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1.
Cell Physiol Biochem ; 53(5): 820-831, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31701729

RESUMO

BACKGROUND/AIMS: MLK4 (KIAA1804) is the second most frequently mutated kinase in microsatellite stable (MSS) colorectal carcinomas (CRC). This molecule is known to regulate different physiological cellular processes, including cell cycle, senescence and apoptosis, and mechanistic evidence has been provided that MLK4 plays a role in carcinogenesis. However, whether this kinase exerts a tumor suppressive role or an oncogenic function is still an object of debate. This study aims to elucidate the role of MLK4 in the pathogenesis of CRC by investigating human tumor specimens. METHODS: This study assessed MLK4 expression levels by immunohistochemistry in surgical tumor samples from 204 early-stage CRC patients and their correlation with various clinical-pathological features and patients' outcomes. In addition, MLK4 mRNA transcription was analysed in an independent cohort of 786 colon cancer samples. RESULTS: Loss of MLK4 staining was associated with poor overall (OS) and progression free survival (PFS) in CRC patients during a univariate analysis (OS:101 vs 164 months, p=0.0002; PFS:85 vs 125 months, p=0.0001), as well as in multivariate analysis (OS:HR=1.70; p=0.001; PFS:HR=1,61; p=0.001). This was confirmed by analysis of MLK4 mRNA in the second independent cohort. A subgroup analysis according to KRAS mutation status showed that MLK4 staining was associated with better OS and PFS in KRAS mutated cases (HR=2.77; p=0.0001 and HR=2.31; p=0.0003, respectively) and microsatellite stable tumors (HR=1.87; p=0.002 and HR=1.06; p=0.006) but not in KRAS wildtype and microsatellite unstable tumors. CONCLUSION: By providing the first report from clinical specimens on the prognostic significance of MLK4, we define an oncogenic loss-of-function of this kinase and suggest a possible role in the interaction with KRAS signaling in determining an aggressive phenotype of CRC. These findings warrant the further investigation of MLK4 in wider cohorts and various clinical settings.


Assuntos
Neoplasias Colorretais/patologia , MAP Quinase Quinase Quinases/metabolismo , Instabilidade de Microssatélites , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Idoso , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Feminino , Humanos , Estimativa de Kaplan-Meier , MAP Quinase Quinase Quinases/genética , Masculino , Mutação , Prognóstico , Intervalo Livre de Progressão , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas p21(ras)/genética
3.
Curr Top Med Chem ; 19(23): 2098-2113, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31475898

RESUMO

Over the past decades, designing therapeutic strategies to target KRAS-mutant cancers, which is one of the most frequent mutant oncogenes among all cancer types, have proven unsuccessful regardless of many concerted attempts. There are key challenges for KRAS-mutant anticancer therapy, as the complex cellular processes involved in KRAS signaling has present. Herein, we highlight the emerging therapeutic approaches for inhibiting KRAS signaling and blocking KRAS functions, in hope to serve as a more effective guideline for future development of therapeutics.


Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Antineoplásicos/química , Humanos , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
4.
Curr Top Med Chem ; 19(23): 2081-2097, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31486755

RESUMO

Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) is the most commonly mutated oncogene in human cancer. The developments of many cancers depend on sustained expression and signaling of KRAS, which makes KRAS a high-priority therapeutic target. Scientists have not successfully developed drugs that target KRAS, although efforts have been made last three decades. In this review, we highlight the emerging experimental strategies of impairing KRAS membrane localization and the direct targeting of KRAS. We also conclude the combinatorial therapies and RNA interference technology for the treatment of KRAS mutant cancers. Moreover, the virtual screening approach to discover novel KRAS inhibitors and synthetic lethality interactors of KRAS are discussed in detail.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Antineoplásicos/química , Humanos , Mutação , Neoplasias/genética , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA
5.
Int J Nanomedicine ; 14: 6589-6600, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31496700

