Johanna Chiche

Hypoxia-Induced Autophagy Is Mediated through Hypoxia-Inducible Factor Induction of BNIP3 and BNIP3L via Their BH3 Domains†

ABSTRACT While hypoxia-inducible factor (HIF) is a major actor in the cell survival response to hypoxia, HIF also is associated with cell death. Several studies implicate the HIF-induced putative BH3-only proapoptotic genes bnip3 and bnip3l in hypoxia-mediated cell death. We, like others, do not support this assertion. Here, we clearly demonstrate that the hypoxic microenvironment contributes to survival rather than cell death by inducing autophagy. The ablation of Beclin1, a major actor of autophagy, enhances cell death under hypoxic conditions. In addition, the ablation of BNIP3 and/or BNIP3L triggers cell death, and BNIP3 and BNIP3L are crucial for hypoxia-induced autophagy. First, while the small interfering RNA-mediated ablation of either BNIP3 or BNIP3L has little effect on autophagy, the combined silencing of these two HIF targets suppresses hypoxia-mediated autophagy. Second, the ectopic expression of both BNIP3 and BNIP3L in normoxia activates autophagy. Third, 20-mer BH3 peptides of BNIP3 or BNIP3L are sufficient in initiating autophagy in normoxia. Herein, we propose a model in which the atypical BH3 domains of hypoxia-induced BNIP3/BNIP3L have been designed to induce autophagy by disrupting the Bcl-2-Beclin1 complex without inducing cell death. Hypoxia-induced autophagy via BNIP3 and BNIP3L is clearly a survival mechanism that promotes tumor progression.

Hypoxia and cancer

A major feature of solid tumours is hypoxia, decreased availability of oxygen, which increases patient treatment resistance and favours tumour progression. How hypoxic conditions are generated in tumour tissues and how cells respond to hypoxia are essential questions in understanding tumour progression and metastasis. Massive tumour-cell proliferation distances cells from the vasculature, leading to a deficiency in the local environment of blood carrying oxygen and nutrients. Such hypoxic conditions induce a molecular response, in both normal and neoplastic cells, that drives the activation of a key transcription factor; the hypoxia-inducible factor. This transcription factor regulates a large panel of genes that are exploited by tumour cells for survival, resistance to treatment and escape from a nutrient-deprived environment. Although now recognized as a major contributor to cancer progression and to treatment failure, the precise role of hypoxia signalling in cancer and in prognosis still needs to be further defined. It is hoped that a better understanding of the mechanisms implicated will lead to alternative and more efficient therapeutic approaches.

Tumour hypoxia induces a metabolic shift causing acidosis: a common feature in cancer

•  Introduction •  Intracellular pH regulation controls energy balance and cell proliferation: chemical and biological proof of principle ‐  Chemical proof of principle ‐  Biological proof of principle: the role of the Na+/H+ exchanger‐1 •  Oncogene activation and transformation cause acidosis ‐  Warburg effect (aerobic glycolysis) ‐  Inhibition of tumour suppressor genes and oncogene activation drive the ‘Warburg effect’ and cause acidosis ‐  Neoplastic transformation drives intracellular alkalinization and extracellular acidification through the activation and up‐regulation of pHi‐regulating systems •  Hypoxia promotes acidosis by shifting from oxidative phosphorylation to glycolytic metabolism ‐  HIF mediates cellular adaptation to low oxygen availability ‐  HIF‐induced metabolic reprogramming in response to tumour hypoxia causes acidosis ‐  Acidosis may affect HIF‐α stabilization and on HIF‐induced gene regulation •  Hypoxia enhances the expression and activity of pHi‐regulating systems to promote cell survival and invasion ‐  Hypoxia increases NHE‐1 expression and activity ‐  The hypoxia‐induced membrane‐associated carbonic anhydrases are key enzymes involved in pH homeostasis, cell survival and migration in a hypoxic/acidic microenvironment ‐  CAIX regulation and expression ‐  CAXII regulation and expression ‐  The activity and functions of CAIX and CAXII ‐  The hypoxia‐induced monocarboxylate transporter MCT4, the constitutively expressed MCT1 and their chaperone CD147 are key plasma‐membrane proteins involved in pH regulation, energy balance, tumour progression and metastasis ‐  MCT regulation, expression, structure and implication of their chaperone CD147 ‐  The MCT1, MCT4 and CD147 activity and functions •  Strategies taking advantage of changes in the oxygen level, energy balance and pH homeostasis to target primary tumours and metastases ‐  Decreasing the pHi of hypoxic cells of the primary tumour by inhibiting key pHi‐regulating systems to collapse ATP production ‐  Increasing pHo and the extracellular buffering capacity in targeting metastasis and reducing multidrug resistance •  Conclusion

