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1.
PLoS One ; 15(10): e0234836, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33064725

RESUMO

The Ras family of proteins is known to play an important role in cellular signal transduction. The oncoprotein Ras is also found to be mutated in ~90% of the pancreatic cancers, of which G12V, G13V, A59G and Q61L are the known hot-spot mutants. These ubiquitous proteins fall in the family of G-proteins, and hence switches between active GTP bound and inactive GDP bound states, which is hindered in most of its oncogenic mutant counterparts. Moreover, Ras being a GTPase has an intrinsic property to hydrolyze GTP to GDP, which is obstructed due to mutations and lends the mutants stuck in constitutively active state leading to oncogenic behavior. In this regard, the present study aims to understand the dynamics involved in the hot-spot mutant A59G-Ras using long 10µs classical MD simulations (5µs for each of the wild-type and mutant systems) and comparing the same with its wild-type counterpart. Advanced analytics using Markov State Model (MSM) based approach has been deployed to comparatively understand the transition path for the wild-type and mutant systems. Roles of crucial residues like Tyr32, Gln61 and Tyr64 have also been established using multivariate PCA analyses. Furthermore, this multivariate PCA analysis also provides crucial features which may be used as reaction coordinates for biased simulations for further studies. The absence of formation of pre-hydrolysis network is also reported for the mutant conformation, using the distance-based analyses (between crucial residues) of the conserved regions. The implications of this study strengthen the hypothesis that the disruption of the pre-hydrolysis network in the mutant A59G ensemble might lead to permanently active oncogenic conformation in the mutant conformers.


Assuntos
Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Mutação , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Humanos , Hidrólise , Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(39): 24258-24268, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32913056

RESUMO

The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as "membrane-distal" and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.


Assuntos
Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases raf/metabolismo , Membrana Celular/metabolismo , Simulação de Dinâmica Molecular
3.
Proc Natl Acad Sci U S A ; 117(33): 20004-20014, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747568

RESUMO

KRAS, NRAS, and BRAF mutations which activate p44/42 mitogen-activated protein kinase (MAPK) signaling are found in half of myeloma patients and contribute to proteasome inhibitor (PI) resistance, but the underlying mechanisms are not fully understood. We established myeloma cell lines expressing wild-type (WT), constitutively active (CA) (G12V/G13D/Q61H), or dominant-negative (DN) (S17N)-KRAS and -NRAS, or BRAF-V600E. Cells expressing CA mutants showed increased proteasome maturation protein (POMP) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) expression. This correlated with an increase in catalytically active proteasome subunit ß (PSMB)-8, PSMB9, and PSMB10, which occurred in an ETS transcription factor-dependent manner. Proteasome chymotrypsin-like, trypsin-like, and caspase-like activities were increased, and this enhanced capacity reduced PI sensitivity, while DN-KRAS and DN-NRAS did the opposite. Pharmacologic RAF or MAPK kinase (MEK) inhibitors decreased proteasome activity, and sensitized myeloma cells to PIs. CA-KRAS, CA-NRAS, and CA-BRAF down-regulated expression of endoplasmic reticulum (ER) stress proteins, and reduced unfolded protein response activation, while DN mutations increased both. Finally, a bortezomib (BTZ)/MEK inhibitor combination showed enhanced activity in vivo specifically in CA-NRAS models. Taken together, the data support the hypothesis that activating MAPK pathway mutations enhance PI resistance by increasing proteasome capacity, and provide a rationale for targeting such patients with PI/RAF or PI/MEK inhibitor combinations. Moreover, they argue these mutations promote myeloma survival by reducing cellular stress, thereby distancing plasma cells from the apoptotic threshold, potentially explaining their high frequency in myeloma.


