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1.
Oncol Res ; 2020 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-32252875

RESUMO

The deubiquitinase cyclindromatosis (CYLD) functions as a tumour suppressor inhibiting cell proliferation in many cancer types including melanoma. Here we present evidence that a proportion of melanoma cells are nonetheless addicted to CYLD for survival. The expression levels of CYLD varied widely in melanoma cell lines and melanomas in vivo, with a subset of melanoma cell lines and melanomas displaying even higher levels of CYLD than melanocyte lines and nevi, respectively. Strikingly, although shRNA knockdown of CYLD promoted, as anticipated, cell proliferation in some melanoma cell lines, it reduced cell viability in a fraction of melanoma cell lines with relatively high levels of CYLD expression and did not impinge on survival and proliferation in a third type of melanoma cell lines. The decrease in cell viability caused by CYLD knockdown was due to induction of apoptosis, as it was associated with activation of the caspase cascade and was abolished by treatment with a general caspase inhibitor. Mechanistic investigation s demonstrated that induction of apoptosis by CYLD knockdown was caused by upregulation of receptor-interacting protein kinase 1 (RIPK1) that was associated with elevated K63-linked polyubiquitination of the protein, indicating that CYLD is critical for controlling RIPK1 expression in these cells. Of note, microRNA (miR) profiling showed that miR-99b-3p that was predicted to target the 3'UTR of the CYLD mRNA was reduced in melanoma cell lines with high levels of CYLD compared with melanocyte lines. Further functional studies confirmed that the reduction in miR-99b-3p expression was responsible for the increased expression of CYLD in a highly cell line-specific manner. Taken together, these results reveal an unexpected role of CYLD in promoting survival of a subset of melanoma cells and uncover the heterogeneity of CYLD expression and its biological significance in melanoma.

2.
Nucleic Acids Res ; 48(6): 3089-3102, 2020 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-32030426

RESUMO

Long non-coding RNAs (lncRNAs) have emerged as important biological tuners. Here, we reveal the role of an uncharacterized lncRNA we call SENEBLOC that is expressed by both normal and transformed cells under homeostatic conditions. SENEBLOC was shown to block the induction of cellular senescence through dual mechanisms that converge to repress the expression of p21. SENEBLOC facilitates the association of p53 with MDM2 by acting as a scaffold to promote p53 turnover and decrease p21 transactivation. Alternatively, SENEBLOC was shown to affect epigenetic silencing of the p21 gene promoter through regulation of HDAC5. Thus SENEBLOC drives both p53-dependent and p53-independent mechanisms that contribute to p21 repression. Moreover, SENEBLOC was shown to be involved in both oncogenic and replicative senescence, and from the perspective of senolytic agents we show that the antagonistic actions of rapamycin on senescence are dependent on SENEBLOC expression.

3.
Histochem Cell Biol ; 153(2): 77-87, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31834485

RESUMO

CD44 is a transmembrane receptor that acts as adhesion protein, fundamentally recognizing hyaluronan, an essential component of the extracellular matrix. It has a well-established functional association with cancer metastasis, particularly the CD44 variant forms which are considered essential markers of cancer stem cells. CD44 itself lacks intrinsic kinase activity but rather engages in signalling through specific interactions with kinases and other signalling components. Proteolysis within its transmembrane region also leads to release of the CD44 cytoplasmic domain, which can translocate to the nucleus and regulate transcription. A third signalling modality has been reported where the intact CD44 receptor translocates to the nucleus. Here, we investigated the latter using imaging techniques together with biochemical analyses. Our findings support observations where CD44 is cleaved prior to nuclear translocation and challenges the evidence for the presence of intact CD44 receptors in the cell nucleus. Conclusions regarding the presence of intact CD44 in the cell nucleus as a signalling modality, therefore, require re-evaluation. We highlight artefacts and common technical issues associated with these experiments that can lead to misinterpretation.