RESUMO

Background: The RAS family of oncogenes (KRAS, HRAS, NRAS) are the most frequent mutations in cancers and regulate key signaling pathways that drive tumor progression. As a result, drug delivery targeting RAS-driven tumors has been a long-standing challenge in cancer therapy. Mutant RAS activates cancer cells to actively take up nutrients, including glucose, lipids, and albumin, via macropinocytosis to fulfill their energetic requirements to survive and proliferate. Purpose: We exploit macropinocytosis pathway to deliver nanoparticles (NPs) in cancer cells harboring activating KRAS mutations. Methods: NPs were synthesized by the desolvation method. The physicochemical properties and stability of NPs were characterized by dynamic light scattering and transmission electron microscopy. Uptake of fluorescently labelled NPs in wild-type and mutant KRAS cells were quantitively determined by flow cytometry and qualitatively by fluorescent microscopy. NP uptake by KRAS-driven macropinocytosis was confirmed by pharmacological inhibition and genetic knockdown. Results: We have synthesized stable albumin NPs that demonstrate significantly greater uptake in cancer cells with activating mutations of KRA S than monomeric albumin (ie, dissociated form of clinically used nab-paclitaxel). From pharmacological inhibition and semi-quantitative fluorescent microscopy studies, these NPs exhibit significantly increased uptake in mutant KRAS cancer cells than wild-type KRAS cells by macropinocytosis. Conclusions: The uptake of albumin nanoparticles is driven by KRAS. This NP-based strategy targeting RAS-driven macropinocytosis is a facile approach toward improved delivery into KRAS-driven cancers.


Assuntos
Espaço Intracelular/metabolismo , Nanopartículas/administração & dosagem , Oncogenes , Pinocitose , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular Tumoral , Fenômenos Químicos , Humanos , Nanopartículas/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , Soroalbumina Bovina/metabolismo
6.
Cell Biochem Funct ; 37(7): 545-552, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31429100

RESUMO

Ras-GTPases regulate many central signalling pathways in the cell. Hypoxia induces nitrosative/oxidative stress and dysregulates Ras-dependent downstream processes. H-Ras possesses two cysteine residues (C181 and C184) in the C-termini, which are palmitoylated once or twice. Palmitoylation is sufficient for promoting stable plasma membrane localization. We hypothesized that high concentrations of hypoxia-formed nitric oxide could induce terminal cysteine S-nitrosylation, followed by depalmitoylation and H-Ras mislocalization. We investigated the action of a 100-µM nitric oxide-donor (sodium nitroprusside [SNP]) and a 100-µM palmitoylation inhibitor (2-bromopalmitate) on the distribution of membrane-bound S-nitrosylated and palmitoylated H-Ras under hypoxic/normoxic conditions in undifferentiated/differentiated pheochromocytoma (PC12) cells. We found that under normoxic conditions, SNP increases membrane-bound H-Ras nitrosylation only in differentiated cells, whereas under hypoxic conditions, SNP stimulates H-Ras nitrosylation in both differentiated and undifferentiated cells. SNP greatly decreases the palmitoylation of H-Ras under hypoxic conditions in both undifferentiated and differentiated cells, while under normoxic conditions, the effect of SNP is more negligible. Furthermore, Western blot analyses have shown that SNP decreases ERK phosphorylation under hypoxic conditions, in parallel with an elevation in hypoxia-induced factor activity and intracellular succinate concentration. We propose that high concentrations of hypoxia-formed nitric oxide can nitrosylate H-Ras terminal cysteines, which induce H-Ras activity dysregulation and alter the cellular response to hypoxia. SIGNIFICANCE OF THE STUDY: To our knowledge, these observations may be important for cancer prevention and therapy because cancer is one of the most prevalent disorders caused by the misregulation of Ras activity by a redox agent. Oncogenic activation of the H-Ras gene has been found in a wide variety of neoplastic transformations, and thus, investigation of the redox regulation of H-Ras activity is significant for cancer research as well.


Assuntos
Diferenciação Celular , Hipóxia/tratamento farmacológico , Nitroprussiato/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Diferenciação Celular/efeitos dos fármacos , Hipóxia/metabolismo , Células PC12 , Proteínas Proto-Oncogênicas p21(ras)/análise , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ratos
7.
Gene ; 716: 144033, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31377313

