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Tumor hypoxia has been associated with cancer progression, angiogenesis, and metastasis via modifications in the release and cargo composition of extracellular vesicles secreted by tumor cells. Indeed, hypoxic extracellular vesicles are known to trigger a variety of angiogenic responses via different mechanisms. We recently showed that hypoxia promotes endosomal signaling in tumor cells via HIF-1α-dependent induction of the guanine exchange factor ALS2, which activates Rab5, leading to downstream events involved in cell migration and invasion. Since Rab5-dependent signaling is required for endothelial cell migration and angiogenesis, we explored the possibility that hypoxia promotes the release of small extracellular vesicles containing ALS2, which in turn activate Rab5 in recipient endothelial cells leading to pro-angiogenic properties. In doing so, we found that hypoxia promoted ALS2 expression and incorporation as cargo within small extracellular vesicles, leading to subsequent transfer to recipient endothelial cells and promoting cell migration, tube formation, and downstream Rab5 activation. Consequently, ALS2-containing small extracellular vesicles increased early endosome size and number in recipient endothelial cells, which was followed by subsequent sequestration of components of the ß-catenin destruction complex within endosomal compartments, leading to stabilization and nuclear localization of ß-catenin. These events converged in the expression of ß-catenin target genes involved in angiogenesis. Knockdown of ALS2 in donor tumor cells precluded its incorporation into small extracellular vesicles, preventing Rab5-downstream events and endothelial cell responses, which depended on Rab5 activity and guanine exchange factor activity of ALS2. These findings indicate that vesicular ALS2, secreted in hypoxia, promotes endothelial cell events leading to angiogenesis. Finally, these events might explain how tumor angiogenesis proceeds in hypoxic conditions.
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Movimiento Celular , Vesículas Extracelulares , Factores de Intercambio de Guanina Nucleótido , Transducción de Señal , beta Catenina , Proteínas de Unión al GTP rab5 , Humanos , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión al GTP rab5/genética , beta Catenina/metabolismo , Vesículas Extracelulares/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Línea Celular TumoralRESUMEN
Nearly 90% of oral cancers are characterized as oral squamous cell carcinoma (OSCC), representing the sixth most common type of cancer. OSCC usually evolves from oral potentially malignant disorders that, in some cases, are histologically consistent with a oral dysplasia. The levels of 1α,25 dihydroxyvitamin D3 (1,25-(OH)2D3; calcitriol), the active form of vitamin D3, have been shown to be decreased in patients with oral dysplasia and OSCC. Moreover, treatment with 1,25-(OH)2D3 has been proven beneficial in OSCC by inhibiting the Wnt/ß-catenin pathway, a signaling route that promotes cell migration, proliferation, and viability. However, whether this inhibition mechanism occurs in oral dysplasia is unknown. To approach this question, we used dysplastic oral keratinocyte cultures and oral explants (ex vivo model of oral dysplasia) treated with 1,25-(OH)2D3 for 48 h. Following treatment with 1,25-(OH)2D3, both in vitro and ex vivo models of oral dysplasia showed decreased levels of nuclear ß-catenin by immunofluorescence (IF) and immunohistochemistry (IHC). Consistently, reduced protein and mRNA levels of the Wnt/ß-catenin target gene survivin were observed after treatment with 1,25-(OH)2D3. Moreover, 1,25-(OH)2D3 promoted membranous localization of E-cadherin and nuclear localization of vitamin D receptor (VDR). Functionally, DOK cells treated with 1,25-(OH)2D3 displayed diminished cell migration and viability in vitro.
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PURPOSE: Oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, referred to as oral dysplasia. We recently reported that oral dysplasia is associated with aberrant activation of the Wnt/ß-catenin pathway, due to overexpression of Wnt ligands in a Porcupine (PORCN)-dependent manner. Pharmacologic inhibition of PORCN precludes Wnt secretion and has been proposed as a potential therapeutic approach to treat established cancers. Nevertheless, there are no studies that explore the effects of PORCN inhibition at the different stages of oral carcinogenesis. EXPERIMENTAL DESIGN: We performed a model of tobacco-induced oral cancer in vitro, where dysplastic oral keratinocytes (DOK) were transformed into oral carcinoma cells (DOK-TC), and assessed the effects of inhibiting PORCN with the C59 inhibitor. Similarly, an in vivo model of oral carcinogenesis and ex vivo samples derived from patients diagnosed with oral dysplasia and OSCC were treated with C59. RESULTS: Both in vitro and ex vivo oral carcinogenesis approaches revealed decreased levels of nuclear ß-catenin and Wnt3a, as observed by immunofluorescence and IHC analyses. Consistently, reduced protein and mRNA levels of survivin were observed after treatment with C59. Functionally, treatment with C59 in vitro resulted in diminished cell migration, viability, and invasion. Finally, by using an in vivo model of oral carcinogenesis, we found that treatment with C59 prevented the development of OSCC by reducing the size and number of oral tumor lesions. CONCLUSIONS: The inhibition of Wnt ligand secretion with C59 represents a feasible treatment to prevent the progression of early oral lesions toward OSCC.
