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1.
J Immunol ; 199(12): 4091-4102, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29127145

RESUMO

T cell expression of TIM-3 following Ag encounter has been associated with a continuum of functional states ranging from effector memory T cells to exhaustion. We have designed an in vitro culture system to specifically address the impact of anti-TIM-3/TIM-3 engagement on human Ag-specific CD8 T cells during a normal response to Ag and found that anti-TIM-3 treatment enhances T cell function. In our in vitro T cell culture system, MART1-specific CD8 T cells were expanded from healthy donors using artificial APCs. To ensure that the T cells were the only source of TIM-3, cells were rechallenged with peptide-loaded artificial APCs in the presence of anti-TIM-3 Ab. In these conditions, anti-TIM-3 treatment promotes generation of effector T cells as shown by acquisition of an activated phenotype, increased cytokine production, enhanced proliferation, and a transcription program associated with T cell differentiation. Activation of mTORC1 has been previously demonstrated to enhance CD8 T cell effector function and differentiation. Anti-TIM-3 drives CD8 T cell differentiation through activation of the mTORC1 as evidenced by increased levels of phosphorylated S6 protein and rhebl1 transcript. Altogether these findings suggest that anti-TIM-3, together with Ag, drives differentiation in favor of effector T cells via the activation of mTOR pathway. To our knowledge, this is the first report demonstrating that TIM-3 engagement during Ag stimulation directly influences T cell differentiation through mTORC1.


Assuntos
Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Memória Imunológica/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Anticorpos Monoclonais/farmacologia , Divisão Celular , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Humanos , Ativação Linfocitária , Linfocinas/biossíntese , Linfocinas/genética , Antígeno MART-1/imunologia , Fosforilação , Processamento de Proteína Pós-Traducional , Especificidade do Receptor de Antígeno de Linfócitos T , Proteínas ras/biossíntese , Proteínas ras/genética
2.
Int J Cancer ; 141(6): 1201-1214, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28580661

RESUMO

Increasing reports show noninflammation underlying HCC, challenging our understanding of the roles of the immune system in hepatocarcinogenesis. By exploring a mouse model of hepatic tumor induced by hepatocyte-specific expression of the Hras12V oncogene without obvious inflammation, we found that the proportion of B cells, but not T cells, progressively and significantly decreased in 3, 5-month-old transgenic mice (Tg) compared with non-transgenic mice. Notably, the proportions of total and activated B and T cells all significantly decreased in 9-month-old Tg with multiple massive hepatic tumors. Together with the decreased B cell proportion, serum IgG1/2 also significantly decreased in 5, 9-month-old Tg. Interestingly, homozygous Tg showed significantly higher B cell proportion and IgG2 levels, accompanied by significantly lower incidences of liver nodules but not adenomas and carcinomas compared with heterozygous Tg. Treatment of Tg with PCI-32765, a potent Bruton's tyrosine kinase (BTK) inhibitor for suppressing B cell proliferation and activation, significantly decreased the B cell proportion and IgG2 levels, accompanied by a significantly higher incidence of liver nodules, but had no effects on adenoma and carcinoma. Treatment of Tg with insulin-like growth factor 1 (IGF-1) significantly increased the B cell proportion and IgG2 levels, accompanied by a significantly lower incidence of liver nodules and carcinoma, but had no effects on adenoma. Conclusively, B cells and IgG2 may play important roles in suppressing hepatic tumorigenesis, but not development. In addition, hepatocyte-specific expression of the ras oncogene may play roles in suppressing B cells, while developed hepatic tumors suppress both B and T cells.


Assuntos
Linfócitos B/imunologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/imunologia , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/imunologia , Imunoglobulina G/sangue , Fator de Crescimento Insulin-Like I/imunologia , Fator de Crescimento Insulin-Like I/farmacologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Oncogênicas/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteínas ras/biossíntese , Proteínas ras/genética
3.
Mol Neurobiol ; 54(1): 779-787, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27021020

