RESUMO
Acute myeloid leukemia (AML) is maintained by self-renewing leukemic stem cells (LSCs). A fundamental problem in treating AML is that conventional therapy fails to eliminate LSCs, which can reinitiate leukemia. Heat shock transcription factor 1 (HSF1), a central regulator of the stress response, has emerged as an important target in cancer therapy. Using genetic Hsf1 deletion and a direct HSF1 small molecule inhibitor, we show that HSF1 is specifically required for the maintenance of AML, while sparing steady-state and stressed hematopoiesis. Mechanistically, deletion of Hsf1 dysregulates multifaceted genes involved in LSC stemness and suppresses mitochondrial oxidative phosphorylation through downregulation of succinate dehydrogenase C (SDHC), a direct HSF1 target. Forced expression of SDHC largely restores the Hsf1 ablation-induced AML developmental defect. Importantly, the growth and engraftment of human AML cells are suppressed by HSF1 inhibition. Our data provide a rationale for developing efficacious small molecules to specifically target HSF1 in AML.
Assuntos
Autorrenovação Celular , Leucemia Mieloide Aguda , Humanos , Autorrenovação Celular/genética , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Succinato Desidrogenase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Heat shock transcription factor 1 (HSF1) orchestrates cellular stress protection by activating or repressing gene transcription in response to protein misfolding, oncogenic cell proliferation, and other environmental stresses. HSF1 is tightly regulated via intramolecular repressive interactions, post-translational modifications, and protein-protein interactions. How these HSF1 regulatory protein interactions are altered in response to acute and chronic stress is largely unknown. To elucidate the profile of HSF1 protein interactions under normal growth and chronic and acutely stressful conditions, quantitative proteomics studies identified interacting proteins in the response to heat shock or in the presence of a poly-glutamine aggregation protein cell-based model of Huntington's disease. These studies identified distinct protein interaction partners of HSF1 as well as changes in the magnitude of shared interactions as a function of each stressful condition. Several novel HSF1-interacting proteins were identified that encompass a wide variety of cellular functions, including roles in DNA repair, mRNA processing, and regulation of RNA polymerase II. One HSF1 partner, CTCF, interacted with HSF1 in a stress-inducible manner and functions in repression of specific HSF1 target genes. Understanding how HSF1 regulates gene repression is a crucial question, given the dysregulation of HSF1 target genes in both cancer and neurodegeneration. These studies expand our understanding of HSF1-mediated gene repression and provide key insights into HSF1 regulation via protein-protein interactions.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Doença de Huntington/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Animais , Fator de Ligação a CCCTC/genética , Células HEK293 , Fatores de Transcrição de Choque Térmico/genética , Humanos , Doença de Huntington/genética , Doença de Huntington/patologia , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Mapas de Interação de ProteínasRESUMO
Heat shock factor 1 (HSF1) is a cellular stress-protective transcription factor exploited by a wide range of cancers to drive proliferation, survival, invasion, and metastasis. Nuclear HSF1 abundance is a prognostic indicator for cancer severity, therapy resistance, and shortened patient survival. The HSF1 gene was amplified, and nuclear HSF1 abundance was markedly increased in prostate cancers and particularly in neuroendocrine prostate cancer (NEPC), for which there are no available treatment options. Despite genetic validation of HSF1 as a therapeutic target in a range of cancers, a direct and selective small-molecule HSF1 inhibitor has not been validated or developed for use in the clinic. We described the identification of a direct HSF1 inhibitor, Direct Targeted HSF1 InhiBitor (DTHIB), which physically engages HSF1 and selectively stimulates degradation of nuclear HSF1. DTHIB robustly inhibited the HSF1 cancer gene signature and prostate cancer cell proliferation. In addition, it potently attenuated tumor progression in four therapy-resistant prostate cancer animal models, including an NEPC model, where it caused profound tumor regression. This study reports the identification and validation of a direct HSF1 inhibitor and provides a path for the development of a small-molecule HSF1-targeted therapy for prostate cancers and other therapy-resistant cancers.
