RESUMO
Epithelial-mesenchymal transition (EMT) and primary ciliogenesis are two cell-biological programs that are essential for development of multicellular organisms and whose abnormal regulation results in many diseases (i.e., developmental anomalies and cancers). Emerging studies suggest an intricate interplay between these two processes. Here, we discuss physiological and pathological contexts in which their interconnections promote normal development or disease progression. We describe underlying molecular mechanisms of the interplay and EMT/ciliary signaling axes that influence EMT-related processes (i.e., stemness, motility and invasion). Understanding the molecular and cellular mechanisms of the relationship between EMT and primary ciliogenesis may provide new insights in the etiology of diseases related to EMT and cilia dysfunction.
Assuntos
Transição Epitelial-Mesenquimal , Neoplasias , Humanos , Transição Epitelial-Mesenquimal/fisiologia , Transdução de Sinais , CíliosRESUMO
MOTIVATION: The principle of Breiman's random forest (RF) is to build and assemble complementary classification trees in a way that maximizes their variability. We propose a new type of random forest that disobeys Breiman's principles and involves building trees with no classification errors in very large quantities. We used a new type of decision tree that uses a neuron at each node as well as an in-innovative half Christmas tree structure. With these new RFs, we developed a score, based on a family of ten new statistical information criteria, called Nguyen information criteria (NICs), to evaluate the predictive qualities of features in three dimensions. RESULTS: The first NIC allowed the Akaike information criterion to be minimized more quickly than data obtained with the Gini index when the features were introduced in a logistic regression model. The selected features based on the NICScore showed a slight advantage compared to the support vector machines-recursive feature elimination (SVM-RFE) method. We demonstrate that the inclusion of artificial neurons in tree nodes allows a large number of classifiers in the same node to be taken into account simultaneously and results in perfect trees without classification errors. AVAILABILITY AND IMPLEMENTATION: The methods used to build the perfect trees in this article were implemented in the 'ROP' R package, archived at https://cran.r-project.org/web/packages/ROP/index.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
RESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Isoxazóis/farmacologia , Piperidinas/farmacologia , Triazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Isoxazóis/química , Modelos Moleculares , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
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.
Assuntos
Morte Celular , Fator de Transcrição E2F1/metabolismo , Proteína bcl-X/metabolismo , Apoptose , Linhagem Celular Tumoral , Fator de Transcrição E2F1/química , Espaço Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transcrição Gênica , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/químicaRESUMO
Survivin overexpression, frequently found in breast cancers and others, is associated with poor prognosis. Its dual regulation of cell division and apoptosis makes it an attractive therapeutic target but its exact functions that are required for tumor maintenance are still elusive. Survivin protects cancer cells from genotoxic agents and this ability is generally assigned to a universal anti-apoptotic function. However, a specific role in cancer cell protection from DNA damage has been overlooked so far. We assessed DNA damage occurrence in Survivin-depleted breast cancer cells using γH2AX staining and comete assay. QPCR data and a gene conversion assay indicated that homologous recombination (HR) was impaired upon Survivin depletion. We conducted the analysis of Survivin and HR genes' expression in breast tumors. We revealed BRCAness phenotype of Survivin-depleted cells using cell death assays combined to PARP targeting. Survivin silencing leads to DNA double-strand breaks in breast cancer cells and functionally reduces HR. Survivin depletion decreases the transcription of a set of genes involved in HR, decreases RAD51 protein expression and impairs the endonuclease complex MUS81/EME1 involved in the resolution of Holliday junctions. Clinically, EME1, RAD51, EXO1, BLM expressions correlate with that of BIRC5 (coding for Survivin) and are of prognostic value. Functionally, Survivin depletion triggers p53 activation and sensitizes cancer cells to of PARP inhibition. We defined Survivin as a constitutive actor of HR in breast cancers, and implies that its inhibition would enhance cell vulnerability upon PARP inhibition.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Reparo do DNA , Recombinação Homóloga , Proteínas Inibidoras de Apoptose/metabolismo , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Resistencia a Medicamentos Antineoplásicos , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Histonas/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Prognóstico , Survivina , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
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Desenho de Fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Relação Estrutura-Atividade , Proteína bcl-X/químicaRESUMO
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.
