Danièle Roux

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.

Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry.

Mitogen‐activated protein kinase (MAPK) modules, composed of three protein kinases activated by successive phosphorylation, are involved in the signal transduction of a wide range of extracellular agents. In mammalian cells, mitogenic stimulation triggers the translocation of p42/p44MAPK from the cytoplasm to the nucleus, whereas the other protein kinases of the module remain cytosolic. Since MAPK has been shown to phosphorylate and activate nuclear targets, such as the transcription factor Elk1, it has been proposed, but not yet demonstrated, that MAPK nuclear translocation could represent a critical step in signal transduction. In this study, we sequestered p42/p44MAPK in the cytoplasm by the expression of a catalytically inactive form of cytoplasmic MAP kinase phosphatase (MKP‐3/Pyst‐1). Sequestering MAPK in the cytoplasm did not alter its activation or its ability to phosphorylate cytoplasmic substrates of MAPK (p90RSK1 or an engineered cytoplasmic form of Elk1). In contrast, prevention of MAPK nuclear translocation strongly inhibited Elk1‐dependent gene transcription and the ability of cells to reinitiate DNA replication in response to growth factors. Thus the relocalization of MAPK to the nucleus appears to be an important regulatory step for mitogen‐induced gene expression and cell cycle re‐entry.

The Oxygen Sensor Factor-Inhibiting Hypoxia-Inducible Factor-1 Controls Expression of Distinct Genes through the Bifunctional Transcriptional Character of Hypoxia-Inducible Factor-1α

The function of the hypoxia-inducible factor-1 (HIF-1), the key transcription factor involved in cellular adaptation to hypoxia, is restricted to low oxygen tension (pO(2)). As such, this transcription factor is central in modulating the tumor microenvironment, sensing nutrient availability, and controlling anaerobic glycolysis, intracellular pH, and cell survival. Degradation and inhibition of the limiting HIF-1alpha subunit are intimately connected in normoxia. Hydroxylation of two proline residues by prolyl hydroxylase domain (PHD) 2 protein earmarks the protein for degradation, whereas hydroxylation of an asparagine residue by factor-inhibiting HIF-1 (FIH-1 or FIH) reduces its transcriptional activity. Indeed, silencing of either PHD2 or FIH in normoxia partially induced hypoxic genes, whereas combined PHD2/FIH silencing generated a full hypoxic gene response. Given the fact that HIF-1alpha possesses two transcriptional activation domains [TAD; NH(2)-terminal (N-TAD) and COOH-terminal (C-TAD)], we hypothesized on a possible bifunctional activity of HIF-1alpha that could be discriminated by FIH, an inhibitor of the C-TAD. In human cell lines engineered to overexpress or silence FIH in response to tetracycline, we show by quantitative reverse transcription-PCR that a set of hypoxic genes (ca9, phd3, pgk1, and bnip3) respond differently toward FIH expression. This finding, extended to 26 hypoxia-induced genes, indicates differential gene expression by the N-TAD and C-TAD in response to the hypoxic gradient. We propose that the oxygen-sensitive attenuator FIH, together with two distinct TADs, is central in setting the gene expression repertoire dictated by the cell pO(2).

Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation

ABSTRACT Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.

Na/H exchange activities in NHE1-transfected OK-cells : cell polarity and regulation