RESUMO

In this study, we investigated the influence of elevated RAS expression on the growth of meningioma in vivo and in vitro. The IOMM-LEE cells, representing a cell line derived from malignant meningioma, were divided into blank control group (cells without any drug treatment), negative control group (cells treated with an equal volume of normal saline to replace drug), and farnesyl thiosalicylic acid (FTS)-treated group (cells treated with FTS). Methyl-thiazole-tetrazolium bromide (MTT) assay and flow cytometer (with cells after FTS (75 µmol/L) treatment for 48 h) were utilized to determine the proliferation and apoptosis, respectively, of IOMM-LEE cells after RAS inhibition. Western blot analysis was used for semi-quantitative analysis of p-ERK and p-AKT levels. Animal model of human meningioma was established with sub-renal capsule transplantation, and mice were divided into two groups: experimental group (50 mg/kg group, 75 mg/kg group, and 100 mg/kg, hypodermic injection with FTS) and control group. Proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry (IHC). Western blot analysis was used for detecting ERK and AKT signal pathway. The proliferation of IOMM-LEE cells decreased dramatically and apoptosis rate increased significantly in FTS-treated group compared to blank control group and negative control group (all P < 0.05). At FTS concentration of 75 µmol/L, the apoptosis rate of IOMM-LEE cells reduced significantly over time (P < 0.05). Cell cycle analysis showed that IOMM-LEE cells exhibited G1-arrest in the FTS-treated group, compared to no cell-cycle arrest in blank control group and the negative control group (P < 0.05). Further, significantly decreased ERK and AKT phosphorylation levels were detected in IOMM-Lee cells after FTS (75 µmol/L) treatment for 48 h, compared to blank control group and negative control group (P < 0.05). The results in vivo experiments showed that after FTS treatment, tumor volume, PCNA LI, and the levels of p-ERK and p-Akt decreased significantly in 75 mg/kg group and 100 mg/kg group when compared with the control group and 50 mg/kg group (all P < 0.05). Our findings provide strong evidence that RAS protein is highly expressed in meningioma cells, and the RAS activity is inhibited by downregulating ERK and AKT signal pathway, which may further inhibit the growth of meningioma.


Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Neoplasias Meníngeas/metabolismo , Meningioma/metabolismo , Proteínas ras/antagonistas & inibidores , Proteínas ras/biossíntese , Adulto , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Método Duplo-Cego , Farneseno Álcool/análogos & derivados , Farneseno Álcool/farmacologia , Farneseno Álcool/uso terapêutico , Feminino , Humanos , Masculino , Neoplasias Meníngeas/tratamento farmacológico , Meningioma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Distribuição Aleatória , Salicilatos/farmacologia , Salicilatos/uso terapêutico , Células Tumorais Cultivadas
4.
Oncotarget ; 7(28): 43868-43893, 2016 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-27270312

RESUMO

Surgical resection supplemented with adjuvant chemotherapy is the current preferred treatment for Stage III colorectal cancer (CRC). However, as many as 48% of patients who undergo curative resection eventually suffer from incurable distant recurrence. To investigate the molecular mechanisms involved in Stage III CRC post-surgical distant recurrence, we identified a total of 146 differentially expressed proteins (DEPs) associated with distant recurrence in Stage III CRC using TMT-based quantitative mass spectrometry. Among these DEPs, the altered expressions of R-Ras and Transgelin were then validated in 192 individual specimens using immunohistochemistry (IHC). Furthermore, Kaplan-Meier analysis revealed that the levels of R-Ras and Transgelin were significantly associated with 5-year overall survival (OS) and disease-free survival (DFS), and multivariate Cox-regression analyses revealed that R-Ras and Transgelin were independent prognostic factors for OS and DFS, respectively. In conclusion, this study identified potential biochemical players involved in distant recurrence and indicates that R-Ras and Transgelin are potential post-surgical prognostic biomarkers for Stage III CRC. This proteomics data have been submitted to Proteome Xchange under accession number PXD002903.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Colorretais/patologia , Proteínas dos Microfilamentos/biossíntese , Proteínas Musculares/biossíntese , Recidiva Local de Neoplasia/patologia , Proteínas ras/biossíntese , Adulto , Idoso , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Proteínas dos Microfilamentos/análise , Pessoa de Meia-Idade , Proteínas Musculares/análise , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/mortalidade , Prognóstico , Modelos de Riscos Proporcionais , Proteômica/métodos , Proteínas ras/análise
5.
Oncotarget ; 7(30): 47720-47737, 2016 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-27351226

RESUMO

The link between oncogenic RAS expression and the acquisition of the invasive phenotype has been attributed to alterations in cellular activities that control degradation of the extracellular matrix. Oncogenic RAS-mediated upregulation of matrix metalloproteinase 2 (MMP-2), MMP-9 and urokinase-type plasminogen activator (uPA) is critical for invasion through the basement membrane and extracellular matrix. The uPA converts cell surface-bound plasminogen to plasmin, a process that is regulated by the binding of plasminogen to specific receptors on the cell surface, however, the identity of the plasminogen receptors that function in this capacity is unclear. We have observed that transformation of cancer cells with oncogenic forms of RAS increases plasmin proteolytic activity by 2- to 4-fold concomitant with a 3-fold increase in cell invasion. Plasminogen receptor profiling revealed RAS-dependent increases in both S100A10 and cytokeratin 8. Oncogenic RAS expression increased S100A10 gene expression which resulted in an increase in S100A10 protein levels. Analysis with the RAS effector-loop mutants that interact specifically with Raf, Ral GDS pathways highlighted the importance of the RalGDS pathways in the regulation of S100A10 gene expression. Depletion of S100A10 from RAS-transformed cells resulted in a loss of both cellular plasmin generation and invasiveness. These results strongly suggest that increases in cell surface levels of S100A10, by oncogenic RAS, plays a critical role in RAS-stimulated plasmin generation, and subsequently, in the invasiveness of oncogenic RAS expressing cancer cells.


