RESUMO
Elevated mitochondrial metabolism promotes tumorigenesis of Embryonal Rhabdomyosarcomas (ERMS). Accordingly, targeting oxidative phosphorylation (OXPHOS) could represent a therapeutic strategy for ERMS. We previously demonstrated that genetic reduction of Staufen1 (STAU1) levels results in the inhibition of ERMS tumorigenicity. Here, we examined STAU1-mediated mechanisms in ERMS and focused on its potential involvement in regulating OXPHOS. We report the novel and differential role of STAU1 in mitochondrial metabolism in cancerous versus non-malignant skeletal muscle cells (NMSkMCs). Specifically, our data show that STAU1 depletion reduces OXPHOS and inhibits proliferation of ERMS cells. Our findings further reveal the binding of STAU1 to several OXPHOS mRNAs which affects their stability. Indeed, STAU1 depletion reduced the stability of OXPHOS mRNAs, causing inhibition of mitochondrial metabolism. In parallel, STAU1 depletion impacted negatively the HIF2α pathway which further modulates mitochondrial metabolism. Exogenous expression of HIF2α in STAU1-depleted cells reversed the mitochondrial inhibition and induced cell proliferation. However, opposite effects were observed in NMSkMCs. Altogether, these findings revealed the impact of STAU1 in the regulation of mitochondrial OXPHOS in cancer cells as well as its differential role in NMSkMCs. Overall, our results highlight the therapeutic potential of targeting STAU1 as a novel approach for inhibiting mitochondrial metabolism in ERMS.
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Rabdomiossarcoma Embrionário , Humanos , Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/tratamento farmacológico , Rabdomiossarcoma Embrionário/metabolismo , Proteínas do Citoesqueleto/metabolismo , Transformação Celular Neoplásica , Carcinogênese/genética , Proliferação de Células/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The development and regeneration of skeletal muscle are mediated by satellite cells (SCs), which ensure the efficient formation of myofibers while repopulating the niche that allows muscle repair following injuries. Pannexin 1 (Panx1) channels are expressed in SCs and their levels increase during differentiation in vitro, as well as during skeletal muscle development and regeneration in vivo. Panx1 has recently been shown to regulate muscle regeneration by promoting bleb-based myoblast migration and fusion. While skeletal muscle is largely influenced in a sex-specific way, the sex-dependent roles of Panx1 in regulating skeletal muscle and SC function remain to be investigated. Here, using global Panx1 knockout (KO) mice, we demonstrate that Panx1 loss reduces muscle fiber size and strength, decreases SC number, and alters early SC differentiation and myoblast fusion in male, but not in female mice. Interestingly, while both male and female Panx1 KO mice display an increase in the number of regenerating fibers following acute injury, the newly formed fibers in male Panx1 KO mice are smaller. Overall, our results demonstrate that Panx1 plays a significant role in regulating muscle development, regeneration, and SC number and function in male mice and reveal distinct sex-dependent functions of Panx1 in skeletal muscle.
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Mioblastos , Células Satélites de Músculo Esquelético , Animais , Diferenciação Celular , Conexinas/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas , Músculo Esquelético , Proteínas do Tecido Nervoso/genéticaRESUMO
Pannexin 1 (Panx1) and Pannexin 3 (Panx3) are single membrane channels recently implicated in myogenic commitment, as well as myoblast proliferation and differentiation in vitro. However, their expression patterns during skeletal muscle development and regeneration had yet to be investigated. Here, we show that Panx1 levels increase during skeletal muscle development becoming highly expressed together with Panx3 in adult skeletal muscle. In adult mice, Panx1 and Panx3 were differentially expressed in fast- and slow-twitch muscles. We also report that Panx1/PANX1 and Panx3/PANX3 are co-expressed in mouse and human satellite cells, which play crucial roles in skeletal muscle regeneration. Interestingly, Panx1 and Panx3 levels were modulated in muscle degeneration/regeneration, similar to the pattern seen during skeletal muscle development. As Duchenne muscular dystrophy is characterized by skeletal muscle degeneration and impaired regeneration, we next used mild and severe mouse models of this disease and found a significant dysregulation of Panx1 and Panx3 levels in dystrophic skeletal muscles. Together, our results are the first demonstration that Panx1 and Panx3 are differentially expressed amongst skeletal muscle types with their levels being highly modulated during skeletal muscle development, regeneration, and dystrophy. These findings suggest that Panx1 and Panx3 channels may play important and distinct roles in healthy and diseased skeletal muscles.