Assuntos
Estresse do Retículo Endoplasmático , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Mieloma Múltiplo/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , GTP Fosfo-Hidrolases/genética , Humanos , Proteínas de Membrana/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/fisiopatologia , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética
4.
Mol Carcinog ; 59(10): 1147-1158, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32805066

RESUMO

Acquired resistance is a barrier to cetuximab efficacy in patients with head and neck squamous cell carcinoma (HNSCC). Secreted phosphoprotein 1 (SPP1) is involved in various biological processes, including immune responses, cancer progression, and prognosis in many cancers, while little is known in HNSCC. Bioinformatics methods were used to identify candidate genes and further in vivo and in vitro experiments were performed to examine and validate the function of SPP1. We found that SPP1 was upregulated and has been found to have an oncogenic role in HNSCC. We further confirmed that overexpression of SPP1 affected proliferation, migration, invasion, and survival, and inhibited apoptosis, whereas silencing of SPP1 yielded opposite results to those of SPP1 overexpression. In addition, activation of the KRAS/MEK pathway contributed to the SPP1-induced malignant progression of HNSCC and resistance to cetuximab. Furthermore, SPP1 knockdown or an MEK inhibitor overcame this cetuximab-resistance pattern. Taken together, our findings for the first time identify the role of SPP1 in tumor promotion, prognostic prediction, and potential therapeutic targeting, as well as resistance to cetuximab in HNSCC.


Assuntos
Biomarcadores Tumorais/metabolismo , Cetuximab/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias de Cabeça e Pescoço/patologia , MAP Quinase Quinase 1/metabolismo , Osteopontina/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Antineoplásicos Imunológicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , MAP Quinase Quinase 1/genética , Camundongos , Camundongos Nus , Invasividade Neoplásica , Osteopontina/genética , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Anticancer Res ; 40(8): 4687-4694, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727793

RESUMO

BACKGROUND/AIM: The Japanese apricot "Prunus mume" is a traditional Japanese medicine. MK615, a compound extract from Prunus mume has been reported to have anti-tumor effects. Herein, we used 3D floating (3DF) culture to evaluate the anticancer effects of MK615 against human colorectal cancer (CRC) cells that contain mutant (mt) KRAS. MATERIALS AND METHODS: HKe3 cells exogenously expressing mtKRAS (HKe3-mtKRAS) were treated with MK615 in 3DF cultures. The protein levels of hypoxia-inducible factor 1 (HIF-1) and E-cadherin were quantified by western blotting. RESULTS: MtKRAS enhanced hypoxia tolerance via up-regulation of HIF-1. The expression of HIF-1 protein was suppressed by constitutive overexpression of E-cadherin in CRC HCT116 spheroids. MK615 increased the expression of E-cadherin and decreased the expression of HIF-1 in HKe3-mtKRAS. These results suggest that MK615 suppresses hypoxia tolerance by up-regulation of E-cadherin in CRC cells with mtKRAS. CONCLUSION: MK615 exhibits properties useful for the potential treatment of CRC patients with mtKRAS.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Hipóxia Celular/fisiologia , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Regulação para Cima/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prunus/química , Ativação Transcricional/efeitos dos fármacos
6.
Cancer Treat Rev ; 89: 102070, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32711246

RESUMO

RAS is the most frequently mutated oncogene in human cancers, with mutations in about 30% of all cancers. RAS exists in three different isoforms (K-RAS, H-RAS and N-RAS) with high sequence homology. K-RAS is the most commonly mutated RAS isoform. The Ras protein is a membrane bound protein with inherent GTPase activity and is activated by numerous extracellular stimuli, cycling between an inactive (GDP-bound) and active (GTP-bound) form. When bound to GTP, it is switched "on" and activates intracellular signaling pathways, critical for cell proliferation and angiogenesis. Mutated RAS is constitutively activated and persistently turned "on" thereby enhancing downstream signaling and leading to tumorigenesis. Various attempts to inhibit Kras in the past were unsuccessful. Recently, several small molecules (AMG510, MRTX849, JNJ-74699157, and LY3499446) have been developed to specifically target K-RAS G12C. Additionally, various other approaches including, SHP2, SOS1 and eIF4 inhibition, have been utilized to abrogate tumor growth in K-RAS mutant cells, resulting in a renewed interest in this pathway. In this review article, we provide an overview on the role of K-RAS in tumorigenesis, past approaches to inhibiting Kras, and current and future prospects for targeting Kras.