RESUMO

Oral squamous cell cancer (OSCC) is one of the causes of death worldwide. The purpose of this project was to define the restoring of microRNA-143 in HN-5 cells and discover molecular apparatuses responsible for the anticancer processes. Firstly, expression levels of miR-143, K-Ras, MMP9 and C-Myc were evaluated in OSCC tissues. Then, microRNA-143 was transfected into HN-5 cells. The cytotoxic effects of microRNA-143 on HN-5 cells were evaluated. To estimate the effects of microRNA-143 on cell migration, wound healing assay was done. The expression levels of microRNA-143, K-Ras, MMP9, C-Myc, ADAMTS and CXCR4 were evaluated via the qRT-PCR method. microRNA-143 mimic inhibited cell migration in HN-5 cell line. microRNA-143 mimic decreased K-Ras, MMP9, C-My, ADAMTS and CXCR4 gene expression. microRNA-143 can inhibit HN-5 cells migration in vitro by down-regulating the expression of invasion-linked genes. Hence, microRNA-143 can be a new diagnostic biomarker and new therapeutic target for OSCC.


Assuntos
MicroRNAs/metabolismo , Neoplasias Bucais/genética , Neoplasias de Células Escamosas/genética , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Metástase Neoplásica , Neoplasias de Células Escamosas/metabolismo , Neoplasias de Células Escamosas/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transfecção
8.
Chem Commun (Camb) ; 55(75): 11183-11186, 2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31465055

RESUMO

Reversible S-palmitoylation is a key regulatory mechanism of protein function and localization. There is increasing evidence that S-acylation is not restricted to palmitate but it includes shorter, longer, and unsaturated fatty acids. However, the diversity of this protein modification has not been fully explored. Herein, we report a chemical probe that combined with MS-based analysis allows the rapid detection and quantification of fatty acids linked to proteins. We have used this approach to profile the S-acylome and to show that the oncogene N-Ras is heterogeneously acylated with palmitate and palmitoleate. Studies on protein distribution in membrane subdomains with semisynthetic proteins revealed that unsaturated N-Ras presents an increased tendency toward clustering and higher insertion kinetic rate constants.


Assuntos
Ácidos Graxos/metabolismo , Corantes Fluorescentes/química , Hidroxilamina/química , Proteínas Proto-Oncogênicas p21(ras)/química , Acilação , Animais , Linhagem Celular Tumoral , Ácidos Graxos/análise , Corantes Fluorescentes/síntese química , Células HEK293 , Células HeLa , Humanos , Hidroxilamina/síntese química , Camundongos , Estrutura Molecular , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
9.
Cancer Sci ; 110(9): 2760-2772, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31325400

RESUMO

Long noncoding RNAs (lncRNAs) are emerging as key regulators in cancer initiation and progression. TP53TG1 is a recently identified lncRNA and several studies have shown that TP53TG1 may play the role of tumor suppressor gene or oncogene in different tumors. Nevertheless, the involvement of TP53TG1 in carcinogenesis of pancreatic ductal adenocarcinoma (PDAC) has not been characterized. In our studies, we identified that TP53TG1 was highly expressed in PDAC and was a novel regulator of PDAC development. Knockdown of TP53TG1 inhibited proliferation, induced apoptosis, and decreased migration and invasion in PDAC cells, whereas enhanced expression of TP53TG1 had the opposite effects. Mechanistically, TP53TG1 could directly bind to microRNA (miR)-96 and effectively function as a sponge for miR-96, thus antagonizing the functions of miR-96 and leading to derepression of its endogenous target KRAS, which is a core oncogene in the initiation and maintenance of PDAC. Taken together, these observations imply that TP53TG1 contributes to the growth and progression of PDAC by acting as a competing endogenous RNA (ceRNA) to competitively bind to miR-96 and regulate KRAS expression, which highlights the importance of the complicated miRNA-lncRNA network in modulating the progression of PDAC.


Assuntos
Carcinoma Ductal Pancreático/genética , Proteínas de Ligação a DNA/metabolismo , MicroRNAs/antagonistas & inibidores , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Longo não Codificante/metabolismo , Carcinogênese/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Invasividade Neoplásica/genética , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Longo não Codificante/genética
10.
Nat Commun ; 10(1): 3055, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296870

