RESUMO
A major pharmacological assumption is that lowering disease-promoting protein levels is generally beneficial. For example, inhibiting metastasis activator BACH1 is proposed to decrease cancer metastases. Testing such assumptions requires approaches to measure disease phenotypes while precisely adjusting disease-promoting protein levels. Here we developed a two-step strategy to integrate protein-level tuning, noise-aware synthetic gene circuits into a well-defined human genomic safe harbor locus. Unexpectedly, engineered MDA-MB-231 metastatic human breast cancer cells become more, then less and then more invasive as we tune BACH1 levels up, irrespective of the native BACH1. BACH1 expression shifts in invading cells, and expression of BACH1's transcriptional targets confirm BACH1's nonmonotone phenotypic and regulatory effects. Thus, chemical inhibition of BACH1 could have unwanted effects on invasion. Additionally, BACH1's expression variability aids invasion at high BACH1 expression. Overall, precisely engineered, noise-aware protein-level control is necessary and important to unravel disease effects of genes to improve clinical drug efficacy.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , Neoplasias da Mama , Humanos , Feminino , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Neoplasias da Mama/metabolismo , Metástase NeoplásicaRESUMO
Raf Kinase Inhibitory Protein (RKIP) maintains cellular robustness and prevents the progression of diseases such as cancer and heart disease by regulating key kinase cascades including MAP kinase and protein kinase A (PKA). Phosphorylation of RKIP at S153 by Protein Kinase C (PKC) triggers a switch from inhibition of Raf to inhibition of the G protein coupled receptor kinase 2 (GRK2), enhancing signaling by the ß-adrenergic receptor (ß-AR) that activates PKA. Here we report that PKA-phosphorylated RKIP promotes ß-AR-activated PKA signaling. Using biochemical, genetic, and biophysical approaches, we show that PKA phosphorylates RKIP at S51, increasing S153 phosphorylation by PKC and thereby triggering feedback activation of PKA. The S51V mutation blocks the ability of RKIP to activate PKA in prostate cancer cells and to induce contraction in primary cardiac myocytes in response to the ß-AR activator isoproterenol, illustrating the functional importance of this positive feedback circuit. As previously shown for other kinases, phosphorylation of RKIP at S51 by PKA is enhanced upon RKIP destabilization by the P74L mutation. These results suggest that PKA phosphorylation at S51 may lead to allosteric changes associated with a higher-energy RKIP state that potentiates phosphorylation of RKIP at other key sites. This allosteric regulatory mechanism may have therapeutic potential for regulating PKA signaling in disease states.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Proteína de Ligação a Fosfatidiletanolamina , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Retroalimentação Fisiológica , Humanos , Masculino , Células PC-3 , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosforilação , Neoplasias da Próstata/metabolismo , Proteína Quinase C/metabolismo , Transdução de SinaisRESUMO
Cancer and heart disease are leading causes of morbidity and mortality worldwide. These diseases have common risk factors, common molecular signaling pathways that are central to their pathogenesis, and even some disease phenotypes that are interdependent. Thus, a detailed understanding of common regulators is critical for the development of new and synergistic therapeutic strategies. The Raf kinase inhibitory protein (RKIP) is a regulator of the cellular kinome that functions to maintain cellular robustness and prevent the progression of diseases including heart disease and cancer. Two of the key signaling pathways controlled by RKIP are the ß-adrenergic receptor (ßAR) signaling to protein kinase A (PKA), particularly in the heart, and the MAP kinase cascade Raf/MEK/ERK1/2 that regulates multiple diseases. The goal of this review is to discuss how we can leverage RKIP to suppress cancer without incurring deleterious effects on the heart. Specifically, we discuss: (1) How RKIP functions to either suppress or activate ßAR (PKA) and ERK1/2 signaling; (2) How we can prevent cancer-promoting kinase signaling while at the same time avoiding cardiotoxicity.
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The spread of SARS-CoV-2 and ongoing COVID-19 pandemic underscores the need for new treatments. Here we report that cannabidiol (CBD) inhibits infection of SARS-CoV-2 in cells and mice. CBD and its metabolite 7-OH-CBD, but not THC or other congeneric cannabinoids tested, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after viral entry, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD inhibits SARS-CoV-2 replication in part by up-regulating the host IRE1α RNase endoplasmic reticulum (ER) stress response and interferon signaling pathways. In matched groups of human patients from the National COVID Cohort Collaborative, CBD (100 mg/ml oral solution per medical records) had a significant negative association with positive SARS-CoV-2 tests. This study highlights CBD as a potential preventative agent for early-stage SARS-CoV-2 infection and merits future clinical trials. We caution against use of non-medical formulations including edibles, inhalants or topicals as a preventative or treatment therapy at the present time.