4.
Nat Commun ; 10(1): 5334, 2019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31767869

RESUMO

Protein products of the regenerating islet-derived (REG) gene family are important regulators of many cellular processes. Here we functionally characterise a non-protein coding product of the family, the long noncoding RNA (lncRNA) REG1CP that is transcribed from a DNA fragment at the family locus previously thought to be a pseudogene. REG1CP forms an RNA-DNA triplex with a homopurine stretch at the distal promoter of the REG3A gene, through which the DNA helicase FANCJ is tethered to the core promoter of REG3A where it unwinds double stranded DNA and facilitates a permissive state for glucocorticoid receptor α (GRα)-mediated REG3A transcription. As such, REG1CP promotes cancer cell proliferation and tumorigenicity and its upregulation is associated with poor outcome of patients. REG1CP is also transcriptionally inducible by GRα, indicative of feedforward regulation. These results reveal the function and regulation of REG1CP and suggest that REG1CP may constitute a target for cancer treatment.

5.
Adv Exp Med Biol ; 1206: 127-149, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31776983

RESUMO

The tumor suppressor gene Tp53 encodes p53, a pivotal transcription factor with a broad target gene repertoire. Induction and stabilization of p53 during DNA damage and oncogene activation function to induce cell cycle arrest, apoptosis, or senescence. These actions are a failsafe to counteract carcinogenesis but Tp53 also plays a key role in regulating different aspects of cell metabolism including autophagy. Autophagy or cellular "self-eating" involves the dismantling and remodeling of cellular components, activities which are fundamental in maintaining cellular homeostasis and in supporting cell growth. After providing an historical overview of Tp53 research, the purpose of this chapter is to review the different mechanistic aspects of Tp53's role in autophagy and to highlight the key challenges which lie ahead. Tp53 functions are regulated by tight control of its cellular levels and notably, Tp53 can be both an activator or inhibitor of autophagy. Under stress conditions such as nutrient depletion or hypoxia, Tp53 contributes to autophagic activation by inhibiting mTOR signaling. Alternatively, p53 can interact with death-associated protein kinase 1 (DAPK1), acting to stabilize nuclear p53 amongst other functions including activation of the key autophagic mediator, Beclin-1. Under normal physiological conditions, Tp53 can inhibit autophagosome formation but stress conditions can also result in Tp53-mediated promotion of autophagy, demonstrating that Tp53 actions are highly context dependent. Tp53 target genes also play key opposing roles in autophagy induction or inhibition such as DRAM and TIGAR, respectively. Finally, the role of Tp53 mutants in autophagy regulation are discussed.


Assuntos
Proteínas Reguladoras de Apoptose , Autofagia , Proteínas de Membrana , Proteína Supressora de Tumor p53 , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
6.
Proteomics ; 19(21-22): e1900059, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31287215

RESUMO

Dysregulation of fascin actin-bundling protein 1 (FSCN1) enhances cell proliferation, invasion, and motility in laryngeal squamous cell carcinoma (LSCC), while the mechanism remains unclear. Here, co-immunoprecipitation and mass spectrometry is utilized to identify potential FSCN1-binding proteins. Functional annotation of FSCN1-binding proteins are performed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Furthermore, the protein-protein interaction network of FSNC1-binding proteins is constructed and the interactions between FSCN1 and novel identified interacting proteins AIMP1 and LTA4H are validated. Moreover, the expression and functional role of AIMP1 and LTA4H in LSCC are investigated. A total of 123 proteins are identified as potential FSCN1-binding proteins, and functional annotation shows that FSCN1-binding proteins are significantly enriched in carcinogenic processes, such as filopodium assembly-regulation and GTPase activity. Co-IP/western blotting and immunofluorescence confirm that AIMP1 and LTA4H bind and colocalize with FSCN1. Furthermore, both AIMP1 and LTA4H are upregulated in LSCC tissues, and knockdown of AIMP1 or LTA4H inhibits LSCC cell proliferation, migration, and invasion. Collectively, the identification of FSCN1-binding partners enhances understanding of the mechanism of FSCN1-mediated malignant phenotypes, and these findings indicate that FSCN1 binds to AIMP1 and LTA4H might promote the progression of LSCC.