Assuntos
Fatores de Transcrição de Choque Térmico/antagonistas & inibidores , Neoplasias da Próstata , Animais , Núcleo Celular/metabolismo , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genéticaRESUMO
The ability of cancer cells to cope with stressful conditions is critical for their survival, proliferation, and metastasis. The heat shock transcription factor 1 (HSF1) protects cells from stresses such as chemicals, radiation, and temperature. These properties of HSF1 are exploited by a broad spectrum of cancers, which exhibit high levels of nuclear, active HSF1. Functions for HSF1 in malignancy extend well beyond its central role in protein quality control. While HSF1 has been validated as a powerful target in cancers by genetic knockdown studies, HSF1 inhibitors reported to date have lacked sufficient specificity and potency for clinical evaluation. We review the roles of HSF1 in cancer, its potential as a prognostic indicator for cancer treatment, evaluate current HSF1 inhibitors and provide guidelines for the identification of selective HSF1 inhibitors as chemical probes and for clinical development.
Assuntos
Antineoplásicos/farmacologia , Fatores de Transcrição de Choque Térmico/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Humanos , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome.
Assuntos
Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Splicing de RNA , Proteínas de Ligação a RNA/genética , Spliceossomos/metabolismo , Asparagina/química , Asparagina/metabolismo , Sítios de Ligação , Clonagem Molecular , Cobre/química , Cobre/metabolismo , Cryptococcus neoformans/metabolismo , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Inteínas , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Spliceossomos/ultraestrutura , Homologia Estrutural de Proteína , Zinco/química , Zinco/metabolismoRESUMO
Copper (Cu) is an essential trace element for growth and development and abnormal Cu levels are associated with anemia, metabolic disease and cancer. Evolutionarily conserved from fungi to humans, the high-affinity Cu+ transporter Ctr1 is crucial for both dietary Cu uptake and peripheral distribution, yet the mechanisms for selective permeation of potentially toxic Cu+ ions across cell membranes are unknown. Here we present X-ray crystal structures of Ctr1 from Salmo salar in both Cu+-free and Cu+-bound states, revealing a homo-trimeric Cu+-selective ion channel-like architecture. Two layers of methionine triads form a selectivity filter, coordinating two bound Cu+ ions close to the extracellular entrance. These structures, together with Ctr1 functional characterization, provide a high resolution picture to understand Cu+ import across cellular membranes and suggest therapeutic opportunities for intervention in diseases characterized by inappropriate Cu accumulation.
Assuntos
Proteínas de Transporte de Cátions/ultraestrutura , Cobre/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular , Transportador de Cobre 1 , Cristalografia por Raios X , Transporte de Íons , Salmo salarRESUMO
Iron is an indispensable micronutrient for all eukaryotic organisms due to its participation as a redox cofactor in many metabolic pathways. Iron imbalance leads to the most frequent human nutritional deficiency in the world. Adaptation to iron limitation requires a global reorganization of the cellular metabolism directed to prioritize iron utilization for essential processes. In response to iron scarcity, the conserved Saccharomyces cerevisiae mRNA-binding protein Cth2, which belongs to the tristetraprolin family of tandem zinc finger proteins, coordinates a global remodeling of the cellular metabolism by promoting the degradation of multiple mRNAs encoding highly iron-consuming proteins. In this work, we identify a critical mechanism for the degradation of Cth2 protein during the adaptation to iron deficiency. Phosphorylation of a patch of Cth2 serine residues within its amino-terminal region facilitates recognition by the SCFGrr1 ubiquitin ligase complex, accelerating Cth2 turnover by the proteasome. When Cth2 degradation is impaired by either mutagenesis of the Cth2 serine residues or deletion of GRR1, the levels of Cth2 rise and abrogate growth in iron-depleted conditions. Finally, we uncover that the casein kinase Hrr25 phosphorylates and promotes Cth2 destabilization. These results reveal a sophisticated posttranslational regulatory pathway necessary for the adaptation to iron depletion.IMPORTANCE Iron is a vital element for many metabolic pathways, including the synthesis of DNA and proteins, and the generation of energy via oxidative phosphorylation. Therefore, living organisms have developed tightly controlled mechanisms to properly distribute iron, since imbalances lead to nutritional deficiencies, multiple diseases, and vulnerability against pathogens. Saccharomyces cerevisiae Cth2 is a conserved mRNA-binding protein that coordinates a global reprogramming of iron metabolism in response to iron deficiency in order to optimize its utilization. Here we report that the phosphorylation of Cth2 at specific serine residues is essential to regulate the stability of the protein and adaptation to iron depletion. We identify the kinase and ubiquitination machinery implicated in this process to establish a posttranscriptional regulatory model. These results and recent findings for both mammals and plants reinforce the privileged position of E3 ubiquitin ligases and phosphorylation events in the regulation of eukaryotic iron homeostasis.