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Conexinas/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Camundongos , Mioblastos/metabolismo , Regeneração/fisiologiaRESUMO
Pannexins are newly discovered channels that are now recognized as mediators of adenosine triphosphate release from several cell types allowing communication with the extracellular environment. Pannexins have been associated with various physiological and pathological processes including apoptosis, inflammation, and cancer. However, it is only recently that our work has unveiled a role for Pannexin 1 and Pannexin 3 as novel regulators of skeletal muscle myoblast proliferation and differentiation. Myoblast differentiation is an ordered multistep process that includes withdrawal from the cell cycle and the expression of key myogenic factors leading to myoblast differentiation and fusion into multinucleated myotubes. Eventually, myotubes will give rise to the diverse muscle fiber types that build the complex skeletal muscle architecture essential for body movement, postural behavior, and breathing. Skeletal muscle cell proliferation and differentiation are crucial processes required for proper skeletal muscle development during embryogenesis, as well as for the postnatal skeletal muscle regeneration that is necessary for muscle repair after injury or exercise. However, defects in skeletal muscle cell differentiation and/or deregulation of cell proliferation are involved in various skeletal muscle pathologies. In this review, we will discuss the expression of pannexins and their post-translational modifications in skeletal muscle, their known functions in various steps of myogenesis, including myoblast proliferation and differentiation, as well as their possible roles in skeletal muscle development, regeneration, and diseases such as Duchenne muscular dystrophy.
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Conexinas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Diferenciação Celular , Proliferação de Células , Conexinas/genética , Expressão Gênica , Humanos , Camundongos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/citologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Mioblastos/citologia , Proteínas do Tecido Nervoso/genética , FosforilaçãoRESUMO
Pannexins constitute a family of three glycoproteins (Panx1, -2, and -3) forming single membrane channels. Recent work demonstrated that Panx1 is expressed in skeletal muscle and involved in the potentiation of contraction. However, Panxs functions in skeletal muscle cell differentiation, and proliferation had yet to be assessed. We show here that Panx1 and Panx3, but not Panx2, are present in human and rodent skeletal muscle, and their various species are differentially expressed in fetal versus adult human skeletal muscle tissue. Panx1 levels were very low in undifferentiated human primary skeletal muscle cells and myoblasts (HSMM) but increased drastically during differentiation and became the main Panx expressed in differentiated cells. Using HSMM, we found that Panx1 expression promotes this process, whereas it was impaired in the presence of probenecid or carbenoxolone. As for Panx3, its lower molecular weight species were prominent in adult skeletal muscle but very low in the fetal tissue and in undifferentiated skeletal muscle cells and myoblasts. Its overexpression (â¼43-kDa species) induced HSMM differentiation and also inhibited their proliferation. On the other hand, a â¼70-kDa immunoreactive species of Panx3, likely glycosylated, sialylated, and phosphorylated, was highly expressed in proliferative myoblasts but strikingly down-regulated during their differentiation. Reduction of its endogenous expression using two Panx3 shRNAs significantly inhibited HSMM proliferation without triggering their differentiation. In summary, our results demonstrate that Panx1 and Panx3 are co-expressed in human skeletal muscle myoblasts and play a pivotal role in dictating the proliferation and differentiation status of these cells.
