Inhibition of PORCN Blocks Wnt Signaling to Attenuate Progression of Oral Carcinogenesis.

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.

Protein kinase B (AKT) upregulation and Thy-1-αvß3 integrin-induced phosphorylation of Connexin43 by activated AKT in astrogliosis.

BACKGROUND: In response to brain injury or inflammation, astrocytes undergo hypertrophy, proliferate, and migrate to the damaged zone. These changes, collectively known as "astrogliosis", initially protect the brain; however, astrogliosis can also cause neuronal dysfunction. Additionally, these astrocytes undergo intracellular changes involving alterations in the expression and localization of many proteins, including αvß3 integrin. Our previous reports indicate that Thy-1, a neuronal glycoprotein, binds to this integrin inducing Connexin43 (Cx43) hemichannel (HC) opening, ATP release, and astrocyte migration. Despite such insight, important links and molecular events leading to astrogliosis remain to be defined. METHODS: Using bioinformatics approaches, we analyzed different Gene Expression Omnibus datasets to identify changes occurring in reactive astrocytes as compared to astrocytes from the normal mouse brain. In silico analysis was validated by both qRT-PCR and immunoblotting using reactive astrocyte cultures from the normal rat brain treated with TNF and from the brain of a hSOD1G93A transgenic mouse model. We evaluated the phosphorylation of Cx43 serine residue 373 (S373) by AKT and ATP release as a functional assay for HC opening. In vivo experiments were also performed with an AKT inhibitor (AKTi). RESULTS: The bioinformatics analysis revealed that genes of the PI3K/AKT signaling pathway were among the most significantly altered in reactive astrocytes. mRNA and protein levels of PI3K, AKT, as well as Cx43, were elevated in reactive astrocytes from normal rats and from hSOD1G93A transgenic mice, as compared to controls. In vitro, reactive astrocytes stimulated with Thy-1 responded by activating AKT, which phosphorylated S373Cx43. Increased pS373Cx43 augmented the release of ATP to the extracellular medium and AKTi inhibited these Thy-1-induced responses. Furthermore, in an in vivo model of inflammation (brain damage), AKTi decreased the levels of astrocyte reactivity markers and S373Cx43 phosphorylation. CONCLUSIONS: Here, we identify changes in the PI3K/AKT molecular signaling network and show how they participate in astrogliosis by regulating the HC protein Cx43. Moreover, because HC opening and ATP release are important in astrocyte reactivity, the phosphorylation of Cx43 by AKT and the associated increase in ATP release identify a potential therapeutic window of opportunity to limit the adverse effects of astrogliosis.

A Brief Report: Comparative Evaluation of Online Spanish and English Content on Pancreatic Cancer Treatment.

The Internet is a major source of patient information on medical subjects. Several studies have evaluated the content of English medical material for patient use. However, few have focused on evaluating other languages, an important gap in a growing Spanish-speaking population. The aim of this study is to evaluate and compare English and Spanish online content related to pancreatic cancer treatment. We conducted a Google web search in English and Spanish using the following terms "pancreatic cancer treatment" and "tratamiento cancer de pancreas." The first 15 educational patient-directed websites for each language were included. Two independent reviewers assessed materials for quality and understandability using the DISCERN and the Patient Education Materials Assessment Tool (PEMAT)-validated tools. Readability was measured using two standardized tests. Wilcoxon rank sum test and unpaired Student's T-test were used for comparisons. Overall, websites in Spanish and English were understandable and had moderate to high quality. There were no significant differences in quality (p = 0.712) and understandability (p = 0.069) between languages. Readability level was significantly higher in English (p < 0.001) with content being at the university level, while Spanish was at the 12th grade level. Patient-directed online content on pancreatic cancer treatments exceeds the recommended reading level in both languages. Material is understandable with reasonable quality. Health content creators should acknowledge readability for information to be easily comprehended by those with lower health literacy.

