RESUMO
BACKGROUND: Triple negative breast cancer (TNBC) remains the most challenging breast cancer subtype so far. Specific therapeutic approaches have rarely achieved clinical improvements in treatment of TNBC patients and effective molecular biomarkers are largely unknown. METHODS: We used paired TNBC samples and high throughput RNA sequencing to identify differentially expressed circRNAs. Sucrose gradient polysome fractionation assay, antibody and Mass spectra were used to validate active circRNA translation. The novel protein function was validated in vitro and in vivo by gain or loss of function assays. Mechanistic results were concluded by immunoprecipitation analyses and kinase activity assay. RESULTS: Circular HER2 RNA (circ-HER2) encoded a novel protein, HER2-103. Unexpectedly, while HER2 mRNA and protein were barely detected, circ-HER2/HER2-103 was expressed in ~ 30% TNBC clinical samples. Circ-HER2/HER2-103 positive TNBC patients harbored worse overall prognosis than circ-HER2/HER2-103 negative patients. Knockdown circ-HER2 inhibited TNBC cells proliferation, invasion and tumorigenesis in vitro and in vivo, suggesting the critical role of circ-HER2/HER2-103 in TNBC tumorigenicity. Mechanistically, HER2-103 promoted homo/hetero dimerization of epidermal growth factor receptor (EGFR)/HER3, sustained AKT phosphorylation and downstream malignant phenotypes. Furthermore, HER2-103 shared most of the same amino acid sequences as HER2 CR1 domain which could be antagonized by Pertuzumab, a clinical used HER2 antibody. Pertuzumab markedly attenuated in vivo tumorigenicity of circ-HER2/HER2-103 expressing TNBC cells but showed no effects in circ-HER2/HER2-103 negative TNBC cells. CONCLUSION: Our results not only demonstrated that certain TNBCs were not truly 'HER2 negative' but also highlighted the clinical implications of Pertuzumab in circ-HER2/HER2-103 expressing TNBC patients.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , RNA Circular/genética , Receptor ErbB-2/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Pessoa de Meia-Idade , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
OBJECTIVES: Droplet digital PCR (ddPCR) has been reported to have a superior validity over PCR with amplification-refractory mutation system (ARMS-PCR) for detecting the BRAF V600E mutation in thyroid nodule fine-needle aspiration (FNA) samples using cytological diagnosis as the reference. However, the added value of ddPCR on surgical decision-making remains to be illustrated when the technique is combined with FNA cytology. METHODS: A total of 277 consecutive patients with thyroid nodules were subjected to FNA cytology and BRAF V600E testing with ARMS-PCR. Within this patient cohort, 90 patients underwent surgical intervention with pathological diagnosis available. BRAF V600E testing with ddPCR was performed retrospectively using FNA frozen DNA specimens. The clinical validity and utility of ddPCR in comparison with ARMS-PCR were compared using surgical pathology as the reference. RESULTS: Overall, 101 BRAF V600E mutations were detected by ddPCR, including five ARMS negative patients, four of whom were confirmed to have papillary thyroid cancer (PTC) by surgical pathology. Of the 90 patients with surgical pathology, which is considered the gold standard, ddPCR BRAF V600E testing yielded a sensitivity of 91.3% and specificity of 100% for PTC diagnosis, higher than that of ARMS (sensitivity 83.1%, specificity 100%). However, ddPCR only identified one more candidate patient for surgical intervention than ARMS when the techniques were combined with cytology. CONCLUSIONS: This study highlighted the superior performance of ddPCR over ARMS in BRAF V600E detection from thyroid nodule FNA samples. Further studies are needed to evaluate the cost-effectiveness of replacing ARMS-PCR with ddPCR for surgical decision-making.