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Diferenciação Celular , Proliferação de Células , Conexinas/fisiologia , Mioblastos Esqueléticos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Carbenoxolona/farmacologia , Diferenciação Celular/efeitos dos fármacos , Conexinas/metabolismo , Glicosilação , Células HEK293 , Humanos , Desenvolvimento Muscular , Músculo Esquelético/citologia , Fosforilação , Probenecid/farmacologia , Processamento de Proteína Pós-Traducional , RatosRESUMO
BACKGROUND: Pulmonary vascular disease (PVD) complicated with pulmonary hypertension (PH) is a leading cause of mortality in congenital diaphragmatic hernia (CDH). Unfortunately, CDH patients are often resistant to PH therapy. Using the nitrogen CDH rat model, we previously demonstrated that CDH-associated PVD involves an induction of elastase and matrix metalloproteinase (MMP) activities, increased osteopontin and epidermal growth factor (EGF) levels, and enhanced smooth muscle cell (SMC) proliferation. Here, we aimed to determine whether the levels of the key members of this proteinase-induced pathway are also elevated in the pulmonary arteries (PAs) of CDH patients. METHODS: Neutrophil elastase (NE), matrix metalloproteinase-2 (MMP-2), epidermal growth factor (EGF), tenascin-C, and osteopontin levels were assessed by immunohistochemistry in the PAs from the lungs of 11 CDH patients and 5 normal age-matched controls. Markers of proliferation (proliferating cell nuclear antigen (PCNA)) and apoptosis (cleaved (active) caspase-3) were also used. RESULTS: While expressed by both control and CDH lungs, the levels of NE, MMP-2, EGF, as well as tenascin-C and osteopontin were significantly increased in the PAs from CDH patients. The percentage of PCNA-positive PA SMCs were also enhanced, while those positive for caspase-3 were slightly decreased. CONCLUSIONS: These results suggest that increased elastase and MMPs, together with elevated tenascin-C and osteopontin levels in an EGF-rich environment may contribute to the PVD in CDH infants. The next step of this study is to expand our analysis to a larger cohort, and determine the potential of targeting this pathway for the treatment of CDH-associated PVD and PH. TYPE OF STUDY: Therapeutic. LEVEL OF EVIDENCE: LEVEL III.
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Hérnias Diafragmáticas Congênitas , Hipertensão Pulmonar , Doenças Vasculares , Humanos , Ratos , Animais , Hérnias Diafragmáticas Congênitas/complicações , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 2 da Matriz/metabolismo , Artéria Pulmonar , Osteopontina/metabolismo , Caspase 3/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Elastase Pancreática/metabolismo , Fator de Crescimento Epidérmico , Tenascina/metabolismo , Pulmão/metabolismo , Hipertensão Pulmonar/complicações , Metaloproteinases da Matriz , Doenças Vasculares/complicações , Éteres Fenílicos/metabolismoRESUMO
BACKGROUND: Duchenne muscular dystrophy (DMD) is associated with impaired muscle regeneration, progressive muscle weakness, damage, and wasting. While the cause of DMD is an X-linked loss of function mutation in the gene encoding dystrophin, the exact mechanisms that perpetuate the disease progression are unknown. Our laboratory has demonstrated that pannexin 1 (Panx1 in rodents; PANX1 in humans) is critical for the development, strength, and regeneration of male skeletal muscle. In normal skeletal muscle, Panx1 is part of a multiprotein complex with dystrophin. We and others have previously shown that Panx1 levels and channel activity are dysregulated in various mouse models of DMD. METHODS: We utilized myoblast cell lines derived from DMD patients to assess PANX1 expression and function. To investigate how Panx1 dysregulation contributes to DMD, we generated a dystrophic (mdx) mouse model that lacks Panx1 (Panx1-/-/mdx). In depth characterization of this model included histological analysis, as well as locomotor, and physiological tests such as muscle force and grip strength assessments. RESULTS: Here, we demonstrate that PANX1 levels and channel function are reduced in patient-derived DMD myoblast cell lines. Panx1-/-/mdx mice have a significantly reduced lifespan, and decreased body weight due to lean mass loss. Their tibialis anterior were more affected than their soleus muscles and displayed reduced mass, myofiber loss, increased centrally nucleated myofibers, and a lower number of muscle stem cells compared to that of Panx1+/+/mdx mice. These detrimental effects were associated with muscle and locomotor functional impairments. In vitro, PANX1 overexpression in patient-derived DMD myoblasts improved their differentiation and fusion. CONCLUSIONS: Collectively, our findings suggest that PANX1/Panx1 dysregulation in DMD exacerbates several aspects of the disease. Moreover, our results suggest a potential therapeutic benefit to increasing PANX1 levels in dystrophic muscles.
