Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3

Hypoxia (lack of oxygen) is a physiological stress often associated with solid tumors. Hypoxia correlates with poor prognosis since hypoxic regions within tumors are considered apoptosisresistant. Autophagy (cellular "self digestion") has been associated with hypoxia during cardiac ischemia and metabolic stress as a survival mechanism. However, although autophagy is best characterized as a survival response, it can also function as a mechanism of programmed cell death. Our results show that autophagic cell death is induced by hypoxia in cancer cells with intact apoptotic machinery. We have analyzed two glioma cell lines (U87, U373), two breast cancer cell lines (MDA-MB-231, ZR75) and one embryonic cell line (HEK293) for cell death response in hypoxia (<1% O(2)). Under normoxic conditions, all five cell lines undergo etoposide-induced apoptosis whereas hypoxia fails to induce these apoptotic responses. All five cell lines induce an autophagic response and undergo cell death in hypoxia. Hypoxia-induced cell death was reduced upon treatment with the autophagy inhibitor 3-methyladenine, but not with the caspase inhibitor z-VAD-fmk. By knocking down the autophagy proteins Beclin-1 or ATG5, hypoxia-induced cell death was also reduced. The pro-cell death Bcl-2 family member BNIP3 (Bcl-2/adenovirus E1B 19kDainteracting protein 3) is upregulated during hypoxia and is known to induce autophagy and cell death. We found that BNIP3 overexpression induced autophagy, while expression of BNIP3 siRNA or a dominant-negative form of BNIP3 reduced hypoxia-induced autophagy. Taken together, these results suggest that prolonged hypoxia induces autophagic cell death in apoptosis-competent cells, through a mechanism involving BNIP3.     

EBV-LMP1 promotes radioresistance by inducing protective autophagy through BNIP3 in nasopharyngeal carcinoma

Studies have indicated that dysfunction of autophagy is involved in the initiation and progression of multiple tumors and their chemoradiotherapy. Epstein–Barr virus (EBV) is a lymphotropic human gamma herpes virus that has been implicated in the pathogenesis of nasopharyngeal carcinoma (NPC). EBV encoded latent membrane protein1 (LMP1) exhibits the properties of a classical oncoprotein. In previous studies, we experimentally demonstrated that LMP1 could increase the radioresistance of NPC. However, how LMP1 contributes to the radioresistance in NPC is still not clear. In the present study, we found that LMP1 could enhance autophagy by upregulating the expression of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3). Knockdown of BNIP3 could increase the apoptosis and decrease the radioresistance mediated by protective autophagy in LMP1-positive NPC cells. The data showed that increased BNIP3 expression is mediated by LMP1 through the ERK/HIF1α signaling axis, and LMP1 promotes the binding of BNIP3 to Beclin1 and competitively reduces the binding of Bcl-2 to Beclin1, thus upregulating autophagy. Furthermore, knockdown of BNIP3 can reduce the radioresistance promoted by protective autophagy in vivo. These data clearly indicated that, through BNIP3, LMP1 induced autophagy, which has a crucial role in the protection of LMP1-positive NPC cells against irradiation. It provides a new basis and potential target for elucidating LMP1-mediated radioresistance.Subject terms: Oncogenes, Head and neck cancer     

Hypoxia signals autophagy in tumor cells via AMPK activity, independent of HIF-1, BNIP3, and BNIP3L

