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Emerging evidence indicates that arginine methylation promotes the stability of arginine-glycine-rich (RGG) motif-containing RNA-binding proteins (RBPs) and regulates gene expression. Here, we report that post-translational modification of FXR1 enhances the binding with mRNAs and is involved in cancer cell growth and proliferation. Independent point mutations in arginine residues of FXR1's nuclear export signal (R386 and R388) and RGG (R453, R455 and R459) domains prevent it from binding to RNAs that form G-quadruplex (G4) RNA structures. Disruption of G4-RNA structures by lithium chloride failed to bind with FXR1, indicating its preference for G4-RNA structure containing mRNAs. Furthermore, loss-of-function of PRMT5 inhibited FXR1 methylation both in vivo and in vitro, affecting FXR1 protein stability, inhibiting RNA-binding activity and cancer cell growth and proliferation. Finally, the enhanced crosslinking and immunoprecipitation (eCLIP) analyses reveal that FXR1 binds with the G4-enriched mRNA targets such as AHNAK, MAP1B, AHNAK2, HUWE1, DYNC1H1 and UBR4 and controls its mRNA expression in cancer cells. Our findings suggest that PRMT5-mediated FXR1 methylation is required for RNA/G4-RNA binding, which promotes gene expression in cancer cells. Thus, FXR1's structural characteristics and affinity for RNAs preferentially G4 regions provide new insights into the molecular mechanism of FXR1 in oral cancer cells.
Assuntos
Arginina , Proliferação de Células , Proteína-Arginina N-Metiltransferases , Proteínas de Ligação a RNA , Humanos , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Arginina/metabolismo , Arginina/genética , Metilação , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Linhagem Celular Tumoral , Ligação Proteica , Quadruplex G , Regulação Neoplásica da Expressão Gênica , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/química , Processamento de Proteína Pós-Traducional , Neoplasias/genética , Neoplasias/metabolismo , Células HEK293 , Estabilidade ProteicaRESUMO
RNA-binding proteins (RBPs) associate with the primary, precursor, and mature microRNAs, which in turn control post-transcriptional gene regulation. Here, by small RNAseq, we show that RBP FXR1 controls the expression of a subset of mature miRNAs, including highly expressed miR301a-3p in oral cancer cells. We also confirm that FXR1 controls the stability of miR301a-3p. Exoribonuclease PNPT1 degrades miR301a-3p in the absence of FXR1 in oral cancer cells, and the degradation is rescued in the FXR1 and PNPT1 co-knockdown cells. In vitro, we show that PNPT1 is unable to bind and degrade the miRNA once the FXR1-miRNA complex forms. Both miR301a-3p and FXR1 cooperatively target the 3'-UTR of p21 mRNA to promote its degradation. Thus, our work illustrates the unique role of FXR1 that is critical for the stability of a subset of mature miRNAs or at least miR301a-3p to target p21 in oral cancer.
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Inibidor de Quinase Dependente de Ciclina p21/genética , MicroRNAs/genética , Neoplasias Bucais/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Neoplasias Bucais/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/genéticaRESUMO
The fragile X-related (FXR) family proteins FMRP, FXR1, and FXR2 are RNA binding proteins that play a critical role in RNA metabolism, neuronal plasticity, and muscle development. These proteins share significant homology in their protein domains, which are functionally and structurally similar to each other. FXR family members are known to play an essential role in causing fragile X mental retardation syndrome (FXS), the most common genetic form of autism spectrum disorder. Recent advances in our understanding of this family of proteins have occurred in tandem with discoveries of great importance to neurological disorders and cancer biology via the identification of their novel RNA and protein targets. Herein, we review the FXR family of proteins as they pertain to FXS, other mental illnesses, and cancer. We emphasize recent findings and analyses that suggest contrasting functions of this protein family in FXS and tumorigenesis based on their expression patterns in human tissues. Finally, we discuss current gaps in our knowledge regarding the FXR protein family and their role in FXS and cancer and suggest future studies to facilitate bench to bedside translation of the findings.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/metabolismo , Neoplasias/metabolismo , Transtornos do Neurodesenvolvimento/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Mutação , Proteínas de Ligação a RNA/genéticaRESUMO
RNA-binding proteins (RBP) regulate numerous aspects of co- and post-transcriptional gene expression in cancer cells. Here, we demonstrate that RBP, fragile X-related protein 1 (FXR1), plays an essential role in cellular senescence by utilizing mRNA turnover pathway. We report that overexpressed FXR1 in head and neck squamous cell carcinoma targets (G-quadruplex (G4) RNA structure within) both mRNA encoding p21 (Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A, Cip1) and the non-coding RNA Telomerase RNA Component (TERC), and regulates their turnover to avoid senescence. Silencing of FXR1 in cancer cells triggers the activation of Cyclin-Dependent Kinase Inhibitors, p53, increases DNA damage, and ultimately, cellular senescence. Overexpressed FXR1 binds and destabilizes p21 mRNA, subsequently reduces p21 protein expression in oral cancer cells. In addition, FXR1 also binds and stabilizes TERC RNA and suppresses the cellular senescence possibly through telomerase activity. Finally, we report that FXR1-regulated senescence is irreversible and FXR1-depleted cells fail to form colonies to re-enter cellular proliferation. Collectively, FXR1 displays a novel mechanism of controlling the expression of p21 through p53-dependent manner to bypass cellular senescence in oral cancer cells.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Senescência Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neoplasias Bucais/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Telomerase/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA , Humanos , Ligação Proteica , RNA/genética , Proteínas de Ligação a RNA/genética , Telomerase/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1006306.].