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Conexinas , Camundongos Endogâmicos mdx , Músculo Esquelético , Distrofia Muscular de Duchenne , Proteínas do Tecido Nervoso , Animais , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Conexinas/genética , Conexinas/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Humanos , Camundongos , Mioblastos/metabolismo , Linhagem Celular , Força Muscular , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Pannexin 1 (PANX1) is expressed in many tissue types including tissues of neural origin. Neuroblastoma (NB) is a neural crest-derived malignancy mainly occurring in children. The majority of NB patients present with high-risk disease for which current therapies are ineffective. Here, we show that while PANX1 is expressed in NB of all stages, high PANX1 expression in high-risk NB is associated with a reduced survival probability. PANX1 channel inhibition using probenecid (PBN) or carbenoxolone (CBX) reduced the proliferation of our panel of high-risk NB cell lines. We show that expression of the Y10F PANX1 mutant, which cannot be phosphorylated on tyrosine 10 and acts in a dominant-negative manner, curtailed NB cell proliferation. Furthermore, PBN and CBX treatment halted the growth of NB spheroids and in some cases triggered the regression of established NB spheroids. Finally, both drugs reduced the progression of high-risk NB in vivo. Together our data indicate that PANX1 channels regulate human NB malignant properties and that the use of PBN or CBX may provide a new therapeutic approach for high-risk NB.
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Pannexin (Panx) 1 and Panx3 are integral membrane proteins that share some sequence homology with the innexin family of invertebrate gap junctions. They are expressed in mammalian skin. Pannexins have been reported to form functional mechanosensitive single-membrane channels, but their importance in regulating cellular function is poorly understood. In this study, Panx1 and Panx3 were detected in the epidermis of 13.5 day embryonic mice. Compared with newborn mice, there was less Panx1 expression in both thin and thick murine skin, whereas Panx3 expression was unchanged. To investigate the role of pannexins in keratinocyte differentiation, we employed rat epidermal keratinocytes (REKs) that have the capacity to differentiate into organotypic epidermis, and engineered them to overexpress Panx1, Panx1-GFP or Panx3. The expression of Panx1 or Panx3 resulted in the increased ability of REKs to take up dye, suggesting that cell-surface channels were formed. Compared with monolayer REKs, endogenous Panx1 levels remained unchanged, whereas the 70 kDa immunoreactive species of Panx3 was greatly increased in the organotypic epidermis. In monolayer cultures, ectopic Panx1 and Panx1-GFP localized to the plasma membrane, whereas Panx3 displayed both intracellular and plasma-membrane profiles. Although both pannexins reduced cell proliferation, only Panx1 disrupted the architecture of the organotypic epidermis and markedly dysregulated cytokeratin 14 expression and localization. Furthermore, ectopic expression of only Panx1 reduced the vital layer thickness of the organotypic epidermis. In summary, Panx1 and Panx3 are coexpressed in the mammalian epidermis, and the regulation of Panx1 plays a key role in keratinocyte differentiation.
Assuntos
Diferenciação Celular , Conexinas/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular , Movimento Celular , Proliferação de Células , Corantes/metabolismo , Epiderme/patologia , Queratinas/metabolismo , Camundongos , Peso Molecular , Transporte Proteico , Ratos , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: Adequate tissue biopsy is essential for diagnosis and risk stratification of neuroblastoma (NB). Historically, NB diagnosis has relied on tissue obtained via surgical biopsy. However, core needle biopsy may provide a safe and adequate method of obtaining tissue in pediatric patients. AIM: The aim of this study is to compare the adequacy and safety between core needle biopsy and surgical biopsy for the diagnosis of NB in children at our institution. METHODS: Institutional approval was obtained. Medical records of patients diagnosed with NB from 2004 - 2019 were retrospectively reviewed. Patients had either core needle biopsy (CNB) or surgical biopsy (SB) including open/minimally invasive biopsy. Data included patient demographics, tumor location and size, sample adequacy for diagnosis and risk stratification, post-biopsy complications, length of hospital stay, and need for repeat biopsy. Statistical analysis was conducted using the Mann-Whitney U test or Student's t-test. RESULTS: Thirty-eight patients were included; 53 biopsies were performed including 41 SB and 12 CNB. Patient and tumor characteristics were similar in both groups, as well as the biopsy adequacy for diagnosis and risk stratification. In all cases, there was no need for repeat biopsy. The CNB group demonstrated reduced length of stay (2 ± 0.4 days vs 5 ± 0.5 days; P < 0.0001) and fewer complications (8%) than the SB group (44%) (P = 0.038). CONCLUSION: Core needle biopsy is an acceptable modality for diagnosis and risk stratification in the pediatric population. Advantages include decreased length of stay and fewer post-procedure complications.