The GPI-Anchored Protein Thy-1/CD90 Promotes Wound Healing upon Injury to the Skin by Enhancing Skin Perfusion.

Wound healing is a highly regulated multi-step process that involves a plethora of signals. Blood perfusion is crucial in wound healing and abnormalities in the formation of new blood vessels define the outcome of the wound healing process. Thy-1 has been implicated in angiogenesis and silencing of the Thy-1 gene retards the wound healing process. However, the role of Thy-1 in blood perfusion during wound closure remains unclear. We proposed that Thy-1 regulates vascular perfusion, affecting the healing rate in mouse skin. We analyzed the time of recovery, blood perfusion using Laser Speckle Contrast Imaging, and tissue morphology from images acquired with a Nanozoomer tissue scanner. The latter was assessed in a tissue sample taken with a biopsy punch on several days during the wound healing process. Results obtained with the Thy-1 knockout (Thy-1-/-) mice were compared with control mice. Thy-1-/- mice showed at day seven, a delayed re-epithelialization, increased micro- to macro-circulation ratio, and lower blood perfusion in the wound area. In addition, skin morphology displayed a flatter epidermis, fewer ridges, and almost no stratum granulosum or corneum, while the dermis was thicker, showing more fibroblasts and fewer lymphocytes. Our results suggest a critical role for Thy-1 in wound healing, particularly in vascular dynamics.

Mitochondrial Dysfunction and the Glycolytic Switch Induced by Caveolin-1 Phosphorylation Promote Cancer Cell Migration, Invasion, and Metastasis.

Cancer cells often display impaired mitochondrial function, reduced oxidative phosphorylation, and augmented aerobic glycolysis (Warburg effect) to fulfill their bioenergetic and biosynthetic needs. Caveolin-1 (CAV1) is a scaffolding protein that promotes cancer cell migration, invasion, and metastasis in a manner dependent on CAV1 phosphorylation on tyrosine-14 (pY14). Here, we show that CAV1 expression increased glycolysis rates, while mitochondrial respiration was reduced by inhibition of the mitochondrial complex IV. These effects correlated with increased reactive oxygen species (ROS) levels that favored CAV1-induced migration and invasion. Interestingly, pY14-CAV1 promoted the metabolic switch associated with increased migration/invasion and augmented ROS-inhibited PTP1B, a phosphatase that controls pY14 levels. Finally, the glycolysis inhibitor 2-deoxy-D-glucose reduced CAV1-enhanced migration in vitro and metastasis in vivo of murine melanoma cells. In conclusion, CAV1 promotes the Warburg effect and ROS production, which inhibits PTP1B to augment CAV1 phosphorylation on tyrosine-14, thereby increasing the metastatic potential of cancer cells.

Caveolin-1-Mediated Tumor Suppression Is Linked to Reduced HIF1α S-Nitrosylation and Transcriptional Activity in Hypoxia.

Caveolin-1 (CAV1) is a well-established nitric oxide synthase inhibitor, whose function as a tumor suppressor is favored by, but not entirely dependent on, the presence of E-cadherin. Tumors are frequently hypoxic and the activation of the hypoxia-inducible factor-1α (HIF1α) promotes tumor growth. HIF1α is regulated by several post-translational modifications, including S-nitrosylation. Here, we evaluate the mechanisms underlying tumor suppression by CAV1 in cancer cells lacking E-cadherin in hypoxia. Our main findings are that CAV1 reduced HIF activity and Vascular Endothelial Growth Factor expression in vitro and in vivo. This effect was neither due to reduced HIF1α protein stability or reduced nuclear translocation. Instead, HIF1α S-nitrosylation observed in hypoxia was diminished by the presence of CAV1, and nitric oxide synthase (NOS) inhibition by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) reduced HIF1α transcriptional activity in cells to the same extent as observed upon CAV1 expression. Additionally, arginase inhibition by (S)-(2-Boronoethyl)-L-cysteine (BEC) partially rescued cells from the CAV1-mediated suppression of HIF1α transcriptional activity. In vivo, CAV1-mediated tumor suppression was dependent on NOS activity. In summary, CAV1-dependent tumor suppression in the absence of E-cadherin is linked to reduced HIF1α transcriptional activity via diminished NOS-mediated HIF1α S-nitrosylation.