Assuntos
Antivirais/farmacologia , Canabidiol/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Células A549 , Animais , Antivirais/química , COVID-19/virologia , Canabidiol/química , Canabidiol/metabolismo , Chlorocebus aethiops , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Células Epiteliais/virologia , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Interferons/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , SARS-CoV-2/fisiologia , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
Targeted strategies against specific driver molecules of cancer have brought about many advances in cancer treatment since the early success of the first small-molecule inhibitor Gleevec. Today, there are a multitude of targeted therapies approved by the Food and Drug Administration for the treatment of cancer. However, the initial efficacy of virtually every targeted treatment is often reversed by tumor resistance to the inhibitor through acquisition of new mutations in the target molecule, or reprogramming of the epigenome, transcriptome, or kinome of the tumor cells. At the core of this clinical problem lies the assumption that targeted treatments will only be efficacious if the inhibitors are used at their maximum tolerated doses. Such aggressive regimens create strong selective pressure on the evolutionary progression of the tumor, resulting in resistant cells. High-dose single agent treatments activate alternative mechanisms that bypass the inhibitor, while high-dose combinatorial treatments suffer from increased toxicity resulting in treatment cessation. Although there is an arsenal of targeted agents being tested clinically and preclinically, identifying the most effective combination treatment plan remains a challenge. In this review, we discuss novel targeted strategies with an emphasis on the recent cross-disciplinary studies demonstrating that it is possible to achieve antitumor efficacy without increasing toxicity by adopting low-dose multitarget approaches to treatment of cancer and metastasis.
Assuntos
Mesilato de Imatinib/uso terapêutico , Proteínas de Neoplasias , Neoplasias , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/metabolismo , Animais , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologiaRESUMO
PURPOSE: To understand how tumor cells alter macrophage biology once they are recruited to triple-negative breast cancer (TNBC) tumors by CCL5. METHOD: Mouse bone marrow derived macrophage (BMDMs) were isolated and treated with recombinant CCL5 protein alone, with tumor cell conditioned media, or with tumor extracellular vesicles (EVs). Media from these tumor EV-educated macrophages (TEMs) was then used to determine how these macrophages affect TNBC invasion. To understand the mechanism, we assayed the cytokine secretion from these macrophages to determine how they impact tumor cell invasion. Tumor CCL5 expression was varied in tumors to determine its role in regulating macrophage biology through EVs. RESULTS: Tumor EVs are a necessary component for programming naïve macrophages toward a pro-metastatic phenotype. CCL5 expression in the tumor cells regulates both EV biogenesis/secretion/cargo and macrophage EV-education toward a pro-metastatic phenotype. Analysis of the tumor EV-educated macrophages (TEMs) showed secretion of a variety of factors including CXCL1, CTLA-4, IFNG, OPN, HGF, TGFB, and CCL19 capable of remodeling the surrounding tumor stroma and immune infiltrate. Injection of tumor cells with macrophages educated by metastatic tumor cell EVs into mice increased tumor metastasis to the lung. CONCLUSION: These results demonstrate that tumor-derived EVs are key mediators of macrophage education and likely play a more complex role in modulating tumor therapeutic response by regulating the tumor immune infiltrate.
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Obesity is associated with increased incidence and severity of triple-negative breast cancer (TNBC); however, mechanisms underlying this relationship are incompletely understood. Here, we show that obesity reprograms mammary adipose tissue macrophages to a pro-inflammatory metabolically activated phenotype (MMe) that alters the niche to support tumor formation. Unlike pro-inflammatory M1 macrophages that antagonize tumorigenesis, MMe macrophages are pro-tumorigenic and represent the dominant macrophage phenotype in mammary adipose tissue of obese humans and mice. MMe macrophages release IL-6 in an NADPH oxidase 2 (NOX2)-dependent manner, which signals through glycoprotein 130 (GP130) on TNBC cells to promote stem-like properties including tumor formation. Deleting Nox2 in myeloid cells or depleting GP130 in TNBC cells attenuates obesity-augmented TNBC stemness. Moreover, weight loss reverses the effects of obesity on MMe macrophage inflammation and TNBC tumor formation. Our studies implicate MMe macrophage accumulation in mammary adipose tissue as a mechanism for promoting TNBC stemness and tumorigenesis during obesity.