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Desenho de Fármacos , Fenóis/farmacologia , Triazóis/farmacologia , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína bcl-X/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fenóis/síntese química , Fenóis/química , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Breast cancer stands as the most prevalent malignancy afflicting women. Despite significant advancements in its diagnosis and treatment, breast cancer metastasis continues to be a leading cause of mortality among women. To metastasize, cancer cells face numerous challenges: breaking away from the primary tumor, surviving in the circulation, establishing in a distant location, evading immune detection and, finally, thriving to initiate a new tumor. Each of these sequential steps requires cancer cells to adapt to a myriad of stressors and develop survival mechanisms. In addition, most patients with breast cancer undergo surgical removal of their primary tumor and have various therapeutic interventions designed to eradicate cancer cells. Despite this plethora of attacks and stresses, certain cancer cells not only manage to persist but also proliferate robustly, giving rise to substantial tumors that frequently culminate in the patient's demise. To enhance patient outcomes, there is an imperative need for a deeper understanding of the molecular and cellular mechanisms that empower cancer cells to not only survive but also expand. Herein, we delve into the intrinsic stresses that cancer cells encounter throughout the metastatic journey and the additional stresses induced by therapeutic interventions. We focus on elucidating the remarkable strategies adopted by cancer cells, such as cell-cell clustering and intricate cell-cell communication mechanisms, to ensure their survival.
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Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Comunicação CelularRESUMO
Triple negative breast cancers (TNBC) present a poor prognosis primarily due to their resistance to chemotherapy. This resistance is known to be associated with elevated expression of certain anti-apoptotic members within the proteins of the BCL-2 family (namely BCL-xL, MCL-1 and BCL-2). These regulate cell death by inhibiting pro-apoptotic protein activation through binding and sequestration and they can be selectively antagonized by BH3 mimetics. Yet the individual influences of BCL-xL, MCL-1, and BCL-2 on the sensitivity of TNBC cells to chemotherapy, and their regulation by cancer-associated fibroblasts (CAFs), major components of the tumor stroma and key contributors to therapy resistance remain to be delineated. Using gene editing or BH3 mimetics to inhibit anti-apoptotic BCL-2 family proteins in TNBC line MDA-MB-231, we show that BCL-xL and MCL-1 promote cancer cell survival through compensatory mechanisms. This cell line shows limited sensitivity to chemotherapy, in line with the clinical resistance observed in TNBC patients. We elucidate that BCL-xL plays a pivotal role in therapy response, as its depletion or pharmacological inhibition heightened chemotherapy effectiveness. Moreover, BCL-xL expression is associated with chemotherapy resistance in patient-derived tumoroids where its pharmacological inhibition enhances ex vivo response to chemotherapy. In a co-culture model of cancer cells and CAFs, we observe that even in a context where BCL-xL reduced expression renders cancer cells more susceptible to chemotherapy, those in contact with CAFs display reduced sensitivity to chemotherapy. Thus CAFs exert a profound pro-survival effect in breast cancer cells, even in a setting highly favoring cell death through combined chemotherapy and absence of the main actor of chemoresistance, BCL-xL.