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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias de la Boca , Humanos , Vía de Señalización Wnt , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , beta Catenina/genética , beta Catenina/metabolismo , Neoplasias de la Boca/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello , Carcinogénesis/genética , Aciltransferasas/metabolismo , Aciltransferasas/farmacología , Proteínas de la Membrana/metabolismoRESUMEN
The salivary peptide histatin-1 was recently described as a novel osteogenic factor that stimulates cell adhesion, migration, and differentiation in bone-lineage cells. Since these cell responses collectively contribute to bone regeneration, we hypothesized that histatin-1 harbors the capacity to enhance bone tissue repair at the preclinical level. By using a model of monocortical bone defect, we explored the effects of histatin-1 in tibial mineralization and organic matrix formation in vivo. To this end, different amounts of histatin-1 were embedded in one-mm3 collagen sponges and then applied to tibial monocortical defects in C57bl/6 mice. After seven days, mice were euthanized, and samples were processed for subsequent analysis. Micro-computed tomography screening showed that histatin-1 increased intraosseous mineralization, and this phenomenon was accompanied by augmented collagen matrix deposition and closure of cortical defect edges, as determined by Hematoxylin-Eosin and Masson's Trichrome staining. Moreover, immunohistochemical analyses showed that histatin-1 increased the expression of the osteogenic marker alkaline phosphatase, which was accompanied by augmented blood vessel formation. Collectively, our findings show that histatin-1 itself promotes bone regeneration in an orthotopic model, proposing this molecule as a therapeutic candidate for use in bone regenerative medicine.
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Histatinas , Osteogénesis , Ratones , Animales , Histatinas/farmacología , Microtomografía por Rayos X , Regeneración Ósea , Colágeno/metabolismo , Proteínas y Péptidos Salivales , Diferenciación CelularRESUMEN
AIM: Angiogenesis contributes to the development of apical periodontitis, periodontitis, and other oral pathologies; however, it remains unclear how this process is triggered. The aim was to evaluate whether lipopolysaccharide (LPS) from Porphyromonas endodontalis and Porphyromonas gingivalis induced angiogenesis-related effects in vitro via TLR2 and TLR4. METHODOLOGY: Porphyromonas endodontalis LPS (ATCC 35406 and clinical isolate) was purified with TRIzol, whereas P. gingivalis LPS was obtained commercially. The effects of the different LPS (24 h) in endothelial cell migration were analysed by Transwell assays, following quantification in an optical microscope (40×). The effects of LPS on FAK Y397 phosphorylation were assessed by Western blotting. Angiogenesis in vitro was determined in an endothelial tube formation assay (14 h) in Matrigel in the absence or presence of either LPS. IL-6 and VEGF-A levels were determined in cell supernatants, following 24 h treatment with LPS, and measured in multiplex bead immunoassay. The involvement of TLR2 and TLR4 was assessed with blocking antibodies. The statistical analysis was performed using STATA 12® (StataCorp LP). RESULTS: The results revealed that P. endodontalis LPS, but not P. gingivalis LPS, stimulated endothelial cell migration. Pre-treatment with anti-TLR2 and anti-TLR4 antibodies prevented P. endodontalis LPS-induced cell migration. P. endodontalis LPS promoted FAK phosphorylation on Y397, as observed by an increased p-FAK/FAK ratio. Both P. gingivalis and P. endodontalis LPS (ATCC 35406) induced endothelial tube formation in a TLR-2 and -4-dependent manner, as shown by using blocking antibodies, however, only TLR2 blocking decreased tube formation induced by P. endodontalis (clinical isolate). Moreover, all LPS induced IL-6 and VEGF-A synthesis in endothelial cells. TLR2 and TLR4 were required for IL-6 induction by P. endodontalis LPS (ATCC 35406), while only TLR4 was involved in IL-6 secretion by the other LPS. Finally, VEGF-A synthesis did not require TLR signalling. CONCLUSION: Porphyromonas endodontalis and P. gingivalis LPS induced angiogenesis via TLR2 and TLR4. Collectively, these data contribute to understanding the role of LPS from Porphyromonas spp. in angiogenesis and TLR involvement.