pH control mechanisms of tumor survival and growth

A distinguishing phenotype of solid tumors is the presence of an alkaline cellular feature despite the surrounding acidic microenvironment. This phenotypic characteristic of tumors, originally described by Otto Warburg, arises due to alterations in metabolism of solid tumors. Hypoxic regions of solid tumors develop due to poor vascularization and in turn regulate the expression of numerous genes via the transcription factor HIF‐1. Ultimately, the tumor microenvironment directs the development of tumor cells adapted to survive in an acidic surrounding where normal cells perish. The provision of unique pH characteristics in tumor cells provides a defining trait that has led to the pursuit of treatments that target metabolism, hypoxia, and pH‐related mechanisms to selectively kill cancer cells. Numerous studies over the past decade involving the cancer‐specific carbonic anhydrase IX have re‐kindled an interest in pH disruption‐based therapies. Although an acidification of the intracellular compartment is established as a means to induce normal cell death, the defining role of acid–base disturbances in tumor physiology and survival remains unclear. The aim of this review is to summarize recent data relating to the specific role of pH regulation in tumor cell survival. We focus on membrane transport and enzyme studies in an attempt to elucidate their respective functions regarding tumor cell pH regulation. These data are discussed in the context of future directions for the field of tumor cell acid–base‐related research. J. Cell. Physiol. 226: 299–308, 2011.

Hypoxic enlarged mitochondria protect cancer cells from apoptotic stimuli

It is well established that cells exposed to the limiting oxygen microenvironment (hypoxia) of tumors acquire resistance to chemotherapy, through mechanisms not fully understood. We noted that a large number of cell lines showed protection from apoptotic stimuli, staurosporine, or etoposide, when exposed to long‐term hypoxia (72 h). In addition, these cells had unusual enlarged mitochondria that were induced in a HIF‐1‐dependent manner. Enlarged mitochondria were functional as they conserved their transmembrane potential and ATP production. Here we reveal that mitochondria of hypoxia‐induced chemotherapy‐resistant cells undergo a HIF‐1‐dependent and mitofusin‐1‐mediated change in morphology from a tubular network to an enlarged phenotype. An imbalance in mitochondrial fusion/fission occurs since silencing of not only the mitochondrial fusion protein mitofusin 1 but also BNIP3 and BNIP3L, two mitochondrial HIF‐targeted genes, reestablished a tubular morphology. Hypoxic cells were insensitive to staurosporine‐ and etoposide‐induced cell death, but the silencing of mitofusin, BNIP3, and BNIP3L restored sensitivity. Our results demonstrate that some cancer cells have developed yet another way to evade apoptosis in hypoxia, by inducing mitochondrial fusion and targeting BNIP3 and BNIP3L to mitochondrial membranes, thereby giving these cells a selective growth advantage. J. Cell. Physiol. 222: 648–657, 2010.

Targeting Hypoxic Tumor Cell Viability with Carbohydrate-Based Carbonic Anhydrase IX and XII Inhibitors

Carbonic anhydrase (CA) enzymes, specifically membrane-bound isozymes CA IX and CA XII, underpin a pH-regulating system that enables hypoxic tumor cell survival and proliferation. CA IX and XII are implicated as potential targets for the development of new hypoxic cancer therapies. To date, only a few small molecules have been characterized in CA-relevant cell and animal model systems. In this paper, we describe the development of a new class of carbohydrate-based small molecule CA inhibitors, many of which inhibit CA IX and XII within a narrow range of low nanomolar K(i) values (5.3-11.2 nM). We evaluate for the first time carbohydrate-based CA inhibitors in cell-based models that emulate the protective role of CA IX in an acidic tumor microenvironment. Our findings identified two inhibitors (compounds 5 and 17) that block CA IX-induced survival and have potential for development as in vivo cancer cell selective inhibitors.

In vivo pH in metabolic-defective Ras-transformed fibroblast tumors: key role of the monocarboxylate transporter, MCT4, for inducing an alkaline intracellular pH.