RESUMO

KRAS mutations are present in over 90% of pancreatic ductal adenocarcinomas (PDAC), and drive their poor outcomes and failure to respond to targeted therapies. Here we show that Leukemia Inhibitory Factor (LIF) expression is induced specifically by oncogenic KRAS in PDAC and that LIF depletion by genetic means or by neutralizing antibodies prevents engraftment in pancreatic xenograft models. Moreover, LIF-neutralizing antibodies synergize with gemcitabine to eradicate established pancreatic tumors in a syngeneic, KrasG12D-driven, PDAC mouse model. The related cytokine IL-6 cannot substitute for LIF, suggesting that LIF mediates KRAS-driven malignancies through a non-STAT-signaling pathway. Unlike IL-6, LIF inhibits the activity of the Hippo-signaling pathway in PDACs. Depletion of YAP inhibits the function of LIF in human PDAC cells. Our data suggest a crucial role of LIF in KRAS-driven pancreatic cancer and that blockade of LIF by neutralizing antibodies represents an attractive approach to improving therapeutic outcomes.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Fator Inibidor de Leucemia/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Feminino , Técnicas de Inativação de Genes , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Fator Inibidor de Leucemia/antagonistas & inibidores , Fator Inibidor de Leucemia/genética , Camundongos , Mutação , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Pharm Res ; 36(9): 133, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289919

RESUMO

PURPOSE: KRAS is the most frequently mutated gene in human cancers. Despite its direct involvement in malignancy and intensive effort, direct inhibition of KRAS via pharmacological inhibitors has been challenging. RNAi induced knockdown using siRNAs against mutant KRAS alleles offers a promising tool for selective therapeutic silencing in KRAS-mutant lung cancers. However, the major bottleneck for clinical translation is the lack of efficient biocompatible siRNA carrier systems. METHODS: Bovine serum albumin (BSA) nanoparticles were prepared by desolvation method to deliver siRNA targeting the KRAS G12S mutation. The BSA nanoparticles were characterized with respect to their size, zeta potential, encapsulation efficiency and nucleic acid release. Nanoparticle uptake, cellular distribution of nucleic acids, cytotoxicity and gene knock down to interfere with cancer hallmarks, uncontrolled proliferation and migration, were evaluated in KRAS G12S mutant A459 cells, a lung adenocarcinoma cell line. RESULTS: BSA nanoparticles loaded with siRNA resulted in nanoparticles smaller than 200 nm in diameter and negative zeta potentials, displaying optimal characteristics for in vivo application. Encapsulating and protecting the siRNA payload well, the nanoparticles enabled transport to A549 cells in vitro, could evade endosomal entrapment and mediated significant sequence-specific KRAS knockdown, resulting in reduced cell growth of siRNA transfected lung cancer cells. CONCLUSIONS: BSA nanoparticles loaded with mutant specific siRNA are a promising therapeutic approach for KRAS-mutant cancers.


Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Interferente Pequeno/farmacologia , Soroalbumina Bovina/química , Células A549 , Animais , Apoptose/efeitos dos fármacos , Bovinos , Sobrevivência Celular , Técnicas de Silenciamento de Genes , Terapia Genética , Humanos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Transfecção
12.
Nat Commun ; 10(1): 2919, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266962

RESUMO

Oncogenic mutations in KRAS or BRAF are frequent in colorectal cancer and activate the ERK kinase. Here, we find graded ERK phosphorylation correlating with cell differentiation in patient-derived colorectal cancer organoids with and without KRAS mutations. Using reporters, single cell transcriptomics and mass cytometry, we observe cell type-specific phosphorylation of ERK in response to transgenic KRASG12V in mouse intestinal organoids, while transgenic BRAFV600E activates ERK in all cells. Quantitative network modelling from perturbation data reveals that activation of ERK is shaped by cell type-specific MEK to ERK feed forward and negative feedback signalling. We identify dual-specificity phosphatases as candidate modulators of ERK in the intestine. Furthermore, we find that oncogenic KRAS, together with ß-Catenin, favours expansion of crypt cells with high ERK activity. Our experiments highlight key differences between oncogenic BRAF and KRAS in colorectal cancer and find unexpected heterogeneity in a signalling pathway with fundamental relevance for cancer therapy.