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Lipopolisacáridos , Receptor Toll-Like 2 , Lipopolisacáridos/farmacología , Receptor Toll-Like 2/metabolismo , Porphyromonas gingivalis/metabolismo , Porphyromonas endodontalis/metabolismo , Factor A de Crecimiento Endotelial Vascular , Células Endoteliales/metabolismo , Anticuerpos Bloqueadores , Interleucina-6 , Receptor Toll-Like 4/metabolismoRESUMEN
SALL2/Sall2 is a transcription factor associated with development, neuronal differentiation, and cancer. Interestingly, SALL2/Sall2 deficiency leads to failure of the optic fissure closure and neurite outgrowth, suggesting a positive role for SALL2/Sall2 in cell migration. However, in some cancer cells, SALL2 deficiency is associated with increased cell migration. To further investigate the role of Sall2 in the cell migration process, we used immortalized Sall2 knockout (Sall2 -/- ) and Sall2 wild-type (Sall2 +/+ ) mouse embryonic fibroblasts (iMEFs). Our results indicated that Sall2 positively regulates cell migration, promoting cell detachment and focal adhesions turnover. Sall2 deficiency decreased cell motility and altered focal adhesion dynamics. Accordingly, restoring Sall2 expression in the Sall2 -/- iMEFs by using a doxycycline-inducible Tet-On system recovered cell migratory capabilities and focal adhesion dynamics. In addition, Sall2 promoted the autophosphorylation of Focal Adhesion Kinase (FAK) at Y397 and increased integrin ß1 mRNA and its protein expression at the cell surface. We demonstrated that SALL2 increases ITGB1 promoter activity and binds to conserved SALL2-binding sites at the proximal region of the ITGB1 promoter, validated by ChIP experiments. Furthermore, the overexpression of integrin ß1 or its blockade generates a cell migration phenotype similar to that of Sall2 +/+ or Sall2 -/- cells, respectively. Altogether, our data showed that Sall2 promotes cell migration by modulating focal adhesion dynamics, and this phenotype is associated with SALL2/Sall2-transcriptional regulation of integrin ß1 expression and FAK autophosphorylation. Since deregulation of cell migration promotes congenital abnormalities, tumor formation, and spread to other tissues, our findings suggest that the SALL2/Sall2-integrin ß1 axis could be relevant for those processes.
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Aging is a gradual and progressive deterioration of integrity across multiple organ systems that negatively affects gingival wound healing. The cellular responses associated with wound healing, such as collagen synthesis, cell migration, proliferation, and collagen contraction, have been shown to be lower in gingival fibroblasts (the most abundant cells from the connective gingival tissue) in aged donors than young donors. Cellular senescence is one of the hallmarks of aging, which is characterized by the acquisition of a senescence-associated secretory phenotype that is characterized by the release of pro-inflammatory cytokines, chemokines, growth factors, and proteases which have been implicated in the recruitment of immune cells such as neutrophils, T cells and monocytes. Moreover, during aging, macrophages show altered acquisition of functional phenotypes in response to the tissue microenvironment. Thus, inflammatory and resolution macrophage-mediated processes are impaired, impacting the progression of periodontal disease. Interestingly, salivary antimicrobial peptides, such as histatins, which are involved in various functions, such as antifungal, bactericidal, enamel-protecting, angiogenesis, and re-epithelization, have been shown to fluctuate with aging. Several studies have associated the presence of Porphyromonas gingivalis, a key pathogen related to periodontitis and apical periodontitis, with the progression of Alzheimer's disease, as well as gut, esophageal, and gastric cancers. Moreover, herpes simplex virus types 1 and 2 have been associated with the severity of periodontal disease, cardiovascular complications, and nervous system-related pathologies. This review encompasses the effects of aging on periodontal tissues, how P. gingivalis and HSV infections could favor periodontitis and their relationship with other pathologies.