Assuntos
Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Humanos , Terapia de Alvo Molecular , Mutação , Neoplasias/enzimologia , Neoplasias/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais
7.
Nat Commun ; 11(1): 3701, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709883

RESUMO

Despite its importance in human cancers, including colorectal cancers (CRC), oncogenic KRAS has been extremely challenging to target therapeutically. To identify potential vulnerabilities in KRAS-mutated CRC, we characterize the impact of oncogenic KRAS on the cell surface of intestinal epithelial cells. Here we show that oncogenic KRAS alters the expression of a myriad of cell-surface proteins implicated in diverse biological functions, and identify many potential surface-accessible therapeutic targets. Cell surface-based loss-of-function screens reveal that ATP7A, a copper-exporter upregulated by mutant KRAS, is essential for neoplastic growth. ATP7A is upregulated at the surface of KRAS-mutated CRC, and protects cells from excess copper-ion toxicity. We find that KRAS-mutated cells acquire copper via a non-canonical mechanism involving macropinocytosis, which appears to be required to support their growth. Together, these results indicate that copper bioavailability is a KRAS-selective vulnerability that could be exploited for the treatment of KRAS-mutated neoplasms.


Assuntos
Neoplasias Colorretais/metabolismo , Cobre/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Disponibilidade Biológica , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , ATPases Transportadoras de Cobre/metabolismo , Feminino , Humanos , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Mutação
8.
Nat Commun ; 11(1): 3018, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541668

RESUMO

Chronic pancreatitis represents a risk factor for the development of pancreatic cancer. We find that heterozygous loss of histone H2A lysine 119 deubiquitinase BAP1 (BRCA1 Associated Protein-1) associates with a history of chronic pancreatitis and occurs in 25% of pancreatic ductal adenocarcinomas and 40% of acinar cell carcinomas. Deletion or heterozygous loss of Bap1 in murine pancreata causes genomic instability, tissue damage, and pancreatitis with full penetrance. Concomitant expression of KrasG12D leads to predominantly intraductal papillary mucinous neoplasms and mucinous cystic neoplasms, while pancreatic intraepithelial neoplasias are rarely detected. These lesions progress to metastatic pancreatic cancer with high frequency. Lesions with histological features mimicking Acinar Cell Carcinomas are also observed in some tumors. Heterozygous mice also develop pancreatic cancer suggesting a haploinsufficient tumor suppressor role for BAP1. Mechanistically, BAP1 regulates genomic stability, in a catalytic independent manner, and its loss confers sensitivity to irradiation and platinum-based chemotherapy in pancreatic cancer.


Assuntos
Neoplasias Pancreáticas/metabolismo , Pancreatite Crônica/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Estudos de Coortes , Regulação Neoplásica da Expressão Gênica , Haploinsuficiência , Humanos , Camundongos , Neoplasias Pancreáticas/genética , Pancreatite Crônica/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética
9.
J Med Chem ; 63(13): 6677-6678, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32568546

RESUMO

KRAS, one of the most prevalent oncogenes and sought-after anticancer targets, has eluded chemists for decades until an irreversible covalent strategy targeting a specific mutation (G12C) paved the way for the first KRAS inhibitors to reach the clinic. MRTX849 is one such clinical candidate with promising initial results in patients harboring the mutation. The impressive optimization story of MRTX849 highlights challenges and solutions in the development of covalent drugs, including the use of an α-fluoroacrylamide electrophile.


Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Humanos , Terapia de Alvo Molecular , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
10.
Nat Commun ; 11(1): 2817, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499547

RESUMO

Both KRAS and EGFR are essential mediators of pancreatic cancer development and interact with Argonaute 2 (AGO2) to perturb its function. Here, in a mouse model of mutant KRAS-driven pancreatic cancer, loss of AGO2 allows precursor lesion (PanIN) formation yet prevents progression to pancreatic ductal adenocarcinoma (PDAC). Precursor lesions with AGO2 ablation undergo oncogene-induced senescence with altered microRNA expression and EGFR/RAS signaling, bypassed by loss of p53. In mouse and human pancreatic tissues, PDAC progression is associated with increased plasma membrane localization of RAS/AGO2. Furthermore, phosphorylation of AGO2Y393 disrupts both the wild-type and oncogenic KRAS-AGO2 interaction, albeit under different conditions. ARS-1620 (G12C-specific inhibitor) disrupts the KRASG12C-AGO2 interaction, suggesting that the interaction is targetable. Altogether, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR-RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression.


Assuntos
Proteínas Argonauta/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Alelos , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Senescência Celular , Progressão da Doença , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Neoplasias Pancreáticas/patologia , Fosforilação , Ligação Proteica , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
11.
Nat Chem Biol ; 16(9): 979-987, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32483379

RESUMO

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Animais , Antineoplásicos/química , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Cristalografia por Raios X , Cisteína/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Peptidilprolil Isomerase de Interação com NIMA/química , Peptidilprolil Isomerase de Interação com NIMA/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
12.
Proc Natl Acad Sci U S A ; 117(23): 13012-13022, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32444490

RESUMO

Metastatic colorectal cancer (mCRC) patients have poor overall survival despite using irinotecan- or oxaliplatin-based chemotherapy combined with anti-EGFR (epidermal growth factor receptor) drugs, especially those with the oncogene mutation of KRAS Metformin has been reported as a potentially novel antitumor agent in many experiments, but its therapeutic activity is discrepant and controversial so far. Inspiringly, the median survival time for KRAS-mutation mCRC patients with diabetes on metformin is 37.8 mo longer than those treated with other hypoglycemic drugs in combination with standard systemic therapy. In contrast, metformin could not improve the survival of mCRC patients with wild-type KRAS Interestingly, metformin is preferentially accumulated in KRAS-mutation mCRC cells, but not wild-type ones, in both primary cell cultures and patient-derived xenografts, which is in agreement with its tremendous effect in KRAS-mutation mCRC. Mechanistically, the mutated KRAS oncoprotein hypermethylates and silences the expression of multidrug and toxic compound extrusion 1 (MATE1), a specific pump that expels metformin from the tumor cells by up-regulating DNA methyltransferase 1 (DNMT1). Our findings provide evidence that KRAS-mutation mCRC patients benefit from metformin treatment and targeting MATE1 may provide a strategy to improve the anticancer response of metformin.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Metformina/farmacologia , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Metformina/uso terapêutico , Camundongos , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Gene ; 751: 144706, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32387386

RESUMO

Skeletal muscle, the most abundant and plasticity tissue in mammals, is essential for various functions such as movement, breathing, maintaining posture and metabolism. Myogenesis is a complex and precise process, which is regulated by the sequential expression of multiple transcription factors, and accumulating evidence have confirmed that multiple lncRNAs are involved in muscle development as the important transcriptional regulator. In this study, a novel lncRNA, named lnc403 was obtained, with a full-length 2689 bp, which had poor coding ability and was mainly expressed in the nucleus of myoblasts and myotubes. The expression of lnc403 was significantly different in the proliferation and differentiation stages of muscle cells. Then we successfully constructed lnc403 loss/gain-function cell models by transfecting silnc403 and pCDNA3.1-EGFP-lnc403 into satellite cells, respectively; and found that lnc403 inhibited skeletal muscle satellite cell differentiation but had no significant effect on cell proliferation, either in the case of lnc403 knockdown or overexpression. In order to further screen the target factors regulated by lncRNA in the process of myogenic differentiation, the RNA-pull down, mass spectrometry and bioinformatics analysis were performed. The results showed that lnc403 negatively regulated the expression of the adjacent gene Myf6 and positively regulated interaction proteins KRAS expression. The above results indicate that lnc403 affects skeletal muscle cell differentiation by affecting the expression of nearby genes and interacting proteins, implying lnc403 might participate in the bovine myoblasts differentiation through multi-pathway network regulation mode. This study provides a new perspective for further understanding of the regulation mechanism of lncRNAs on bovine myogenic process.


