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

Abstract

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.

Date of Award	2 Dec 2013
Original language	French
Awarding Institution	University of Namur
Supervisor	Carine Michiels (Supervisor), Thierry Arnould (Co-Supervisor), Jean-Pierre Gillet (Jury), Martine Raes (President), Julien Verrax (Jury) & Nathalie MAZURE (Jury)