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Enfermedades Periodontales , Periodontitis , Humanos , Encía/patología , Porphyromonas gingivalis , Periodoncio , Enfermedades Periodontales/metabolismoRESUMEN
Histatin-1 is a salivary peptide with antimicrobial and wound healing promoting activities, which was previously shown to stimulate angiogenesis in vitro and in vivo via inducing endothelial cell migration. The mechanisms underlying the proangiogenic effects of Histatin-1 remain poorly understood and specifically, the endothelial receptor for this peptide, is unknown. Based on the similarities between Histatin-1-dependent responses and those induced by the prototypical angiogenic receptor, vascular endothelial growth factor receptor 2 (VEGFR2), we hypothesized that VEGFR2 is the Histatin-1 receptor in endothelial cells. First, we observed that VEGFR2 is necessary for Histatin-1-induced endothelial cell migration, as shown by both pharmacological inhibition studies and siRNA-mediated ablation of VEGFR2. Moreover, Histatin-1 co-immunoprecipitated and co-localized with VEGFR2, associating spatial proximity between these proteins with receptor activation. Indeed, pulldown assays with pure, tagged and non-tagged proteins showed that Histatin-1 and VEGFR2 directly interact in vitro. Optical tweezers experiments permitted estimating kinetic parameters and rupture forces, indicating that the Histatin-1-VEGFR2 interaction is transient, but specific and direct. Sequence alignment and molecular modeling identified residues Phe26, Tyr30 and Tyr34 within the C-terminal domain of Histatin-1 as relevant for VEGFR2 binding and activation. This was corroborated by mutation and molecular dynamics analyses, as well as in direct binding assays. Importantly, these residues were required for Histatin-1 to induce endothelial cell migration and angiogenesis in vitro. Taken together, our findings reveal that VEGFR2 is the endothelial cell receptor of Histatin-1 and provide insights to the mechanism by which this peptide promotes endothelial cell migration and angiogenesis.
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Células Endoteliales , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Proteínas Portadoras/metabolismo , Movimiento Celular , Células Endoteliales/metabolismo , Histatinas/metabolismo , Histatinas/farmacología , Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Background: Oral cancer represents the sixth most common cancer in the world and is associated with 40-50%survival at 5 years. Within oral malignancies, oral squamous cell carcinoma (OSCC) is commonly preceded bypotentially malignant lesions, which, according to histopathological criteria, are referred to as oral dysplasia andtheir diagnosis are associated with higher rates of malignant transformation towards cancer. We recently reportedthat aberrant activation of the Wnt/β catenin pathway is due to overexpression of Wnt ligands in oral dysplasia.However, the expression of other regulators of this pathway, namely components of the β-catenin destructioncomplex has not been explored in oral dysplasia.Material and Methods: Using immunohistochemical analyses, we evaluated nuclear expression of β catenin andits association with Wnt3a and Wnt5a. Likewise, components of the β-catenin destruction complex, includingAdenomatous Polyposis Coli (APC), Axin and Glycogen Synthase Kinase 3 beta (GSK-3β) were also evaluatedin oral dysplasia and OSCC biopsies.Results: We found that moderate and severe dysplasia samples, which harbored increased expression of nuclearβ catenin, depicted augmented cytoplasmic expression of GSK 3β, Axin and APC, in comparison with OSCCsamples. Also, GSK-3β was found nuclear in mild dysplasia and OSCC samples, when compared with other studysamples.Conclusions: Cytoplasmic levels of components of the β-catenin destruction complex are increased in oral dyspla-sia and might be responsible of augmented nuclear β catenin.(AU)
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Humanos , Masculino , Femenino , Neoplasias de la Boca , beta Catenina , Complejo de Señalización de la Axina , Carcinoma de Células Escamosas , Vía de Señalización WntRESUMEN
Aging is a gradual biological process characterized by a decrease in cellular and organism functions. Aging-related processes involve changes in the expression and activity of several proteins. Here, we identified the transmembrane protease serine 11a (TMPRSS11a) as a new age-specific protein that plays an important role in skin wound healing. TMPRSS11a levels increased with age in rodent and human skin and gingival samples. Strikingly, overexpression of TMPRSS11a decreased cell migration and spreading, and inducing cellular senescence. Mass spectrometry, bioinformatics, and functional analyses revealed that TMPRSS11a interacts with integrin ß1 through an RGD sequence contained within the C-terminal domain and that this motif was relevant for cell migration. Moreover, TMPRSS11a was associated with cellular senescence, as shown by overexpression and downregulation experiments. In agreement with tissue-specific expression of TMPRSS11a, shRNA-mediated downregulation of this protein improved wound healing in the skin, but not in the skeletal muscle of old mice, where TMPRSS11a is undetectable. Collectively, these findings indicate that TMPRSS11a is a tissue-specific factor relevant for wound healing, which becomes elevated with aging, promoting cellular senescence and inhibiting cell migration and skin repair.