7.
Cell Metab ; 30(1): 157-173.e7, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31155494

RESUMO

We report that circACC1, a circular RNA derived from human ACC1, plays a critical role in cellular responses to metabolic stress. CircACC1 is preferentially produced over ACC1 in response to serum deprivation by the transcription factor c-Jun. It functions to stabilize and promote the enzymatic activity of the AMPK holoenzyme by forming a ternary complex with the regulatory ß and γ subunits. The cellular levels of circACC1 modulate both fatty acid ß-oxidation and glycolysis, resulting in profound changes in cellular lipid storage. In a tumor xenograft model, silencing or enforced expression of circACC1 resulted in growth inhibition and enhancement, respectively. Moreover, increased AMPK activation in colorectal cancer tissues was frequently associated with elevated circACC1 expression. We conclude that circACC1 serves as an economic means to elicit AMPK activation and moreover propose that cancer cells exploit circACC1 during metabolic reprogramming.

8.
Proteomics ; 19(21-22): e1900020, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31169343

RESUMO

This study intends to investigate the transcriptional regulatory role of miR-145-5p in laryngeal squamous cell carcinoma (LSCC). LSCC cell line TU-177 is transfected with miR-145-5p mimics, generating miR-145-5p-overexpression LSCC cells. Whole transcriptome microarrays are used to investigate the differentially expressed lncRNAs, circRNAs, mRNAs, and miRNAs. The target genes of miRNAs are predicted and performed functional annotation. Additionally, the circRNAs, lncRNAs, and mRNAs that interact with miRNAs are predicted, and then the competing endogenous RNAs (ceRNAs) are predicted. Microarray analysis identifies 26 miRNAs, 248 mRNAs, 1118 lncRNAs, and 382 circRNAs differentially expressed in miR-145-5p overexpressed LSCC cells. Overall, 675 target genes are identified for the differentially expressed miRNAs, which involved in cell adhesion associated gene ontology (GO) terms, and MAPK and FoxO signaling pathways. The up-regulated mRNAs involved in the pathway of ABC transporters, while the down-regulated mRNAs involved in pathway of olfactory transduction. Moreover, 149 ceRNAs are predicted, which are associated with apoptosis, Wnt pathway, and metabolic pathway. Furthermore, qPCR results confirm that miR-145-5p affects expression of lncRNAs, miRNAs, mRNAs, and circRNAs in LSCC cells. Collectively, miR-145-5p may be inhibits LSCC progression via ceRNA-mediated pathways, such as WNT2B-miR-145-5p-NONHSAT127539.2, CASP10-miR-145-5p-NONHSAT127539.2, CASP10-miR-145-5p-circ_0003519, and TPO-miR-145-5p-circ_0003519.

9.
Transl Oncol ; 12(7): 925-931, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31085408

RESUMO

Cancer can be considered the result of a series of genetic variations that lead to a normal cell being transformed into a malignant one while avoiding cell death-atypical characteristics of tumor development. Although a large number of genomics and epigenetic alterations have been identified in cells undergoing apoptotic, autophagic or necrotic cell death, the treatment of cancer remains thought-provoking. Pyroptosis is differentiated from other types of programmed cell death and is mainly activated by Caspase-1. To initiate pyroptosis, cells receive specific "death" messages, produce cytokines, swell, burst, and ultimately die. The deficiency of Caspase-1 expression may lead to inflammation-mediated tumor progression. Hence, the molecular mechanisms for the Caspase-1 activation in tumor tissues are yet to be exploited extensively. This review aims to summarise the latest discoveries about pyroptosis and its new exciting role in inducing cancer cell death.

