ABSTRACT
A small library of new piperidine-triazole hybrids with 3-aryl isoxazole side chains has been designed and synthesized. Their cytotoxicity against a panel of seven cancer cell lines has been established. For the most promising compound, an IC50 value of 3.8 µM on PUMA/Bcl-xL interaction in live cancer cells was established through BRET analysis. A rationale was proposed for these results through complete molecular modelling studies.
Subject(s)
Antineoplastic Agents/pharmacology , Isoxazoles/pharmacology , Piperidines/pharmacology , Triazoles/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Isoxazoles/chemistry , Models, Molecular , Molecular Structure , Piperidines/chemical synthesis , Piperidines/chemistry , Structure-Activity Relationship , Triazoles/chemical synthesis , Triazoles/chemistryABSTRACT
BACKGROUND: Glioblastoma (GBM) are highly heterogeneous on the cellular and molecular basis. It has been proposed that glutamine metabolism of primary cells established from human tumors discriminates aggressive mesenchymal GBM subtype to other subtypes. METHODS: To study glutamine metabolism in vivo, we used a human orthotopic mouse model for GBM. Tumors evolving from the implanted primary GBM cells expressing different molecular signatures were analyzed using mass spectrometry for their metabolite pools and enrichment in carbon 13 (13C) after 13C-glutamine infusion. RESULTS: Our results showed that mesenchymal GBM tumors displayed increased glutamine uptake and utilization compared to both control brain tissue and other GBM subtypes. Furthermore, both glutamine synthetase and transglutaminase-2 were expressed accordingly to GBM metabolic phenotypes. CONCLUSION: Thus, our results outline the specific enhanced glutamine flux in vivo of the aggressive mesenchymal GBM subtype.
ABSTRACT
A fascinating but uncharacterized action of antimitotic chemotherapy is to collectively prime cancer cells to apoptotic mitochondrial outer membrane permeabilization (MOMP), while impacting only on cycling cell subsets. Here, we show that a proapoptotic secretory phenotype is induced by activation of cGAS/STING in cancer cells that are hit by antimitotic treatment, accumulate micronuclei and maintain mitochondrial integrity despite intrinsic apoptotic pressure. Organotypic cultures of primary human breast tumors and patient-derived xenografts sensitive to paclitaxel exhibit gene expression signatures typical of type I IFN and TNFα exposure. These cytokines induced by cGAS/STING activation trigger NOXA expression in neighboring cells and render them acutely sensitive to BCL-xL inhibition. cGAS/STING-dependent apoptotic effects are required for paclitaxel response in vivo, and they are amplified by sequential, but not synchronous, administration of BH3 mimetics. Thus anti-mitotic agents propagate apoptotic priming across heterogeneously sensitive cancer cells through cytosolic DNA sensing pathway-dependent extracellular signals, exploitable by delayed MOMP targeting.
Subject(s)
Antimitotic Agents/pharmacology , Apoptosis/drug effects , Breast Neoplasms/pathology , Membrane Proteins/metabolism , Paracrine Communication/drug effects , Animals , Breast Neoplasms/metabolism , Cell Line , Female , Gene Knockout Techniques , Humans , Interferon Type I/genetics , Interferon Type I/metabolism , Membrane Proteins/genetics , Mice , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Paclitaxel/pharmacology , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction/drug effects , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Xenograft Model Antitumor Assays , bcl-X Protein/antagonists & inhibitors , bcl-X Protein/metabolismABSTRACT
We describe the first examples of small molecules able to disrupt the nanomolar interaction between the pro-apoptotic protein PUMA and its anti-apoptotic counterpart BcL-xL in malignant cells. Based on molecular modelling studies, we propose a rationale to this result, through a new "bottle-opener"-type strategy which could be of general use in the area of protein-protein interaction studies.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Models, Molecular , Proto-Oncogene Proteins/metabolism , bcl-X Protein/metabolism , Animals , Apoptosis/drug effects , Humans , Neoplasms/drug therapy , Neoplasms/pathology , Protein Interaction Domains and Motifs/drug effectsABSTRACT
Protein-protein interactions are attractive targets because they control numerous cellular processes. In oncology, apoptosis regulating Bcl-2 family proteins are of particular interest. Apoptotic cell death is controlled via PPIs between the anti-apoptotic proteins hydrophobic groove and the pro-apoptotic proteins BH3 domain. In ovarian carcinoma, it has been previously demonstrated that Bcl-xL and Mcl-1 cooperate to protect tumor cells against apoptosis. Moreover, Mcl-1 is a key regulator of cancer cell survival and is a known resistance factor to Bcl-2/Bcl-xL pharmacological inhibitors making it an attractive therapeutic target. Here, using a structure-guided design from the oligopyridine lead Pyridoclax based on Noxa/Mcl-1 interaction we identified a new derivative, active at lower concentration as compared to Pyridoclax. This new derivative selectively binds to the Mcl-1 hydrophobic groove and releases Bak and Bim from Mcl-1 to induce cell death and sensitize cancer cells to Bcl-2/Bcl-xL targeting strategies.