The human fibroblast, “amiloride-sensitive” Na/H exchanger (NHE1) was transfected into opossum kidney cells (OK cells) (OK/NHE1 cells). Northern blot analysis confirmed that the NHE1 message is expressed in OK/NHE1 cells. In immunoblot analysis, an anti-human NHE1 antibody labelled a membrane protein only present in OK/NHE1 cells. In contrast to the parental cell line containing only an apically located, “amilorideresistant” Na/H exchange activity, OK/NHE1 cells contain apically and basolaterally located Na/H exchange activities, the apical activity being “amiloride resistant” and the basolateral being “amiloride sensitive”. Parathyroid hormone (PTH) inhibited apical transport activity (OK and OK/NHE1 cells) but had no effect on basolateral transport activity (OK/NHE1 cells). Pharmacological activation of protein kinase A (forskolin) decreased both apical and basolateral Na/H exchange activity. Incubation with phorbol ester (exogenous activation of protein kinase C) reduced apical Na/H exchange activity (OK and OK/NHE1 cells) but had only a moderate, inhibitory effect on basolateral Na/H exchange activity (OK/NHE1 cells). These results indicate that transfection of OK cells with human fibroblast NHE1 cDNA encoding an “amiloride-sensitive” form of the Na/H exchanger results in expression of basolaterally located “NHE1-related” transport activity. Regulatory control of intracellular Na/H exchange activities (apically versus basolaterally located) and intercellular Na/H exchange activities (NHE1-related) differs. This may relate to cell-specific properties as well as to exchanger-specific properties.

Abstract 3225: Growth inhibition of glycolytic tumors by targeting basigin/lactate-H+ symporters (MCTs): Metformin sensitizes MCT inhibition

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Intense conversion of glucose to lactic acid via glycolysis is a feature of rapidly growing cells often encountered in hypoxic tumor microenvironments. To survive and expand, tumor cells must efficiently export lactic acid to maintain intracellular pH. Cells possess several systems for lactic acid extrusion. A family of H+-linked MonoCarboxylate Transporters (MCTs) is represented by the ubiquitously expressed MCT1, a H+/lactate symporter that operates in both directions. MCT4, a close relative of MCT1, is up-regulated by HIF-1 and is highly expressed in aggressive malignant tumors. In addition, the functional expression of MCT1/MCT4 requires the interaction with the glycoprotein CD147/Basigin also known as EMMPRIN, a protumoral protein involved in invasion. Objectives: 1) Demonstrate that tumor growth is dependent on lactic acid export and that both transporters MCT1/MCT4 represent key anticancer targets. 2) Demonstrate whether the protumoral function of Basigin/CD147 is primary linked to lactic acid export or to other invasive functions. 3) Demonstrate whether Metformin, an inhibitor of mitochondrial complex I, sensitizes glycolytic tumor cells to MCTs inhibitors. Methods: We exploited two tumoral models. i) Ras transformed fibroblasts expressing only MCT1/MCT2 and ii) the human colon adenocarcinoma cell line LS174T expressing MCT1 and MCT4. We inhibited MCT1/MCT2 with the specific astraZeneca compound AR-C155858 and knocked-down MCT1, MCT4 and CD147 with inducible shRNAs. In addition we knocked-out mct4 or basigin with Zinc Finger Nucleases in LS174T cells. Results: First we demonstrated that silencing or pharmacological blockage of MCTs, reduced pHi, the rate of glycolysis and tumor growth in mice xenografts. This tumor growth inhibition was recapitulated by a single silencing or knock out of basigin/CD147 gene that also reduced the plasma membrane expression of MCT1 and MCT4 and lactate transport up to 10-fold. Second, to gain insight into CD147/Basigin function, we uncoupled MCTs from Basigin expression. Inhibition of MCT1 in MCT4-null, Basigin-high expressors, suppressed tumor growth. Conversely in Basigin-null cells, in which MCT activity had been maintained, tumorigenicity was not affected. Thirdly, we showed that tumor cells lacking Basigin or MCT4 become highly sensitive to MCT1 inhibition when treated with Metformin. This synthetic lethality demonstrated in vitro and currently being tested in vivo will be discussed. Conclusions: These findings highlight that a major protumoral action of CD147/Basigin is to control the energetics of glycolytic tumors via MCT1/MCT4 activity and that blocking lactic acid export provides an efficient anticancer approach. Furthermore, we reveal that Metformin, by sensitizing normoxic cells to inhibitors of lactic export (MCTs), could offer an interesting novel anticancer strategy for rapidly growing tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3225. doi:1538-7445.AM2012-3225