Assuntos
Análise Mutacional de DNA , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas B-raf/genética , Nódulo da Glândula Tireoide/genética , Nódulo da Glândula Tireoide/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha Fina , Análise Mutacional de DNA/métodos , Análise Mutacional de DNA/normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Estudos Retrospectivos , Glândula Tireoide/patologia , Nódulo da Glândula Tireoide/diagnóstico , Nódulo da Glândula Tireoide/terapia , Adulto JovemRESUMO
The aim of this study was to evaluate the association of functional expression of TRPM7 with nasopharyngeal carcinoma (NPC) growth. We examined the correlation of TRPM7 expression with cell growth and proliferation, cell cycle, and apoptosis in vitro in NPC cell lines and NPC tumorigenesis in mice by conducting experiments in mice and by further analyzing the tumor volume and growth. We further explored to see whether there is any positive correlation with the TRPM7 knockdown in NPC cells with their sensitivity to radiation. We found that the functional expression of TRPM7 in nasopharyngeal carcinoma is a critical requirement for physiological processes such as cell cycle, resistance to apoptosis, and cell proliferation. TRPM7 knockdown also enhanced sensitivity to radiotherapy of nasopharyngeal carcinoma. Moreover, we identified TRPM7 as a novel potential regulator of cell proliferation in NPC, through signal transducer and activator of transcription 3 (STAT3)-mediated signaling pathway and other anti-apoptotic factors. TRPM7 and STAT3 activation might be critical for the growth of NPC cells and could be an effective target for treatment of nasopharyngeal carcinoma.
Assuntos
Carcinogênese/genética , Neoplasias Nasofaríngeas/genética , Proteínas Serina-Treonina Quinases/genética , Canais de Cátion TRPM/genética , Animais , Apoptose/genética , Carcinogênese/patologia , Carcinoma , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patologia , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Carga Tumoral/genéticaRESUMO
Glycogen synthase kinase-3ß (GSK-3ß), a key regulator of neuronal apoptosis, is inhibited by the phosphorylation of Ser-9/Ser-389 and was recently shown to be cleaved by calpain at the N terminus, leading to its subsequent activation. In this study calpain was found to cleave GSK-3ß not only at the N terminus but also at the C terminus, and cleavage sites were identified at residues Thr-38-Thr-39 and Ile-384-Gln-385. Furthermore, the cleavage of GSK-3ß occurred in tandem with Ser-9 dephosphorylation during cerebellar granule neuron apoptosis. Increasing Ser-9 phosphorylation of GSK-3ß by inhibiting phosphatase 1/2A or pretreating with purified active Akt inhibited calpain-mediated cleavage of GSK-3ß at both N and C termini, whereas non-phosphorylatable mutant GSK-3ß S9A facilitated its cleavage. In contrast, Ser-389 phosphorylation selectively inhibited the cleavage of GSK-3ß at the C terminus but not the N terminus. Calpain-mediated cleavage resulted in three truncated products, all of which contained an intact kinase domain: ΔN-GSK-3ß (amino acids 39-420), ΔC-GSK-3ß (amino acids 1-384), and ΔN/ΔC-GSK-3ß (amino acids 39-384). All three truncated products showed increased kinase and pro-apoptotic activity, with ΔN/ΔC-GSK-3ß being the most active form. This observation suggests that the GSK-3ß C terminus acts as an autoinhibitory domain similar to the N terminus. Taken together, these findings demonstrate that calpain-mediated cleavage activates GSK-3ß by removing its N- and C-terminal autoinhibitory domains and that Ser-9 phosphorylation inhibits the cleavage of GSK-3ß at both termini. In contrast, Ser-389 phosphorylation inhibits only C-terminal cleavage but not N-terminal cleavage. These findings also identify a mechanism by which site-specific phosphorylation and calpain-mediated cleavage operate in concert to regulate GSK-3ß activity.