Src-family kinase inhibitors block early steps of caveolin-1-enhanced lung metastasis by melanoma cells.

In advanced stages of cancer disease, caveolin-1 (CAV1) expression increases and correlates with increased migratory and invasive capacity of the respective tumor cells. Previous findings from our laboratory revealed that specific ECM-integrin interactions and tyrosine-14 phosphorylation of CAV1 are required for CAV1-enhanced melanoma cell migration, invasion and metastasis in vivo. In this context, CAV1 phosphorylation on tyrosine-14 mediated by non-receptor Src-family tyrosine kinases seems to be important; however, the effect of Src-family kinase inhibitors on CAV1-enhanced metastasis in vivo has not been studied. Here, we evaluated the effect of CAV1 and c-Abl overexpression, as well as the use of the Src-family kinase inhibitors, PP2 and dasatinib (more specific for Src/Abl) in lung metastasis of B16F10 melanoma cells. Overexpression of CAV1 and c-Abl enhanced CAV1 phosphorylation and the metastatic potential of the B16F10 murine melanoma cells. Alternatively, treatment with PP2 or dasatinib for 2 h reduced CAV1 tyrosine-14 phosphorylation and levels recovered fully within 12 h of removing the inhibitors. Nonetheless, pre-treatment of cells with these inhibitors for 2 h sufficed to prevent migration, invasion and trans-endothelial migration in vitro. Importantly, the transient decrease in CAV1 phosphorylation by these kinase inhibitors prevented early steps of CAV1-enhanced lung metastasis by B16F10 melanoma cells injected into the tail vein of mice. In conclusion, this study underscores the relevance of CAV1 tyrosine-14 phosphorylation by Src-family kinases during the first steps of the metastatic sequence promoted by CAV1. These findings open up potential options for treatment of metastatic tumors in patients in which Src-family kinase activation and CAV1 overexpression favor dissemination of cancer cells to secondary sites.

The non-receptor tyrosine phosphatase type 14 blocks caveolin-1-enhanced cancer cell metastasis.

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.

Thy-1 (CD90)-Induced Metastatic Cancer Cell Migration and Invasion Are ß3 Integrin-Dependent and Involve a Ca2+/P2X7 Receptor Signaling Axis.

Cancer cell adhesion to the vascular endothelium is an important step in tumor metastasis. Thy-1 (CD90), a cell adhesion molecule expressed in activated endothelial cells, has been implicated in melanoma metastasis by binding to integrins present in cancer cells. However, the signaling pathway(s) triggered by this Thy-1-Integrin interaction in cancer cells remains to be defined. Our previously reported data indicate that Ca2+-dependent hemichannel opening, as well as the P2X7 receptor, are key players in Thy-1-αVß3 Integrin-induced migration of reactive astrocytes. Thus, we investigated whether this signaling pathway is activated in MDA-MB-231 breast cancer cells and in B16F10 melanoma cells when stimulated with Thy-1. In both cancer cell types, Thy-1 induced a rapid increase in intracellular Ca2+, ATP release, as well as cell migration and invasion. Connexin and Pannexin inhibitors decreased cell migration, implicating a requirement for hemichannel opening in Thy-1-induced cell migration. In addition, cell migration and invasion were precluded when the P2X7 receptor was pharmacologically blocked. Moreover, the ability of breast cancer and melanoma cells to transmigrate through an activated endothelial monolayer was significantly decreased when the ß3 Integrin was silenced in these cancer cells. Importantly, melanoma cells with silenced ß3 Integrin were unable to metastasize to the lung in a preclinical mouse model. Thus, our results suggest that the Ca2+/hemichannel/ATP/P2X7 receptor-signaling axis triggered by the Thy-1-αVß3 Integrin interaction is important for cancer cell migration, invasion and transvasation. These findings open up the possibility of therapeutically targeting the Thy-1-Integrin signaling pathway to prevent metastasis.