Assuntos
Adaptação Fisiológica , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Tristetraprolina/metabolismo , Regulação Fúngica da Expressão Gênica , Ferro/metabolismo , Mutagênese , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Serina/genética , Tristetraprolina/genéticaRESUMO
Heat shock factor 1 (HSF1) is a stress-responsive transcription factor that regulates expression of protein chaperones and cell survival factors. In cancer, HSF1 plays a unique role, hijacking the normal stress response to drive a cancer-specific transcriptional program. These observations suggest that HSF1 inhibitors could be promising therapeutics. However, HSF1 is activated through a complex mechanism, which involves release of a negative regulatory domain, leucine zipper 4 (LZ4), from a masked oligomerization domain (LZ1-3), and subsequent binding of the oligomer to heat shock elements (HSEs) in HSF1-responsive genes. Recent crystal structures have suggested that HSF1 oligomers are held together by extensive, buried contact surfaces, making it unclear whether there are any possible binding sites for inhibitors. Here, we have rationally designed a series of peptide-based molecules based on the LZ4 and LZ1-3 motifs. Using a plate-based, fluorescence polarization (FP) assay, we identified a minimal region of LZ4 that suppresses binding of HSF1 to the HSE. Using this information, we converted this peptide into a tracer and used it to understand how binding of LZ4 to LZ1-3 suppresses HSF1 activation. Together, these results suggest a previously unexplored avenue in the development of HSF1 inhibitors. Furthermore, the findings highlight how native interactions can inspire the design of inhibitors for even the most challenging protein-protein interactions (PPIs).
Assuntos
Desenho de Fármacos , Fatores de Transcrição de Choque Térmico/antagonistas & inibidores , Peptídeos/química , Sequência de Aminoácidos , Sítios de Ligação , Polarização de Fluorescência , Fatores de Transcrição de Choque Térmico/metabolismo , Humanos , Zíper de Leucina , Peptídeos/síntese química , Peptídeos/metabolismoRESUMO
The heat shock transcription factors (HSFs) were discovered over 30 years ago as direct transcriptional activators of genes regulated by thermal stress, encoding heat shock proteins. The accepted paradigm posited that HSFs exclusively activate the expression of protein chaperones in response to conditions that cause protein misfolding by recognizing a simple promoter binding site referred to as a heat shock element. However, we now realize that the mammalian family of HSFs comprises proteins that independently or in concert drive combinatorial gene regulation events that activate or repress transcription in different contexts. Advances in our understanding of HSF structure, post-translational modifications and the breadth of HSF-regulated target genes have revealed exciting new mechanisms that modulate HSFs and shed new light on their roles in physiology and pathology. For example, the ability of HSF1 to protect cells from proteotoxicity and cell death is impaired in neurodegenerative diseases but can be exploited by cancer cells to support their growth, survival and metastasis. These new insights into HSF structure, function and regulation should facilitate the development tof new disease therapeutics to manipulate this transcription factor family.