Subject(s)
Drug Design , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Pyridines/pharmacology , Cell Death/drug effects , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Dynamics Simulation , Molecular Structure , Myeloid Cell Leukemia Sequence 1 Protein/chemistry , Protein Binding , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/chemistry , Pyridines/chemical synthesis , Pyridines/chemistry , Structure-Activity Relationship , Tumor Cells, Cultured , bcl-X Protein/antagonists & inhibitors , bcl-X Protein/metabolismABSTRACT
Phosphorylation of Ser/Thr residues is a well-established modulating mechanism of the pro-apoptotic function of the BH3-only protein Bim. However, nothing is known about the putative tyrosine phosphorylation of this Bcl-2 family member and its potential impact on Bim function and subsequent Bax/Bak-mediated cytochrome c release and apoptosis. As we have previously shown that the tyrosine kinase Lyn could behave as an anti-apoptotic molecule, we investigated whether this Src family member could directly regulate the pro-apoptotic function of Bim. In the present study, we show that Bim is phosphorylated onto tyrosine residues 92 and 161 by Lyn, which results in an inhibition of its pro-apoptotic function. Mechanistically, we show that Lyn-dependent tyrosine phosphorylation of Bim increases its interaction with anti-apoptotic members such as Bcl-xL, therefore limiting mitochondrial outer membrane permeabilization and subsequent apoptosis. Collectively, our data uncover one molecular mechanism through which the oncogenic tyrosine kinase Lyn negatively regulates the mitochondrial apoptotic pathway, which may contribute to the transformation and/or the chemotherapeutic resistance of cancer cells.
Subject(s)
Apoptosis/genetics , Bcl-2-Like Protein 11/physiology , src-Family Kinases/physiology , Animals , Bcl-2-Like Protein 11/antagonists & inhibitors , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Cells, Cultured , Drug Resistance, Neoplasm/genetics , HEK293 Cells , HeLa Cells , Humans , K562 Cells , Mice , Mitochondria/genetics , Mitochondria/metabolism , Oncogenes/physiology , Signal Transduction/genetics , src-Family Kinases/geneticsABSTRACT
E2F1 is the main pro-apoptotic effector of the pRB-regulated tumor suppressor pathway by promoting the transcription of various pro-apoptotic proteins. We report here that E2F1 partly localizes to mitochondria, where it favors mitochondrial outer membrane permeabilization. E2F1 interacts with BCL-xL independently from its BH3 binding interface and induces a stabilization of BCL-xL at mitochondrial membranes. This prevents efficient control of BCL-xL over its binding partners, in particular over BAK resulting in the induction of cell death. We thus identify a new, non-BH3-binding regulator of BCL-xL localization dynamics that influences its anti-apoptotic activity.
Subject(s)
Cell Death , E2F1 Transcription Factor/metabolism , bcl-X Protein/metabolism , Apoptosis , Cell Line, Tumor , E2F1 Transcription Factor/chemistry , Extracellular Space/metabolism , Gene Expression Regulation/drug effects , Humans , Mitochondria/metabolism , Protein Binding , Protein Transport , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Transcription, Genetic , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-X Protein/chemistryABSTRACT
In tumours, accumulation of chemoresistant cells that express high levels of anti-apoptotic proteins such as BCL-XL is thought to result from the counter selection of sensitive, low expresser clones during progression and/or initial treatment. We herein show that BCL-XL expression is selectively advantageous to cancer cell populations even in the absence of pro-apoptotic pressure. In transformed human mammary epithelial cells BCL-XL favours full activation of signalling downstream of constitutively active RAS with which it interacts in a BH4-dependent manner. Comparative proteomic analysis and functional assays indicate that this is critical for RAS-induced expression of stemness regulators and maintenance of a cancer initiating cell (CIC) phenotype. Resistant cancer cells thus arise from a positive selection driven by BCL-XL modulation of RAS-induced self-renewal, and during which apoptotic resistance is not necessarily the directly selected trait.