Assuntos
Anexina A2/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas S100/metabolismo , Proteínas ras/genética , Células A549 , Animais , Anexina A2/genética , Linhagem Celular Tumoral , Ativação Enzimática , Fibrinolisina/genética , Fibrinolisina/metabolismo , Genes ras , Células HCT116 , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Células NIH 3T3 , Peptídeo Hidrolases/genética , Proteínas S100/genética , Transfecção , Proteínas ras/biossíntese
6.
Cancer Biol Ther ; 17(1): 91-103, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26574927

RESUMO

We previously investigated MET and its oncogenic mutants relevant to lung cancer in C. elegans. The inactive orthlogues of the receptor tyrosine kinase Eph and MET, namely vab-1 and RB2088 respectively, the temperature sensitive constitutively active form of KRAS, SD551 (let-60; GA89) and the inactive c-CBL equivalent mutants in sli-1 (PS2728, PS1258, and MT13032) when subjected to chronic exposure of nicotine resulted in a significant loss in egg-laying capacity and fertility. While the vab-1 mutant revealed increased circular motion in response to nicotine, the other mutant strains failed to show any effect. Overall locomotion speed increased with increasing nicotine concentration in all tested mutant strains except in the vab-1 mutants. Moreover, chronic nicotine exposure, in general, upregulated kinases and phosphatases. Taken together, these studies provide evidence in support of C. elegans as initial in vivo model to study nicotine and its effects on oncogenic mutations identified in humans.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Neoplasias/genética , Nicotina/toxicidade , Receptores Proteína Tirosina Quinases/genética , Sequência de Aminoácidos/genética , Animais , Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Ciclo Celular/biossíntese , Fertilidade/genética , Humanos , Locomoção/efeitos dos fármacos , Locomoção/genética , Mutação , Neoplasias/induzido quimicamente , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-met/biossíntese , Proteínas Proto-Oncogênicas c-met/genética , Proteínas ras/biossíntese
7.
Tumour Biol ; 37(4): 5097-105, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26546438

RESUMO

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancer worldwide. However, the mechanism underlying the HCC development remains unclear. Ras-related associated with diabetes (RRAD) is a small Ras-related GTPase which has been implicated in metabolic disease and several types of cancer, yet its functions in HCC remain unknown. A tissue microarray constructed by 90 paired HCC tissues and adjacent non-cancerous liver tissues was used to examine the protein levels of RRAD, and the messenger RNA (mRNA) expression of RRAD was also detected in a subset of this cohort. The prognostic significance of RRAD was estimated by the Kaplan-Meier analysis and Cox regression. The glucose utilization assay and lactate production assay were performed to measure the role of RRAD in HCC glycolysis. The effect of RRAD in HCC invasion and metastasis was analyzed by transwell assays. Our results suggested that the expression of RRAD was downregulated in HCC tissues compared to the adjacent non-tumorous liver tissues both in mRNA and protein levels and lower RRAD expression served as an independent prognostic indicator for the survival of HCC patients. Moreover, RRAD inhibited hepatoma cell aerobic glycolysis by negatively regulating the expression of glucose transporter 1 (GLUT1) and hexokinase II (HK-II). In addition, RRAD inhibition dramatically increased hepatoma cell invasion and metastasis. In conclusion, our study revealed that RRAD expression was decreased in HCC tumor tissues and predicted poor clinical outcome for HCC patients and played an important role in regulating aerobic glycolysis and cell invasion and metastasis and may represent potential targets for improving the treatment of HCC.