Assuntos
Regulação da Expressão Gênica/genética , Fatores de Transcrição de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Transcrição Gênica/genética , Animais , Resposta ao Choque Térmico/genética , Humanos , Processamento de Proteína Pós-Traducional/genéticaRESUMO
Copper (Cu) ions serve as catalytic cofactors to drive key biochemical processes, and yet Cu levels that exceed cellular homeostatic control capacity are toxic. The underlying mechanisms for Cu toxicity are poorly understood. During pulmonary infection by the fungal pathogen Cryptococcus neoformans, host alveolar macrophages compartmentalize Cu to the phagosome, and the ability to detoxify Cu is critical for its survival and virulence. Here, we report that iron-sulfur (Fe-S) clusters are critical targets of Cu toxicity in both Saccharomyces cerevisiae and C. neoformans in a manner that depends on the accessibility of Cu to the Fe-S cofactor. To respond to this Cu-dependent Fe-S stress, C. neoformans induces the transcription of mitochondrial ABC transporter Atm1, which functions in cytosolic-nuclear Fe-S protein biogenesis in response to Cu and in a manner dependent on the Cu metalloregulatory transcription factor Cuf1. As Atm1 functions in exporting an Fe-S precursor from the mitochondrial matrix to the cytosol, C. neoformans cells depleted for Atm1 are sensitive to Cu even while the Cu-detoxifying metallothionein proteins are highly expressed. We provide evidence for a previously unrecognized microbial defense mechanism to deal with Cu toxicity, and we highlight the importance for C. neoformans of having several distinct mechanisms for coping with Cu toxicity which together could contribute to the success of this microbe as an opportunistic human fungal pathogen.IMPORTANCEC. neoformans is an opportunistic pathogen that causes lethal meningitis in over 650,000 people annually. The severity of C. neoformans infections is further compounded by the use of toxic or poorly effective systemic antifungal agents as well as by the difficulty of diagnosis. Cu is a natural potent antimicrobial agent that is compartmentalized within the macrophage phagosome and used by innate immune cells to neutralize microbial pathogens. While the Cu detoxification machinery of C. neoformans is essential for virulence, little is known about the mechanisms by which Cu kills fungi. Here we report that Fe-S cluster-containing proteins, including members of the Fe-S protein biogenesis machinery itself, are critical targets of Cu toxicity and therefore that this biosynthetic process provides an important layer of defense against high Cu levels. Given the role of Cu ionophores as antimicrobials, understanding how Cu is toxic to microorganisms could lead to the development of effective, broad-spectrum antimicrobials. Moreover, understanding Cu toxicity could provide additional insights into the pathophysiology of human diseases of Cu overload such as Wilson's disease.
Assuntos
Cobre/metabolismo , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Estresse Fisiológico , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Cryptococcus neoformans/patogenicidade , Citosol/metabolismo , Regulação Fúngica da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Proteínas Ferro-Enxofre/genética , Macrófagos/imunologia , Macrófagos/microbiologia , Metalotioneína/genética , Metalotioneína/metabolismo , Camundongos , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , VirulênciaRESUMO
Huntington's Disease (HD) is a neurodegenerative disease caused by poly-glutamine expansion in the Htt protein, resulting in Htt misfolding and cell death. Expression of the cellular protein folding and pro-survival machinery by heat shock transcription factor 1 (HSF1) ameliorates biochemical and neurobiological defects caused by protein misfolding. We report that HSF1 is degraded in cells and mice expressing mutant Htt, in medium spiny neurons derived from human HD iPSCs and in brain samples from patients with HD. Mutant Htt increases CK2α' kinase and Fbxw7 E3 ligase levels, phosphorylating HSF1 and promoting its proteasomal degradation. An HD mouse model heterozygous for CK2α' shows increased HSF1 and chaperone levels, maintenance of striatal excitatory synapses, clearance of Htt aggregates and preserves body mass compared with HD mice homozygous for CK2α'. These results reveal a pathway that could be modulated to prevent neuronal dysfunction and muscle wasting caused by protein misfolding in HD.