Subject(s)
Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Neoplastic Stem Cells/cytology , Signal Transduction , bcl-X Protein/metabolism , ras Proteins/metabolism , Animals , Apoptosis , Cell Line, Tumor , Drug Resistance, Neoplasm , Female , HMGA2 Protein/metabolism , Humans , MCF-7 Cells , Mass Spectrometry , Mice , Mice, Nude , Neoplasm Recurrence, Local , Phenotype , Plasmids/metabolism , Proteomics , Proto-Oncogene Proteins c-fos/metabolismABSTRACT
Purpose: Glioblastoma (GBM) is the most common and malignant form of primary human brain tumor in adults, with an average survival at diagnosis of 18 months. Metabolism is a new attractive therapeutic target in cancer; however, little is known about metabolic heterogeneity and plasticity within GBM tumors. We therefore aimed to investigate metabolic phenotyping of primary cultures in the context of molecular tumor heterogeneity to provide a proof of concept for personalized metabolic targeting of GBM.Experimental Design: We have analyzed extensively several primary GBM cultures using transcriptomics, metabolic phenotyping assays, and mitochondrial respirometry.Results: We found that metabolic phenotyping clearly identifies 2 clusters, GLNHigh and GLNLow, mainly based on metabolic plasticity and glutamine (GLN) utilization. Inhibition of glutamine metabolism slows the in vitro and in vivo growth of GLNHigh GBM cultures despite metabolic adaptation to nutrient availability, in particular by increasing pyruvate shuttling into mitochondria. Furthermore, phenotypic and molecular analyses show that highly proliferative GLNHigh cultures are CD133neg and display a mesenchymal signature in contrast to CD133pos GLNLow GBM cells.Conclusions: Our results show that metabolic phenotyping identified an essential metabolic pathway in a GBM cell subtype, and provide a proof of concept for theranostic metabolic targeting. Clin Cancer Res; 23(20); 6292-304. ©2017 AACR.
Subject(s)
Brain Neoplasms/metabolism , Glioblastoma/metabolism , Glutamine/metabolism , Mitochondria/metabolism , Animals , Biomarkers , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cluster Analysis , Computational Biology/methods , Disease Models, Animal , Energy Metabolism , Gene Expression Profiling , Glioblastoma/genetics , Glioblastoma/pathology , Glucose/metabolism , Heterografts , Humans , Metabolomics/methods , Mice , Models, Biological , PhenotypeABSTRACT
Anti-apoptotic BCL-2 family members bind to BH3-only proteins and multidomain BAX/BAK to preserve mitochondrial integrity and maintain survival. Whereas inhibition of these interactions is the biological basis of BH3-mimetic anti-cancer therapy, the actual response of membrane-bound protein complexes to these compounds is currently ill-defined. Here, we find that treatment with BH3 mimetics targeting BCL-xL spares subsets of cells with the highest levels of this protein. In intact cells, sequestration of some pro-apoptotic activators (including PUMA and BIM) by full-length BCL-xL is much more resistant to derepression than previously described in cell-free systems. Alterations in the BCL-xL C-terminal anchor that impacts subcellular membrane-targeting and localization dynamics restore sensitivity. Thus, the membrane localization of BCL-xL enforces its control over cell survival and, importantly, limits the pro-apoptotic effects of BH3 mimetics by selectively influencing BCL-xL binding to key pro-apoptotic effectors.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis/genetics , Mitochondria/genetics , Neoplasms/genetics , bcl-X Protein/genetics , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11/genetics , Cell Survival/genetics , Cell-Free System , HCT116 Cells , Humans , Mitochondria/metabolism , Neoplasms/drug therapy , Peptide Fragments/administration & dosage , Proto-Oncogene Proteins/administration & dosage , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , bcl-X Protein/metabolismABSTRACT
The Bcl-2 family includes 26 proteins involved in apoptosis. Cancer cells can develop the ability to avoid apoptosis through the upregulation and/or down regulation of such proteins Bax, Bcl-xL or Mcl-1, especially during chemoresistance progress. These proteins engaged in a network of dynamic interactions that control apoptosis triggering have become attractive therapeutic targets in cancers including melanoma. Among them, the Bax/Bcl-xL interaction appears critical in maintaining mitochondria integrity. Therefore a series of mixed polyphenol-heterocyclic molecules, were rationally designed by molecular docking as Bax/Bcl-xL inhibitors. It has been screened against B16-F10 melanoma cancer cells for a preliminary investigation of their cytotoxicity. All these compounds exhibited a significant cytotoxicity against these cancer cells, in the 0.3-6 .M range. A pyrazole-type molecule, which had a submicromolar IC50 value with an excellent selectivity index (14), is the most promising derivative for further development.