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The oral mucosal epithelium is typically insulted during chemotherapy and ionizing radiation (IR) therapy and disposed to mucositis, which creates painful inflammation and ulceration in the oral cavity. Oral mucositis alters gene expression patterns, inhibits cellular growth, and initiates cell death in the oral epithelial compartments. Such alterations are governed by several different factors, including transcription factors, RNA-binding proteins, and microRNAs. IR-induced post-transcriptional regulation of RNA-binding proteins exists but is poorly studied in clinically relevant settings. We herein report that the RNA-binding protein human antigen R (HuR) undergoes cleavage modification by caspase-3 following IR-induced oral mucositis and subsequently promotes the expression of the pro-apoptotic factor BAX (Bcl-2-associated X protein), as well as cell death. Further analyses revealed that the HuR cleavage product-1 (HuR-CP1) directly associates and stabilizes the BAX mRNA and concurrently activates the apoptotic pathway. On the other hand, a noncleavable isoform of HuR promotes the clonogenic capacity of primary oral keratinocytes and decreases the effect of IR-induced cell death. Additionally, specific inhibition of caspase-3 by a compound, NSC321205, increases the clonogenic capacity of primary oral keratinocytes and causes increased basal layer cellularity, thickened mucosa, and elevated epithelial cell growth in the tongues of mice with oral mucositis. This protective effect of NSC321205 is mediated by a decrease in caspase-3 activity and the consequent inhibition of HuR cleavage, which reduces the expression of BAX in mice with IR-induced oral mucositis. Thus, we have identified a new molecular mechanism of HuR in the regulation of mRNA turnover and apoptosis in oral mucositis, and our data suggest that blocking the cleavage of HuR enhances cellular growth in the oral epithelial compartment.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Proteínas ELAV/metabolismo , Raios gama/efeitos adversos , Inibidores de Proteases/farmacologia , Lesões Experimentais por Radiação/prevenção & controle , Estomatite/prevenção & controle , Animais , Linhagem Celular , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , RNA Mensageiro/metabolismo , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Estomatite/etiologia , Estomatite/metabolismo , Estomatite/patologia , Proteína X Associada a bcl-2/metabolismoRESUMO
Hu Antigen R, also known as ELAVL1 (HuR), is a key posttranscriptional regulator in eukaryotic cells. HuR overexpression promotes several malignancies, including head and neck squamous cell carcinoma (HNSCC). However, its immune dysfunction-associated tumorigenesis pathways remain unknown. We examined HuR's effects on oral malignancies and immune cell function in vitro and in vivo using oral carcinoma cells and transgenic HuR knockout (KO) mice. CRISPR/Cas9-mediated HuR deletion in mice syngeneic oral cancer cells eliminated colony formation and tumor development. HuR-KO tumors had a lower tumor volume, fewer CD4+CD25+FoxP3+ regulatory T cells, and more CD8+ T cells, suggesting that HuR may suppress the immune response during oral cancer progression. In contrast, HuR KO oral epithelial tissues are resistant to 4NQO-induced oral malignancies compared to control tumor-bearing mice. HuR KO mice showed fewer Tregs and greater IFN levels than WT tumor-bearing mice, suggesting anticancer activity. Finally, the HuR inhibitor pyrvinium pamoate lowers tumor burden by enhancing CD8+ infiltration at the expense of CD4+, suggesting anticancer benefits. Thus, HuR-dependent oral neoplasia relies on immunological dysfunction, suggesting that decreasing HuR may boost antitumor potential and offer a novel HNSCC therapy.