Assuntos
Fibroblastos Associados a Câncer , Resistencia a Medicamentos Antineoplásicos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neoplasias de Mama Triplo Negativas , Proteína bcl-X , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteína bcl-X/metabolismo , Proteína bcl-X/genética , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Resistencia a Medicamentos Antineoplásicos/genética , Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidoresRESUMO
BACKGROUND: Robust molecular subtyping of triple-negative breast cancer (TNBC) is a prerequisite for the success of precision medicine. Today, there is a clear consensus on three TNBC molecular subtypes: luminal androgen receptor (LAR), basal-like immune-activated (BLIA), and basal-like immune-suppressed (BLIS). However, the debate about the robustness of other subtypes is still open. METHODS: An unprecedented number (n = 1942) of TNBC patient data was collected. Microarray- and RNAseq-based cohorts were independently investigated. Unsupervised analyses were conducted using k-means consensus clustering. Clusters of patients were then functionally annotated using different approaches. Prediction of response to chemotherapy and targeted therapies, immune checkpoint blockade, and radiotherapy were also screened for each TNBC subtype. RESULTS: Four TNBC subtypes were identified in the cohort: LAR (19.36%); mesenchymal stem-like (MSL/MES) (17.35%); BLIA (31.06%); and BLIS (32.23%). Regarding the MSL/MES subtype, we suggest renaming it to mesenchymal-like immune-altered (MLIA) to emphasize its specific histological background and nature of immune response. Treatment response prediction results show, among other things, that despite immune activation, immune checkpoint blockade is probably less or completely ineffective in MLIA, possibly caused by mesenchymal background and/or an enrichment in dysfunctional cytotoxic T lymphocytes. TNBC subtyping results were included in the bc-GenExMiner v5.0 webtool ( http://bcgenex.ico.unicancer.fr ). CONCLUSION: The mesenchymal TNBC subtype is characterized by an exhausted and altered immune response, and resistance to immune checkpoint inhibitors. Consensus for molecular classification of TNBC subtyping and prediction of cancer treatment responses helps usher in the era of precision medicine for TNBC patients.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/terapia , Neoplasias de Mama Triplo Negativas/genética , Feminino , Biomarcadores Tumorais/metabolismo , Pessoa de Meia-Idade , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Receptores Androgênicos/metabolismo , Perfilação da Expressão Gênica , Inibidores de Checkpoint Imunológico/uso terapêutico , Prognóstico , Adulto , Idoso , Medicina de Precisão/métodosRESUMO
Cilia are hair-like projections that assemble at the surface of cells in various tissues of multicellular organisms through a complex cell biological process called ciliogenesis. Cilia can assemble as single structures per cell (i.e. non-motile primary cilia), which act as cell signaling centers that dictate cell fate, or can be assembled in distinct cell types as many copies per cell (i.e. motile cilia) that beat to move fluids at the cell surface. The mechanisms that orchestrate formation and function of cilia, which are dysregulated in pathological settings such as ciliopathies, remain incompletely understood. Stem cell-derived organoids represent valuable models to study the mechanisms of ciliogenesis, ciliary signaling, and ciliary beating that collectively promote tissue development and homeostasis. Here, we present a comprehensive protocol for the growth of mammary organoids derived from mouse mammary stem cells and for immunofluorescence staining of primary cilia in these three-dimensional structures.
Assuntos
Cílios , Transdução de Sinais , Animais , Camundongos , Cílios/metabolismo , Diferenciação CelularRESUMO
Introduction: Tumor Associated Macrophages (TAM) are a major component of the tumor environment and their accumulation often correlates with poor prognosis by contributing to local inflammation, inhibition of anti-tumor immune response and resistance to anticancer treatments. In this study, we thus investigated the anti-cancer therapeutic interest to target ChemR23, a receptor of the resolution of inflammation expressed by macrophages, using an agonist monoclonal antibody, αChemR23. Methods: Human GM-CSF, M-CSF and Tumor Associated Macrophage (TAM)-like macrophages were obtained by incubation of monocytes from healthy donors with GM-CSF, M-CSF or tumor cell supernatants (Breast cancer (BC) or malignant pleural mesothelioma (MPM) cells). The effects of αChemR23 on macrophages were studied at the transcriptomic, protein and functional level. Datasets from The Cancer Genome Atlas (TCGA) were used to study CMKLR1 expression, coding for ChemR23, in BC and MPM tumors. In vivo, αChemR23 was evaluated on overall survival, metastasis development and transcriptomic modification of the metastatic niche using a model of resected triple negative breast cancer. Results: We show that ChemR23 is expressed at higher levels in M-CSF and tumor cell supernatant differentiated macrophages (TAM-like) than in GM-CSF-differentiated macrophages. ChemR23 activation triggered by αChemR23 deeply modulates M-CSF and TAM-like macrophages including profile of cell surface markers, cytokine secretion, gene mRNA expression and immune functions. The expression of ChemR23 coding gene (CMKLR1) strongly correlates to TAM markers in human BC tumors and MPM and its histological detection in these tumors mainly corresponds to TAM expression. In vivo, treatment with αChemR23 agonist increased mouse survival and decreased metastasis occurrence in a model of triple-negative BC in correlation with modulation of TAM phenotype in the metastatic niche. Conclusion: These results open an attractive opportunity to target TAM and the resolution of inflammation pathways through ChemR23 to circumvent TAM pro-tumoral effects.