Assuntos
Apoptose/fisiologia , Calpaína/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Degeneração Neural/metabolismo , Neurônios/enzimologia , Animais , Cerebelo/citologia , Glutamina/metabolismo , Quinase 3 da Glicogênio Sintase/química , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Isoleucina/metabolismo , Mutagênese/fisiologia , Degeneração Neural/patologia , Neurônios/citologia , Fosforilação/fisiologia , Estrutura Terciária de Proteína , Coelhos , Ratos , Serina/metabolismo , Treonina/metabolismoRESUMO
PURPOSE: Overexpression of glucose transporters has been identified in a variety of human cancers. However, the expression status of Sodium dependent Glucose Transporter 1 (SGLT1) in ovarian carcinoma has not been investigated. METHODS: In our study, protein expression levels of SGLT1 were explored by semiquantitative immunohistochemical staining on archival formalin-fixed paraffin-embedded pathologic specimen consisting of 178 epithelial ovarian tumors. Receiver operating characteristic curve analysis, Spearman's rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model were utilized to analyze the data. RESULTS: The threshold for high expression of SGLT1 was determined to be above 40% (areas under curve = 0.683, P = 0.003) based on the area under curves. Significantly overexpression of SGLT1 was observed in 39.7% invasive carcinomas, 11.5% borderline tumors, 10% cystadenomas but in none of the normal ovaries (0%). In ovarian carcinomas, SGLT1 overexpression was positively correlated with later pT status (P = 0.029) and advanced FIGO stage (P = 0.024). By univariate survival analysis on the ovarian carcinoma cohorts, overexpression of SGLT1 was associated with shortened patient survival (mean 70.5 months in tumors with overexpression of SGLT1 versus 89.3 months in tumors with normal levels of SGLT1; P = 0.019). By multivariate analysis, SGLT1 protein expression remained as a significant and independent prognostic factor for the prediction of patient survival (P = 0.033). CONCLUSIONS: SGLT1 overexpression, as examined by immunohistochemistry, is an independent biomarker for poor prognosis of patients with ovarian carcinoma.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma/metabolismo , Neoplasias Ovarianas/metabolismo , Transportador 1 de Glucose-Sódio/metabolismo , Adulto , Idoso , Carcinoma/mortalidade , Estudos de Casos e Controles , China/epidemiologia , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neoplasias Ovarianas/mortalidade , Prognóstico , Adulto JovemRESUMO
Glycogen synthase kinase-3 (GSK-3) plays a critical role in neuronal apoptosis. The two mammalian isoforms of the kinase, GSK-3α and GSK-3ß, are inhibited by phosphorylation at Ser-21 and Ser-9, respectively. Depolarization, which is vital for neuronal survival, causes both an increase in Ser-21/9 phosphorylation and an inhibition of GSK-3α/ß. However, the role of GSK-3 phosphorylation in depolarization-dependent neuron survival and the signaling pathway contributing to GSK-3 phosphorylation during depolarization remain largely unknown. Using several approaches, we showed that both isoforms of GSK-3 are important for mediating neuronal apoptosis. Nonphosphorylatable GSK-3α/ß mutants (S21A/S9A) promoted apoptosis, whereas a peptide encompassing Ser-9 of GSK-3ß protected neurons in a phosphorylation-dependent manner; these results indicate a critical role for Ser-21/9 phosphorylation on depolarization-dependent neuron survival. We found that Ser-21/9 phosphorylation of GSK-3 was mediated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) but not by Akt/PKB, PKA, or p90(RSK). CaMKII associated with and phosphorylated GSK-3α/ß. Furthermore, the pro-survival effect of CaMKII was mediated by GSK-3 phosphorylation and inactivation. These findings identify a novel Ca(2+)/calmodulin/CaMKII/GSK-3 pathway that couples depolarization to neuronal survival.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Polaridade Celular/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Neurônios/enzimologia , Transdução de Sinais/fisiologia , Substituição de Aminoácidos , Animais , Apoptose/fisiologia , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Calmodulina/genética , Calmodulina/metabolismo , Sobrevivência Celular/fisiologia , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Neurônios/citologia , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