Assuntos
Neoplasias do Colo/enzimologia , Mucosa Intestinal/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Especificidade da Espécie
13.
Nat Commun ; 10(1): 2914, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266968

RESUMO

The deubiquitylase OTUD3 plays a suppressive role in breast tumorigenesis through stabilizing PTEN protein, but its role in lung cancer remains unclear. Here, we demonstrate that in vivo deletion of OTUD3 indeed promotes breast cancer development in mice, but by contrast, it slows down KrasG12D-driven lung adenocarcinoma (ADC) initiation and progression and markedly increases survival in mice. Moreover, OTUD3 is highly expressed in human lung cancer tissues and its higher expression correlates with poorer survival of patients. Further mechanistic studies reveal that OTUD3 interacts with, deubiquitylates and stabilizes the glucose-regulated protein GRP78. Knockdown of OTUD3 results in a decrease in the level of GRP78 protein, suppression of cell growth and migration, and tumorigenesis in lung cancer. Collectively, our results reveal a previously unappreciated pro-oncogenic role of OTUD3 in lung cancer and indicate that deubiquitylases could elicit tumor-suppressing or tumor-promoting activities in a cell- and tissue-dependent context.


Assuntos
Proteínas de Choque Térmico/metabolismo , Neoplasias Pulmonares/enzimologia , Proteases Específicas de Ubiquitina/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Proteínas de Choque Térmico/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteases Específicas de Ubiquitina/genética
14.
Int J Mol Sci ; 20(12)2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31207989

RESUMO

Colon cancer is one of the major causes of cancer death worldwide. The five-year survival rate for the early-stage patients is more than 90%, and only around 10% for the later stages. Moreover, half of the colon cancer patients have been clinically diagnosed at the later stages. It is; therefore, of importance to enhance the ability for the early diagnosis of colon cancer. Taking advantages from our previous studies, there are several potential biomarkers which have been associated with the early diagnosis of the colon cancer. In order to investigate these early diagnostic biomarkers for colon cancer, human chromogranin-A (CHGA) was further analyzed among the most powerful diagnostic biomarkers. In this study, we used a logistic regression-based meta-analysis to clarify associations of CHGA expression with colon cancer diagnosis. Both healthy populations and the normal mucosa from the colon cancer patients were selected as the double normal controls. The results showed decreased expression of CHGA in the early stages of colon cancer as compared to the normal controls. The decline of CHGA expression in the early stages of colon cancer is probably a new diagnostic biomarker for colon cancer diagnosis with high predicting possibility and verification performance. We have also compared the diagnostic powers of CHGA expression with the typical oncogene KRAS, classic tumor suppressor TP53, and well-known cellular proliferation index MKI67, and the CHGA showed stronger ability to predict early diagnosis for colon cancer than these other cancer biomarkers. In the protein-protein interaction (PPI) network, CHGA was revealed to share some common pathways with KRAS and TP53. CHGA might be considered as a novel, promising, and powerful biomarker for early diagnosis of colon cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Cromogranina A/metabolismo , Neoplasias do Colo/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/normas , Cromogranina A/genética , Cromogranina A/normas , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Redes Reguladoras de Genes , Humanos , Metanálise como Assunto , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sensibilidade e Especificidade , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
15.
Nat Commun ; 10(1): 2607, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197133

RESUMO

Inhibiting the RAS oncogenic protein has largely been through targeting the switch regions that interact with signalling effector proteins. Here, we report designed ankyrin repeat proteins (DARPins) macromolecules that specifically inhibit the KRAS isoform by binding to an allosteric site encompassing the region around KRAS-specific residue histidine 95 at the helix α3/loop 7/helix α4 interface. We show that these DARPins specifically inhibit KRAS/effector interactions and the dependent downstream signalling pathways in cancer cells. Binding by the DARPins at that region influences KRAS/effector interactions in different ways, including KRAS nucleotide exchange and inhibiting KRAS dimerization at the plasma membrane. These results highlight the importance of targeting the α3/loop 7/α4 interface, a previously untargeted site in RAS, for specifically inhibiting KRAS function.