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Envejecimiento/patología , Movimiento Celular , Fibroblastos/patología , Proteínas de la Membrana/metabolismo , Serina Proteasas/metabolismo , Piel/patología , Cicatrización de Heridas , Adolescente , Adulto , Anciano , Envejecimiento/metabolismo , Animales , Proliferación Celular , Fibroblastos/metabolismo , Encía/metabolismo , Encía/patología , Humanos , Proteínas de la Membrana/genética , Ratones , Persona de Mediana Edad , Serina Proteasas/genética , Transducción de Señal , Piel/metabolismo , Adulto JovenRESUMEN
Neuroblastoma is a highly metastatic tumor that emerges from neural crest cell progenitors. Focal Adhesion Kinase (FAK) is a regulator of cell migration that binds to the receptor Neogenin-1 and is upregulated in neuroblastoma. Here, we show that Netrin-1 ligand binding to Neogenin-1 leads to FAK autophosphorylation and integrin ß1 activation in a FAK dependent manner, thus promoting neuroblastoma cell migration. Moreover, Neogenin-1, which was detected in all tumor stages and was required for neuroblastoma cell migration, was found in a complex with integrin ß1, FAK, and Netrin-1. Importantly, Neogenin-1 promoted neuroblastoma metastases in an immunodeficient mouse model. Taken together, these data show that Neogenin-1 is a metastasis-promoting protein that associates with FAK, activates integrin ß1 and promotes neuroblastoma cell migration.
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Integrina beta1 , Neuroblastoma , Animales , Adhesión Celular , Movimiento Celular , Quinasa 1 de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal , Proteínas de la Membrana , Ratones , Netrina-1RESUMEN
Histatin-1 is a salivary antimicrobial peptide involved in the maintenance of enamel and oral mucosal homeostasis. Moreover, Histatin-1 has been shown to promote re-epithelialization in soft tissues, by stimulating cell adhesion and migration in oral and dermal keratinocytes, gingival and skin fibroblasts, endothelial cells and corneal epithelial cells. The broad-spectrum activity of Histatin-1 suggests that it behaves as a universal wound healing promoter, although this is far from being clear yet. Here, we report that Histatin-1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation. Specifically, Histatin-1 promoted cell adhesion, spreading, and migration of SAOS-2 cells and MC3T3-E1 preosteoblasts in vitro, when placed on a fibronectin matrix. Besides, Histatin-1 induced the expression of osteogenic genes, including osteocalcin, osteopontin, and Runx2, and increased both activity and protein levels of alkaline phosphatase. Furthermore, Histatin-1 promoted mineralization in vitro, as it augmented the formation of calcium deposits in both SAOS-2 and MC3T3-E1 cells. Mechanistically, although Histatin-1 failed to activate ERK1/2, FAK, and Akt, which are signaling proteins associated with osteogenic differentiation or cell migration, it triggered nuclear relocalization of ß-catenin. Strikingly, the effects of Histatin-1 were recapitulated in cells that are nonosteogenically committed, since it promoted surface adhesion, migration, and the acquisition of osteogenic markers in primary mesenchymal cells derived from the apical papilla and dental pulp. Collectively, these observations indicate that Histatin-1 is a novel osteogenic factor that promotes bone cell differentiation, surface adhesion and migration, as crucial events required for bone tissue regeneration.
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Diferenciación Celular , Movimiento Celular , Histatinas/farmacología , Osteogénesis , Animales , Calcificación Fisiológica/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Macroautophagy/autophagy is an intracellular process involved in the breakdown of macromolecules and organelles. Recent studies have shown that PKD2/PC2/TRPP2 (polycystin 2, transient receptor potential cation channel), a nonselective cation channel permeable to Ca2+ that belongs to the family of transient receptor potential channels, is required for autophagy in multiple cell types by a mechanism that remains unclear. Here, we report that PKD2 forms a protein complex with BECN1 (beclin 1), a key protein required for the formation of autophagic vacuoles, by acting as a scaffold that interacts with several co-modulators via its coiled-coil domain (CCD). Our data identified a physical and functional interaction between PKD2 and BECN1, which depends on one out of two CCD domains (CC1), located in the carboxy-terminal tail of PKD2. In addition, depletion of intracellular Ca2+ with BAPTA-AM not only blunted starvation-induced autophagy but also disrupted the PKD2-BECN1 complex. Consistently, PKD2 overexpression triggered autophagy by increasing its interaction with BECN1, while overexpression of PKD2D509V, a Ca2+ channel activity-deficient mutant, did not induce autophagy and manifested diminished interaction with BECN1. Our findings show that the PKD2-BECN1 complex is required for the induction of autophagy, and its formation depends on the presence of the CC1 domain of PKD2 and on intracellular Ca2+ mobilization by PKD2. These results provide new insights regarding the molecular mechanisms by which PKD2 controls autophagy.Abbreviations: ADPKD: autosomal dominant polycystic kidney disease; ATG: autophagy-related; ATG14/ATG14L: autophagy related 14; Baf A1: bafilomycin A1; BCL2/Bcl-2: BCL2 apoptosis regulator; BCL2L1/BCL-XL: BCL2 like 1; BECN1: beclin 1; CCD: coiled-coil domain; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GOLGA2/GM130: golgin A2; GST: glutathione s-transferase; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1 autophagy cargo receptor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PKD2/PC2: polycystin 2, transient receptor potential cation channel; RTN4/NOGO: reticulon 4; RUBCN/RUBICON: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; UVRAG: UV radiation resistance associated; WIPI2: WD repeat domain, phosphoinositide interacting 2.