Assuntos
Desenvolvimento Muscular/genética , Mioblastos Esqueléticos/metabolismo , Fatores de Regulação Miogênica/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Longo não Codificante/metabolismo , Animais , Bovinos , Proliferação de Células , Células Cultivadas , Regulação da Expressão Gênica , Fatores de Regulação Miogênica/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células Satélites de Músculo Esquelético/citologia
14.
Proc Natl Acad Sci U S A ; 117(22): 12101-12108, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414921

RESUMO

Membrane anchoring of farnesylated KRAS is critical for activation of RAF kinases, yet our understanding of how these proteins interact on the membrane is limited to isolated domains. The RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF engage KRAS and the plasma membrane, unleashing the kinase domain from autoinhibition. Due to experimental challenges, structural insight into this tripartite KRAS:RBD-CRD:membrane complex has relied on molecular dynamics simulations. Here, we report NMR studies of the KRAS:CRAF RBD-CRD complex. We found that the nucleotide-dependent KRAS-RBD interaction results in transient electrostatic interactions between KRAS and CRD, and we mapped the membrane interfaces of the CRD, RBD-CRD, and the KRAS:RBD-CRD complex. RBD-CRD exhibits dynamic interactions with the membrane through the canonical CRD lipid-binding site (CRD ß7-8), as well as an alternative interface comprising ß6 and the C terminus of CRD and ß2 of RBD. Upon complex formation with KRAS, two distinct states were observed by NMR: State A was stabilized by membrane association of CRD ß7-8 and KRAS α4-α5 while state B involved the C terminus of CRD, ß3-5 of RBD, and part of KRAS α5. Notably, α4-α5, which has been proposed to mediate KRAS dimerization, is accessible only in state B. A cancer-associated mutation on the state B membrane interface of CRAF RBD (E125K) stabilized state B and enhanced kinase activity and cellular MAPK signaling. These studies revealed a dynamic picture of the assembly of the KRAS-CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD-CRD, and the membrane.


Assuntos
Membrana Celular/metabolismo , Cisteína/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sítios de Ligação , Cisteína/química , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética
15.
Mol Carcinog ; 59(8): 886-896, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32291806

RESUMO

Inhibiting the disease progression in KRAS-driven cancers after diagnosis has been a difficult task for clinicians to manage due to the lack of effective intervention/preventive therapies. KRAS-driven cancers depend on sustained KRAS signaling. Although developing inhibitors of KRAS signaling has proven difficult in the past, the quest for identifying newer agents has not stopped. Based on studies showing terpenoids as modulators of KRAS-regulated downstream molecular pathways, we asked if this chemical family has an affinity of inhibiting KRAS protein activity. Using crystal structure as a bait in silico, we identified 20 terpenoids for their KRAS protein-binding affinity. We next carried out biological validation of in silico data by employing in situ, in vitro, patient-derived explant ex vivo, and KPC transgenic mouse models. In this report, we provide a comprehensive analysis of a lup-20(29)-en-3b-ol (lupeol) as a KRAS inhibitor. Using nucleotide exchange, isothermal titration calorimetry, differential scanning fluorimetry, and immunoprecipitation assays, we show that lupeol has the potential to reduce the guanosine diphosphate/guanosine triphosphate exchange of KRAS protein including mutant KRASG12V . Lupeol treatment inhibited the KRAS activation in KRAS-activated cell models (NIH-panel, colorectal, lung, and pancreatic intraepithelial neoplasia) and patient tumor explants ex vivo. Lupeol reduced the three-dimensional growth of KRAS-activated cells. The pharmacokinetic analysis showed the bioavailability of lupeol after consumption via oral and intraperitoneal routes in animals. Tested under prevention settings, the lupeol consumption inhibited the development of pancreatic intraepithelial neoplasia in LSL-KRASG12D/Pdx-cre mice (pancreatic ductal adenocarcinoma progression model). These data suggest that the selected members of the triterpene family (such as lupeol) could be exploited as clinical agents for preventing the disease progression in KRAS-driven cancers which however warrants further investigation.