Assuntos
Neoplasias da Mama , Carcinoma , Receptores de Quimiocinas , Animais , Feminino , Humanos , Camundongos , Carcinoma/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Inflamação/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos , Fenótipo , Receptores de Quimiocinas/metabolismoRESUMO
Oncogene-induced senescence (OIS) is a tumor suppressor response that induces permanent cell cycle arrest in response to oncogenic signaling. Through the combined activation of the p53-p21 and p16-Rb suppressor pathways, OIS leads to the transcriptional repression of proliferative genes. Although this protective mechanism has been essentially described in primary cells, we surprisingly observed in this study that the OIS program is conserved in established colorectal cell lines. In response to the RAS oncogene and despite the inactivation of p53 and p16(INK4), HT29 cells enter senescence, up-regulate p21(WAF1), and induce senescence-associated heterochromatin foci formation. The same effect was observed in response to B-RAF(v600E) in LS174T cells. We also observed that p21(WAF1) prevents the expression of the CDC25A and PLK1 genes to induce cell cycle arrest. Using ChIP and luciferase experiments, we have observed that p21(WAF1) binds to the PLK1 promoter to induce its down-regulation during OIS induction. Following 4-5 weeks, several clones were able to resume proliferation and escape this tumor suppressor pathway. Tumor progression was associated with p21(WAF1) down-regulation and CDC25A and PLK1 reexpression. In addition, OIS and p21(WAF1) escape was associated with an increase in DNA damage, an induction of the epithelial-mesenchymal transition program, and an increase in the proportion of cells expressing the CD24(low)/CD44(high) phenotype. Results also indicate that malignant cells having escaped OIS rely on survival pathways induced by Bcl-xL/MCL1 signaling. In light of these observations, it appears that the transcriptional functions of p21(WAF1) are active during OIS and that the inactivation of this protein is associated with cell dedifferentiation and enhanced survival.
Assuntos
Desdiferenciação Celular , Senescência Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Mutação de Sentido Incorreto , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais/genética , Fatores de Tempo , Transcrição Gênica/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/genética , Proteína bcl-X/genética , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo , Quinase 1 Polo-LikeRESUMO
INTRODUCTION: Inappropriate Notch signaling, downstream of γ-secretase activity, is understood to have tumor-promoting function and to be associated with poor outcome in cancer, of the breast in particular. The molecular basis of antitumoral effects of its inhibitors, however, remains poorly characterized. Moreover, the effects of their combination with the pro-apoptotic pharmacologic inhibitor of Bcl-2/Bcl-xL, ABT-737, have never been evaluated. In this study, we thus specifically addressed the biologic consequences of targeting γ-secretase and Bcl-2/Bcl-xL, alone or simultaneously, in breast cancer cell lines as well as in a novel human breast cancer ex vivo assay. METHODS: By using in vitro 2D or 3D cultures of breast cancer cells plus a novel preclinical short-term ex vivo assay that correctly maintains human mammary tissue integrity and preserves tumor microenvironment, we tested the effects of the pharmacologic γ-secretase inhibitor GSIXII used as a single agent or in combination with ABT-737. RESULTS: We show herein that the γ-secretase inhibitor, GSIXII, efficiently induces apoptosis in breast cancer cell lines by a process that relies on the induction of Noxa, a pro-apoptotic Bcl2-homology 3 domain (BH3)-only protein of the Bcl-2 family that functions as an inhibitor of antiapoptotic Mcl1. GSIXII also targets mammary cancer stem-like cells because it dramatically prevents in vitro mammosphere formation. Moreover, combining GSIXII treatment with ABT-737, a BH3-mimetic inhibitor of additional antiapoptotic proteins, such as Bcl-2 and Bcl-xL, leads to both a synergistic apoptotic response in breast cancer cells and to an inhibitory effect