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Autofagia , Beclina-1/fisiología , Canales Catiónicos TRPP/fisiología , Beclina-1/metabolismo , Western Blotting , Técnica del Anticuerpo Fluorescente , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Canales Catiónicos TRPP/metabolismoRESUMEN
Oral squamous cell carcinoma, the most common type of oral cancer, affects more than 275,000 people per year worldwide. Oral squamous cell carcinoma is very aggressive, as most patients die after 3 to 5 years post-diagnosis. The initiation and progression of oral squamous cell carcinoma are multifactorial: smoking, alcohol consumption, and human papilloma virus infection are among the causes that promote its development. Although oral squamous cell carcinoma involves abnormal growth and migration of oral epithelial cells, other cell types such as fibroblasts and immune cells form the carcinoma niche. An underlying inflammatory state within the oral tissue promotes differential stress-related responses that favor oral squamous cell carcinoma. Autophagy is an intracellular degradation process that allows cancer cells to survive under stress conditions. Autophagy degrades cellular components by sequestering them in vesicles called autophagosomes, which ultimately fuse with lysosomes. Although several autophagy markers have been associated with oral squamous cell carcinoma, it remains unclear whether up- or down-regulation of autophagy favors its progression. Autophagy levels during oral squamous cell carcinoma are both timing- and cell-specific. Here we discuss how autophagy is required to establish a new cellular microenvironment in oral squamous cell carcinoma and how autophagy drives the phenotypic change of oral squamous cell carcinoma cells by promoting crosstalk between carcinoma cells, fibroblasts, and immune cells.
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Tumor hypoxia and the hypoxia inducible factor-1, HIF-1, play critical roles in cancer progression and metastasis. We previously showed that hypoxia activates the endosomal GTPase Rab5, leading to tumor cell migration and invasion, and that these events do not involve changes in Rab protein expression, suggesting the participation of intermediate activators. Here, we identified ALS2, a guanine nucleotide exchange factor that is upregulated in cancer, as responsible for increased Rab5-GTP loading, cell migration and metastasis in hypoxia. Specifically, hypoxia augmented ALS2 mRNA and protein levels, and these events involved HIF-1α-dependent transcription, as shown by RNAi, pharmacological inhibition, chromatin immunoprecipitation and bioinformatics analyses, which identified a functional HIF-1α-binding site in the proximal promoter region of ALS2. Moreover, ALS2 and Rab5 activity were elevated both in a model of endogenous HIF-1α stabilization (renal cell carcinoma) and by following expression of stable non-hydroxylatable HIF-1α. Strikingly, ALS2 upregulation in hypoxia was required for Rab5 activation, tumor cell migration and invasion, as well as experimental metastasis in C57BL/6 mice. Finally, immunohistochemical analyses in patient biopsies with renal cell carcinoma showed that elevated HIF-1α correlates with increased ALS2 expression. Hence, this study identifies ALS2 as a novel hypoxia-inducible gene associated with tumor progression and metastasis.