Subject(s)
Antineoplastic Agents/pharmacology , Catechols/pharmacology , Pyrazoles/pharmacology , Antineoplastic Agents/chemical synthesis , Apoptosis/drug effects , Bioluminescence Resonance Energy Transfer Techniques , Catechols/chemical synthesis , Cell Line, Tumor , HeLa Cells , Humans , Melanoma, Experimental , Molecular Docking Simulation , Pyrazoles/chemical synthesis , bcl-2-Associated X Protein/antagonists & inhibitors , bcl-X Protein/antagonists & inhibitorsABSTRACT
Translationally Controlled Tumor Protein (TCTP) is anti-apoptotic, key in development and cancer, however without the typical Bcl2 family members' structure. Here we report that TCTP contains a BH3-like domain and forms heterocomplexes with Bcl-xL. The crystal structure of a Bcl-xL deletion variant-TCTP11-31 complex reveals that TCTP refolds in a helical conformation upon binding the BH3-groove of Bcl-xL, although lacking the h1-subregion interaction. Experiments using in vitro-vivo reconstituted systems and TCTP(+/-) mice indicate that TCTP activates the anti-apoptotic function of Bcl-xL, in contrast to all other BH3-proteins. Replacing the non-conserved h1 of TCTP by that of Bax drastically increases the affinity of this hybrid for Bcl-xL, modifying its biological properties. This work reveals a novel class of BH3-proteins potentiating the anti-apoptotic function of Bcl-xL.
Subject(s)
Biomarkers, Tumor/metabolism , Protein Interaction Domains and Motifs , bcl-X Protein/metabolism , Amino Acid Sequence , Animals , Apoptosis , BH3 Interacting Domain Death Agonist Protein/metabolism , Biomarkers, Tumor/chemistry , Cell Membrane Permeability , Mice , Models, Molecular , Multiprotein Complexes/metabolism , Protein Binding , Protein Conformation , Protein Multimerization , Tumor Protein, Translationally-Controlled 1 , bcl-2-Associated X Protein/metabolism , bcl-X Protein/chemistryABSTRACT
Inhibition of Bcl-2 family protein-protein interactions (PPI) is a very promising direction in cancer chemotherapy. Hence over the last decade, many medicinal chemistry studies endeavoured to discover drug candidates, and a wealth of chemical scaffolds with striking chemical diversity was reported as Bcl-xL inhibitors. This raises the question of whether all these molecules could occupy a unique binding site, or rather discrete pockets of the protein surface. To test if small and chemically diverse Bcl-xL inhibitors are likely to bind a single pocket, and to identify which pocket, we used a battery of computational and modeling approaches. We first checked that the large dataset of Bcl-xL inhibitors we built can actually fit to a universal pharmacophore. Then we defined the probable binding hot spots of interaction through comparison of crystal structures, as well as virtual fragment screening. Finally, new analogues of small polyphenol derivatives were synthesized to precisely probe a hydrogen bond suggested by docking experiments. Bcl-xL inhibition potency of these products confirmed the predicted binding mode. This combination of X-ray structure exploration, molecular modeling studies and medicinal chemistry supports that all these small Bcl-xL inhibitors occupy the same hot spot of interaction. The identification of this binding site should help the design and optimization of small PPI Bcl-xL inhibitors.