on mammosphere formation. These effects are also found when a Notch transcriptional inhibitor, SAHM1, is used. Finally, we evaluated individual human tumor responses to γ-secretase inhibition alone or in combination with ABT-737 in ex vivo assays. Analysis of a series of 30 consecutive tumors indicated that a majority of tumors are sensitive to apoptosis induction by GSIXII and that association of GSIXII with ABT-737 leads to an enhanced induction of apoptosis in tumor cells. CONCLUSIONS: We thus provide evidence that γ-secretase, and downstream Notch signaling, are relevant targets in breast cancer. GSIXII, used as single agent or in combination with clinically relevant BH3-mimetics, is a promising innovative proapoptotic strategy to treat mammary tumors.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Dipeptídeos/farmacologia , Nitrofenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Neoplasias da Mama , Linhagem Celular Tumoral , Feminino , Humanos , Células MCF-7 , Proteína de Sequência 1 de Leucemia de Células Mieloides , Fragmentos de Peptídeos/antagonistas & inibidores , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , RNA Interferente Pequeno , Receptores Notch/metabolismo , Microambiente Tumoral , Proteína bcl-X/antagonistas & inibidoresRESUMO
Cancer-associated fibroblasts (CAF) are a major cellular component of epithelial tumors. In breast cancers in particular these stromal cells have numerous tumorigenic effects in part due to their acquisition of a myofibroblastic phenotype. Breast CAFs (bCAFs) typically express MCL-1. We show here that pharmacological inhibition or knock down of this regulator of mitochondrial integrity in primary bCAFs directly derived from human samples mitigates myofibroblastic features. This decreases expression of genes involved in actomyosin organization and contractility (associated with a cytoplasmic retention of the transcriptional regulator, yes-associated protein-YAP) and decreases bCAFs ability to promote cancer cells invasion in 3D coculture assays. Our findings underscore the usefulness of targeting MCL-1 in breast cancer ecosystems, not only to favor death of cancer cells but also to counteract the tumorigenic activation of fibroblasts with which they co-evolve. Mechanistically, pharmacological inhibition of MCL-1 with a specific BH3 mimetic promotes mitochondrial fragmentation in bCAFs. Inhibition of the mitochondrial fission activity of DRP-1, which interacts with MCL-1 upon BH3 mimetic treatment, allows the maintenance of the myofibroblastic phenotype of bCAFs.
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Neoplasias da Mama , Fibroblastos Associados a Câncer , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Ecossistema , Feminino , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , FenótipoRESUMO
Since the Nobel Prize award more than twenty years ago for discovering the core apoptotic pathway in C. elegans, apoptosis and various other forms of regulated cell death have been thoroughly characterized by researchers around the world. Although many aspects of regulated cell death still remain to be elucidated in specific cell subtypes and disease conditions, many predicted that research into cell death was inexorably reaching a plateau. However, this was not the case since the last decade saw a multitude of cell death modalities being described, while harnessing their therapeutic potential reached clinical use in certain cases. In line with keeping research into cell death alive, francophone researchers from several institutions in France and Belgium established the French Cell Death Research Network (FCDRN). The research conducted by FCDRN is at the leading edge of emerging topics such as non-apoptotic functions of apoptotic effectors, paracrine effects of cell death, novel canonical and non-canonical mechanisms to induce apoptosis in cell death-resistant cancer cells or regulated forms of necrosis and the associated immunogenic response. Collectively, these various lines of research all emerged from the study of apoptosis and in the next few years will increase the mechanistic knowledge into regulated cell death and how to harness it for therapy.