Assuntos
Biomarcadores Tumorais/biossíntese , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas ras/biossíntese , Adulto , Aerobiose , Idoso , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Transportador de Glucose Tipo 1/biossíntese , Glicólise/genética , Células Hep G2 , Hexoquinase/biossíntese , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Prognóstico , Análise Serial de Tecidos , Proteínas ras/genética
8.
Mol Med Rep ; 13(2): 1275-80, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26676380

RESUMO

Osteosarcoma is the most common type of primary malignant tumor of the bone. However, mechanisms underlying osteosarcoma cell proliferation are poorly understood. The present study shows that RBEL1, a newly identified Rab-like GTPase, may be a key regulator of osteosarcoma cell proliferation. Knockdown of RBEL1 in osteosarcoma cells resulted in impaired colony formation and cell proliferation. Cell cycle analysis suggested that RBEL1 depletion induced G1-S arrest in osteosarcoma cells. Furthermore, it was demonstrated that retinoblastoma 1 (Rb) was upregulated and activated following RBEL1 knockdown. In addition, Rb inhibitory downstream targets, such as cyclin A2, cyclin D1, c-Myc and cyclin-dependent kinase 2, were downregulated. Rb knockdown reversed RBEL1 depletion-induced tumor suppressive effects. In conclusion, the present results suggest that RBEL1 modulates cell proliferation and G1­S transition by inhibiting Rb in osteosarcoma. These results suggest a potential therapeutic target in osteosarcoma.


Assuntos
Proliferação de Células/genética , Osteossarcoma/genética , Proteína do Retinoblastoma/biossíntese , Proteínas ras/genética , Linhagem Celular Tumoral , Ciclina A2/biossíntese , Ciclina D1/biossíntese , Quinase 2 Dependente de Ciclina/biossíntese , Técnicas de Silenciamento de Genes , Humanos , Osteossarcoma/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteína do Retinoblastoma/genética , Proteínas ras/biossíntese
9.
Biomed Res Int ; 2015: 414250, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26649302

RESUMO

Overexpression of human epidermal growth factor receptor 2 (HER2) drives the biology of 30% of breast cancer cases. As a transducer of HER2 signaling, RAS/RAF/MAPK pathway plays a pivotal role in the development of breast cancer. In this study, we examined the molecular mechanisms underlying the chemopreventive effects of black rice anthocyanins (BRACs) extract and identified their molecular targets in HER2(+) breast cancer cells. Treatment of MDA-MB-453 cells (HER2(+)) with BRACs inhibited cell migration and invasion, suppressed the activation of mitogen-activated protein kinase kinase kinase (RAF), mitogen-activated protein kinase kinase (MEK), and c-Jun N-terminal kinase (JNK), and downregulated the secretion of matrix metalloproteinase 2 (MMP2) and MMP9. BRACs also weakened the interactions of HER2 with RAF, MEK, and JNK proteins, respectively, and decreased the mRNA expression of raf, mek, and jnk. Further, we found combined treatment with BRACs and RAF, MEK, or JNK inhibitors could enhance the antimetastatic activity, compared with that of each treatment. Transient transfection with small interfering RNAs (siRNAs) specific for raf, mek, and jnk inhibited their mRNA expression in MDA-MB-453 cells. Moreover, cotreatment with BRACs and siRNA induces a more remarkable inhibitory effect than that by either substance alone. In summary, our study suggested that BRACs suppress metastasis in breast cancer cells by targeting the RAS/RAF/MAPK pathway.


Assuntos
Antocianinas/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , MAP Quinase Quinase Quinase 1/biossíntese , Quinases raf/biossíntese , Proteínas ras/biossíntese , Antocianinas/química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase Quinase 1/genética , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Invasividade Neoplásica/genética , Metástase Neoplásica , Oryza/química , Receptor ErbB-2/genética , Transdução de Sinais/efeitos dos fármacos , Quinases raf/genética , Proteínas ras/genética
10.
Genet Mol Res ; 14(3): 8810-8, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26345812

RESUMO

The aim of this study was to investigate the expression of miR-21 in esophageal cancer and the impact of miR-21 on apoptosis, invasion, and the expression of target genes in esophageal cancer cells. Fluorescence quantitative polymerase chain reaction analysis was used to detect the expression of miR-21 in human esophageal tissues, adjacent tissues, and an esophageal cancer cell line (TE-13). The antisense miR-21 oligonucleotide was generated commercially using the solid-phase chemical synthesis method. Transient transfection was used to transfect esophageal cancer cells (TE-13 antisense and TE-13 control cells). Flow cytometry and Transwell cell assays were used to detect the apoptosis and invasion of esophageal cancer cells, respectively. The western blot method was used to detect the expression of PTEN, PDCD4, and K-ras proteins. These analyses determined that mir-21 expression significantly increased in esophageal cancer tissues and in TE-13 cells, and that this phenomenon was not associated with staging or lymph node metastasis. The apoptosis rate of TE-13 control cells was lower than that of antisense TE-13 cells indicating an enhanced invasive ability. In tissues adjacent to esophageal cancer and in TE-13 antisense cells, the expression of PTEN and PDCD4 was found to be higher than that in the control group, whereas the expression of K-ras showed the opposite pattern. Together, these results suggest that miR- 21 might be involved in the development and metastasis of esophageal cancer, through interaction with its PDCD4 and K-ras target genes.


Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , MicroRNAs/biossíntese , Idoso , Apoptose/genética , Proteínas Reguladoras de Apoptose/biossíntese , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , PTEN Fosfo-Hidrolase/biossíntese , Proteínas de Ligação a RNA/biossíntese , Transfecção , Proteínas ras/biossíntese
11.
Cancer Res ; 75(17): 3623-35, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26130651

RESUMO

Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy, and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS-MAPK pathway circumvents Shh pathway dependency, drives tumor growth, and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together, these findings reveal a critical role of the RAS-MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors.


Assuntos
Carcinoma Basocelular/genética , Proteínas Hedgehog/genética , Meduloblastoma/genética , Receptores Acoplados a Proteínas-G/biossíntese , Proteínas ras/genética , Anilidas/administração & dosagem , Animais , Carcinoma Basocelular/tratamento farmacológico , Carcinoma Basocelular/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Piridinas/administração & dosagem , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/biossíntese
12.
J Hepatol ; 63(2): 429-36, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25828473

RESUMO

BACKGROUND & AIMS: Overexpression of FoxM1 correlates with poor prognosis in hepatocellular carcinoma (HCC). Moreover, the Ras-signaling pathway is found to be ubiquitously activated in HCC through epigenetic silencing of the Ras-regulators. We investigated the roles of FoxM1 in Ras-driven HCC, and on HCC cells with stem-like features. METHODS: We employed a transgenic mouse model that expresses the oncogenic Ras in the liver. That strain was crossed with a strain that harbor floxed alleles of FoxM1 and the MxCre gene that allows conditional deletion of FoxM1. FoxM1 alleles were deleted after development of HCC, and the effects on the tumors were analyzed. Also, FoxM1 siRNA was used in human HCC cell lines to determine its role in the survival of the HCC cells with stem cell features. RESULTS: Ras-driven tumors overexpress FoxM1. Deletion of FoxM1 inhibits HCC progression. There was increased accumulation of reactive oxygen species (ROS) in the FoxM1 deleted HCC cells. Moreover, FoxM1 deletion caused a disproportionate loss of the CD44+ and EpCAM+ HCC cells in the tumors. We show that FoxM1 directly activates expression of CD44 in human HCC cells. Moreover, the human HCC cells with stem cell features are addicted to FoxM1 for ROS-regulation and survival. CONCLUSION: Our results provide genetic evidence for an essential role of FoxM1 in the progression of Ras-driven HCC. In addition, FoxM1 is required for the expression of CD44 in HCC cells. Moreover, FoxM1 plays a critical role in the survival of the HCC cells with stem cell features by regulating ROS.


Assuntos
Carcinogênese/genética , Carcinoma Hepatocelular/genética , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Células-Tronco/patologia , Proteínas ras/genética , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/biossíntese , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prognóstico , Transdução de Sinais , Células-Tronco/metabolismo , Proteínas ras/biossíntese
13.
BMC Cancer ; 15: 301, 2015 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-25896895

RESUMO

BACKGROUND: The impact of KRAS signaling on cancerous inhibitor of protein phosphatase 2A (CIP2A) expression has not yet been explored. We investigated the impact of KRAS on CIP2A expression in colorectal cancer patients after colorectal liver metastasectomy. METHODS: We examined CIP2A expression by immunohistochemistry (IHC) and used direct sequencing to identify the mutational status of KRAS exon 2 (codon 12 and 13). The association between CIP2A expression, KRAS genotype, clinicopathological parameters and survival were examined by the Kaplan-Meier method and the Cox proportional hazards model. A combination of immunoblotting and proliferation assays were employed to elucidate the role of CIP2A in signal transduction pathways in wild-type KRAS Caco-2 cells. RESULTS: A total of 220 colorectal cancer patients who had undergone colorectal liver metastasectomy were included in the study. The mutant KRAS genotype was associated with CIP2A overexpression. CIP2A expression was an independent prognostic marker in patients with wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy (relative risk = 1.873, P = 0.019). Targeted silencing of CIP2A in Caco-2 cells (wild-type KRAS) led to decreased expression of pERK/ERK and decreased cell proliferation. Overexpression of mutant KRAS G12D in Caco-2 cells led to an increase in CIP2A expression and cell proliferation. In Caco-2 cells with the KRAS G12D, KRAS overexpression preserved the regulation effect of CIP2A in KRAS and abrogated the impact of CIP2A regulation on pERK/ERK and cell proliferation. CIP2A inhibition also increased the efficacy of cetuximab in Caco-2 cells. CONCLUSIONS: CIP2A is an independent prognostic marker in patients with wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy.