Macroautophagy (called autophagy hereafter) is a catabolic process activated by various types of stress, most notably by nutrient deprivation. The autophagic degradation of intracellular macromolecules provides metabolic support for the cell; however, this physiological process can also initiate a form of cell death (type 2 programmed cell death). Here we report that oxygen deprivation can activate the autophagic pathway in human cancer cell lines. We observed that hypoxia induced distinct cellular changes characteristic of autophagy such as an increase in cytoplasmic acidic vesicles, and processing and cellular localization of microtubule-associated protein-1 light chain 3. Oxygen deprivation-induced autophagy did not require nutrient deprivation, hypoxia-inducible factor-1 (HIF-1) activity, or expression of the HIF-1 target gene BNIP3 (Bcl-2 adenovirus E1a nineteen kilodalton interacting protein 3) or BNIP3L (BNIP3 like protein). Hypoxia-induced autophagy involved the activity of 5'-AMP-activated protein kinase (AMPK). Finally, we determined that cells lacking the autophagy gene ATG5 were unable to activate the autophagic machinery in hypoxia, had decreased oxygen consumption and increased glucose uptake under hypoxia, had increased survival in hypoxic environments, and exhibited accelerated growth as xenografted tumors. Together, these findings suggest that the autophagic degradation of cellular macromolecules contributes to the energetic balance governed by AMPK, and that suppression of autophagy in transformed cells can increase both resistance to hypoxic stress and tumorigenicity.     

Regulation of mitochondrial integrity, autophagy and cell survival by BNIP3

Understanding the role of BNIP3 in the systemic response to hypoxia has been complicated by conflicting results that indicate on the one hand that BNIP3 promotes cell death, and other data, including our own that BNIP3 is not sufficient for cell death, but rather plays a critical role in hypoxia-induced autophagy. This work suggests that rather than promoting death, BNIP3 may actually allow survival either by preventing ATP depletion or by eliminating damaged mitochondria. However, the function of BNIP3 may be subverted under unusual conditions associated with acidosis that arise following extended periods of hypoxia and anaerobic glycolysis. Despite this novel insight into BNIP3 function, much remains to be done in terms of pinning down a molecular activity for BNIP3 that explains both its role in autophagy and how this may be subverted to induce cell death. As a target of the RB tumor suppressor, our work also places BNIP3 at the center of efforts to exploit autophagy to better treat human cancers in which tumor hypoxia is implicated as a progression factor.     

Regulation of Mitochondrial Integrity,Autophagy and Cell Survival by BNIP3

Understanding the role of BNIP3 in the systemic response to hypoxia has been complicated by conflicting results that indicate on the one hand that BNIP3 promotes cell death, and other data, including our own that BNIP3 is not sufficient for cell death, but rather plays a critical role in hypoxia-induced autophagy. This work suggests that rather than promoting death, BNIP3 may actually allow survival either by preventing ATP depletion or by eliminating damaged mitochondria. However, the function of BNIP3 may be subverted under unusual conditions associated with acidosis that arise following extended periods of hypoxia and anaerobic glycolysis. Despite this novel insight into BNIP3 function, much remains to be done in terms of pinning down a molecular activity for BNIP3 that explains both its role in autophagy and how this may be subverted to induce cell death. As a target of the RB tumor suppressor, our work also places BNIP3 at the center of efforts to exploit autophagy to better treat human cancers in which tumor hypoxia is implicated as a progression factor.

Addendum to:

BNIP3 is an RB/E2F Target Gene Required for Hypoxia-Induced Autophagy

K. Tracy, B.C. Dibling, BT. Spike, J. Knabb, P. Schumacker and K.F. Macleod

Mol Cell Biol 2007; In press     

Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy

BNIP3 is a dual function protein, able to activate autophagy and induce cell death. Upon expression of BNIP3, which is upregulated by hypoxia, the protein induces mitochondrial dysfunction, often leading to cell death. However, some highly respiring cells and cancer cells tolerate BNIP3 expression, suggesting that a yet unknown mechanism exists to restrain the lethal effects of BNIP3 on mitochondria. Here we present evidence that BNIP3 undergoes several phosphorylation events at its C-terminus, adjacent to the transmembrane domain. Phosphorylation at these residues inhibits BNIP3-induced mitochondrial damage, preventing a loss of mitochondrial mass and mitochondrial membrane potential, as well as preventing an increase in reactive oxygen species. This decrease in mitochondrial damage, as well as the reduction of cell death upon C-terminal BNIP3 phosphorylation, can be explained by a diminished interaction between BNIP3 and OPA1, a key regulator of mitochondrial fusion and mitochondrial inner membrane structure. Importantly, phosphorylation of these C-terminal BNIP3 residues blocks cell death without preventing autophagy, providing evidence that the two functional roles of BNIP3 can be regulated independently. These findings establish phosphorylation as a switch to determine the pro-survival and pro-death effects of the protein. Our findings also suggest a novel target for the regulation of these activities in transformed cells where BNIP3 is often highly expressed.     