We present an investigation of tumor pH regulation, designed to support a new anticancer therapy concept that we had previously proposed. Our study uses a tumor model of ras‐transformed hamster fibroblasts, CCL39, xenografted in the thighs of nude mice. We demonstrate, for the first time, that genetic modifications of specific mechanisms of proton production and/or proton transport result in distinct, reproducible changes in intracellular and extracellular tumor pH that can be detected and quantified noninvasively in vivo, simultaneously with determinations of tumor energetic status and necrosis in the same experiment. The CCL39 variants used were deficient in the sodium/proton exchanger, NHE‐1, and/or in the monocarboxylate transporter, MCT4; further, variants were deficient in glycolysis or respiration. MCT4 expression markedly increased the gradient between intracellular and extracellular pH from 0.14 to 0.43 when compared to CCL39 wild‐type tumors not expressing MCT4. The other genetic modifications studied produced smaller but significant increases in intracellular and decreases in extracellular pH. In general, increased pH gradients were paralleled by increased tumor growth performance and diminished necrotic regions, and 50% of the CCL39 variant expressing neither MCT4 nor NHE‐1, but possessing full genetic capacity for glycolysis and oxidative phosphorylation, underwent regression before reaching a 1‐cm diameter. Except for CCL39 wild‐type tumors, no significant HIF‐1α expression was detected. Our in vivo results support a multipronged approach to tumor treatment based on minimizing intracellular pH by targeting several proton production and proton transport processes, among which the very efficient MCT4 proton/lactate co‐transport deserves particular attention.

Protective mitochondrial transfer from bone marrow stromal cells to acute myeloid leukemic cells during chemotherapy

Here we demonstrate that in a niche-like coculture system, cells from both primary and cultured acute myeloid leukemia (AML) sources take up functional mitochondria from murine or human bone marrow stromal cells. Using different molecular and imaging approaches, we show that AML cells can increase their mitochondrial mass up to 14%. After coculture, recipient AML cells showed a 1.5-fold increase in mitochondrial adenosine triphosphate production and were less prone to mitochondrial depolarization after chemotherapy, displaying a higher survival. This unidirectional transfer enhanced by some chemotherapeutic agents required cell-cell contacts and proceeded through an endocytic pathway. Transfer was greater in AML blasts compared with normal cord blood CD34(+) cells. Finally, we demonstrate that mitochondrial transfer was observed in vivo in an NSG immunodeficient mouse xenograft model and also occurred in human leukemia initiating cells and progenitors. As mitochondrial transfer provides a clear survival advantage following chemotherapy and a higher leukemic long-term culture initiating cell potential, targeting mitochondrial transfer could represent a future therapeutic target for AML treatment.

Membrane-bound carbonic anhydrases are key pH regulators controlling tumor growth and cell migration.

Hypoxia and acidosis in the tumor microenvironment are critical in driving tumor growth and metastasis. Hypoxia, a low level of oxygenation below normal, results from excessive cancer cell proliferation distant from oxygen-carrying blood vessels. Acidosis, a low pH, results from enhanced glucose uptake and metabolism to lactic acid by cancer cells, which is exacerbate by inefficient removal of lactic acid and CO2 by a deficient vasculature in the tumor mass (Brahimi-Horn et al., 2007a,b; Brahimi-Horn and Pouyssegur, 2007). Since variations in the pHi, as low as 0.1 units, disrupt multiple biological functions such as ATP production, proliferation, migration, invasion and metastasis, drug resistance and apoptosis, cells must regulate their pHi so as to survive, proliferate and migrate (Chambard and Pouyssegur, 1986; Pouyssegur et al., 1984; Roos and Boron, 1981). However, hypoxic tumor cells have developed key strategies to regulate their pHi, which thereby protects the cytosol from acidosis and allow cells to survive hypoxia. Under hypoxic conditions the transcription factor hypoxia-inducible factor-1 (HIF-1) is activated and regulates pH homeostasis by enhancing expression of membrane located transporters, exchangers, pumps and ecto-enzymes (Brahimi-Horn and Pouyssegur, 2007). The pHi-regulating system of tumor cells actively exports acids via the Na þ/Hþ exchanger (NHE-1) (Cardone et al., 2005; Counillon and Pouyssegur, 2000; Sardet et al., 1989; Shimoda et al., 2006) and the monocarboxylate transporters (MCTs) (Ullah et al., 2006), and transports HCO3 into the cells through Cl /HCO3 exchangers (AE) (Karumanchi et al., 2001) for cytoplasmic alkalinization. NHE1 is known to play a key role in vivo in tumor development, in particular when highly glycolytic cells produce large amounts of lactic acid (Pouyssegur et al., 2001). Ubiquitously expressed Cl /HCO3 exchangers play an important role in cellular alkalinization by pumping the weak base HCO3 , which traps intracellular Hþ and thus maintains a permissive pHi that favors cell survival (Izumi et al., 2003). Another family of proteins, the carbonic anhydrases (CAs), also contributes to cellular alkalinization by catalyzing the reversible hydration of cell-generated carbon dioxide into protons and bicarbonate

Targeting tumour hypoxia to prevent cancer metastasis: from biology, biosensing and technology to drug development : the METOXIA consortium

Abstract The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation–deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009–2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [18F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O2), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.