Assuntos
Autoantígenos/biossíntese , Biomarcadores Tumorais/biossíntese , Neoplasias Colorretais/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/biossíntese , Metastasectomia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas ras/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Células CACO-2 , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/cirurgia , Feminino , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)
14.
APMIS ; 123(4): 298-304, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25708741

RESUMO

Hyperplastic polyps (HP) of the colon and rectum were previously considered benign. Newer studies have suggested that colorectal HP are different entities. The aim of this study was to reclassify lesions from a 5-year period previously classified as colorectal HP into traditional hyperplastic polyp (THP), sessile serrated lesions (SSL), and other lesions. All patients were confirmed in the Danish National Pathology Database for the occurrence of metachronous polyps/adenomas, colorectal cancer (CRC), and other gastrointestinal malignancies. Molecular pathology of the CRC were characterized and correlated with the index lesion. In total, 591 HP biopsy specimens were obtained from 480 patients. The lesions were reclassified as: 358 THP, 109 SSL, 35 TA, 81 unspecified non-neoplastic lesions, four traditional serrated adenoma, and 4 SSL with cytological dysplasia. Seven patients developed CRC in the follow-up period (1 patient had SSL, 4 had THP, and 2 had unspecified non-neoplastic lesions). Ten patients developed other gastrointestinal malignancies. The patient with SSL as index lesions who developed CRC harbored V600E BRAF mutation in both index lesion and the carcinoma. Sixteen percent of patients with SSL subsequently developed a neoplastic lesion. Further studies are needed to clarify the cancer risk of SSL.


Assuntos
Pólipos do Colo/classificação , Pólipos do Colo/genética , Neoplasias Colorretais/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas/biossíntese , Proteínas ras/biossíntese , Adenoma/genética , Adenoma/patologia , Sequência de Bases , Colo/patologia , Pólipos do Colo/patologia , Neoplasias Colorretais/patologia , Reparo do DNA/genética , Feminino , Humanos , Hiperplasia/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Patologia Molecular , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Reto/patologia , Sistema de Registros , Estudos Retrospectivos , Análise de Sequência de DNA , Proteínas ras/genética
15.
Int J Biochem Cell Biol ; 61: 120-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25701793

RESUMO

Recent evidence has indicated that miRNAs play important roles in carcinogenesis. The identification of dysregulated miRNAs and the target genes they regulate might enhance our understanding of the molecular mechanisms of nasopharyngeal carcinoma (NPC). A microarray analysis was performed to identify dysregulated miRNAs in NPC tissue samples, and protein-coding genes targeted by three or more downregulated miRNAs were selected using miRWalk and used in a pathway enrichment analysis. Nineteen KEGG pathways were selected by DAVID, including the MAPK, focal adhesion, gap junction, ECM-receptor interaction, TGF-beta, and p53 signalling pathways, most of which are involved in NPC carcinogenesis and progression. MiR-143 was significantly downregulated in NPC cell lines and clinical samples. The ectopic expression of miR-143 suppressed NPC cell viability, colony formation, and anchorage-independent growth in vitro, and it inhibited xenograft tumour growth in vivo. Furthermore, KRAS was confirmed as a direct target of miR-143, and silencing KRAS expression suppressed NPC cell viability and proliferation. The miR-143/KRAS pathway provides new insight into the molecular mechanisms that regulate the development and progression of NPC, and it provides novel therapeutic targets for NPC.


Assuntos
MicroRNAs/genética , Neoplasias Nasofaríngeas/genética , Animais , Carcinoma , Estudos de Casos e Controles , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Feminino , Perfilação da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Genes ras , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/metabolismo , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Inclusão em Parafina , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais , Transfecção , Proteínas ras/biossíntese , Proteínas ras/genética
16.
Oncotarget ; 6(6): 3947-62, 2015 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-25714130

RESUMO

Increase in aerobic glycolysis and mitochondrial dysfunction are important biochemical features observed in human cancers. Recent studies suggest oncogenic K-Ras can cause suppression of mitochondrial respiration and up-regulation of glycolytic activity through a yet unknown mechanism. Here we employed proteomic approach and used a K-RasG12V inducible cell system to investigate the impact of oncogenic K-Ras on mitochondria and cell metabolism. Mitochondria isolated from cells before and after K-Ras induction were subjected to protein analysis using stable isotope labeling with amino acids (SILAC) and liquid chromatography coupled with mass spectrometry (LC-MS). 70 mitochondrial proteins with significant expression alteration after K-Ras induction were identified. A majority of these proteins were involved in energy metabolism. Five proteins with significant decrease belong to mitochondrial respiratory chain complex I. NADH dehydrogenase 1 alpha subcomplex assembly factor 1 (NDUFAF1) showed most significant decrease by 50%. Such decrease was validated in primary human pancreatic cancer tissues. Knockdown of NDUFAF1 by siRNA caused mitochondrial respiration deficiency, accumulation of NADH and subsequent increase of glycolytic activity. Our study revealed that oncogenic K-Ras is able to induce significant alterations in mitochondrial protein expression, and identified NDUFAF1 as an important molecule whose low expression contributes to mitochondrial dysfunction induced by K-Ras.