BNIP3 and Nix: Atypical regulators of cell fate

Since their discovery nearly 25 years ago, the BCL-2 family members BNIP3 and BNIP3L (aka Nix) have been labelled ‘atypical’. Originally, this was because BNIP3 and Nix have divergent BH3 domains compared to other BCL-2 proteins. In addition, this atypical BH3 domain is dispensable for inducing cell death, which is also unusual for a ‘death gene’. Instead, BNIP3 and Nix utilize a transmembrane domain, which allows for dimerization and insertion into and through organelle membranes to elicit cell death. Much has been learned regarding the biological function of these two atypical death genes, including their role in metabolic stress, where BNIP3 is responsive to hypoxia, while Nix responds variably to hypoxia and is also down-stream of PKC signaling and lipotoxic stress. Interestingly, both BNIP3 and Nix respond to signals related to cell atrophy. In addition, our current view of regulated cell death has expanded to include forms of necrosis such as necroptosis, pyroptosis, ferroptosis, and permeability transition-mediated cell death where BNIP3 and Nix have been shown to play context- and cell-type specific roles. Perhaps the most intriguing discoveries in recent years are the results demonstrating roles for BNIP3 and Nix outside of the purview of death genes, such as regulation of proliferation, differentiation/maturation, mitochondrial dynamics, macro- and selective-autophagy. We provide a historical and unbiased overview of these ‘death genes’, including new information related to alternative splicing and post-translational modification. In addition, we propose to redefine these two atypical members of the BCL-2 family as versatile regulators of cell fate.     

Expression and Functional Characterization of the BNIP3 Protein in Renal Cell Carcinomas

BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3) is a BH3-only protein that regulates apoptosis and autophagy. BNIP3 plays also an important role in hypoxia-induced cell response and is regulated by HIF1. Here, we studied a possible association of BNIP3 expression and the prognosis of patients with renal cell carcinomas (RCCs) and examined the functional relevance of BNIP3 in the regulation of cell survival and apoptosis of renal carcinoma cells. BNIP3 expression was determined by immunohistochemistry in RCC tumor tissue samples of 569 patients using a tissue microarray. Functional characterization of BNIP3 in renal carcinoma cells indicates prosurvival effects. In human RCC tumor samples, high cytoplasmic BNIP3 expression was associated with high-grade RCCs and regional lymph node metastasis. BNIP3 expression correlated negatively with disease-specific survival. Multivariate Cox regression analysis retained BNIP3 expression as an independent prognostic factor in patients without distant metastasis. Together, our studies imply that BNIP3 regulates cell survival in RCCs and its expression is an independent prognostic marker in patients with localized RCCs.     

BH3-Only Proteins and BH3 Mimetics Induce Autophagy by Competitively Disrupting the Interaction between Beclin 1 and Bcl-2/Bcl-XL