10.
Biochem Biophys Res Commun ; 514(3): 625-631, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31076104

RESUMO

Fat1 cadherin is broadly expressed throughout the nervous system and has been implicated in neuronal differentiation. Here we examined the functional contribution of FAT1 during neuronal differentiation of the Ntera2 cell line model. FAT1 expression was increased during the retinoic acid (RA)-induced differentiation of NTera2 cells. Depletion of FAT1 with siRNA decreased the number of neurites produced after RA treatment. Moreover, FAT1 silencing also led to decreased Ser127-phosphorylation of YAP along with transcriptional increases in the Hippo target genes CTGF and ANKRD1, suggesting FAT1 alters Hippo signalling during differentiation. In the context of the Ntera2 model, FAT1 is required for efficient neuritogenesis, acting as a regulator of neurite formation during the early stages of differentiation.

11.
Mol Ther ; 27(2): 365-379, 2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30341010

RESUMO

Laryngeal squamous cell carcinoma (LSCC) is a common form of head and neck cancer with poor prognosis. However, the mechanism underlying the pathogenesis of LSCC remains unclear. Here, we demonstrated increased expression of fascin actin-bundling protein 1 (FSCN1) and decreased expression of microRNA-145-5p (miR-145-5p) in a clinical cohort of LSCC. Luciferase assay revealed that miR-145-5p is a negative regulator of FSCN1. Importantly, low miR-145-5p expression was correlated with TNM (tumor, node, metastasis) status and metastasis. Moreover, cases with low miR-145-5p/high FSCN1 expression showed poor prognosis, and these characteristics together served as independent prognostic indicators of survival. Gain- and loss-of-function studies showed that miR-145-5p overexpression or FSCN1 knockdown inhibited LSCC migration, invasion, and growth by suppressing the epithelial-mesenchymal transition along with inducing cell-cycle arrest and apoptosis. Additionally, hypermethylation of the miR-145-5p promoter suggested that repression of miR-145-5p arises through epigenetic inactivation. LSCC tumor growth in vivo could be inhibited by using miR-145-5p agomir or FSCN1 small interfering RNA (siRNA), which highlights the potential for clinical translation. Collectively, our findings indicate that miR-145-5p plays critical roles in inhibiting the progression of LSCC by suppressing FSCN1. Both miR-145-5p and FSCN1 are important potential prognostic markers and therapeutic targets for LSCC.


Assuntos
Proteínas de Transporte/metabolismo , Metilação de DNA/fisiologia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/metabolismo , MicroRNAs/genética , Proteínas dos Microfilamentos/metabolismo , Regiões Promotoras Genéticas/genética , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proteínas de Transporte/genética , Linhagem Celular , Linhagem Celular Tumoral , Metilação de DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/fisiologia , Proteínas dos Microfilamentos/genética
13.
Proc Natl Acad Sci U S A ; 115(50): E11661-E11670, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30478051

RESUMO

Long noncoding RNAs (lncRNAs) function through a diverse array of mechanisms that are not presently fully understood. Here, we sought to find lncRNAs differentially regulated in cancer cells resistant to either TNF-related apoptosis-inducing ligand (TRAIL) or the Mcl-1 inhibitor UMI-77, agents that act through the extrinsic and intrinsic apoptotic pathways, respectively. This work identified a commonly up-regulated lncRNA, ovarian adenocarcinoma-amplified lncRNA (OVAAL), that conferred apoptotic resistance in multiple cancer types. Analysis of clinical samples revealed OVAAL expression was significantly increased in colorectal cancers and melanoma in comparison to the corresponding normal tissues. Functional investigations showed that OVAAL depletion significantly inhibited cancer cell proliferation and retarded tumor xenograft growth. Mechanically, OVAAL physically interacted with serine/threonine-protein kinase 3 (STK3), which, in turn, enhanced the binding between STK3 and Raf-1. The ternary complex OVAAL/STK3/Raf-1 enhanced the activation of the RAF protooncogene serine/threonine-protein kinase (RAF)/mitogen-activated protein kinase kinase 1 (MEK)/ERK signaling cascade, thus promoting c-Myc-mediated cell proliferation and Mcl-1-mediated cell survival. On the other hand, depletion of OVAAL triggered cellular senescence through polypyrimidine tract-binding protein 1 (PTBP1)-mediated p27 expression, which was regulated by competitive binding between OVAAL and p27 mRNA to PTBP1. Additionally, c-Myc was demonstrated to drive OVAAL transcription, indicating a positive feedback loop between c-Myc and OVAAL in controlling tumor growth. Taken together, these results reveal that OVAAL contributes to the survival of cancer cells through dual mechanisms controlling RAF/MEK/ERK signaling and p27-mediated cell senescence.