Assuntos
Encéfalo/metabolismo , Fatores de Transcrição de Choque Térmico/metabolismo , Doença de Huntington/metabolismo , Neurônios/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Células HEK293 , Fatores de Transcrição de Choque Térmico/genética , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células PC12 , RatosRESUMO
Copper is an essential metal ion for embryonic development, iron acquisition, cardiac function, neuropeptide biogenesis, and other critical physiological processes. Ctr1 is a high affinity Cu(+) transporter on the plasma membrane and endosomes that exists as a full-length protein and a truncated form of Ctr1 lacking the methionine- and histidine-rich metal-binding ectodomain, and it exhibits reduced Cu(+) transport activity. Here, we identify the cathepsin L/B endolysosomal proteases functioning in a direct and rate-limiting step in the Ctr1 ectodomain cleavage. Cells and mice lacking cathepsin L accumulate full-length Ctr1 and hyper-accumulate copper. As Ctr1 also transports the chemotherapeutic drug cisplatin via direct binding to the ectodomain, we demonstrate that the combination of cisplatin with a cathepsin L/B inhibitor enhances cisplatin uptake and cell killing. These studies identify a new processing event and the key protease that cleaves the Ctr1 metal-binding ectodomain, which functions to regulate cellular Cu(+) and cisplatin acquisition.
Assuntos
Catepsinas/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cisplatino/metabolismo , Cobre/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Transportador de Cobre 1 , Humanos , Camundongos , Camundongos Knockout , Proteólise , Homologia de Sequência de AminoácidosRESUMO
Heat-shock transcription factor (HSF) family members function in stress protection and in human diseases including proteopathies, neurodegeneration and cancer. The mechanisms that drive distinct post-translational modifications, cofactor recruitment and target-gene activation for specific HSF paralogs are unknown. We present crystal structures of the human HSF2 DNA-binding domain (DBD) bound to DNA, revealing an unprecedented view of HSFs that provides insights into their unique biology. The HSF2 DBD structures resolve a new C-terminal helix that directs wrapping of the coiled-coil domain around DNA, thereby exposing paralog-specific sequences of the DBD surface for differential post-translational modifications and cofactor interactions. We further demonstrate a direct interaction between HSF1 and HSF2 through their coiled-coil domains. Together, these features provide a new model for HSF structure as the basis for differential and combinatorial regulation, which influences the transcriptional response to cellular stress.
Assuntos
Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição de Choque Térmico , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , SumoilaçãoRESUMO
Divalent metal-ion transporter-1 (DMT1) is a widely expressed iron-preferring membrane-transport protein that serves a critical role in erythroid iron utilization. We have investigated its role in intestinal metal absorption by studying a mouse model lacking intestinal DMT1 (i.e., DMT1(int/int)). DMT1(int/int) mice exhibited a profound hypochromic-microcytic anemia, splenomegaly, and cardiomegaly. That the anemia was due to iron deficiency was demonstrated by the following observations in DMT1(int/int) mice: 1) blood iron and tissue nonheme-iron stores were depleted; 2) mRNA expression of liver hepcidin (Hamp1) was depressed; and 3) intraperitoneal iron injection corrected the anemia, and reversed the changes in blood iron, nonheme-iron stores, and hepcidin expression levels. We observed decreased total iron content in multiple tissues from DMT1(int/int) mice compared with DMT1(+/+) mice but no meaningful change in copper, manganese, or zinc. DMT1(int/int) mice absorbed (64)Cu and (54)Mn from an intragastric dose to the same extent as did DMT1(+/+) mice but the absorption of (59)Fe was virtually abolished in DMT1(int/int) mice. This study reveals a critical function for DMT1 in intestinal nonheme-iron absorption for normal growth and development. Further, this work demonstrates that intestinal DMT1 is not required for the intestinal transport of copper, manganese, or zinc.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Absorção Intestinal/fisiologia , Ferro/metabolismo , Manganês/metabolismo , Anemia Hipocrômica/genética , Anemia Hipocrômica/patologia , Animais , Proteínas de Transporte de Cátions/genética , Transportador de Cobre 1 , Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , Camundongos , Camundongos Knockout , Zinco/metabolismoRESUMO
Cancer cells often have increased levels of reactive oxygen species (ROS); however, acquisition of redox adaptive mechanisms allows for evasion of ROS-mediated death. Inflammatory breast cancer (IBC) is a distinct, advanced BC subtype characterized by high rates of residual disease and recurrence despite advances in multimodality treatment. Using a cellular model of IBC, we identified an oxidative stress response (OSR) signature in surviving IBC cells after administration of an acute dose of an ROS inducer. Metagene analysis of patient samples revealed significantly higher OSR scores in IBC tumor samples compared to normal or non-IBC tissues, which may contribute to the poor response of IBC tumors to common treatment strategies, which often rely heavily on ROS induction. To combat this adaptation, we utilized a potent redox modulator, the FDA-approved small molecule Disulfiram (DSF), alone and in combination with copper. DSF forms a complex with copper (DSF-Cu) increasing intracellular copper concentration both in vitro and in vivo, bypassing the need for membrane transporters. DSF-Cu antagonized NFκB signaling, aldehyde dehydrogenase activity and antioxidant levels, inducing oxidative stress-mediated apoptosis in multiple IBC cellular models. In vivo, DSF-Cu significantly inhibited tumor growth without significant toxicity, causing apoptosis only in tumor cells. These results indicate that IBC tumors are highly redox adapted, which may render them resistant to ROS-inducing therapies. DSF, through redox modulation, may be a useful approach to enhance chemo- and/or radio-sensitivity for advanced BC subtypes where therapeutic resistance is an impediment to durable responses to current standard of care.