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Esclerosis Amiotrófica Lateral/genética , Carcinoma de Células Renales/genética , Factores de Intercambio de Guanina Nucleótido/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Activación Transcripcional/genética , Hipoxia Tumoral , Proteínas de Unión al GTP rab5/genéticaRESUMEN
Oral carcinogenesis is a complex and multifactorial process that involves cumulative genetic and molecular alterations, leading to uncontrolled cell proliferation, impaired DNA repair and defective cell death. At the early stages, the onset of potentially malignant lesions in the oral mucosa, or oral dysplasia, is associated with higher rates of malignant progression towards carcinoma in situ and invasive carcinoma. Efforts have been made to get insights about signaling pathways that are deregulated in oral dysplasia, as these could be translated into novel markers and might represent promising therapeutic targets. In this context, recent evidence underscored the relevance of the Wnt/ß-catenin signaling pathway in oral dysplasia, as this pathway is progressively "switched on" through the different grades of dysplasia (mild, moderate and severe dysplasia), with the consequent nuclear translocation of ß-catenin and expression of target genes associated with the maintenance of representative traits of oral dysplasia, namely cell proliferation and viability. Intriguingly, recent studies provide an unanticipated connection between active ß-catenin signaling and deregulated endosome trafficking in oral dysplasia, highlighting the relevance of endocytic components in oral carcinogenesis. This review summarizes evidence about the role of the Wnt/ß-catenin signaling pathway and the underlying mechanisms that account for its aberrant activation in oral carcinogenesis.
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Carcinogénesis/metabolismo , Carcinoma/etiología , Neoplasias de la Boca/etiología , Vía de Señalización Wnt , beta Catenina/metabolismo , Carcinoma/metabolismo , Humanos , Neoplasias de la Boca/metabolismoRESUMEN
Caveolin-1 (CAV1) enhanced migration, invasion, and metastasis of cancer cells is inhibited by co-expression of the glycoprotein E-cadherin. Although the two proteins form a multiprotein complex that includes ß-catenin, it remained unclear how this would contribute to blocking the metastasis promoting function of CAV1. Here, we characterized by mass spectrometry the protein composition of CAV1 immunoprecipitates from B16F10 murine melanoma cells expressing or not E-cadherin. The novel protein tyrosine phosphatase PTPN14 was identified by mass spectrometry analysis exclusively in co-immunoprecipitates of CAV1 with E-cadherin. Interestingly, PTPN14 is implicated in controlling metastasis, but only few known PTPN14 substrates exist. We corroborated by western blotting experiments that PTPN14 and CAV1 co-inmunoprecipitated in the presence of E-cadherin in B16F10 melanoma and other cancer cells. Moreover, the CAV1(Y14F) mutant protein was shown to co-immunoprecipitate with PTPN14 even in the absence of E-cadherin, and overexpression of PTPN14 reduced CAV1 phosphorylation on tyrosine-14, as well as suppressed CAV1-enhanced cell migration, invasion and Rac-1 activation in B16F10, metastatic colon [HT29(US)] and breast cancer (MDA-MB-231) cell lines. Finally, PTPN14 overexpression in B16F10 cells reduced the ability of CAV1 to induce metastasis in vivo. In summary, we identify here CAV1 as a novel substrate for PTPN14 and show that overexpression of this phosphatase suffices to reduce CAV1-induced metastasis.
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Cadherinas/genética , Caveolina 1/genética , Melanoma Experimental/genética , Proteínas Tirosina Fosfatasas no Receptoras/genética , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Melanoma Experimental/patología , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Fosforilación/genética , beta Catenina/genéticaRESUMEN
Potentially malignant lesions, commonly referred to as dysplasia, are associated with malignant transformation by mechanisms that remain unclear. We recently reported that increased Wnt secretion promotes the nuclear accumulation of ß-catenin and expression of target genes in oral dysplasia. However, the mechanisms accounting for nuclear re-localization of ß-catenin in oral dysplasia remain unclear. In this study, we show that endosomal sequestration of the ß-catenin destruction complex allows nuclear accumulation of ß-catenin in oral dysplasia, and that these events depended on the endocytic protein Rab5. Tissue immunofluorescence analysis showed aberrant accumulation of enlarged early endosomes in oral dysplasia biopsies, when compared with healthy oral mucosa. These observations were confirmed in cell culture models, by comparing dysplastic oral keratinocytes (DOK) and non-dysplastic oral keratinocytes (OKF6). Intriguingly, DOK depicted higher levels of active Rab5, a critical regulator of early endosomes, when compared with OKF6. Increased Rab5 activity in DOK was necessary for nuclear localization of ß-catenin and Tcf/Lef-dependent transcription, as shown by expression of dominant negative and constitutively active mutants of Rab5, along with immunofluorescence, subcellular fractionation, transcription, and protease protection assays. Mechanistically, elevated Rab5 activity in DOK accounted for endosomal sequestration of components of the destruction complex, including GSK3ß, Axin, and adenomatous polyposis coli (APC), as observed in Rab5 dominant negative experiments. In agreement with these in vitro observations, tissue immunofluorescence analysis showed increased co-localization of GSK3ß, APC, and Axin, with early endosome antigen 1- and Rab5-positive early endosomes in clinical samples of oral dysplasia. Collectively, these data indicate that increased Rab5 activity and endosomal sequestration of the ß-catenin destruction complex leads to stabilization and nuclear accumulation of ß-catenin in oral dysplasia.