Assuntos
Tecido Adiposo/patologia , Macrófagos/metabolismo , Obesidade/patologia , Neoplasias de Mama Triplo Negativas/patologia , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Receptor gp130 de Citocina/metabolismo , Dieta , Feminino , Humanos , Interleucina-6/metabolismo , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Humanas/patologia , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/patologia , Fenótipo , Redução de PesoRESUMO
Mitochondrial metabolism is an attractive target for cancer therapy1,2. Reprogramming metabolic pathways could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options, such as triple-negative breast cancer (TNBC)1,3. Here we show that BTB and CNC homology1 (BACH1)4, a haem-binding transcription factor that is increased in expression in tumours from patients with TNBC, targets mitochondrial metabolism. BACH1 decreases glucose utilization in the tricarboxylic acid cycle and negatively regulates transcription of electron transport chain (ETC) genes. BACH1 depletion by shRNA or degradation by hemin sensitizes cells to ETC inhibitors such as metformin5,6, suppressing growth of both cell line and patient-derived tumour xenografts. Expression of a haem-resistant BACH1 mutant in cells that express a short hairpin RNA for BACH1 rescues the BACH1 phenotype and restores metformin resistance in hemin-treated cells and tumours7. Finally, BACH1 gene expression inversely correlates with ETC gene expression in tumours from patients with breast cancer and in other tumour types, which highlights the clinical relevance of our findings. This study demonstrates that mitochondrial metabolism can be exploited by targeting BACH1 to sensitize breast cancer and potentially other tumour tissues to mitochondrial inhibitors.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Hemina/uso terapêutico , Metformina/uso terapêutico , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Transporte de Elétrons/genética , Feminino , Glucose/metabolismo , Hemina/metabolismo , Xenoenxertos , Humanos , Metformina/metabolismo , Camundongos , Camundongos Nus , Mitocôndrias/genética , Proteólise , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Raf Kinase Inhibitory Protein (RKIP) is a highly conserved kinase inhibitor that functions as a metastasis suppressor in a variety of cancers. Since RKIP can reprogram tumor cells to a non-metastatic state by rewiring kinase networks, elucidating the mechanism by which RKIP acts not only reveals molecular mechanisms that regulate metastasis, but also represents an opportunity to target these signaling networks therapeutically. Although RKIP is often lost during metastatic progression, the mechanism by which this occurs in tumor cells is complex and not well understood. In this review, we summarize our current understanding of RKIP regulation in tumors and consider experimental and computational strategies for recovering or mimicking its function by targeting mediators of metastasis.
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The surrounding microenvironment has been implicated in the progression of breast tumors to metastasis. However, the degree to which metastatic breast tumors locally reprogram stromal cells as they disrupt tissue boundaries is not well understood. We used species-specific RNA sequencing in a mouse xenograft model to determine how the metastasis suppressor RKIP influences transcription in a panel of paired tumor and stroma tissues. We find that gene expression in metastatic breast tumors is pervasively correlated with gene expression in local stroma of both mouse xenografts and human patients. Changes in stromal gene expression elicited by tumors better predicts subtype and patient survival than tumor gene expression, and genes with coordinated expression in both tissues predict metastasis-free survival. These observations support the use of stroma-based strategies for the diagnosis and prognosis of breast cancer.