Assuntos
Caenorhabditis elegans , Neoplasias , Animais , Apoptose , Morte Celular , Humanos , NecroseRESUMO
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.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Neoplasias da Mama , Agregação Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Everolimo , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas de Membrana/genética , Complexos Multiproteicos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Regiões Promotoras Genéticas , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , Transdução de Sinais , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
The level of the Mcl-1 pro-survival protein is highly regulated, and the down-regulation of Mcl-1 expression favors the apoptotic process. Mcl-1 physically interacts with different BH3-only proteins; particularly, Noxa is involved in the modulation of Mcl-1 expression. In this study, we demonstrated that Noxa triggers the degradation of Mcl-1 at the mitochondria according to the exclusive location of Noxa at this compartment. The Noxa-induced degradation of Mcl-1 required the E3 ligase Mule, which is responsible for the polyubiquitination of Mcl-1. Because the USP9X deubiquitinase was recently demonstrated to be involved in Mcl-1 protein turnover by preventing its degradation through the removal of conjugated ubiquitin, we investigated whether Noxa affected the deubiquitination process. Interestingly, Noxa over-expression caused a decrease in the USP9X/Mcl-1 interaction associated with an increase in the Mcl-1 polyubiquitinated forms. Additionally, Noxa over-expression triggered an increase in the Mule/Mcl-1 interaction in parallel with the decrease in Mule/USP9X complex formation. Taken together, these modifications result in the degradation of Mcl-1 by the proteasome machinery. The implication of Noxa in the regulation of Mcl-1 proteasomal degradation adds complexity to this process, which is governed by multiple interactions.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Supressoras de TumorRESUMO
We are taking advantage of the launch of the latest version (v4.6) of our web-based data mining tool "breast cancer gene-expression miner" (bc-GenExMiner) to take stock of its position within the oncology research landscape and to present an activity report ten years after its establishment (http://bcgenex.ico.unicancer.fr). bc-GenExMiner is an open-access, user-friendly tool for statistical mining on breast tumor transcriptomes, annotated with more than 20 clinicopathologic and molecular characteristics. The database comprises more than 16,000 patients from 64 cohorts - including TCGA, METABRIC and SCAN-B - for whom several thousands of genes have been quantified by microarrays or RNA-seq. Correlation, expression and prognostic analyses are available for targeted, exhaustive or customized explorations of queried genes. bc-GenExMiner facilitates the validation, investigation, and prioritization of discoveries and hypotheses on genes of interest. It allows users to analyse large databases, create data visualizations, and obtain robust statistical analysis, thereby accelerating biomarker discovery. Ten years after its launch, judging by the number of visits, analyses, and scientific citations of bc-GenExMiner, we conclude that this web resource serves its purpose in the international scientific community working in breast cancer research, with a never-ending rise in its use.
Assuntos
Neoplasias da Mama/genética , Mineração de Dados/métodos , Bases de Dados Genéticas , Perfilação da Expressão Gênica/métodos , Neoplasias da Mama/química , Bases de Dados Genéticas/estatística & dados numéricos , Feminino , Marcadores Genéticos , Humanos , Intervenção Baseada em Internet , Prognóstico , Fatores de Tempo , TranscriptomaRESUMO
'Breast cancer gene-expression miner' (bc-GenExMiner) is a breast cancer-associated web portal (http://bcgenex.ico.unicancer.fr). Here, we describe the development of a new statistical mining module, which permits several differential gene expression analyses, i.e. 'Expression' module. Sixty-two breast cancer cohorts and one healthy breast cohort with their corresponding clinicopathological information are included in bc-GenExMiner v4.5 version. Analyses are based on microarray or RNAseq transcriptomic data. Thirty-nine differential gene expression analyses, grouped into 13 categories, according to clinicopathological and molecular characteristics ('Targeted' and 'Exhaustive') and gene expression ('Customized'), have been developed. Output results are visualized in four forms of plots. This new statistical mining module offers, among other things, the possibility to compare gene expression in healthy (cancer-free), tumour-adjacent and tumour tissues at once and in three triple-negative breast cancer subtypes (i.e. C1: molecular apocrine tumours; C2: basal-like tumours infiltrated by immune suppressive cells and C3: basal-like tumours triggering an ineffective immune response). Several validation tests showed that bioinformatics process did not alter the pathobiological information contained in the source data. In this work, we developed and demonstrated that bc-GenExMiner 'Expression' module can be used for exploratory and validation purposes. Database URL: http://bcgenex.ico.unicancer.fr.