Assuntos
Senescência Celular/genética , Senescência Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Sistema de Sinalização das MAP Quinases , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Xenoenxertos , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Nus , Estabilidade Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
14.
Cancer Res ; 78(23): 6666-6679, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30301840

RESUMO

: Cancer cells in quiescence (G0 phase) are resistant to death, and re-entry of quiescent cancer cells into the cell-cycle plays an important role in cancer recurrence. Here we show that two p53-responsive miRNAs utilize distinct but complementary mechanisms to promote cancer cell quiescence by facilitating stabilization of p27. Purified quiescent B16 mouse melanoma cells expressed higher levels of miRNA-27b-3p and miRNA-455-3p relative to their proliferating counterparts. Induction of quiescence resulted in increased levels of these miRNAs in diverse types of human cancer cell lines. Inhibition of miRNA-27b-3p or miRNA-455-3p reduced, whereas its overexpression increased, the proportion of quiescent cells in the population, indicating that these miRNAs promote cancer cell quiescence. Accordingly, cancer xenografts bearing miRNA-27b-3p or miRNA-455-3p mimics were retarded in growth. miRNA-27b-3p targeted cyclin-dependent kinase regulatory subunit 1 (CKS1B), leading to reduction in p27 polyubiquitination mediated by S-phase kinase-associated protein 2 (Skp2). miRNA-455-3p targeted CDK2-associated cullin domain 1 (CAC1), which enhanced CDK2-mediated phosphorylation of p27 necessary for its polyubiquitination. Of note, the gene encoding miRNA-27b-3p was embedded in the intron of the chromosome 9 open reading frame 3 gene that was transcriptionally activated by p53. Similarly, the host gene of miRNA-455-3p, collagen alpha-1 (XXVII) chain, was also a p53 transcriptional target. Collectively, our results identify miRNA-27b-3p and miRNA-455-3p as important regulators of cancer cell quiescence in response to p53 and suggest that manipulating miRNA-27b-3p and miRNA-455-3p may constitute novel therapeutic avenues for improving outcomes of cancer treatment. SIGNIFICANCE: Two novel p53-responsive microRNAs whose distinct mechanisms of action both stabilize p27 to promote cell quiescence and may serve as therapeutic avenues for improving outcomes of cancer treatment.


Assuntos
Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , MicroRNAs/genética , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Senescência Celular/genética , Genes Reporter , Genes cdc , Humanos , Camundongos , Modelos Biológicos , Fosforilação , Interferência de RNA , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo
15.
Nat Commun ; 9(1): 2372, 2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29985391

RESUMO

Dysregulation of the Hippo signaling pathway and the consequent YAP1 activation is a frequent event in human malignancies, yet the underlying molecular mechanisms are still poorly understood. A pancancer analysis of core Hippo kinases and their candidate regulating molecules revealed few alterations in the canonical Hippo pathway, but very frequent genetic alterations in the FAT family of atypical cadherins. By focusing on head and neck squamous cell carcinoma (HNSCC), which displays frequent FAT1 alterations (29.8%), we provide evidence that FAT1 functional loss results in YAP1 activation. Mechanistically, we found that FAT1 assembles a multimeric Hippo signaling complex (signalome), resulting in activation of core Hippo kinases by TAOKs and consequent YAP1 inactivation. We also show that unrestrained YAP1 acts as an oncogenic driver in HNSCC, and that targeting YAP1 may represent an attractive precision therapeutic option for cancers harboring genomic alterations in the FAT1 tumor suppressor genes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Caderinas/fisiologia , Neoplasias de Cabeça e Pescoço/genética , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caderinas/genética , Caderinas/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Fatores de Transcrição
16.
Oncogene ; 37(29): 4033-4045, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29706658