Midkine (MDK) expression is associated with the proliferation of many cancers, including glioma. However, the upstream signaling that leads to MDK accumulation remains elusive. This study investigates the molecular mechanism that induces MDK overexpression in human glioma. The Repository for Molecular Brain Neoplasia Data was analyzed to identify potential MDK regulators. Expression of MDK and specificity protein 1 (SP1) was compared in glioma specimens. Chromatin immunoprecipitation assay was used to confirm the transcriptional regulation. MDK-force-expressed, SP1-silenced glioma cells were used to test rescue effects in vitro and in vivo. MDK and SP1 expression in gliomas was significantly higher than in adjacent tissues and was positively correlated in glioma clinical samples and cell lines. The promoter of the human MDK gene has a putative SP1 binding site. SP1 binds to the promoter of the MDK gene and directly regulates MDK expression. MDK or SP1 gene silencing inhibited the proliferation of glioma cells and reduced the tumor volume in nude mice. Overexpression of MDK in SP1-silenced cells could partially rescue the SP1 inhibition effects in vivo and in vitro. SP1 directly up-regulated the expression of MDK, and the SP1-MDK axis cooperated in glioma tumorigenesis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Glioma/genética , Fatores de Crescimento Neural/genética , Fator de Transcrição Sp1/metabolismo , Ativação Transcricional , Animais , Linhagem Celular Tumoral , Proliferação de Células , Glioma/metabolismo , Humanos , Camundongos , Camundongos Nus , Midkina , Regiões Promotoras Genéticas , Regulação para CimaRESUMO
Heroin has been shown to cause spongiform leukoencephalopathy (SLE) in heroin addicts. In this study, we found that heroin could induce apoptosis of primary cultured cerebellar granule cells (CGC) and c-Jun N-terminal kinase (JNK) pathway is activated during CGCs apoptosis. Inhibiting JNK with a specific inhibitor, SP600125, reduced the levels of c-Jun phosphorylation and caspase-3 activation. We also showed that use the JNK inhibitor SP600125, caspase inhibitor z-VAD, or use SP600125 and z-VAD together significantly suppressed cell death induced by heroin. These results indicate that JNK pathway is an important mediator of the neurotoxic effects of heroin and inhibiting JNK activity may represent a new and effective strategy to treat heroin-induced SLE.
Assuntos
Apoptose/fisiologia , Caspase 3/metabolismo , Dependência de Heroína/enzimologia , Dependência de Heroína/patologia , Heroína/toxicidade , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Neurônios/enzimologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Caspase 3/fisiologia , Inibidores de Caspase , Cerebelo/enzimologia , Cerebelo/patologia , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/patologia , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Entorpecentes/toxicidade , Neurônios/patologia , Cultura Primária de Células , Ratos , Ratos Sprague-DawleyRESUMO
In cerebellar granule neurons, a BH3-only Bcl-2 family member, death protein 5/harakiri, is up-regulated in a JNK-dependent manner during apoptosis induced by potassium deprivation. However, it is not clear whether c-Jun is directly involved in the induction of dp5. Here, we showed that the up-regulation of dp5, but not fas ligand and bim, after potassium deprivation was suppressed by the expression of a dominant negative form of c-Jun. Deletion analysis of the 5'-flanking sequence of the dp5 gene revealed that a major responsive element responsible for the induction by potassium deprivation is an ATF binding site located at -116 to -109 relative to the transcriptional start site. Mutation of this site completely abolished promoter activation. Furthermore, a gel shift assay showed that a specific complex containing c-Jun and ATF2 recognized this site and increased in potassium-deprived cerebellar granule neurons. Chromatin immunoprecipitation demonstrated that c-Jun was able to bind to this site in vivo. Finally, we demonstrated that knockdown of Dp5 by small interfering RNA rescued neurons from potassium deprivation-induced apoptosis. Taken together, these results suggest that dp5 is a target gene of c-Jun and plays a critical role in potassium deprivation-induced apoptosis in cerebellar granule neurons.