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Neuroblastoma , Biópsia/métodos , Biópsia com Agulha de Grande Calibre/efeitos adversos , Biópsia com Agulha de Grande Calibre/métodos , Criança , Humanos , Tempo de Internação , Neuroblastoma/diagnóstico , Neuroblastoma/patologia , Neuroblastoma/cirurgia , Estudos RetrospectivosRESUMO
Horseshoe lung (HL) is a rare congenital anomaly that has been classically associated with Scimitar syndrome. Very few cases have been described in the context of the VACTERL spectrum. We present a case of a newborn girl with mesocardia, tracheoesophageal fistula, and imperforated anus, who required O2 support at birth and during hospitalization. A chest CT angiography revealed a HL as an incidental finding. We suspect that HL and the VACTERL spectrum, are not separated entities but likely a further expansion of VACTERL-associated symptoms. HL might be underdiagnosed in asymptomatic patients as Chest CT angiography is not part of the routine work up for patients with VACTERL association.
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Rhabdomyosarcoma (RMS) is a deadly cancer of skeletal muscle origin. Pannexin 1 (PANX1) is down-regulated in RMS and increasing its levels drastically inhibits RMS progression. PANX1 upregulation thus represents a prospective new treatment strategy for this malignancy. However, the mechanisms regulating PANX1 expression, in RMS and other contexts, remain largely unknown. Here we show that both RMS and normal skeletal muscle express a comparable amount of PANX1 mRNAs, but surprisingly the canonical 5' untranslated region (5' UTR) or 5' leader of the transcript is completely lost in RMS. We uncover that quercetin, a natural plant flavonoid, increases PANX1 protein levels in RMS by inducing re-expression of a 5' leader-containing PANX1 transcript variant that is efficiently translated. This particular PANX1 mRNA variant is also present in differentiated human skeletal muscle myoblasts (HSMM) that highly express PANX1. Mechanistically, abolishing ETV4 transcription factor binding sites in the PANX1 promoter significantly reduced the luciferase reporter activities and PANX1 5' UTR levels, and both quercetin treatment in RMS cells and induction of differentiation in HSMM enriched the binding of ETV4 to its consensus element in the PANX1 promoter. Notably, quercetin treatment promoted RMS differentiation in a PANX1-dependent manner. Further showing its therapeutic potential, quercetin treatment prevented RMS in vitro tumor formation while inducing complete regression of established spheroids. Collectively, our results demonstrate the tumor-suppressive effects of quercetin in RMS and present a hitherto undescribed mechanism of PANX1 regulation via ETV4-mediated transcription of a translationally functional 5' leader-containing PANX1 mRNA.
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PURPOSE: Recent work has highlighted the therapeutic potential of targeting autophagy to modulate cell survival in a variety of diseases including cancer. Recently, we found that the RNA-binding protein Staufen1 (STAU1) is highly expressed in alveolar rhabdomyosarcoma (ARMS) and that this abnormal expression promotes tumorigenesis. Here, we asked whether STAU1 is involved in the regulation of autophagy in ARMS cells. METHODS: We assessed the impact of STAU1 expression modulation in ARMS cell lines (RH30 and RH41), non-transformed skeletal muscle cells (C2C12) and STAU1-transgenic mice using complementary techniques. RESULTS: We found that STAU1 silencing reduces autophagy in the ARMS cell lines RH30 and RH41, while increasing their apoptosis. Mechanistically, this inhibitory effect was found to be caused by a direct negative impact of STAU1 depletion on the stability of Beclin-1 (BECN1) and ATG16L1 mRNAs, as well as by an indirect inhibition of JNK signaling via increased expression of Dual specificity phosphatase 8 (DUSP8). Pharmacological activation of JNK or expression silencing of DUSP8 was sufficient to restore autophagy in STAU1-depleted cells. By contrast, we found that STAU1 downregulation in non-transformed skeletal muscle cells activates autophagy in a mTOR-dependent manner, without promoting apoptosis. A similar effect was observed in skeletal muscles obtained from STAU1-overexpressing transgenic mice. CONCLUSIONS: Together, our data indicate an effect of STAU1 on autophagy regulation in ARMS cells and its differential role in non-transformed skeletal muscle cells. Our findings suggest a cancer-specific potential of targeting STAU1 for the treatment of ARMS.