Assuntos
Antineoplásicos/farmacologia , Cobre/metabolismo , Dissulfiram/farmacologia , Neoplasias Inflamatórias Mamárias/tratamento farmacológico , Ionóforos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Inflamatórias Mamárias/genética , Neoplasias Inflamatórias Mamárias/metabolismoRESUMO
Copper (Cu) is an essential metal for growth and development that has the potential to be toxic if levels accumulate beyond the ability of cells to homeostatically balance uptake with detoxification. One system for Cu acquisition is the integral membrane Cu(+) transporter, Ctr1, which has been quite well characterized in terms of its function and physiology. The mammalian Ctr2 protein has been a conundrum for the copper field, as it is structurally closely related to the high affinity Cu transporter Ctr1, sharing important motifs for Cu transport activity. However, in contrast to mammalian Ctr1, Ctr2 fails to suppress the Cu-dependent growth phenotype of yeast cells defective in Cu(+) import, nor does it appreciably stimulate Cu acquisition when over-expressed in mammalian cells, underscoring important functional dissimilarities between the two proteins. Several roles for the mammalian Ctr2 have been suggested both in vitro and in vivo. Here, we summarize and discuss current insights into the Ctr2 protein and its interaction with Ctr1, its functions in mammalian Cu homeostasis and platinum-based chemotherapy.
Assuntos
Antineoplásicos/farmacocinética , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Modelos Biológicos , Compostos Organoplatínicos/farmacocinética , Compostos de Platina/farmacocinética , Motivos de Aminoácidos , Animais , Antineoplásicos/uso terapêutico , Transporte Biológico , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Transportador de Cobre 1 , Regulação da Expressão Gênica , Homeostase , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Compostos Organoplatínicos/uso terapêutico , Compostos de Platina/uso terapêutico , Conformação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas SLC31RESUMO
Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors, and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i.
Assuntos
Benzimidazóis/química , Benzimidazóis/farmacologia , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Ácidos Nipecóticos/química , Ácidos Nipecóticos/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Benzimidazóis/metabolismo , Benzimidazóis/farmacocinética , Disponibilidade Biológica , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Proteínas de Choque Térmico HSP70/química , Humanos , Camundongos , Modelos Moleculares , Ácidos Nipecóticos/metabolismo , Ácidos Nipecóticos/farmacocinética , Permeabilidade , Piperidinas/metabolismo , Piperidinas/farmacocinética , Agregados Proteicos/efeitos dos fármacos , Estrutura Terciária de Proteína , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The heat shock transcription factor 1 (HSF1) activates expression of a variety of genes involved in cell survival, including protein chaperones, the protein degradation machinery, anti-apoptotic proteins, and transcription factors. Although HSF1 activation has been linked to amelioration of neurodegenerative disease, cancer cells exhibit a dependence on HSF1 for survival. Indeed, HSF1 drives a program of gene expression in cancer cells that is distinct from that activated in response to proteotoxic stress, and HSF1 DNA binding activity is elevated in cycling cells as compared with arrested cells. Active HSF1 homotrimerizes and binds to a DNA sequence consisting of inverted repeats of the pentameric sequence nGAAn, known as heat shock elements (HSEs). Recent comprehensive ChIP-seq experiments demonstrated that the architecture of HSEs is very diverse in the human genome, with deviations from the consensus sequence in the spacing, orientation, and extent of HSE repeats that could influence HSF1 DNA binding efficacy and the kinetics and magnitude of target gene expression. To understand the mechanisms that dictate binding specificity, HSF1 was purified as either a monomer or trimer and used to evaluate DNA-binding site preferences in vitro using fluorescence polarization and thermal denaturation profiling. These results were compared with quantitative chromatin immunoprecipitation assays in vivo. We demonstrate a role for specific orientations of extended HSE sequences in driving preferential HSF1 DNA binding to target loci in vivo. These studies provide a biochemical basis for understanding differential HSF1 target gene recognition and transcription in neurodegenerative disease and in cancer.
