RESUMO
BACKGROUND: RAS-to-ERK signaling is crucial for the onset and progression of advanced thyroid carcinoma, and blocking ERK dimerization provides a therapeutic benefit in several human carcinomas. Here we analyzed the effects of DEL-22379, a relatively specific ERK dimerization inhibitor, on the activation of the RAS-to-ERK signaling cascade and on tumor-related processes in vitro and in vivo. METHODS: We used a panel of four human anaplastic thyroid carcinoma (ATC) cell lines harboring BRAF or RAS mutations to analyze ERK dynamics and tumor-specific characteristics. We also assessed the impact of DEL-22379 on the transcriptional landscape of ATC cell lines using RNA-sequencing and evaluated its therapeutic efficacy in an orthotopic mouse model of ATC. RESULTS: DEL-22379 impaired upstream ERK activation in BRAF- but not RAS-mutant cells. Cell viability and metastasis-related processes were attenuated by DEL-22379 treatment, but mostly in BRAF-mutant cells, whereas in vivo tumor growth and dissemination were strongly reduced for BRAF-mutant cells and mildly reduced for RAS-mutant cells. Transcriptomics analyses indicated that DEL-22379 modulated the transcriptional landscape of BRAF- and RAS-mutant cells in opposite directions. CONCLUSIONS: Our findings establish that BRAF- and RAS-mutant thyroid cells respond differentially to DEL-22379, which cannot be explained by the previously described mechanism of action of the inhibitor. Nonetheless, DEL-22379 demonstrated significant anti-tumor effects against BRAF-mutant cells in vivo with an apparent lack of toxicity, making it an interesting candidate for the development of combinatorial treatments. Our data underscore the differences elicited by the specific driver mutation for thyroid cancer onset and progression, which should be considered for experimental and clinical approaches.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Animais , Linhagem Celular Tumoral , Dimerização , Humanos , Camundongos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genéticaRESUMO
K-Ras mutations are frequent in colorectal cancer (CRC), albeit K-Ras is the only Ras isoform that can elicit apoptosis. Here, we show that mutant K-Ras directly binds to the tumor suppressor RASSF1A to activate the apoptotic MST2-LATS1 pathway. In this pathway LATS1 binds to and sequesters the ubiquitin ligase Mdm2 causing stabilization of the tumor suppressor p53 and apoptosis. However, mutant Ras also stimulates autocrine activation of the EGF receptor (EGFR) which counteracts mutant K-Ras-induced apoptosis. Interestingly, this protection requires the wild-type K-Ras allele, which inhibits the MST2 pathway in part via AKT activation. Confirming the pathophysiological relevance of the molecular findings, we find a negative correlation between K-Ras mutation and MST2 expression in human CRC patients and CRC mouse models. The small number of tumors with co-expression of mutant K-Ras and MST2 has elevated apoptosis rates. Thus, in CRC, mutant K-Ras transformation is supported by the wild-type allele.
Assuntos
Apoptose , Neoplasias Colorretais/genética , Genes ras/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Alelos , Animais , Apoptose/genética , Humanos , Camundongos , Proteínas Mutantes/genética , Proteínas Serina-Treonina Quinases/genética , Serina-Treonina Quinase 3RESUMO
The involvement of Ras-GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H- and N-Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K-Ras in cellular processes involved in the development of fibrosis: proliferation, migration, and extracellular matrix (ECM) proteins synthesis. K-Ras knockout (KO) mouse embryonic fibroblasts (K-ras(-/-) ) stimulated with transforming growth factor-ß1 (TGF-ß1) exhibited reduced proliferation and impaired mobility than wild-type fibroblasts. Moreover, an increase on ECM production was observed in K-Ras KO fibroblasts in basal conditions. The absence of K-Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF-ß1-induced Smad signaling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K-ras but reduced migration and ECM proteins expression only in wild-type fibroblasts, while the PI3K-AKT inhibitor LY294002 decreased cell proliferation, migration, and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K-Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K-Ras may be a crucial mediator in TGF-ß1-mediated effects in this cell type. J. Cell. Physiol. 231: 2224-2235, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Movimento Celular , Proliferação de Células , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Animais , Butadienos/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Camundongos , Camundongos Knockout , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/deficiência , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Signals transmitted by ERK MAP kinases regulate the functions of multiple substrates present in the nucleus and in the cytoplasm. ERK signals are optimized by scaffold proteins that modulate their intensity and spatial fidelity. Once phosphorylated, ERKs dimerize, but how dimerization impacts on the activation of the different pools of substrates and whether it affects scaffolds functions as spatial regulators are unknown aspects of ERK signaling. Here we demonstrate that scaffolds and ERK dimers are essential for the activation of cytoplasmic but not nuclear substrates. Dimerization is critical for connecting the scaffolded ERK complex to cognate cytoplasmic substrates. Contrarily, nuclear substrates associate to ERK monomers. Furthermore, we show that preventing ERK dimerization is sufficient for attenuating cellular proliferation, transformation, and tumor development. Our results disclose a functional relationship between scaffold proteins and ERK dimers and identify dimerization as a key determinant of the spatial specificity of ERK signals.