Subject(s)
Drug Design , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , bcl-X Protein/antagonists & inhibitors , bcl-X Protein/metabolism , Amino Acid Sequence , Binding Sites , Computer Simulation , Humans , Models, Molecular , Molecular Sequence Data , Protein Binding , Structure-Activity Relationship , bcl-X Protein/chemistryABSTRACT
Apoptosis control defects such as the deregulation of Bcl-2 family member expression are frequently involved in chemoresistance. In ovarian carcinoma, we previously demonstrated that Bcl-xL and Mcl-1 cooperate to protect cancer cells against apoptosis and their concomitant inhibition leads to massive apoptosis even in the absence of chemotherapy. Whereas Bcl-xL inhibitors are now available, Mcl-1 inhibition, required to sensitize cells to Bcl-xL-targeting strategies, remains problematic. In this context, we designed and synthesized oligopyridines potentially targeting the Mcl-1 hydrophobic pocket, evaluated their capacity to inhibit Mcl-1 in live cells, and implemented a functional screening assay to evaluate their ability to sensitize ovarian carcinoma cells to Bcl-xL-targeting strategies. We established structure-activity relationships and focused our attention on MR29072, named Pyridoclax. Surface plasmon resonance assay demonstrated that pyridoclax directly binds to Mcl-1. Without cytotoxic activity when administered as a single agent, pyridoclax induced apoptosis in combination with Bcl-xL-targeting siRNA or with ABT-737 in ovarian, lung, and mesothelioma cancer cells.
Subject(s)
Molecular Targeted Therapy , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Pyridines/pharmacology , bcl-X Protein/antagonists & inhibitors , Apoptosis/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Female , Humans , Models, Molecular , Molecular Structure , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Ovarian Neoplasms/pathology , Pyridines/chemical synthesis , Pyridines/chemistry , Quantitative Structure-Activity Relationship , Quantum Theory , Tumor Cells, Cultured , bcl-X Protein/metabolismABSTRACT
We describe the synthesis of a series of new molecules containing phenol and triazoles moieties, compounds which have been evaluated for their ability to inhibit Bax/Bcl-xL interactions in cancer cells, by using BRET assays, and to induce cell death. Several derivatives exhibit a very promising activity, being more potent than the reference compounds acylpyrogallol A and ABT-737. These preliminary results demonstrate that derivatives of this family can be attractive to develop new molecules with potent anticancer activity.
Subject(s)
Drug Design , Phenols/pharmacology , Triazoles/pharmacology , bcl-2-Associated X Protein/antagonists & inhibitors , bcl-X Protein/antagonists & inhibitors , Cell Death/drug effects , Dose-Response Relationship, Drug , HeLa Cells , Humans , Molecular Docking Simulation , Molecular Structure , Phenols/chemical synthesis , Phenols/chemistry , Structure-Activity Relationship , Triazoles/chemical synthesis , Triazoles/chemistry , bcl-2-Associated X Protein/metabolism , bcl-X Protein/metabolismABSTRACT
Cancer cells are subject to many apoptotic stimuli that would kill them were it not for compensatory prosurvival alterations. BCL-2-like (BCL-2L) proteins contribute to such aberrant behaviour by engaging a network of interactions that is potent at promoting survival but that is also fragile: inhibition of a restricted number of interactions may suffice to trigger cancer cell death. Currently available and novel compounds that inhibit these interactions could be efficient therapeutic agents if this phenotype of BCL-2L dependence was better understood at a molecular, cellular and systems level and if it could be diagnosed by relevant biomarkers.