Asunto(s)
Apraxias/metabolismo , Núcleo Celular/metabolismo , beta Catenina/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Línea Celular , Endosomas/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Focal adhesion kinase (FAK) is a central regulator of integrin-dependent cell adhesion and migration and has recently been shown to co-localize with endosomal proteins. The early endocytic protein Rab5 controls integrin trafficking, focal adhesion disassembly, and cell migration and has been shown to be activated upon integrin engagement by mechanisms that remain unclear. Because FAK is a critical regulator of integrin-dependent signaling and Rab5 recapitulates FAK-mediated effects, we evaluated the possibility that FAK activates Rab5 and contributes to cell migration. Pulldown assays revealed that Rab5-GTP levels are decreased upon treatment with a pharmacological inhibitor of FAK, PF562,271, in resting A549 cells. These events were associated with decreased peripheral Rab5 puncta and a reduced number of early endosome antigen 1 (EEA1)-positive early endosomes. Accordingly, as indicated by FAK inhibition experiments and in FAK-null fibroblasts, adhesion-induced FAK activity increased Rab5-GTP levels. In fact, expression of WT FAK and FAK/Y180A/M183A (open conformation), but not FAK/Arg454 (kinase-dead), augmented Rab5-GTP levels in FAK-null fibroblasts and A549 cells. Moreover, expression of a GDP-bound Rab5 mutant (Rab5/S34N) or shRNA-mediated knockdown of endogenous Rab5 prevented FAK-induced A549 cell migration, whereas expression of WT or GTP-bound Rab5 (Rab5/Q79L), but not Rab5/S34N, promoted cell migration in FAK-null fibroblasts. Mechanistically, FAK co-immunoprecipitated with the GTPase-activating protein p85α in a phosphorylation (Tyr397)-dependent manner, preventing Rab5-GTP loading, as shown by knockdown and transfection recovery experiments. Taken together, these results reveal that FAK activates Rab5, leading to cell migration.
Asunto(s)
Movimiento Celular , Quinasa 1 de Adhesión Focal/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Células A549 , Humanos , Células Tumorales CultivadasRESUMEN
OBJECTIVES: To evaluate the localization of ß-catenin in oral dysplastic cells, the expression of target genes upregulated in oral dysplasia, and the role of Wnt ligands in these events. MATERIALS AND METHODS: Subcellular localization of total and non-phosphorylated (transcriptionally active) ß-catenin was evaluated by immunofluorescence and biochemical fractionation in dysplastic oral keratinocytes (DOK), non-dysplastic oral keratinocytes (OKF6), oral squamous carcinoma cells (CAL27) and primary oral keratinocytes. Tcf/Lef-dependent transcription was measured by luciferase reporter assays. Expression of target genes, survivin and cyclin D1, was evaluated by RT-qPCR and Western blotting. Wnt secretion was inhibited with the inhibitor of porcupine, C59. Wnt3a and ß-catenin were evaluated in biopsies by tissue immunofluorescence. RESULTS: Immunofluorescence and fractionation experiments showed augmented nuclear ß-catenin (total and transcriptionally active) in DOK, when compared with OKF6 and CAL27 cells. Intriguingly, conditioned medium from DOK promoted nuclear accumulation of ß-catenin and Tcf/Lef-dependent transcription in OKF6 and primary oral keratinocytes, suggesting the participation of secreted factors. Treatment of DOK with C59 decreased Wnt3a secretion, nuclear ß-catenin and the expression of survivin and cyclin D1 at both mRNA and protein levels. Accordingly, DOK secreted higher Wnt3a levels than OKF6, and inhibition of Wnt3a secretion prevented DOK-induced Tcf/Lef-dependent transcription in OKF6. These observations were confirmed in clinical samples, since tissue immunofluorescence analysis showed simultaneous expression of Wnt3a and nuclear ß-catenin in oral dysplasia, but not in healthy mucosa biopsies. CONCLUSION: These data indicate that secretion of Wnt ligands is critical for ß-catenin nuclear localization and expression of target genes in oral dysplasia.