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CELF1 RNA-binding protein, otherwise called CUGBP1, associates and coordinates the degradation of GU-rich element (GRE) containing mRNA's encoding factors important for cell growth, migration and apoptosis. Although many substrates of CELF1 have been identified, the biological significance of CELF1-mediated mRNA decay remains unclear. As the processes modulated by CELF1 are frequently disrupted in cancer, we investigated the expression and role of CELF1 in oral squamous cancer cells (OSCCs). We determined that CELF1 is reproducibly overexpressed in OSCC tissues and cell lines. Moreover, depletion of CELF1 reduced proliferation and increased apoptosis in OSCCs, but had negligible effect in non-transformed cells. We found that CELF1 associates directly with the 3'UTR of mRNAs encoding the pro-apoptotic factors BAD, BAX and JunD and mediates their rapid decay. Specifically, 3'UTR fragment analysis of JunD revealed that the GRE region is critical for binding with CELF1 and expression of JunD in oral cancer cells. In addition, silencing of CELF1 rendered BAD, BAX and JunD mRNAs stable and increased their protein expression in oral cancer cells. Taken together, these results support a critical role for CELF1 in modulating apoptosis and implicate this RNA-binding protein as a cancer marker and potential therapeutic target.
Assuntos
Apoptose , Neoplasias Bucais/patologia , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteínas CELF1 , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Bucais/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Altered expression of RNA-binding proteins modulates gene expression in association with mRNAs encoding many proto-oncogenes, cytokines, chemokines, and proinflammatory factors. Hu antigen R (HuR), a ubiquitously expressed protein, controls a range of cellular functions such as tumor progression, apoptosis, invasion, and metastasis by stabilizing the AU-rich element located at the 3'-untranslated region (UTR) of target mRNAs. Although significant progress has been made in understanding HuR regulation in gene expression, little is known about how HuR undergoes post-translational modifications and recruits target mRNAs during hypoxic stress. Here, we report that during CoCl(2)-induced hypoxic stress, HuR is significantly overexpressed and undergoes caspase-dependent cleavage in head and neck squamous cell carcinoma cells. Unexpectedly, the HuR-cleavage product 1 (HuR-CP1) was found to strongly associate with the 3'-UTR of c-myc mRNA and block mRNA translation. The binding efficiency of HuR to the 3'-UTR of c-myc mRNA was confirmed using ribonucleoprotein immunoprecipitation and site-directed mutagenesis at the AU-rich element sequences of the c-myc mRNA. Overexpression of a non-cleavable isoform, HuR-D226A, revealed a potent dominant-negative effect, repressing cleavage of endogenous HuR and promoting cell viability. Surprisingly, under hypoxia, siRNA knockdown of HuR elevated c-Myc protein expression. These findings suggest an important role for HuR in hypoxia, and we may have revealed a novel post-transcriptional mechanism that controls c-Myc expression in oral cancer progression.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Proteínas ELAV/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Bucais/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Estresse Fisiológico , Regiões 3' não Traduzidas/genética , Carcinoma de Células Escamosas/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Cobalto/farmacologia , Proteínas ELAV/genética , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Bucais/genética , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
The control of eukaryotic gene expression occurs at multiple levels, from transcription to messenger RNA processing, transport, localization, turnover, and translation. RNA-binding proteins control gene expression and are involved in different stages of mRNA processing, including splicing, maturation, turnover, and translation. A ubiquitously expressed RBP Human antigen R is engaged in the RNA processes mentioned above but, most importantly, controls mRNA stability and turnover. Dysregulation of HuR is linked to many diseases, including cancer and other immune-related disorders. HuR targets mRNAs containing AU-rich elements at their 3'untranslated region, which encodes proteins involved in cell growth, proliferation, tumor formation, angiogenesis, immune evasion, inflammation, invasion, and metastasis. HuR overexpression has been reported in many tumor types, which led to a poor prognosis for patients. Hence, HuR is considered an appealing drug target for cancer treatment. Therefore, multiple attempts have been made to identify small molecule inhibitors for blocking HuR functions. This article reviews the current prospects of drugs that target HuR in numerous cancer types, their mode of action, and off-target effects. Furthermore, we will summarize drugs that interfered with HuR-RNA interactions and established themselves as novel therapeutics. We will also highlight the significance of HuR overexpression in multiple cancers and discuss its role in immune functions. This review provides evidence of a new era of HuR-targeted small molecules that can be used for cancer therapeutics either as a monotherapy or in combination with other cancer treatment modalities.