Beclin 1 has recently been identified as novel BH3-only protein, meaning that it carries one Bcl-2-homology-3 (BH3) domain. As other BH3-only proteins, Beclin 1 interacts with anti-apoptotic multidomain proteins of the Bcl-2 family (in particular Bcl-2 and its homologue Bcl-X_L) by virtue of its BH3 domain, an amphipathic α-helix that binds to the hydrophobic cleft of Bcl-2/Bcl-X_L. The BH3 domains of other BH3-only proteins such as Bad, as well as BH3-mimetic compounds such as ABT737, competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-X_L. This causes autophagy of mitochondria (mitophagy) but not of the endoplasmic reticulum (ER-phagy). Only ER-targeted (not mitochondrion-targeted) Bcl-2/Bcl-X_L can inhibit autophagy induced by Beclin 1, and only Beclin 1-Bcl-2/Bcl-X_L complexes present in the ER (but not those present on heavy membrane fractions enriched in mitochondria) are disrupted by ABT737. These findings suggest that the Beclin 1-Bcl-2/Bcl-X_L complexes that normally inhibit autophagy are specifically located in the ER and point to an organelle-specific regulation of autophagy. Furthermore, these data suggest a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrion-permeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-X_L at the level of the endoplasmic reticulum.

Addendum to:

Functional and Physical Interaction Between Bcl-X_L and a BH3-Like Domain in Beclin-1

M.C. Maiuri, G. Le Toumelin, A. Criollo, J.-C. Rain, F. Gautier, P. Juin, E. Tasdemir, G. Pierron, K. Troulinaki, N. Tavernarakis, J.A. Hickman, O. Geneste and G. Kroemer

EMBO J 2007; In press     

Platinum‐based combination chemotherapy triggers cancer cell death through induction of BNIP3 and ROS,but not autophagy

These days, cancer can still not be effectively cured because cancer cells readily develop resistance to anticancer drugs. Therefore, an effective combination of drugs with different mechanisms to prevent drug resistance has become a very important issue. Furthermore, the BH3‐only protein BNIP3 is involved in both apoptotic and autophagic cell death. In this study, lung cancer cells were treated with a chemotherapy drug alone or in combination to identify the role of BNIP3 and autophagy in combination chemotherapy for treating cancer. Our data revealed that various combinational treatments of two drugs could increase cancer cell death and cisplatin in combination with rapamycin or LBH589, which triggered the cell cycle arrest at the S phase. Cells with autophagosome and pEGFP‐LC3 puncta increased when treated with drugs. To confirm the role of autophagy, cancer cells were pre‐treated with the autophagy inhibitor 3‐methyladenine (3‐MA). 3‐MA sensitized cancer cells to chemotherapy drug treatments. These results suggest that autophagy may be responsible for cell survival in combination chemotherapy for lung cancer. Moreover, BNIP3 was induced and localized in mitochondria when cells were treated with drugs. The transfection of a dominant negative transmembrane deletion construct of BNIP3 (BNIP3ΔTM) and treatment of a reactive oxygen species (ROS) inhibitor suppressed chemo drug‐induced cell death. These results indicate that BNIP3 and ROS may be involved in combination chemo drug‐induced cell death. However, chemo drug‐induced autophagy may protect cancer cells from drug cytotoxicity. As a result, inhibiting autophagy may improve the effects of combination chemotherapy when treating lung cancer.     

Neuroprotective role of BNIP3 under oxidative stress through autophagy in neuroblastoma cells

Reactive oxygen species (ROS) are produced due to oxidative stress which has wide range of affiliation with different diseases including cancer, heart failure, diabetes and neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease, ischemic and hemorrhagic diseases. This study shows the involvement of BNIP3 in the amplification of metabolic pathways related to cellular quality control and cellular self defence mechanism in the form of autophagy. We used conventional methods to induce autophagy by treating the cells with H₂O₂. MTT assay was performed to observe the cellular viability in stressed condition. MDC staining was carried out for detection of autophagosomes formation which confirmed the autophagy. Furthermore, expression of BNIP3 was validated by western blot analysis with LC3 antibody. From these results it is clear that BNIP3 plays a key role in defence mechanism by removing the misfolded proteins through autophagy. These results enhance the practical application of BNIP3 in neuroblastoma cells and are helpful in reducing the chances of neurodegenerative diseases. Although, the exact mode of action is still unknown but these findings unveil a molecular mechanism for the role of autophagy in cell death and provide insight into complex relationship between ROS and non-apoptotic programmed cell death.