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , MAP Quinases Reguladas por Sinal Extracelular , Estrutura Quaternária de Proteína , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Citoplasma/metabolismo , Proteínas do Citoesqueleto/genética , Dimerização , MAP Quinases Reguladas por Sinal Extracelular/química , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo IV/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Nus , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Lysine methylation has been traditionally associated with histones and epigenetics. Recently, lysine methyltransferases and demethylases - which are involved in methylation of non-histone substrates - have been frequently found deregulated in human tumours. In this realm, a new discovery has unveiled the methyltransferase SMYD3 as an enhancer of Ras-driven cancer. SMYD3 is up-regulated in different types of tumours. SMYD3-mediated methylation of MAP3K2 increases mutant K-Ras-induced activation of ERK1/2. Methylation of MAP3K2 prevents it from binding to the phosphatase PP2A, thereby impeding the impact of this negative regulator on Ras-ERK1/2 signals, leading to the formation of lung and pancreatic adenocarcinomas. Furthermore, depletion of SMYD3 synergises with a MEK inhibitor, currently in clinical trials, to block Ras-driven pancreatic neoplasia. These results underscore the importance of lysine methylation in the regulation of signalling pathways relevant for tumourigenesis and endorse the development of drugs targeting unregulated lysine methylation as therapeutic agents in the struggle against cancer.
Assuntos
Adenocarcinoma/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias Pulmonares/metabolismo , Lisina/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Antineoplásicos/uso terapêutico , Histona-Lisina N-Metiltransferase/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinase 2 , MAP Quinase Quinase Quinases/genética , Metilação , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/uso terapêutico , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de SinaisRESUMO
In addition to their role as oncogenes, Ras GTPases are key regulators of cell function. There is a proven relationship between the signaling pathways of transforming growth factor-ß1 (TGF- ß1) and Ras GTPases. Each of the Ras isoforms (H, N and K) exhibits specific modulatory activity on different cellular pathways. Our purpose has been to study some of the mechanisms involved in the development of renal fibrosis, assessing the individual role of N-Ras in basal and TGF-ß1-mediated extracellular matrix (ECM) synthesis, proliferation, and migration in immortalized N-Ras deficient fibroblasts (N-ras(-/-)). Compared to normal counterparts, fibroblasts deficient for N-Ras exhibited higher basal activity levels of phosphatidylinositol-3-kinase (PI3K)/Akt and MEK/Erk, accompanied by upregulated collagen synthesis and diminished proliferation and migration rates. We found that the absence of N-Ras did not affect TGF-ß1-induced proliferation and migration, which required PI3K/Akt but not Erk1/2 activation. Similar effector pathway dependence was found for fibronectin and collagen type I expression. Our results indicate that N-Ras might contribute to renal fibrosis through the down-regulation of ECM synthesis and up-regulation proliferation and migration modulating Akt activation. N-Ras also regulates TGF-ß1-induced collagen I and fibronectin expression through Erk-independent pathways.
Assuntos
Movimento Celular , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/enzimologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Animais , Proliferação de Células , Células Clonais , Ativação Enzimática , Proteínas da Matriz Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Antígeno Ki-67/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
The mevalonate pathway plays an important role in breast cancer and other tumor types. However, many issues remain obscure as yet regarding its mechanism of regulation and action. In the present study, we report that the expression of mevalonate pathway enzymes is mediated by the RHO guanosine nucleotide exchange factors VAV2 and VAV3 in a RAC1- and sterol regulatory element-binding factor (SREBF)-dependent manner in breast cancer cells. Furthermore, in vivo tumorigenesis experiments indicated that the two most upstream steps of this metabolic pathway [3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR)] are important for primary tumorigenesis, angiogenesis, and cell survival in breast cancer cells. HMGCR, but not HMGCS1, is also important for the extravasation and subsequent fitness of breast cancer cells in the lung parenchyma. Genome-wide expression analyses revealed that HMGCR influences the expression of gene signatures linked to proliferation, metabolism, and immune responses. The HMGCR-regulated gene signature predicts long-term tumor recurrence but not metastasis in cohorts of nonsegregated and chemotherapy-resistant breast cancer patients. These results reveal a hitherto unknown, VAV-catalysis-dependent mechanism involved in the regulation of the mevalonate pathway in breast cancer cells. They also identify specific mevalonate-pathway-dependent processes that contribute to the malignant features of breast cancer cells.