Assuntos
Proteína Semelhante a ELAV 1 , Neoplasias , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Humanos , Neoplasias/patologia , Neovascularização Patológica , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Mitochondria and endoplasmic reticulum (ER) share structural and functional networks and activate well-orchestrated signaling processes to shape cells' fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS promotes mitochondrial function. Strategies to boost antitumor T-cell function by targeting ER-mitochondria cross-talk have not yet been exploited. Here, we used carbon monoxide (CO), a short-lived gaseous molecule, to test whether engaging moderate ERS conditions can improve mitochondrial and antitumor functions in T cells. In melanoma antigen-specific T cells, CO-induced transient activation of ERS sensor protein kinase R-like endoplasmic reticulum kinase (PERK) significantly increased antitumor T-cell function. Furthermore, CO-induced PERK activation temporarily halted protein translation and induced protective autophagy, including mitophagy. The use of LC3-GFP enabled differentiation between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter the process (LC3-GFPneg). LC3-GFPpos T cells showed strong antitumor potential, whereas LC3-GFPneg cells exhibited a T regulatory-like phenotype, harbored dysfunctional mitochondria, and accumulated abnormal metabolite content. These anomalous ratios of metabolites rendered the cells with a hypermethylated state and distinct epigenetic profile, limiting their antitumor activity. Overall, this study shows that ERS-activated autophagy pathways modify the mitochondrial function and epigenetically reprogram T cells toward a superior antitumor phenotype to achieve robust tumor control. SIGNIFICANCE: Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.
Assuntos
Monóxido de Carbono , eIF-2 Quinase , Apoptose , Autofagia , Monóxido de Carbono/farmacologia , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Linfócitos T/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
MAPK phosphatase-1 (MKP-1)/dual specificity protein phosphatase-1 (DUSP-1) is a negative regulator of the host inflammatory response to infection. However, the mechanisms underlying the regulation of cytokine expression by MKP-1, especially at the post-transcriptional level, have not been fully delineated. In the current study, MKP-1 specifically dephosphorylated activated MAPK responses and attenuated LPS-induced IL-6, IL-10, and TNF-α expression. In addition, MKP-1 was important in destabilizing cytokine mRNAs. In LPS-stimulated rat macrophages with overexpressed MKP-1, half-lives of IL-6, IL-10 and TNF-α mRNAs were significantly reduced compared to controls. Conversely, half-lives of IL-6, IL-10, and TNF-α mRNAs were significantly increased in bone marrow macrophages derived from MKP-1 knock out (KO) mice compared with macrophages derived from MKP-1 wild type (WT) mice. Furthermore, MKP-1 promoted translocation of RNA-binding protein (RNA-BP) ARE/poly-(U) binding degradation factor 1 (AUF1) from the nucleus to the cytoplasm in response to LPS stimulation as evidenced by Western blot and immunofluorescent staining. Knockdown AUF1 mRNA expression by AUF1 siRNA in MKP-1 WT bone marrow macrophages significantly delayed degradation of IL-6, IL-10 and TNF- α mRNAs compared with controls. Finally, AUF1 was immunoprecipitated with the RNA complex in cellular lysates derived from bone marrow macrophages of MKP-1 KO vs. WT mice, which had increased AUF1-bound target mRNAs, including IL-6, IL-10, and TNF-α in WT macrophages compared with MKP-1 KO macrophages. Thus, this work provides new mechanistic insight of MKP-1 signaling and regulation of cytokine mRNA stability through RNA binding proteins in response to inflammatory stimuli.