Assuntos
Sítio Alostérico/efeitos dos fármacos , Antineoplásicos/farmacologia , Desenho de Drogas , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Repetição de Anquirina , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Histidina/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/genética , Neoplasias/patologia , Biblioteca de Peptídeos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
BMC Cancer ; 19(1): 530, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151422

RESUMO

BACKGROUND: Histone H2AX phosphorylation at the site of Tyr-142 can participates in multiple biological progressions, which is including DNA repair. Ras pathway is closely involved in human cancers. Our study investigated the effects of Ras pathway via regulating H2AX.Y142ph. METHODS: Gastric cancer cell line SNU-16 and MKN1 cells were transfected with Ras for G12D and T35S site mutation. The phosphorylation of H2A.XY142 and ERK1/2, WSTF and MDM2 was detected by western blot. Cell viability, cell colonies and migration was analyzed by MTT assay, soft-agar colony formation assay, and Transwell assay, respectively. The expression of Ras pathway related downstream factors, EYA3 and WSTF was detected by qRT-PCR. The relationship between Ras and downstream factors were detected by ChIP. The cell cycle progression was measured by flow cytometry. RESULTS: RasG12D/T35V transection decreased the phosphorylation of H2A.XY142 and activated phosphorylation of ERK-1/2. H2A.XY142 inhibited cell viability, colonies and migration. H2A.XY142ph altered the expression of Ras downstream factors. CHIP assay revealed that RasG12D/T35V could bind to the promoters of these Ras pathway downstream factors. Silence of EYA3 increased H2A.XY142ph and inhibited cell viability, migration and percent cells in S stage. Furthermore, silence of EYA3 also changed the downstream factors expression. WSTF and H2A.XY142ph revealed the similar trend and MDM2 on the opposite. CONCLUSION: Ras/ERK signal pathway decreased H2A.XY142ph and promoted cell growth and metastasis. This Ras regulation process was down-regulated by the cascade of MDM2-WSTF-EYA3 to decrease H2A.XY142ph in SNU-16 cells.


Assuntos
Histonas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias Gástricas/metabolismo , Fatores de Transcrição/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Humanos , 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 , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Gástricas/patologia , Fatores de Transcrição/genética
17.
ScientificWorldJournal ; 2019: 5632859, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31097925

RESUMO

Coelomic fluid of Lumbricus rubellus (CFL) has attracted interest due to its pharmacological properties, including antitumor effect. Furthermore, it is necessary to evaluate the response to treatment with new cancer therapeutic agents. This study aims to investigate whether the combination of CFL and 5-fluorouracil could reduce FAK protein level and iCa2+ and enhance p21 level. Furthermore, it is necessary to evaluate the response to treatment with new cancer therapeutic agents. After 24 hours of treatment, it was necessary to assess the percentage of apoptosis, FAK, and p21 protein expression by flow cytometry. iCa2+ concentration was measured using immunofluorescence. The combination therapy of CFL with 5-fluorouracil potently suppressed six treatment groups were included in this study. HT-29 cell lines were cultured and divided into six groups: group 1 was treated with vehicle (negative control), groups 2-5 were treated with 5-fluorouracil, groups 3-5 were treated with either CFL 5, 10, or 20 µg/ml immediately after 5-fluorouracil, and group 6 was treated with CFL 20 µg/ml, the progression of colorectal cancer. Combination of CFL and 5-fluorouracil significantly decreased FAK expression (p<0.05), iCa2+ (p<0.05), and increased p21 expression (p<0.05) in HT-29 cells. Our results suggest that CFL has an anticancer potential in colorectal cancer when combined with 5-fluorouracil.


Assuntos
Antineoplásicos/farmacologia , Citotoxinas/farmacologia , Fluoruracila/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Oligoquetos/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Células HT29 , Humanos
18.
Phys Chem Chem Phys ; 21(22): 12021-12028, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31135801