Assuntos
Anti-Inflamatórios/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Modelos Animais de Doenças , Neoplasias Pancreáticas/tratamento farmacológico , Triterpenos Pentacíclicos/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Apoptose , Proliferação de Células , Transformação Celular Neoplásica/patologia , Progressão da Doença , Feminino , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Nat Commun ; 11(1): 1659, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32246016

RESUMO

Oncogenic Ras mutations occur in various leukemias. It was unclear if, besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation-related effect of KRAS contributes to the disease. Here, we identify a functional link between oncogenic KrasG12D and NLRP3 inflammasome activation in murine and human cells. Mice expressing active KrasG12D in the hematopoietic system developed myeloproliferation and cytopenia, which is reversed in KrasG12D mice lacking NLRP3 in the hematopoietic system. Therapeutic IL-1-receptor blockade or NLRP3-inhibition reduces myeloproliferation and improves hematopoiesis. Mechanistically, KrasG12D-RAC1 activation induces reactive oxygen species (ROS) production causing NLRP3 inflammasome-activation. In agreement with our observations in mice, patient-derived myeloid leukemia cells exhibit KRAS/RAC1/ROS/NLRP3/IL-1ß axis activity. Our findings indicate that oncogenic KRAS not only act via its canonical oncogenic driver function, but also enhances the activation of the pro-inflammatory RAC1/ROS/NLRP3/IL-1ß axis. This paves the way for a therapeutic approach based on immune modulation via NLRP3 blockade in KRAS-mutant myeloid malignancies.


Assuntos
Inflamassomos/imunologia , Transtornos Mieloproliferativos/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Proliferação de Células , Expressão Gênica , Hematopoese , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Leucemia Mieloide/etiologia , Leucemia Mieloide/genética , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Células Mieloides/metabolismo , Proteínas NLR/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
17.
Oncogene ; 39(20): 3997-4013, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32242147

RESUMO

Pancreatic cancer is one of the world's leading causes of cancer-related death. Activation of STAT3 has been reported as a major contributor in pancreatic cancer tumorigenesis and chemoresistance. However, treatment of advanced pancreatic cancer patients with STAT3 inhibitors often meets drug resistance and heterogeneous response. We found that EGFR activation is a main cause for resistance to STAT3 inhibitors in pancreatic cancer cells, regardless of KRAS mutation status. Mechanistically, inhibition of STAT3 promotes STAT1- and STAT4-mediated TGF-α expression, leading to activation of the EGFR pathway. Combined treatment of pancreatic cancer cells with EGFR and STAT3 inhibitors persistently blocks EGFR and STAT3 signaling, and exerts synergistic antitumor activity both in vitro and in vivo, with or without KRAS mutation. Our results indicate that reciprocal cross-talk between STAT3 and EGFR pathways is a key molecular mechanism leading to resistance in pancreatic cancer cells. Furthermore, the study shows that combined inhibition of both EGFR and STAT3 might overcome drug resistance encountered during treatment with single agent alone. This study suggests an improved therapeutic strategy, through combined treatment with STAT3 and EGFR inhibitors, for pancreatic cancer patients.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Camundongos , Mutação , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Fator de Transcrição STAT3/genética
18.
Cancer Genomics Proteomics ; 17(3): 277-290, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32345669