Assuntos
Citocinas/fisiologia , Fosfatase 1 de Especificidade Dupla/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Animais , Western Blotting , Linhagem Celular , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Ribonucleoproteína Nuclear Heterogênea D0 , Camundongos , Camundongos Knockout , Transporte Proteico , Ratos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Exosomes are naturally occurring nanoparticles with unique structure, surface biochemistry, and mechanical characteristics. These distinct nanometer-sized bioparticles are secreted from the surfaces of oral epithelial cells into saliva and are of interest as oral-cancer biomarkers. We use high- resolution AFM to show single-vesicle quantitative differences between exosomes derived from normal and oral cancer patient's saliva. Compared to normal exosomes (circular, 67.4 ± 2.9 nm), our findings indicate that cancer exosome populations are significantly increased in saliva and display irregular morphologies, increased vesicle size (98.3 ± 4.6 nm), and higher intervesicular aggregation. At the single-vesicle level, cancer exosomes exhibit significantly (P < 0.05) increased CD63 surface densities. To our knowledge, it represents the first report detecting single-exosome surface protein variations. Additionally, high-resolution AFM imaging of cancer saliva samples revealed discrete multivesicular bodies with intraluminal exosomes enclosed. We discuss the use of quantitative, nanoscale ultrastructural and surface biomolecular analysis of saliva exosomes at single-vesicle- and single-protein-level sensitivities as a potentially new oral cancer diagnostic.
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Exossomos/química , Nanoestruturas/química , Saliva/química , Humanos , Microscopia de Força Atômica , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/patologia , Propriedades de SuperfícieRESUMO
Control of gene expression is critical in shaping the pro-and eukaryotic organisms' genotype and phenotype. The gene expression regulatory pathways solely rely on protein-protein and protein-nucleic acid interactions, which determine the fate of the nucleic acids. RNA-protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA-protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA-protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.
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Centrosomal P4.1-associated protein (CPAP) plays a critical role in restricting the centriole length in human cells. Here, we report a novel, positive regulatory influence for CPAP on endocytic vesicular transport (EVT) and lysosome targeting of internalized-cell surface receptor EGFR. We observed that higher CPAP levels cause an increase in the abundance of multi-vesicular body (MVB) and EGFR is detectable in CPAP-overexpression induced puncta. The surface and cellular levels of EGFR are higher under CPAP deficiency and lower under CPAP overexpression. While ligand-engagement induced internalization or routing of EGFR into early endosomes is not influenced by cellular levels of CPAP, we found that targeting of ligand-activated, internalized EGFR to lysosome is impacted by CPAP levels. Transport of ligand-bound EGFR from early endosome to late endosome/MVB and lysosome is diminished in CPAP-depleted cells. Moreover, CPAP depleted cells appear to show a diminished ability to form MVB structures upon EGFR activation. These observations suggest a positive regulatory effect of CPAP on EVT of ligand-bound EGFR-like cell surface receptors to MVB and lysosome. Overall, identification of a non-centriolar function of CPAP in endocytic trafficking provides new insights in understanding the non-canonical cellular functions of CPAP.
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Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Vesículas Transportadoras/metabolismo , Linhagem Celular Tumoral , Endocitose , Endossomos/metabolismo , Receptores ErbB/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligantes , Proteínas Associadas aos Microtúbulos/genética , Corpos Multivesiculares/metabolismo , Transporte ProteicoRESUMO
Higher epidermal growth factor receptor (EGFR) signaling can contribute to tumor metastasis and resistance to therapies in oral squamous cell carcinoma (OSCC). EGFR signaling can promote epithelial-mesenchymal transition (EMT) in OSCC. EMT is a process by which epithelial cells acquire invasive properties and it can contribute to tumor metastasis. Not only do the abnormal functions of microtubule and microtubule-organizing centers (MTOC) such as centrosomes lead to cancers, but also the malignant tissues are characterized by aberrant centriolar features and amplified centrosomes. Microtubule inhibition therapies increase the sensitivity to EGFR targeting drugs in various cancers. In this study, we show that the loss of expression of a microtubule/tubulin binding protein, centrosomal protein 4.1-associated protein (CPAP), which is critical for centriole biogenesis and normal functioning of the centrosome, caused an increase in the EGFR levels and its signaling and, enhanced the EMT features and invasiveness of OSCC cells. Further, depletion of CPAP enhanced the tumorigenicity of these cells in a xeno-transplant model. Importantly, CPAP loss-associated EMT features and invasiveness of multiple OSCC cells were attenuated upon depletion of EGFR in them. On the other hand, we found that CPAP protein levels were higher in EGF treated OSCC cells as well as in oral cancer tissues, suggesting that the frequently reported aberrant centriolar features of tumors are potentially a consequence, but not the cause, of tumor progression. Overall, our novel observations show that, in addition to its known indispensable role in centrosome biogenesis, CPAP also plays a vital role in suppressing tumorigenesis in OSCC by facilitating EGFR homeostasis.
