de ces trois senseurs du stress au RE et la régulation de l’autophagie ou de l’apoptose ne fut mis en évidence. Cependant, l’UPR régule la sécrétion du VEGF (vascular Endothelial Growth Factor) induite par le taxol uniquement dans les cellules incubées en hypoxie. Les résultats montrent aussi qu’ATF4 (activating transcription factor 4) favorise l’accomplissement de l’autophagie et est impliqué dans les mécanismes qui permettent l’adaptation des cellules cancéreuses et la résistance à l’apoptose induite par le taxol. Enfin, les résultats montrent que l’expression d’ATF4, en association avec l’expression de gènes induits en hypoxie, peut être utilisée comme biomarqueur reflétant un mauvais pronostic pour les patients atteints d’un cancer du sein. Ces résultats renforcent la conclusion qu’ATF4 peut jouer un rôle dans l’adaptation et la résistance des cellules cancéreuses du sein contre la chimiothérapie, en hypoxie.(DOCSC03) -- FUNDP, 201

Autophagy as a mediator of chemotherapy-induced cell death in cancer

International audienceSince the 1940's, chemotherapy has been the treatment of choice for metastatic disease. Chemotherapeutic agents target proliferating cells, inducing cell death. For most of the history of chemotherapy, apoptosis was thought to be the only mechanism of drug-induced cell death. More recently, a second type of cell death pathway has emerged: autophagy, also called programmed type II cell death. Autophagy is a tightly-regulated process by which selected components of a cell are degraded. It primarily functions as a cell survival adaptive mechanism during stress conditions. However, persistent stress can also promote extensive autophagy, leading to cell death, hence its name. Alterations in the autophagy pathway have been described in cancer cells that suggest a tumor-suppressive function in early tumorigenesis, but a tumor-promoting function in established tumors. Moreover, accumulating data indicate a role for autophagy in chemotherapy-induced cancer cell death. Here, we discuss some of the evidence showing autophagy-dependent cell death induced by anti-neoplastic agents in different cancer models. On the other hand, in some other examples, autophagy dampens treatment efficacy, hence providing a therapeutic target to enhance cancer cell killing. In this paper, we propose a putative mechanism that could reconcile these two opposite observations

Autophagy as a mediator of chemotherapy-induced cell death in cancer

International audienceSince the 1940's, chemotherapy has been the treatment of choice for metastatic disease. Chemotherapeutic agents target proliferating cells, inducing cell death. For most of the history of chemotherapy, apoptosis was thought to be the only mechanism of drug-induced cell death. More recently, a second type of cell death pathway has emerged: autophagy, also called programmed type II cell death. Autophagy is a tightly-regulated process by which selected components of a cell are degraded. It primarily functions as a cell survival adaptive mechanism during stress conditions. However, persistent stress can also promote extensive autophagy, leading to cell death, hence its name. Alterations in the autophagy pathway have been described in cancer cells that suggest a tumor-suppressive function in early tumorigenesis, but a tumor-promoting function in established tumors. Moreover, accumulating data indicate a role for autophagy in chemotherapy-induced cancer cell death. Here, we discuss some of the evidence showing autophagy-dependent cell death induced by anti-neoplastic agents in different cancer models. On the other hand, in some other examples, autophagy dampens treatment efficacy, hence providing a therapeutic target to enhance cancer cell killing. In this paper, we propose a putative mechanism that could reconcile these two opposite observations

Effect of BH3-only proteins silencing on the etoposide-induced cell death

<p>. HepG2 cells were transfected with 50 nM BIM (A, B), NOXA (C, D) or BAD (E, F) siRNAs, or 25 nM BIM combined with 25 nM NOXA (G, H), or 50 nM RISC-free (RF) control siRNA or left untransfected (CTL) for 24 hours. 6 hours later (or 30 hours later for A and B), cells were incubated under normoxia (N, 21% O₂) or hypoxia (H, 1% O₂) with (ETO) or without (CTL) etoposide (50 µM) for 16 (A, C, E, G) or 40 hours (B, D, F, H). (A, C, E, G) Caspase-3/7 activity was assayed by measuring the fluorescence of free AFC released from the cleavage of the caspase-3/7 specific substrate Ac-DEVD-AFC. Results are expressed in relative fluorescence units (RFU) as means ±1 SD (n  = 3). (B, D, F, H) LDH release was assessed. Results are presented in percentages as means ±1 SD (n  = 4, but n  = 3 in B for the condition HE CTL and in <b>f</b> for the condition H BAD). (A-H) Statistical analysis was carried out with ANOVA 1. ns: non-significant; *: P<0.05; **: P<0.01; ***: P<0.001.</p