Assuntos
Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Células Estromais/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Prognóstico , RNA Neoplásico/química , RNA Neoplásico/isolamento & purificação , RNA Neoplásico/metabolismo , Análise de Sequência de RNA , Taxa de Sobrevida , Transplante HeterólogoRESUMO
Triple-negative breast cancer (TNBC) patients have the highest risk of recurrence and metastasis. Because they cannot be treated with targeted therapies, and many do not respond to chemotherapy, they represent a clinically underserved group. TNBC is characterized by reduced expression of metastasis suppressors such as Raf kinase inhibitory protein (RKIP), which inhibits tumor invasiveness. Mechanisms by which metastasis suppressors alter tumor cells are well characterized; however, their ability to regulate the tumor microenvironment and the importance of such regulation to metastasis suppression are incompletely understood. Here, we use species-specific RNA sequencing to show that RKIP expression in tumors markedly reduces the number and metastatic potential of infiltrating tumor-associated macrophages (TAM). TAMs isolated from nonmetastatic RKIP(+) tumors, relative to metastatic RKIP(-) tumors, exhibit a reduced ability to drive tumor cell invasion and decreased secretion of prometastatic factors, including PRGN, and shed TNFR2. RKIP regulates TAM recruitment by blocking HMGA2, resulting in reduced expression of numerous macrophage chemotactic factors, including CCL5. CCL5 overexpression in RKIP(+) tumors restores recruitment of prometastatic TAMs and intravasation, whereas treatment with the CCL5 receptor antagonist Maraviroc reduces TAM infiltration. These results highlight the importance of RKIP as a regulator of TAM recruitment through chemokines such as CCL5. The clinical significance of these interactions is underscored by our demonstration that a signature comprised of RKIP signaling and prometastatic TAM factors strikingly separates TNBC patients based on survival outcome. Collectively, our findings identify TAMs as a previously unsuspected mechanism by which the metastasis-suppressor RKIP regulates tumor invasiveness, and further suggest that TNBC patients with decreased RKIP activity and increased TAM infiltration may respond to macrophage-based therapeutics.
Assuntos
Quimiocinas/fisiologia , Quimiotaxia , Macrófagos/imunologia , Neoplasias Mamárias Experimentais/imunologia , Metástase Neoplásica/imunologia , Proteínas de Neoplasias/fisiologia , Proteína de Ligação a Fosfatidiletanolamina/fisiologia , Neoplasias de Mama Triplo Negativas/imunologia , Microambiente Tumoral/imunologia , Animais , Linhagem Celular Tumoral/transplante , Quimiocina CCL5/biossíntese , Quimiocina CCL5/genética , Quimiocina CCL5/fisiologia , Cicloexanos/farmacologia , Cicloexanos/uso terapêutico , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteína HMGA2/fisiologia , Xenoenxertos/imunologia , Humanos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Maraviroc , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Receptores CCR5/efeitos dos fármacos , Análise de Sequência de RNA , Triazóis/farmacologia , Triazóis/uso terapêutico , Neoplasias de Mama Triplo Negativas/mortalidadeRESUMO
There has been increasing awareness in the wider biological community of the role of clonal phenotypic heterogeneity in playing key roles in phenomena such as cellular bet-hedging and decision making, as in the case of the phage-λ lysis/lysogeny and B. Subtilis competence/vegetative pathways. Here, we report on the effect of stochasticity in growth rate, cellular memory/intermittency, and its relation to phenotypic heterogeneity. We first present a linear stochastic differential model with finite auto-correlation time, where a randomly fluctuating growth rate with a negative average is shown to result in exponential growth for sufficiently large fluctuations in growth rate. We then present a non-linear stochastic self-regulation model where the loss of coherent self-regulation and an increase in noise can induce a shift from bounded to unbounded growth. An important consequence of these models is that while the average change in phenotype may not differ for various parameter sets, the variance of the resulting distributions may considerably change. This demonstrates the necessity of understanding the influence of variance and heterogeneity within seemingly identical clonal populations, while providing a mechanism for varying functional consequences of such heterogeneity. Our results highlight the importance of a paradigm shift from a deterministic to a probabilistic view of clonality in understanding selection as an optimization problem on noise-driven processes, resulting in a wide range of biological implications, from robustness to environmental stress to the development of drug resistance.