RESUMO

The actin crosslinking protein α-actinin-4 (ACTN4) is emerging as an important contributor to the pathogenesis of cancer. This has largely been attributed to its role in regulating cytoskeleton organization and its involvement in transcriptional regulation of gene expression. Here we report a novel function of ACTN4 as a scaffold necessary for stabilization of receptor-interacting protein kinase 1 (RIPK1) that we have recently found to be an oncogenic driver in melanoma. ACTN4 bound to RIPK1 and cellular inhibitor of apoptosis protein 1 (cIAP1) with its actin-binding domain at the N-terminus and the CaM-like domain at the C-terminus, respectively. This facilitated the physical association between RIPK1 and cIAP1 and was critical for stabilization of RIPK1 that in turn activated NF-κB. Functional investigations showed that silencing of ACTN4 suppressed melanoma cell proliferation and retarded melanoma xenograft growth. In contrast, overexpression of ACTN4 promoted melanocyte and melanoma cell proliferation and moreover, prompted melanocyte anchorage-independent growth. Of note, the expression of ACTN4 was transcriptionally activated by NF-κB. Taken together, our findings identify ACTN4 as an oncogenic regulator through driving a feedforward signaling axis of ACTN4-RIPK1-NF-κB, with potential implications for targeting ACTN4 in the treatment of melanoma.


Assuntos
Actinina/metabolismo , Melanoma/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , Masculino , Melanócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , NF-kappa B/metabolismo , Oncogenes/fisiologia , Transdução de Sinais/fisiologia , Transcrição Genética/fisiologia , Ativação Transcricional/fisiologia
17.
Sci Rep ; 8(1): 8135, 2018 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-29802376

RESUMO

The neurotrophic tyrosine kinase receptor TrkA (NTRK1) and its ligand nerve growth factor (NGF) are emerging promoters of tumor progression. In lung cancer, drugs targeting TrkA are in clinical trials, but the clinicopathological significance of TrkA and NGF, as well as that of the precursor proNGF, the neurotrophin co-receptor p75NTR and the proneurotrophin co-receptor sortilin, remains unclear. In the present study, analysis of these proteins was conducted by immunohistochemistry and digital quantification in a series of 204 lung cancers of different histological subtypes versus 121 normal lung tissues. TrkA immunoreactivity was increased in squamous cell carcinoma compared with benign and other malignant lung cancer histological subtypes (p < 0.0001). NGF and proNGF were also increased in squamous cell carcinoma, as well as in adenocarcinoma (p < 0.0001). In contrast, p75NTR was increased across all lung cancer histological subtypes compared to normal lung (p < 0.0001). Sortilin was higher in adenocarcinoma and small cell carcinoma (p < 0.0001). Nerves in the tumor microenvironment were negative for TrkA, NGF, proNGF, p75NTR and sortilin. In conclusion, these data suggest a preferential therapeutic value of targeting the NGF-TrkA axis in squamous cell carcinomas of the lung.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Pulmonares/metabolismo , Fator de Crescimento Neural/metabolismo , Receptor trkA/metabolismo , Carcinoma de Células Escamosas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Ligantes , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Microambiente Tumoral
18.
J Circ Biomark ; 7: 1849454418766966, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29662552