Assuntos
Genes ras , Mitocôndrias/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Proteínas ras/biossíntese , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Proteômica , Ativação Transcricional , Transcriptoma , Proteínas ras/genética
17.
Tumour Biol ; 36(6): 4861-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25631751

RESUMO

Increasingly, in castration-resistant prostate cancer, patients are often treated with docetaxel and the bisphosphonate zoledronic acid concurrently, yet there is still a paucity in the literature regarding the molecular basis of how this drug combination works. The study was performed on the hormone-resistant cell line PC-3. Cells were treated with clinically relevant concentrations of docetaxel and zoledronic acid either as single agents or in sequence and combination. Cell viability and apoptosis were assessed along with the prenylation status of the GTPases Ras and RhoA. Following 1-mM zoledronic acid treatment, inhibition of the prenylation of H-Ras and Rho A was observed along with an increase in the unprenylated form in the cytoplasm. Docetaxel 1 nM and zoledronic acid 1 mM also showed an increase in the unprenylated form of both small GTP-binding proteins in the cytoplasm and a reduction of protein in the membrane fraction. Overall, zoledronic acid followed by docetaxel was the best regimen producing the greatest reduction in cell viability and increase in apoptosis. At the highest concentrations of zoledronic acid and docetaxel, zoledronic acid followed by docetaxel was also the most effective at reducing the prenylation of both H-Ras and RhoA at the membrane. We have demonstrated that clinically achievable concentrations of zoledronic acid and docetaxel cause a reduction in the prenylation of both H-Ras and Rho A and a reduction of protein movement into the membrane. The most effective regimen overall was high-dose zoledronic acid followed by docetaxel, suggesting that this regimen may be of benefit in clinical practice.


Assuntos
Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Proteínas ras/biossíntese , Proteína rhoA de Ligação ao GTP/biossíntese , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Difosfonatos/administração & dosagem , Docetaxel , Regulação Neoplásica da Expressão Gênica , Humanos , Imidazóis/administração & dosagem , Masculino , Neoplasias da Próstata/patologia , Taxoides/administração & dosagem , Ácido Zoledrônico
18.
Cancer Res ; 75(4): 687-97, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25524901

RESUMO

Translation initiation factor eIF4E mediates normal cell proliferation, yet induces tumorigenesis when overexpressed. The mechanisms by which eIF4E directs such distinct biologic outputs remain unknown. We found that mouse mammary morphogenesis during pregnancy and lactation is accompanied by increased cap-binding capability of eIF4E and activation of the eIF4E-dependent translational apparatus, but only subtle oscillations in eIF4E abundance. Using a transgenic mouse model engineered so that lactogenic hormones stimulate a sustained increase in eIF4E abundance in stem/progenitor cells of lactogenic mammary epithelium during successive pregnancy/lactation cycles, eIF4E overexpression increased self-renewal, triggered DNA replication stress, and induced formation of premalignant and malignant lesions. Using complementary in vivo and ex vivo approaches, we found that increasing eIF4E levels rescued cells harboring oncogenic c-Myc or H-RasV12 from DNA replication stress and oncogene-induced replication catastrophe. Our findings indicate that distinct threshold levels of eIF4E govern its biologic output in lactating mammary glands and that eIF4E overexpression in the context of stem/progenitor cell population expansion can initiate malignant transformation by enabling cells to evade DNA damage checkpoints activated by oncogenic stimuli. Maintaining eIF4E levels below its proneoplastic threshold is an important anticancer defense in normal cells, with important implications for understanding pregnancy-associated breast cancer.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Fator de Iniciação 4E em Eucariotos/biossíntese , Glândulas Mamárias Humanas/metabolismo , Animais , Neoplasias da Mama/patologia , Proliferação de Células/genética , Replicação do DNA/genética , Fator de Iniciação 4E em Eucariotos/genética , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Humanas/patologia , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Gravidez , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas ras/biossíntese
19.
Lung Cancer ; 86(3): 324-8, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25453846