Assuntos
Evolução Biológica , Células Clonais/citologia , Modelos Biológicos , Fenótipo , Processos Estocásticos , Resistencia a Medicamentos Antineoplásicos , Aptidão Genética , Humanos , Terapia Neoadjuvante , Neoplasias/patologia , Neoplasias/terapia , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Dinâmica não Linear , Distribuição Normal , Saccharomyces cerevisiae/crescimento & desenvolvimento , Seleção Genética , Fatores de TempoRESUMO
Cancer and healthy cells have distinct distributions of molecular properties and thus respond differently to drugs. Cancer drugs ideally kill cancer cells while limiting harm to healthy cells. However, the inherent variance among cells in both cancer and healthy cell populations increases the difficulty of selective drug action. Here we formalize a classification framework based on the idea that an ideal cancer drug should maximally discriminate between cancer and healthy cells. More specifically, this discrimination should be performed on the basis of measurable cell markers. We divide the problem into three parts which we explore with examples. First, molecular markers should discriminate cancer cells from healthy cells at the single-cell level. Second, the effects of drugs should be statistically predicted by these molecular markers. Third, drugs should be optimized for classification performance. We find that expression levels of a handful of genes suffice to discriminate well between individual cells in cancer and healthy tissue. We also find that gene expression predicts the efficacy of some cancer drugs, suggesting that these cancer drugs act as suboptimal classifiers using gene profiles. Finally, we formulate a framework that defines an optimal drug, and predicts drug cocktails that may target cancer more accurately than the individual drugs alone. Conceptualizing cancer drugs as solving a discrimination problem in the high-dimensional space of molecular markers promises to inform the design of new cancer drugs and drug cocktails.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias/metabolismo , Antineoplásicos/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Modelos Químicos , Neoplasias/tratamento farmacológico , Curva ROCRESUMO
The sources and consequences of nongenetic variability in metastatic progression are largely unknown. To address these questions, we characterized a transcriptional regulatory network for the metastasis suppressor Raf kinase inhibitory protein (RKIP). We previously showed that the transcription factor BACH1 is negatively regulated by RKIP and promotes breast cancer metastasis. Here we demonstrate that BACH1 acts in a double-negative (overall positive) feedback loop to inhibit RKIP transcription in breast cancer cells. BACH1 also negatively regulates its own transcription. Analysis of the BACH1 network reveals the existence of an inverse relationship between BACH1 and RKIP involving both monostable and bistable transitions that can potentially give rise to nongenetic variability. Single-cell analysis confirmed monostable and bistable-like behavior. Treatment with histone deacetylase inhibitors or depletion of the polycomb repressor enhancer of zeste homolog 2 altered relative RKIP and BACH1 levels in a manner consistent with a prometastatic state. Together, our results suggest that the mutually repressive relationship between metastatic regulators such as RKIP and BACH1 can play a key role in determining metastatic progression in cancer.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Motivos de Aminoácidos , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Progressão da Doença , Retroalimentação Fisiológica , Feminino , Variação Genética , Humanos , Células MCF-7 , Modelos Teóricos , Metástase Neoplásica , Estresse Oxidativo , Regiões Promotoras Genéticas , Fatores de Tempo , Transcrição GênicaRESUMO
Cancer is one of the deadliest diseases worldwide, accounting for about 8 million deaths a year. For solid tumors, cancer patients die as a result of the metastatic spread of the tumor to the rest of the body. Therefore, there is a clinical need for understanding the molecular and cellular basis of metastasis, identifying patients whose tumors are more likely to metastasize, and developing effective therapies against metastatic progression. Over the years, Raf kinase inhibitory protein (RKIP) has emerged as a natural suppressor of the metastatic process, constituting a tool for studying metastasis and its clinical outcomes. Here, we review RKIP's role as a metastasis suppressor and the signaling networks and genes regulated by RKIP in metastatic, triple-negative breast cancer. We also highlight the clinical implications and power of building gene signatures based on RKIP-regulated signaling modules in identifying cancer patients that are at higher risk for metastases. Finally, we highlight the potential of RKIP as a tool for developing new therapeutic strategies in cancer treatment.
Assuntos
Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Genes Supressores de Tumor , Metástase Neoplásica/genética , Proteína de Ligação a Fosfatidiletanolamina/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Neoplasias/genética , Neoplasias/patologia , Transdução de Sinais/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Raf kinase inhibitory protein (RKIP) is a highly conserved regulator of many signaling networks whose loss or inactivation can lead to a variety of disease states. The multifaceted roles played by RKIP are enabled by an allosteric structure that is controlled through phosphorylation of RKIP and dynamics in the RKIP pocket loop. Perhaps the most striking feature of RKIP is that it can assume multiple functional states. Specifically, phosphorylation redirects RKIP from a state that binds and inhibits Raf-1 to a state that binds and inhibits GRK2. Recent evidence suggests the presence of a third functional state that facilitates RKIP phosphorylation. Here, we present a three-state model to explain the RKIP functional switch and discuss the role of the pocket loop in regulating RKIP activity.