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AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway. However, whether and how arginine methylation directly regulates AKT kinase activity remain unknown. Here we report that protein arginine methyltransferase 5 (PRMT5), but not other PRMTs, promotes AKT activation by catalyzing symmetric dimethylation of AKT1 at arginine 391 (R391). Mechanistically, AKT1-R391 methylation cooperates with phosphatidylinositol 3,4,5 trisphosphate (PIP3) to relieve the pleckstrin homology (PH)-in conformation, leading to AKT1 membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the mechanistic target of rapamycin complex 2 (mTORC2). As a result, deficiency in AKT1-R391 methylation significantly suppresses AKT1 kinase activity and tumorigenesis. Lastly, we show that PRMT5 inhibitor synergizes with AKT inhibitor or chemotherapeutic drugs to enhance cell death. Altogether, our study suggests that R391 methylation is an important step for AKT activation and its oncogenic function.
Assuntos
Arginina/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Antineoplásicos/farmacologia , Biocatálise/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Metilação/efeitos dos fármacos , Camundongos Nus , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína-Arginina N-Metiltransferases/deficiência , Proteínas Proto-Oncogênicas c-akt/química , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The RNA-binding protein fragile-X mental retardation autosomal 1 (FXR1) is upregulated in head and neck squamous cell carcinomas (HNSCCs) and expressed as at least seven isoforms in humans. Only two of these isoforms are capable of binding to RNA containing G-quadruplex structures. We suggest that these unique isoforms play a role in the pathogenesis of HNSCC.
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Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Músculos/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , HumanosRESUMO
RNA-binding proteins (RBP) and noncoding RNAs (ncRNA), such as long noncoding RNAs (lncRNA) and microRNAs (miRNA), control co- and posttranscriptional gene regulation (PTR). At the PTR level, RBPs and ncRNAs contribute to pre-mRNA processing, mRNA maturation, transport, localization, turnover, and translation. Deregulation of RBPs and ncRNAs promotes the onset of cancer progression and metastasis. Both RBPs and ncRNAs are altered by signaling cascades to cooperate or compete with each other to bind their nucleic acid targets. Most importantly, transforming growth factor-beta (TGFß) signaling plays a significant role in controlling gene expression patterns by targeting RBPs and ncRNAs. Because of TGFß signaling in cancer, RBP-RNA or RNA-RNA interactions are altered and cause enhanced cell growth and tumor cell dissemination. This review focuses on the emerging concepts of TGFß signaling on posttranscriptional gene regulation and highlights the implications of RBPs and ncRNAs in cancer progression and metastasis. Mol Cancer Res; 16(4); 567-79. ©2018 AACR.
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MicroRNAs/genética , Neoplasias/genética , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Neoplásica , Neoplasias/metabolismo , Processamento Pós-Transcricional do RNA , Transdução de SinaisRESUMO
PURPOSE: Human papillomavirus (HPV) infected oropharyngeal squamous cell carcinoma (OPSCC) patients have a better prognosis compared to HPV(-) counterparts. However, a subset of HPV(+) patients with a smoking history fail to respond to the standard of care treatments such as radiation and chemotherapy. To understand the underlying mechanism driving HPV(+) OPSCC patient resistance to treatment and recurrence, we sought to identify and characterize the differentially expressed miRNAs and their target genes in HPV(+) smokers and non-smokers. EXPERIMENTAL DESIGN: MicroRNA expression analysis was performed using Nanostring in tumor tissues isolated from a prospective cohort of HPV(+) smoking (n = 9) and HPV(+) (n = 13) non-smoking OPSCC patients. Identified miRNAs of interest were further validated using qRT-PCR in cigarette smoke extract (CSE) treated HPV(+) and E6/E7 overexpressing HPV(-) cells. RESULTS: In comparison to OPSCC HPV(+) non-smokers, 38 miRNAs were significantly altered in the HPV(+) smoker patients cohort and out of that 9 were downregulated. Altered miRNA expression was also detected in the serum and metastatic lymph nodes of HPV(+) smokers versus non-smokers. Expression of miR-133a-3p was significantly downregulated in OPSCC smokers, HPV(+) cells and E6/E7 overexpressing HPV(-) cells treated with CSE. Reduction of miR-133a-3p induced the upregulation of miR-133a-3p target mRNAs EGFR and HuR. CONCLUSIONS: Our results indicate that miR-133a-3p is a target of smoking-induced changes in HPV(+) patients and alters the expression of EGFR and HuR which may promote HPV associated oropharyngeal cancer. Therefore, future treatment strategies for HPV(+) OPSCC smokers should focus on EGFR inhibition and the development of selective therapies to target HuR.