RESUMO

PI3Kα is a principal Ras effector that phosphorylates PIP2 to PIP3 in the PI3K/Akt/mTOR pathway. How Ras activates PI3K has been unclear: is Ras' role confined to PI3K recruitment to the membrane or does Ras activation also involve allostery? Recently, we determined the mechanism of PI3Kα activation at the atomic level. We showed the vital role and significance of conformational change in PI3Kα activation. Here, by a 'best-match for hydrogen-bonding pair' (BMHP) computational protocol and molecular dynamics (MD) simulations, we model the atomic structure of KRas4B in complex with the Ras binding domain (RBD) of PI3Kα, striving to understand the mechanism of PI3Kα activation by Ras. Point mutations T208D, K210E, and K227E disrupt the KRas4B-RBD interface in the models, in line with the experiments. We identify allosteric signaling pathways connecting Ras to RBD in the p110α subunit. However, the observed weak allosteric signals coupled with the detailed mechanism of PI3Kα activation make us conclude that the dominant mechanistic role of Ras is likely to be recruitment and restriction of the PI3Kα population at the membrane. Thus, RTK recruits the PI3Kα to the membrane and activates it by relieving its autoinhibition exerted by the nSH2 domain, leading to exposure of the kinase domain, which permits PIP2 binding. Ras recruitment can shift the PI3Kα ensemble toward a population where the kinase domain surface and the active site position and orientation favor PIP2 insertion. This work helps elucidate Ras-mediated PI3K activation and explores the structural basis for Ras-PI3Kα drug discovery.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Regulação Alostérica , Classe I de Fosfatidilinositol 3-Quinases/química , Classe I de Fosfatidilinositol 3-Quinases/genética , Ativação Enzimática , Humanos , Simulação de Dinâmica Molecular , Mutação Puntual , Ligação Proteica , Conformação Proteica em Folha beta , Domínios Proteicos , Proteínas Proto-Oncogênicas p21(ras)/química
19.
PLoS Genet ; 15(5): e1008139, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31050682

RESUMO

Accumulating evidence indicates that paternal age correlates with disease risk in children. De novo gain-of-function mutations in the FGF-RAS-MAPK signaling pathway are known to cause a subset of genetic diseases associated with advanced paternal age, such as Apert syndrome, achondroplasia, Noonan syndrome, and Costello syndrome. It has been hypothesized that adult spermatogonial stem cells with pathogenic mutations are clonally expanded over time and propagate the mutations to offspring. However, no model system exists to interrogate mammalian germline stem cell competition in vivo. In this study, we created a lineage tracing system, which enabled undifferentiated spermatogonia with endogenous expression of HrasG12V, a known pathogenic gain-of-function mutation in RAS-MAPK signaling, to compete with their wild-type counterparts in the mouse testis. Over a year of fate analysis, neither HrasG12V-positive germ cells nor sperm exhibited a significant expansion compared to wild-type neighbors. Short-term stem cell capacity as measured by transplantation analysis was also comparable between wild-type and mutant groups. Furthermore, although constitutively active HRAS was detectable in the mutant cell lines, they did not exhibit a proliferative advantage or an enhanced response to agonist-evoked pERK signaling. These in vivo and in vitro results suggest that mouse spermatogonial stem cells are functionally resistant to a heterozygous HrasG12V mutation in the endogenous locus and that mechanisms could exist to prevent such harmful mutations from being expanded and transmitted to the next generation.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Espermatogônias/metabolismo , Células-Tronco Germinativas Adultas/fisiologia , Animais , Mutação com Ganho de Função/genética , Mutação em Linhagem Germinativa/genética , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Mutação/genética , Idade Paterna , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Seleção Genética/genética , Transdução de Sinais/genética , Espermatogônias/fisiologia , Espermatozoides/metabolismo , Testículo/metabolismo
20.
Genes Dev ; 33(11-12): 641-655, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31048544

RESUMO

Pancreatic adenocarcinoma (PDA) is an aggressive disease driven by oncogenic KRAS and characterized by late diagnosis and therapeutic resistance. Here we show that deletion of the ataxia-telangiectasia group D-complementing (Atdc) gene, whose human homolog is up-regulated in the majority of pancreatic adenocarcinoma, completely prevents PDA development in the context of oncogenic KRAS. ATDC is required for KRAS-driven acinar-ductal metaplasia (ADM) and its progression to pancreatic intraepithelial neoplasia (PanIN). As a result, mice lacking ATDC are protected from developing PDA. Mechanistically, we show ATDC promotes ADM progression to PanIN through activation of ß-catenin signaling and subsequent SOX9 up-regulation. These results provide new insight into PDA initiation and reveal ATDC as a potential target for preventing early tumor-initiating events.


Assuntos
Carcinogênese , Carcinoma Ductal Pancreático/fisiopatologia , Neoplasias Pancreáticas/fisiopatologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Transcrição/fisiologia , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Carcinoma in Situ/patologia , Carcinoma in Situ/fisiopatologia , Carcinoma Ductal Pancreático/patologia , Transdiferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Metaplasia , Camundongos , Camundongos Transgênicos , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo
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