Assuntos
Modelos Biológicos , Proteína de Ligação a Fosfatidiletanolamina/química , Proteína de Ligação a Fosfatidiletanolamina/fisiologia , Humanos , Modelos Moleculares , Fosfolipídeos/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-raf/metabolismo , Relação Estrutura-AtividadeRESUMO
Although triple negative breast cancers (TNBC) are the most aggressive subtype of breast cancer, they currently lack targeted therapies. Because this classification still includes a heterogeneous collection of tumors, new tools to classify TNBCs are urgently required in order to improve our prognostic capability for high risk patients and predict response to therapy. We previously defined a gene expression signature, RKIP Pathway Metastasis Signature (RPMS), based upon a metastasis-suppressive signaling pathway initiated by Raf Kinase Inhibitory Protein (RKIP). We have now generated a new BACH1 Pathway Metastasis gene signature (BPMS) that utilizes targets of the metastasis regulator BACH1. Specifically, we substituted experimentally validated target genes to generate a new BACH1 metagene, developed an approach to optimize patient tumor stratification, and reduced the number of signature genes to 30. The BPMS significantly and selectively stratified metastasis-free survival in basal-like and, in particular, TNBC patients. In addition, the BPMS further stratified patients identified as having a good or poor prognosis by other signatures including the Mammaprint® and Oncotype® clinical tests. The BPMS is thus complementary to existing signatures and is a prognostic tool for high risk ER-HER2- patients. We also demonstrate the potential clinical applicability of the BPMS as a single sample predictor. Together, these results reveal the potential of this pathway-based BPMS gene signature to identify high risk TNBC patients that can respond effectively to targeted therapy, and highlight BPMS genes as novel drug targets for therapeutic development.
Assuntos
Transcriptoma , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Intervalo Livre de Doença , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Metástase Neoplásica , Recidiva Local de Neoplasia/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Prognóstico , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Resultado do TratamentoRESUMO
MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Ativação Enzimática , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/antagonistas & inibidores , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Angiogenesis inhibition is an important therapeutic strategy for advanced stage prostate cancer. Previous work from our laboratory showed that sustained stimulation of Rap1 by 8-pCPT-2'-O-Me-cAMP (8CPT) via activation of Epac, a Rap1 GEF, or by expression of a constitutively active Rap1 mutant (cRap1) suppresses endothelial cell chemotaxis and subsequent angiogenesis. When we tested this model in the context of a prostate tumor xenograft, we found that 8CPT had no significant effect on prostate tumor growth alone. However, in cells harboring cRap1, 8CPT dramatically inhibited not only prostate tumor growth but also VEGF expression and angiogenesis within the tumor microenvironment. Subsequent analysis of the mechanism revealed that, in prostate tumor epithelial cells, 8CPT acted via stimulation of PKA rather than Epac/Rap1. PKA antagonizes Rap1 and hypoxic induction of 1α protein expression, VEGF production and, ultimately, angiogenesis. Together these findings provide evidence for a novel interplay between Rap1, Epac, and PKA that regulates tumor-stromal induction of angiogenesis.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/análogos & derivados , Neovascularização Patológica/metabolismo , Neoplasias da Próstata/fisiopatologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Análise de Variância , Animais , Western Blotting , Linhagem Celular Tumoral , AMP Cíclico/farmacologia , Ensaio de Imunoadsorção Enzimática , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/fisiopatologia , Neoplasias da Próstata/tratamento farmacológico , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas rap1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
Cancer lethality is mainly caused by metastasis. Therefore, understanding the nature of the genes involved in this process has become a priority. Given the heterogeneity of mutations in cancer cells, considerable focus has been directed toward characterizing metastasis genes in the context of relevant signaling pathways rather than treating genes as independent and equal entities. One signaling cascade implicated in the regulation of cell growth, invasion and metastasis is the MAP kinase pathway. Raf kinase inhibitory protein (RKIP) functions as an inhibitor of the MAP kinase pathway and is a metastasis suppressor in different cancer models. By utilizing statistical analysis of clinical data integrated with experimental validation, we recently identified components of the RKIP signaling pathway relevant to breast cancer metastasis. Using the RKIP pathway as an example, we show how prior biological knowledge can be efficiently combined with genome-wide patient data to identify gene regulatory mechanisms that control metastasis.