Subject(s)
Antineoplastic Agents/therapeutic use , Neoplasms/etiology , Neoplasms/prevention & control , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/physiology , Antineoplastic Agents/pharmacology , Humans , Molecular Targeted Therapy , Neoplasms/metabolismABSTRACT
Embelin is a natural product, inhibitor of XIAP (X-chromosome-linked Inhibitor of APoptosis) with strong proapoptotic properties on cancer cells. In order to clarify the role of two OH groups on benzoquinone core, we have prepared by hemisynthesis close analogs of embelin, where these OH groups have been replaced in a systematic manner by OMe and OAc groups. Proapoptotic activities of six embelin derivatives have been studied as single agent, or in combination with TRAIL, and their abilities to interact with XIAP have been evaluated by Surface Plasmon Biacore. Our results show that these new embelin analogs have good proapoptotic properties against selected cancer cells, often higher than the natural product itself. Further, this activity is not directly mediated by XIAP. Altogether these preliminary results demonstrate that for active embelin analogs, the two OH groups are not absolutely required for anticancer activity, opening new possibilities for the design of proapoptotic derivatives in these series.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Benzoquinones/chemical synthesis , Benzoquinones/pharmacology , Antineoplastic Agents/chemical synthesis , Benzoquinones/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Humans , Molecular Structure , Structure-Activity RelationshipABSTRACT
This paper describes the synthesis of nine selected diaryl/heteroaryl-containing phenol and polyphenol derivatives which have been evaluated against Bax/Bcl-xL interaction in comparison with ABT-737. Using a BRET assay, six of these derivatives exhibit activity comparable to ABT-737 to disrupt Bax/Bcl-xL interaction. These preliminary results demonstrate that such polyphenol-derived molecules are attractive compounds regarding anticancer activity and that the phenol at position 3 is important regarding disruption of Bax/Bcl-xL interaction.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Polyphenols/chemistry , Polyphenols/pharmacology , bcl-2-Associated X Protein/metabolism , bcl-X Protein/metabolism , Apoptosis/drug effects , HeLa Cells , Humans , Protein Binding/drug effects , Structure-Activity RelationshipABSTRACT
Elevated activation of the platelet-derived growth factor (PDGF) pathway, apoptosis evasion phenotype, and global DNA hypomethylation are hallmarks frequently observed in cancers, such as in low-grade glioma (LGG). However, the orchestration of these malignant functions is not fully elucidated in LGG. Our study reveals that the co-presence of these hallmarks in the same LGG is frequent and confers poor prognosis in patients with LGG. Our data also indicate that the apoptosis evasion phenotype of these cells harboring a hypomethylation-induced activation of the PDGF pathway is associated with a hypomethylation of the bcl-xl and bcl-w genes and the phosphorylation and/or downregulation of three major pro-apoptotic BH3-only proteins: PUMA, Bad, and Bim. Consistent with this, we demonstrate that the use of folate, a DNA-methylating agent, promotes the reprogramming of the sensitivity of glioma cells to ABT-737/etoposide-induced apoptosis and reduces the dose of ABT-737 required to promote etoposide-induced apoptosis. This work supports the idea that the inclusion of folate and/or ABT-737 could be a promising adjuvant in the design of anti-glioma therapeutic protocols in clinical studies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13148-011-0035-5) contains supplementary material, which is available to authorized users.
ABSTRACT
BACKGROUND: Anti-apoptotic signals induced downstream of HER2 are known to contribute to the resistance to current treatments of breast cancer cells that overexpress this member of the EGFR family. Whether or not some of these signals are also involved in tumor maintenance by counteracting constitutive death signals is much less understood. To address this, we investigated what role anti- and pro-apoptotic Bcl-2 family members, key regulators of cancer cell survival, might play in the viability of HER2 overexpressing breast cancer cells. METHODS: We used cell lines as an in vitro model of HER2-overexpressing cells in order to evaluate how anti-apoptotic Bcl-2, Bcl-xL and Mcl-1, and pro-apoptotic Puma and Bim impact on their survival, and to investigate how the constitutive expression of these proteins is regulated. Expression of the proteins of interest was confirmed using lysates from HER2-overexpressing tumors and through analysis of publicly available RNA expression data. RESULTS: We show that the depletion of Mcl-1 is sufficient to induce apoptosis in HER2-overexpressing breast cancer cells. This Mcl-1 dependence is due to Bim expression and it directly results from oncogenic signaling, as depletion of the oncoprotein c-Myc, which occupies regions of the Bim promoter as evaluated in ChIP assays, decreases Bim levels and mitigates Mcl-1 dependence. Consistently, a reduction of c-Myc expression by inhibition of mTORC1 activity abrogates occupancy of the Bim promoter by c-Myc, decreases Bim expression and promotes tolerance to Mcl-1 depletion. Western blot analysis confirms that naïve HER2-overexpressing tumors constitutively express detectable levels of Mcl-1 and Bim, while expression data hint on enrichment for Mcl-1 transcripts in these tumors. CONCLUSIONS: This work establishes that, in HER2-overexpressing tumors, it is necessary, and maybe sufficient, to therapeutically impact on the Mcl-1/Bim balance for efficient induction of cancer cell death.