RESUMO
ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases) are tightly regulated by the cellular microenvironment in which they operate. Mxi2 is a p38α splice isoform capable of binding to ERK1/2 and ensuring their translocation to the nucleus. Therein Mxi2 sustains ERK1/2 phosphorylation levels and, as a consequence, ERK1/2 nuclear signals are enhanced. However, the molecular mechanisms underlying this process are still unclear. In the present study, we show that Mxi2 prevents nuclear but not cytoplasmic phosphatases from binding to and dephosphorylating ERK1/2, disclosing an unprecedented mechanism for the spatial regulation of ERK1/2 activation. We also demonstrate that the kinetics of ERK1/2 extranuclear signals can be significantly altered by artificially tethering Mxi2 to the cytoplasm. In this case, Mxi2 abolishes ERK1/2 inactivation by cytoplasmic phosphatases and potentiates ERK1/2 functions at this compartment. These results highlight Mxi2 as a key spatial regulator of ERK1/2 functions, playing a pivotal role in the balance between ERK1/2 nuclear and cytoplasmic signals.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Células COS , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Cães , Células HEK293 , Humanos , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Transdução de SinaisRESUMO
The transcription factor MYC regulates cell proliferation, transformation, and survival in response to growth factor signaling that is mediated in part by the kinase activity of ERK2. Because ERK2 can also bind to DNA to modify gene expression, we investigated whether it more directly regulates MYC transcription. We identified ERK2 binding sites in the MYC promoter and detected ERK2 at the promoter in various serum-stimulated cell types. Expression of nuclear-localized ERK2 constructs in serum-starved cells revealed that ERK2 in the nucleus-regardless of its kinase activity-increased MYC mRNA expression and MYC protein abundance. ERK2 bound to the promoter through its amino-terminal insert domain and to the cyclin-dependent kinase CDK9 (which activates RNA polymerase II) through its carboxyl-terminal conserved docking domain. Both interactions were essential for ERK2-induced MYC expression, and depleting ERK impaired CDK9 occupancy and RNA polymerase II progression at the MYC promoter. Artificially tethering CDK9 to the MYC promoter by fusing it to the ERK2 insert domain was sufficient to stimulate MYC expression in serum-starved cells. Our findings demonstrate a role for ERK2 at the MYC promoter acting as a kinase-independent anchor for the recruitment of CDK9 to promote MYC expression.
Assuntos
RNA Polimerase II , Fatores de Transcrição , RNA Polimerase II/genética , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Fosforilação , Fatores de Transcrição/metabolismo , Quinases Ciclina-Dependentes/genética , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
RAS-ERK (extracellular signal-regulated kinase) pathway signals are modulated by scaffold proteins that assemble the components of different kinase tiers into a sequential phosphorylation cascade. In the prevailing model scaffold proteins function as isolated entities, where the flux of phosphorylation events progresses downstream linearly, to achieve ERK phosphorylation. We show that different types of scaffold proteins, specifically KSR1 (kinase suppressor of Ras 1) and IQGAP1 (IQ motif-containing guanosine triphosphatase activating protein 1), can bind to each other, forming a complex whereby phosphorylation reactions occur across both species. MEK (mitogen-activated protein kinase kinase) bound to IQGAP1 can phosphorylate ERK docked at KSR1, a process that we have named "trans-phosphorylation." We also reveal that ERK trans-phosphorylation participates in KSR1-regulated adipogenesis, and it also underlies the modest cytotoxicity exhibited by KSR-directed inhibitors. Overall, we identify interactions between scaffold proteins and trans-phosphorylation as an additional level of regulation in the ERK cascade, with broad implications in signaling and the design of scaffold protein-aimed therapeutics.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular , Sistema de Sinalização das MAP Quinases , Fosforilação , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transdução de SinaisRESUMO
Recent discoveries have suggested the concept that intracellular signals are the sum of multiple, site-specified subsignals, rather than single, homogeneous entities. In the context of cancer, searching for compounds that selectively block subsignals essential for tumor progression, but not those regulating "house-keeping" functions, could help in producing drugs with reduced side effects compared to compounds that block signaling completely. The Ras-ERK pathway has become a paradigm of how space can differentially shape signaling. Today, we know that Ras proteins are found in different plasma membrane microdomains and endomembranes. At these localizations, Ras is subject to site-specific regulatory mechanisms, distinctively engaging effector pathways and switching-on diverse genetic programs to generate different biological responses. The Ras effector pathway leading to ERKs activation is also under strict, space-related regulatory processes. These findings may open a gate for aiming at the Ras-ERK pathway in a spatially restricted fashion, in our quest for new anti-tumor therapies.