RESUMO

Enumeration of circulating microvesicles (MVs) by conventional flow cytometry is accomplished by the addition of a known amount of counting beads and calculated from the formula: MV/µl = (MV count/bead count) × final bead concentration. We sought to optimize each variable in the equation by determining the best parameters for detecting 'MV count' and examining the effects of different bead preparations and concentrations on the final calculation. Three commercially available bead preparations (TruCount, Flow-Count and CountBright) were tested, and MV detection on a BD FACSCanto was optimized for gating by either forward scatter (FSC) or side scatter (SSC); the results were compared by calculating different subsets of MV on a series of 74 typical patient plasma samples. The relationship between the number of beads added to each test and the number of beads counted by flow cytometry remained linear over a wide range of bead concentrations (R2 ≥ 0.997). However, TruCount beads produced the most consistent (concentration variation = 3.8%) calculated numbers of plasma CD41+/Annexin V+ MV, which were significantly higher from that calculated using either Flow-Count or CountBright (p < 0.001). The FACSCanto was able to resolve 0.5 µm beads by FSC and 0.16 µm beads by SSC, but there were significantly more background events using SSC compared with FSC (3113 vs. 470; p = 0.008). In general, sample analysis by SSC resulted in significantly higher numbers of MV (p < 0.0001) but was well correlated with enumeration by FSC for all MV subtypes (ρ = 0.62-0.89, p < 0.0001). We conclude that all counting beads provided linear results at concentrations ranging from 6 beads/µl to 100 beads/µl, but TruCount was the most consistent. Using SSC to gate MV events produced high background which negatively affected counting bead enumeration and overall MV calculations. Strategies to reduce SSC background should be employed in order to reliably use this technique.

19.
Nat Cell Biol ; 20(4): 492-502, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29593331

RESUMO

The list of long non-coding RNAs (lncRNAs) involved in the p53 pathway of the DNA damage response is rapidly expanding, but whether lncRNAs have a role in maintaining the de novo structure of DNA is unknown. Here, we demonstrate that the p53-responsive lncRNA GUARDIN is important for maintaining genomic integrity under steady-state conditions and after exposure to exogenous genotoxic stress. GUARDIN is necessary for preventing chromosome end-to-end fusion through maintaining the expression of telomeric repeat-binding factor 2 (TRF2) by sequestering microRNA-23a. Moreover, GUARDIN also sustains breast cancer 1 (BRCA1) stability by acting as an RNA scaffold to facilitate the heterodimerization of BRCA1 and BRCA1-associated RING domain protein 1 (BARD1). As such, GUARDIN silencing triggered apoptosis and senescence, enhanced cytotoxicity of additional genotoxic stress and inhibited cancer xenograft growth. Thus, GUARDIN may constitute a target for cancer treatment.


Assuntos
Instabilidade Genômica , Neoplasias/genética , RNA Longo não Codificante/genética , Proteína Supressora de Tumor p53/genética , Células A549 , Animais , Antineoplásicos/farmacologia , Apoptose , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proliferação de Células , Senescência Celular , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Instabilidade Genômica/efeitos dos fármacos , Células HCT116 , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Conformação de Ácido Nucleico , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Carga Tumoral , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
20.
Proc Natl Acad Sci U S A ; 115(7): E1465-E1474, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29378948

RESUMO

The oncoprotein c-Myc plays an important role in regulating glycolysis under normoxia; yet, in cancer cells, HIF1α, which is essential for driving glycolysis under hypoxia, is often up-regulated even in the presence of oxygen. The relationship between these two major regulators of the Warburg effect remains to be fully defined. Here we demonstrate that regulation of a long noncoding RNA (lncRNA), named IDH1-AS1, enables c-Myc to collaborate with HIF1α in activating the Warburg effect under normoxia. c-Myc transcriptionally repressed IDH1-AS1, which, upon expression, promoted homodimerization of IDH1 and thus enhanced its enzymatic activity. This resulted in increased α-KG and decreased ROS production and subsequent HIF1α down-regulation, leading to attenuation of glycolysis. Hence, c-Myc repression of IDH1-AS1 promotes activation of the Warburg effect by HIF1α. As such, IDH1-AS1 overexpression inhibited cell proliferation, whereas silencing of IDH1-AS1 promoted cell proliferation and cancer xenograft growth. Restoring IDH1-AS1 expression may therefore represent a potential metabolic approach for cancer treatment.


Assuntos
Glicólise/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isocitrato Desidrogenase/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Antissenso/genética , RNA Longo não Codificante/genética , Neoplasias do Colo do Útero/prevenção & controle , Animais , Proliferação de Células , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/genética , Ácidos Cetoglutáricos/metabolismo , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-myc/genética , Ativação Transcricional , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
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