RESUMO

BACKGROUND/AIM: Early-stage gastric cancer has a high risk of recurrence, despite trimodality therapy with surgery, chemotherapy and radiation. To improve patient selection for adjuvant chemoradiotherapy, we evaluated the prognostic significance of immunohistochemical and genetic biomarkers in patients with resected gastric adenocarcinoma. PATIENTS AND METHODS: Tumors from 119 patients were subjected to immunohistochemistry for 12 protein biomarkers, as well as next-generation sequencing. Clinical and biomarker data were available for 91 patients. RESULTS: EBV-positive tumors and tumors with mutations had higher intratumoral CD8 tumor-infiltrating lymphocyte density (p=0.009 and p=0.017, respectively). PIK3CA mutations were correlated with VEGFA overexpression (p=0.042), while KRAS mutations and HER2 expression were mutually exclusive (p=0.036). PTEN expression univariately confirmed longer overall survival (HR=0.27; p=0.046), while there was a trend between the presence of KRAS mutations and inferior disease-free and overall survival. CONCLUSION: PTEN protein expression and KRAS mutations may predict disease outcome in early-stage gastric cancer. These results need to be further validated in larger cohorts.


Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/terapia , Biomarcadores Tumorais/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/terapia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Biomarcadores Tumorais/genética , Quimiorradioterapia Adjuvante , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Estudos Retrospectivos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Taxa de Sobrevida
19.
Oncogene ; 39(21): 4257-4270, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32291414

RESUMO

The mitochondrial HSP70 chaperone mortalin (HSPA9/GRP75) is often upregulated and mislocalized in MEK/ERK-deregulated tumors. Here, we show that mortalin depletion can selectively induce death of immortalized normal fibroblasts IMR90E1A when combined with K-RasG12V expression, but not with wild-type K-Ras expression, and that K-RasG12V-driven MEK/ERK activity is necessary for this lethality. This cell death was attenuated by knockdown or inhibition of adenine nucleotide translocase (ANT), cyclophilin D (CypD), or mitochondrial Ca2+ uniporter (MCU), which implicates a mitochondria-originated death mechanism. Indeed, mortalin depletion increased mitochondrial membrane permeability and induced cell death in KRAS-mutated human pancreatic ductal adenocarcinoma (PDAC) and colon cancer lines, which were attenuated by knockdown or inhibition of ANT, CypD, or MCU, and occurred independently of TP53 and p21CIP1. Intriguingly, JG-98, an advanced MKT-077 derivative, phenocopied the lethal effects of mortalin depletion in K-RasG12V-expressing IMR90E1A and KRAS-mutated tumor cell lines in vitro. Moreover, JG-231, a JG-98 analog with improved microsomal stability effectively suppressed the xenograft of MIA PaCa-2, a K-RasG12C-expressing human PDAC line, in athymic nude mice. These data demonstrate that oncogenic KRAS activity sensitizes cells to the effects of mortalin depletion, suggesting that mortalin has potential as a selective therapeutic target for KRAS-mutated tumors.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Antineoplásicos/farmacologia , Morte Celular , Feminino , Células HCT116 , Proteínas de Choque Térmico HSP70/genética , Humanos , Camundongos , Camundongos Nus , Membranas Mitocondriais/patologia , Proteínas Mitocondriais/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Permeabilidade , Proteínas Proto-Oncogênicas p21(ras)/genética , Ensaios Antitumorais Modelo de Xenoenxerto
20.
J Vis Exp ; (155)2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-32009649

RESUMO

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is processed by farnesylation and carboxymethylation due to the presence of a 'CAAX' box motif at the C-terminus. An engineered baculovirus system was used to express farnesylated and carboxymethylated KRAS4b in insect cells and has been described previously. Here, we describe the detailed, practical purification and biochemical characterization of the protein. Specifically, affinity and ion exchange chromatography were used to purify the protein to homogeneity. Intact and native mass spectrometry was used to validate the correct modification of KRAS4b and to verify nucleotide binding. Finally, membrane association of farnesylated and carboxymethylated KRAS4b to liposomes was measured using surface plasmon resonance spectroscopy.


Assuntos
Prenilação de Proteína , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Baculoviridae , Linhagem Celular , Cromatografia por Troca Iônica , Lipossomos , Espectrometria de Massas , Metilação , Reprodutibilidade dos Testes , Ressonância de Plasmônio de Superfície
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