Assuntos
Autofagia/genética , Proteínas do Citoesqueleto/genética , Perfilação da Expressão Gênica/métodos , Músculo Esquelético/metabolismo , Proteínas de Ligação a RNA/genética , Rabdomiossarcoma Alveolar/genética , Animais , Apoptose/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Músculo Esquelético/citologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismoRESUMO
Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is an aggressive cancer with a poor prognosis. Despite current management, the 5-year survival rate for patients with metastatic RMS is â¼30%; underscoring the need to develop better treatment strategies. We have recently reported that pannexin 1 (PANX1) levels are downregulated in RMS and that restoring its expression inhibits RMS progression. Here, we have surveyed and characterized the molecular changes induced by PANX1 re-expression in RMS. We cataloged transcriptomic changes in this context by RNA sequencing. At the protein level, we unveiled PANX1 interactors using BioID, complemented by co-immunoprecipitation coupled to high-performance liquid chromatography/electrospray ionization tandem mass spectrometry performed in PANX1-enriched fractions. Using these data, we generated searchable public databases for the PANX1 interactome and changes to the RMS transcriptome occurring when PANX1 expression is restored. STRING network analyses revealed a PANX1 interactome involving plasma membrane and cytoskeleton-associated proteins including the previously undescribed interactor AHNAK. Indeed, AHNAK knockdown abrogated the PANX1-mediated reduction in RMS cell viability and migration. Using these unbiased approaches, we bring insight to the mechanisms by which PANX1 inhibits RMS progression, identifying the cell migration protein AHNAK as a key modifier of PANX1-mediated changes in RMS malignant properties.
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Conexinas/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Rabdomiossarcoma/genética , Transcriptoma/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Mapas de Interação de Proteínas/genética , RNA-Seq , Rabdomiossarcoma/patologia , Sequenciamento do ExomaRESUMO
Residual cell-intrinsic innate immunity in cancer cells hampers infection with oncolytic viruses. Translational control of mRNA is an important feature of innate immunity, yet the identity of translationally regulated mRNAs functioning in host defense remains ill-defined. We report the translatomes of resistant murine "4T1" breast cancer cells infected with three of the most clinically advanced oncolytic viruses: herpes simplex virus 1, reovirus, and vaccinia virus. Common among all three infections are translationally de-repressed mRNAs, including Inpp5e, encoding an inositol 5-phosphatase that modifies lipid second messenger signaling. We find that viral infection induces the expression of an Inpp5e mRNA variant that lacks repressive upstream open reading frames (uORFs) within its 5' leader and is efficiently translated. Furthermore, we show that INPP5E contributes to antiviral immunity by altering virus attachment. These findings uncover a role for translational control through alternative 5' leader expression and assign an antiviral function to the ciliopathy gene Inpp5e.
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Regiões 5' não Traduzidas/genética , Neoplasias Mamárias Animais/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/patogenicidade , Monoéster Fosfórico Hidrolases/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Animais , Feminino , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/virologia , Camundongos , Fases de Leitura Aberta , Monoéster Fosfórico Hidrolases/genética , RNA Mensageiro/genética , Ribossomos/metabolismoRESUMO
Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma of childhood thought to arise from impaired differentiation of skeletal muscle progenitors. We have recently identified Pannexin 1 (PANX1) channels as a novel regulator of skeletal myogenesis. In the present study, we determined that PANX1 transcript and protein levels are down-regulated in embryonal (eRMS) and alveolar RMS (aRMS) patient-derived cell lines and primary tumor specimens as compared to differentiated skeletal muscle myoblasts and tissue, respectively. While not sufficient to overcome the inability of RMS to reach terminal differentiation, ectopic expression of PANX1 in eRMS (Rh18) and aRMS (Rh30) cells significantly decreased their proliferative and migratory potential. Furthermore, ectopic PANX1 abolished 3D spheroid formation in eRMS and aRMS cells and induced regression of established spheroids through induction of apoptosis. Notably, PANX1 expression also significantly reduced the growth of human eRMS and aRMS tumor xenografts in vivo. Interestingly, PANX1 does not form active channels when expressed in eRMS (Rh18) and aRMS (Rh30) cells and the addition of PANX1 channel inhibitors did not alter or reverse the PANX1-mediated reduction of cell proliferation and migration. Moreover, expression of channel-defective PANX1 mutants not only disrupted eRMS and aRMS 3D spheroids, but also inhibited in vivo RMS tumor growth. Altogether our findings suggest that PANX1 alleviates RMS malignant properties in vitro and in vivo through a process that is independent of its canonical channel function.