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Transdução de Sinais/fisiologia , Proteínas ras/fisiologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Modelos Biológicos , Neoplasias/genética , Neoplasias/terapia , Transdução de Sinais/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Metastatic melanoma is a highly immunogenic tumor with very poor survival rates due to immune system escape-mechanisms. Immune checkpoint inhibitors (ICIs) targeting the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and the programmed death-1 (PD1) receptors, are being used to impede immune evasion. This immunotherapy entails an increment in the overall survival rates. However, melanoma cells respond with evasive molecular mechanisms. ERK cascade inhibitors are also used in metastatic melanoma treatment, with the RAF activity blockade being the main therapeutic approach for such purpose, and in combination with MEK inhibitors improves many parameters of clinical efficacy. Despite their efficacy in inhibiting ERK signaling, the rewiring of the melanoma cell-signaling results in disease relapse, constituting the reinstatement of ERK activation, which is a common cause of some resistance mechanisms. Recent studies revealed that the combination of RAS-ERK pathway inhibitors and ICI therapy present promising advantages for metastatic melanoma treatment. Here, we present a recompilation of the combined therapies clinically evaluated in patients.
Assuntos
Antineoplásicos , Melanoma , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Sistema de Sinalização das MAP Quinases , Melanoma/patologia , Imunoterapia/métodos , Antineoplásicos/farmacologiaRESUMO
It is known that Rho GTPases control different aspects of the biology of skin stem cells (SSCs). However, little information is available on the role of their upstream regulators under normal and tumorigenic conditions in this process. To address this issue, we have used here mouse models in which the activity of guanosine nucleotide exchange factors of the Vav subfamily has been manipulated using both gain- and loss-of-function strategies. These experiments indicate that Vav2 and Vav3 regulate the number, functional status, and responsiveness of hair follicle bulge stem cells. This is linked to gene expression programs related to the reinforcement of the identity and the quiescent state of normal SSCs. By contrast, in the case of cancer stem cells, they promote transcriptomal programs associated with the identity, activation state, and cytoskeletal remodeling. These results underscore the role of these Rho exchange factors in the regulation of normal and tumor epidermal stem cells.
Assuntos
Proteínas Proto-Oncogênicas c-vav , Pele , Células-Tronco , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Células Epidérmicas/citologia , Células Epidérmicas/metabolismo , Epiderme/metabolismo , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Pele/citologia , Pele/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Signals conveyed through the RAS-ERK pathway constitute a pivotal regulatory element in cancer-related cellular processes. Recently, RAS dimerization has been proposed as a key step in the relay of RAS signals, critically contributing to RAF activation. RAS clustering at plasma membrane microdomains and endomembranes facilitates RAS dimerization in response to stimulation, promoting RAF dimerization and subsequent activation. Remarkably, inhibiting RAS dimerization forestalls tumorigenesis in cellular and animal models. Thus, the pharmacological disruption of RAS dimers has emerged as an additional target for cancer researchers in the quest for a means to curtail aberrant RAS activity.
Assuntos
Carcinogênese/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Multimerização Proteica , Proteínas ras/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
Metallothionein-3 has poorly characterized functions in neuroblastoma. Cisplatin-based chemotherapy is a major regimen to treat neuroblastoma, but its clinical efficacy is limited by chemoresistance. We investigated the impact of human metallothionein-3 (hMT3) up-regulation in neuroblastoma cells and the mechanisms underlying the cisplatin-resistance. We confirmed the cisplatin-metallothionein complex formation using mass spectrometry. Overexpression of hMT3 decreased the sensitivity of neuroblastoma UKF-NB-4 cells to cisplatin. We report, for the first time, cisplatin-sensitive human UKF-NB-4 cells remodelled into cisplatin-resistant cells via high and constitutive hMT3 expression in an in vivo model using chick chorioallantoic membrane assay. Comparative proteomic analysis demonstrated that several biological pathways related to apoptosis, transport, proteasome, and cellular stress were involved in cisplatin-resistance in hMT3 overexpressing UKF-NB-4 cells. Overall, our data confirmed that up-regulation of hMT3 positively correlated with increased cisplatin-chemoresistance in neuroblastoma, and a high level of hMT3 could be one of the causes of frequent tumour relapses.
Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Metalotioneína 3/biossíntese , Proteínas de Neoplasias/biossíntese , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Metalotioneína 3/genética , Proteínas de Neoplasias/genéticaRESUMO
The survival rate in lung cancer remains stubbornly low and there is an urgent need for the identification of new therapeutic targets. In the last decade, several members of the SWI/SNF chromatin remodeling complexes have been described altered in different tumor types. Nevertheless, the precise mechanisms of their impact on cancer progression, as well as the application of this knowledge to cancer patient management are largely unknown. In this study, we performed targeted sequencing of a cohort of lung cancer patients on genes involved in chromatin structure. In addition, we studied at the protein level the expression of these genes in cancer samples and performed functional experiments to identify the molecular mechanisms linking alterations of chromatin remodeling genes and tumor development. Remarkably, we found that 20% of lung cancer patients show ARID2 protein loss, partially explained by the presence of ARID2 mutations. In addition, we showed that ARID2 deficiency provokes profound chromatin structural changes altering cell transcriptional programs, which bolsters the proliferative and metastatic potential of the cells both in vitro and in vivo. Moreover, we demonstrated that ARID2 deficiency impairs DNA repair, enhancing the sensitivity of the cells to DNA-damaging agents. Our findings support that ARID2 is a bona fide tumor suppressor gene in lung cancer that may be exploited therapeutically.
Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Fatores de Transcrição/deficiência , Células A549 , Animais , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Taxa de Sobrevida , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
RAS mutations are the second most common genetic alteration in thyroid tumors. However, the extent to which they are associated with the most aggressive phenotypes is still controversial. Regarding their malignancy, the majority of RAS mutant tumors are classified as undetermined, which complicates their clinical management and can lead to undesired under- or overtreatment. Using the chick embryo spontaneous metastasis model, we herein demonstrate that the aggressiveness of HRAS-transformed thyroid cells, as determined by the ability to extravasate and metastasize at distant organs, is orchestrated by HRAS subcellular localization. Remarkably, aggressiveness inversely correlates with tumor size. In this respect, we also show that RAS site-specific capacity to regulate tumor growth and dissemination is dependent on VEGF-B secretion. Furthermore, we have identified the acyl protein thioesterase APT-1 as a determinant of thyroid tumor growth versus dissemination. We show that alterations in APT-1 expression levels can dramatically affect the behavior of thyroid tumors, based on its role as a regulator of HRAS sublocalization at distinct plasma membrane microdomains. In agreement, APT-1 emerges in thyroid cancer clinical samples as a prognostic factor. As such, APT-1 levels could serve as a biomarker that could help in the stratification of HRAS mutant thyroid tumors based on their aggressiveness.
RESUMO
RHO GTPases are key regulators of the cytoskeletal architecture, which impact a broad range of biological processes in malignant cells including motility, invasion, and metastasis, thereby affecting tumor progression. One of the constraints during cell migration is the diameter of the pores through which cells pass. In this respect, the size and shape of the nucleus pose a major limitation. Therefore, enhanced nuclear plasticity can promote cell migration. Nuclear morphology is determined in part through the cytoskeleton, which connects to the nucleoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Here, we unravel the role of RAC1 as an orchestrator of nuclear morphology in melanoma cells. We demonstrate that activated RAC1 promotes nuclear alterations through its effector PAK1 and the tubulin cytoskeleton, thereby enhancing migration and intravasation of melanoma cells. Disruption of the LINC complex prevented RAC1-induced nuclear alterations and the invasive properties of melanoma cells. Thus, RAC1 induces nuclear morphology alterations through microtubules and the LINC complex to promote an invasive phenotype in melanoma cells.
Assuntos
Núcleo Celular/metabolismo , Melanoma/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Forma do Núcleo Celular/fisiologia , Embrião de Galinha , Citoesqueleto/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Invasividade Neoplásica/genética , Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Ras GTPases convey signals from different types of membranes. At these locations, different Ras isoforms, interactors and regulators generate different biochemical signals and biological outputs. The study of Ras localisation-specific signal transduction networks has been hampered by our inability to specifically activate each of these Ras pools. Here, we describe a new set of site-specific tethered exchange factors, engineered by fusing the RasGRF1 CDC25 domain to sub-localisation-defining cues, whereby Ras pools at specific locations can be precisely activated. We show that the CDC25 domain has a high specificity for activating HRas but not NRas and KRas. This unexpected finding means that our constructs mainly activate endogenous HRas. Hence, their use enabled us to identify distinct pathways regulated by HRas in endomembranes and plasma membrane microdomains. Importantly, these new constructs unveil different patterns of HRas activity specified by their subcellular localisation. Overall, the targeted GEFs described herein constitute ideal tools for dissecting spatially-defined HRas biochemical and biological functions.