Taxol-induced unfolded protein response activation in breast cancer cells exposed to hypoxia: ATF4 activation regulates autophagy and inhibits apoptosis.

Understanding the mechanisms responsible for the resistance against chemotherapy-induced cell death is still of great interest since the number of patients with cancer increases and relapse is commonly observed. Indeed, the development of hypoxic regions as well as UPR (unfolded protein response) activation is known to promote cancer cell adaptive responses to the stressful tumor microenvironment and resistance against anticancer therapies. Therefore, the impact of UPR combined to hypoxia on autophagy and apoptosis activation during taxol exposure was investigated in MDA-MB-231 and T47D breast cancer cells. The results showed that taxol rapidly induced UPR activation and that hypoxia modulated taxol-induced UPR activation differently according to the different UPR pathways (PERK, ATF6, and IRE1α). The putative involvement of these signaling pathways in autophagy or in apoptosis regulation in response to taxol exposure was investigated. However, while no link between the activation of these three ER stress sensors and autophagy or apoptosis regulation could be evidenced, results showed that ATF4 activation, which occurs independently of UPR activation, was involved in taxol-induced autophagy completion. In addition, an ATF4-dependent mechanism leading to cancer cell adaptation and resistance against taxol-induced cell death was evidenced. Finally, our results demonstrate that expression of ATF4, in association with hypoxia-induced genes, can be used as a biomarker of a poor prognosis for human breast cancer patients supporting the conclusion that ATF4 might play an important role in adaptation and resistance of breast cancer cells to chemotherapy in hypoxic tumors.

Local mitochondrial-endolysosomal microfusion cleaves voltage-dependent anion channel 1 to promote survival in hypoxia.

The oxygen-limiting (hypoxic) microenvironment of tumors induces metabolic reprogramming and cell survival, but the underlying mechanisms involving mitochondria remain poorly understood. We previously demonstrated that hypoxia-inducible factor 1 mediates the hyperfusion of mitochondria by inducing Bcl-2/adenovirus E1B 19-kDa interacting protein 3 and posttranslational truncation of the mitochondrial ATP transporter outer membrane voltage-dependent anion channel 1 in hypoxic cells. In addition, we showed that truncation is associated with increased resistance to drug-induced apoptosis and is indicative of increased patient chemoresistance. We now show that silencing of the tumor suppressor TP53 decreases truncation and increases drug-induced apoptosis. We also show that TP53 regulates truncation through induction of the mitochondrial protein Mieap. While we found that truncation was independent of mitophagy, we observed local microfusion between mitochondria and endolysosomes in hypoxic cells in culture and in patients' tumor tissues. Since we found that the endolysosomal asparagine endopeptidase was responsible for truncation, we propose that it is a readout of mitochondrial-endolysosomal microfusion in hypoxia. These novel findings provide the framework for a better understanding of hypoxic cell metabolism and cell survival through mitochondrial-endolysosomal microfusion regulated by hypoxia-inducible factor 1 and TP53.

Hypoxia-induced modulation of apoptosis and BCL-2 family proteins in different cancer cell types.

Hypoxia plays an important role in the resistance of tumour cells to chemotherapy. However, the exact mechanisms underlying this process are not well understood. Moreover, according to the cell lines, hypoxia differently influences cell death. The study of the effects of hypoxia on the apoptosis induced by 5 chemotherapeutic drugs in 7 cancer cell types showed that hypoxia generally inhibited the drug-induced apoptosis. In most cases, the effect of hypoxia was the same for all the drugs in one cell type. The expression profile of 93 genes involved in apoptosis as well as the protein level of BCL-2 family proteins were then investigated. In HepG2 cells that are strongly protected against cell death by hypoxia, hypoxia decreased the abundance of nearly all the pro-apoptotic BCL-2 family proteins while none of them are decreased in A549 cells that are not protected against cell death by hypoxia. In HepG2 cells, hypoxia decreased NOXA and BAD abundance and modified the electrophoretic mobility of BIM(EL). BIM and NOXA are important mediators of etoposide-induced cell death in HepG2 cells and the hypoxia-induced modification of these proteins abundance or post-translational modifications partly account for chemoresistance. Finally, the modulation of the abundance and/or of the post-translational modifications of most proteins of the BCL-2 family by hypoxia involves p53-dependent and -independent pathways and is cell type-dependent. A better understanding of these cell-to-cell variations is crucial in order to overcome hypoxia-induced resistance and to ameliorate cancer therapy.

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

Since the 1940s, 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 type II programmed 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.

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

BACKGROUND: Hypoxia is a hallmark of solid tumors and is associated with metastases, therapeutic resistance and poor patient survival. RESULTS: 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. CONCLUSIONS: 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.