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BACKGROUND/PURPOSE: Pulmonary vascular disease (PVD) is a leading cause of congenital diaphragmatic hernia (CDH) mortality. Progression of PVD involves extracellular matrix remodeling by elastases and matrix metalloproteinases (MMP), concomitant with proliferation of smooth muscle cells in a growth factor-enriched environment. Blockade of this pathway reversed primary pulmonary hypertension and improved survival. This study was designed to determine whether a similar pathway is induced in PVD secondary to CDH. METHODS: Fetal rats exposed to nitrofen at gestational day 9 developed left-sided CDH and were compared at term to their non-CDH littermates by assessing histologic and biochemical features of PVD. RESULTS: Rats with CDH displayed right ventricle hypertrophy, increased pulmonary artery medial wall thickness and muscularization, and decreased lumen size. As revealed by in situ zymography and immunohistochemistry, this was associated with an induction of elastolytic and MMP activities as well as an elevation of epidermal growth factor and osteopontin levels in the diseased lung vasculature. CONCLUSIONS: CDH-associated PVD involves an induction of elastase and MMP activities and increased osteopontin deposition in an epidermal growth factor-rich environment. Inhibition of this pathway may thus represent a novel therapeutic approach for the treatment of CDH-associated PVD. LEVEL OF EVIDENCE: Level I (Basic Science Study).
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Hérnias Diafragmáticas Congênitas/complicações , Hipertensão Pulmonar/etiologia , Metaloproteinases da Matriz/metabolismo , Elastase Pancreática/metabolismo , Animais , Biomarcadores/metabolismo , Feminino , Hérnias Diafragmáticas Congênitas/induzido quimicamente , Hérnias Diafragmáticas Congênitas/enzimologia , Hipertensão Pulmonar/enzimologia , Osteopontina/metabolismo , Éteres Fenílicos , Ratos , Ratos Sprague-DawleyRESUMO
Rhabdomyosarcoma is the most common soft tissue sarcoma in children and young adults. Rhabdomyosarcomas are skeletal muscle-like tumours that typically arise in muscle beds, and express key myogenic regulatory factors. However, their developmental program remains blocked in the proliferative phase with cells unable to exit the cell cycle to fuse into myotubes. Recently, we uncovered a key role for the RNA-binding protein Staufen1 during myogenic differentiation through the regulation of c-myc translation. Given the known implication of c-myc in rhabdomyosarcoma, we hypothesized in the current work that Staufen1 controls rhabdomyosarcoma tumorigenesis. Here, we report for the first time the novel role of Staufen1 in cancer, specifically in rhabdomyosarcoma. We demonstrate that Staufen1 is markedly upregulated in human rhabdomyosarcoma tumours and cell lines as compared to normal skeletal muscle. Moreover, we show that Staufen1 promotes the tumorigenesis of embryonal and alveolar rhabdomyosarcoma subtypes both in cell culture and in animal models. Finally, our data demonstrate that Staufen1 has differential roles in embryonal versus alveolar rhabdomyosarcoma through the control of proliferative and apoptotic pathways, respectively. Together, these results provide the first evidence for Staufen1's direct implication in cancer biology. Accordingly, Staufen1 thus represents a novel target for the development of future therapeutic strategies for rhabdomyosarcoma.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ligação a RNA/metabolismo , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Embrionário/metabolismo , Animais , Apoptose , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos SCID , Invasividade Neoplásica , Rabdomiossarcoma Alveolar/patologia , Rabdomiossarcoma Embrionário/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Rhabdomyosarcoma (RMS), a malignant neoplasm of presumed mesenchymal origin, is the most common soft tissue cancer of childhood. Despite aggressive treatment, resistance to current therapies remains a challenge. The success of most cytotoxic chemotherapies requires intact programmed cell death (apoptosis) pathways. Defects in the cellular apoptotic program play a key role in the pathogenesis of RMS and contribute to chemotherapeutic resistance to current treatments. Targeting and engaging apoptotic pathways using small-molecule IAP antagonists, death-inducing ligands, reestablishing pannexin channel expression and activity, immunotherapies, or a combination of these approaches is expected to improve outcomes in RMS patients. There is a clear need to better understand the molecular basis of apoptotic resistance in RMS, which may provide an opportunity to identify the patients most likely to benefit from targeted treatments, and for the discovery of novel therapies.