Assuntos
Proteínas de Ligação a DNA/química , Fatores de Transcrição/química , Sequência de Bases , Sítios de Ligação , DNA/química , DNA/genética , Proteínas de Ligação a DNA/fisiologia , Genoma Humano , Células HEK293 , Fatores de Transcrição de Choque Térmico , Resposta ao Choque Térmico , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estabilidade Proteica , Elementos de Resposta , Fatores de Transcrição/fisiologia , Temperatura de TransiçãoRESUMO
Recalcitrant microbial infections demand new therapeutic options. Here we present an approach that exploits two prongs of the host immune cell antimicrobial response: the oxidative burst and the compartmentalization of copper (Cu) within phagolysosomes. The prochelator QBP is a nontoxic protected form of 8-hydroxyquinoline (8HQ) in which a pinanediol boronic ester blocks metal ion coordination by 8HQ. QBP is deprotected via reactive oxygen species produced by activated macrophages, creating 8HQ and eliciting Cu-dependent killing of the fungal pathogen Cryptococcus neoformans in vitro and in mouse pulmonary infection. 8HQ ionophoric activity increases intracellular Cu, overwhelming the Cu-resistance mechanisms of C. neoformans to elicit fungal killing. The Cu-dependent antimicrobial activity of 8HQ against a spectrum of microbial pathogens suggests that this strategy may have broad utility. The conditional activation of Cu ionophores by innate immune cells intensifies the hostile antimicrobial environment and represents a promising approach to combat infectious disease.
Assuntos
Antifúngicos/farmacologia , Cobre/farmacologia , Criptococose/tratamento farmacológico , Cryptococcus/efeitos dos fármacos , Imunidade Inata , Compostos Organometálicos/farmacologia , Animais , Antifúngicos/síntese química , Antifúngicos/química , Células Cultivadas , Cobre/química , Relação Dose-Resposta a Droga , Feminino , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Relação Estrutura-AtividadeRESUMO
The BRAF kinase is mutated, typically Val 600âGlu (V600E), to induce an active oncogenic state in a large fraction of melanomas, thyroid cancers, hairy cell leukaemias and, to a smaller extent, a wide spectrum of other cancers. BRAF(V600E) phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate and activate the ERK1 and ERK2 kinases, stimulating the mitogen-activated protein kinase (MAPK) pathway to promote cancer. Targeting MEK1/2 is proving to be an important therapeutic strategy, given that a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma, an effect that is increased when administered together with a BRAF(V600E) inhibitor. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction. Here we show decreasing the levels of CTR1 (Cu transporter 1), or mutations in MEK1 that disrupt Cu binding, decreased BRAF(V600E)-driven signalling and tumorigenesis in mice and human cell settings. Conversely, a MEK1-MEK5 chimaera that phosphorylated ERK1/2 independently of Cu or an active ERK2 restored the tumour growth of murine cells lacking Ctr1. Cu chelators used in the treatment of Wilson disease decreased tumour growth of human or murine cells transformed by BRAF(V600E) or engineered to be resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat cancers containing the BRAF(V600E) mutation.