RESUMO

BACKGROUND: No target therapies are presently available in the treatment of small-cell lung cancer (SCLC). We investigated the presence of potentially drugable mutations in the EGFR, c-MET, BRAF, KRAS, PDGFRa and c-KIT genes in a retrospective series of SCLC from 2 Italian Institutions. Correlations with immunohistochemical, clinical and outcome features were evaluated. MATERIALS AND METHODS: Genes were studied by direct sequencing of DNA extracted from formalin-fixed paraffin-embedded tissues. Immunohistochemical expression of TTF-1, p63, chromogranin, synaptophysin, CD56 and bcl-2 was assessed. RESULTS: Samples from 113 SCLC patients were analyzed. All cases were wild-type for BRAF, KRAS, PDGFRa and c-KIT (data available for 82 patients). Two (1.8%) patients were EGFR-mutated (exon 19 delE746-A750 and exon 21 L858R); both were females, non-smoker and had limited disease. Overall survival of EGFR-mutated patients was 21 months as compared to 11 months in wild-type. Five (4.4%) patients were c-MET-mutated (4 on exon 14: 2 R988C, 1 D990N, 1 D102Y; 1 on exon 17 R1166Q); all were smokers, 3 were males and 4 had extensive disease. Their OS was comparable to wild-type cases (12 vs. 11 months). EGFR and c-MET mutations were mutually exclusive. Gene mutations did not correlate with immunophenotype. CONCLUSIONS: Targetable mutations are uncommon in SCLC. EGFR-mutated patients tended to be female and non-smoker and experienced a prolonged OS suggesting a possible positive prognostic effect. c-MET mutations did not affect survival. Target therapy might be considered in EGFR and c-MET-mutated patients.


Assuntos
Receptores ErbB/genética , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-met/genética , Carcinoma de Pequenas Células do Pulmão/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Receptores ErbB/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/biossíntese , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-kit/biossíntese , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-met/biossíntese , Proteínas Proto-Oncogênicas p21(ras) , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossíntese , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/patologia , Proteínas ras/biossíntese , Proteínas ras/genética
20.
J Biomed Sci ; 21: 95, 2014 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-25287248

RESUMO

BACKGROUND: Two mature miRNA species may be generated from the 5' and 3' arms of a pre-miRNA precursor. In most cases, only one species remains while the complementary species is degraded. However, co-existence of miRNA-5p and -3p species is increasingly being reported. In this work, we aimed to systematically investigate co-expression of miRNA-5p/3p in colon cancer cells in a genome-wide analysis, and to examine cross-targeting of the dysregulated miRNAs and 5p/3p species. RESULTS: Four colon cancer cell lines were examined relative to two normal colon tissues. Of the 1,190 miRNAs analyzed, 92 and 36 were found to be up- or down-regulated, respectively, in cancer cells. Nineteen co-expressed miRNA-5p/3p pairs were further identified suggesting frequent 5p/3p co-accumulation in colon cancer cells. Of these, 14 pairs were co-up-regulated and 3 pairs were co-down-regulated indicating concerted 5p/3p dysregulation. Nine dysregulated miRNA pairs fell into three miRNA gene families, namely let-7, mir-8/200 and mir-17, which showed frequent cross-targeting in the metastasis process. Focusing on the let-7d-5p/3p pair, the respectively targeted IGF1R and KRAS were shown to be in a reverse relationship with expression of the respective miRNA, which was confirmed in transient transfection assays using let-7d mimic or inhibitor. Targeting of KRAS by let-7d was previous reported; targeting of IGF1R by let-7d-5p was confirmed in luciferase assays in this study. The findings of let-7d-5p/3p and multiple other miRNAs targeting IGF1R, KRAS and other metastasis-related factors suggest that 5p/3p miRNAs contribute to cross-targeting of multiple cancer-associated factors and processes possibly to evade functional abolishment when any one of the crucial factors are inactivated. CONCLUSIONS: miRNA-5p/3p species are frequently co-expressed and are coordinately regulated in colon cancer cells. In cancer cells, multiple cross-targeting by the miRNAs, including the co-existing 5p/3p species, frequently occurs in an apparent safe-proof scheme of miRNA regulation of important tumorigenesis processes. Further systematic analysis of co-existing miRNA-5p/3p pairs in clinical tissues is important in elucidating 5p/3p contributions to cancer pathogenesis.


Assuntos
Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/biossíntese , RNA Neoplásico/biossíntese , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Estudo de Associação Genômica Ampla , Humanos , MicroRNAs/genética , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , RNA Neoplásico/genética , Receptor IGF Tipo 1/biossíntese , Receptor IGF Tipo 1/genética , Proteínas ras/biossíntese , Proteínas ras/genética
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