AT2 Receptor Mediated Activation of the Tyrosine Phosphatase PTP1B Blocks Caveolin-1 Enhanced Migration, Invasion and Metastasis of Cancer Cells.

: The renin-angiotensin receptor AT2R controls systemic blood pressure and is also suggested to modulate metastasis of cancer cells. However, in the latter case, the mechanisms involved downstream of AT2R remain to be defined. We recently described a novel Caveolin-1(CAV1)/Ras-related protein 5A (Rab5)/Ras-related C3 botulinum toxin substrate 1 (Rac1) signaling axis that promotes metastasis in melanoma, colon, and breast cancer cells. Here, we evaluated whether the antimetastatic effect of AT2R is connected to inhibition of this pathway. We found that murine melanoma B16F10 cells expressed AT2R, while MDAMB-231 human breast cancer cells did not. AT2R activation blocked migration, transendothelial migration, and metastasis of B16F10(cav-1) cells, and this effect was lost when AT2R was silenced. Additionally, AT2R activation reduced transendothelial migration of A375 human melanoma cells expressing CAV1. The relevance of AT2R was further underscored by showing that overexpression of the AT2R in MDA-MB-231 cells decreased migration. Moreover, AT2R activation increased non-receptor protein tyrosine phosphatase 1B (PTP1B) activity, decreased phosphorylation of CAV1 on tyrosine-14 as well as Rab5/Rac1 activity, and reduced lung metastasis of B16F10(cav-1) cells in C57BL/6 mice. Thus, AT2R activation reduces migration, invasion, and metastasis of cancer cells by PTP1B-mediated CAV1 dephosphorylation and inhibition of the CAV1/Rab5/Rac-1 pathway. In doing so, these observations open up interesting, novel therapeutic opportunities to treat metastatic cancer disease.

Estado de desarrollo psicomotor en niños sanos que asisten a un centro infantil en Santo Domingo, Ecuador / Psychomotor development status in healthy children attending a child center in Santo Domingo, Ecuador / Estado do desenvolvimento psicomotor em crianças saudáveis que participam a um centro da SAUDÁVEIS QUE PARTICIPAM A UM centro da infância em Santo Domingo, Equador

Resumen: El objetivo de este artículo es describir los puntajes de desarrollo psicomotor en niños y niñas que acuden un centro infantil, mediante el formulario 028 que corresponde al Test de Denver II, estandarizado internacionalmente. Se trata de un estudio con un enfoque cuantitativo de carácter descriptivo-transversal. Se reclutaron 42 niños de 18-36 meses, que acuden al centro infantil en Santo Domingo de los Tsáchilas, Ecuador, para la aplicación del test de Denver II. Se excluyeron los niños con patologías graves con impedimento para contestar el instrumento por déficit en su capacidad física y mental. Mediante los puntajes obtenidos por la aplicación del test de Denver se observó que el déficit en el Área Personal-Social se da más en los niños (20%) que en las niñas (5%). En el Área Motriz Fino-Adaptativo el déficit se presenta tanto en los niños (10%) como en las niñas (9%). En el Área de Lenguaje se observó que existe un retardo muy marcado, se presentó más en los niños (35%) que en las niñas (18%). Al igual se observó que el déficit en el Área Motriz Grande se da más en los niños (15%) que en las niñas (5%). Destaca la alta frecuencia de déficit en el desarrollo psicomotor en la población evaluada, especialmente en los niños, donde el área más significativa fue la de Lenguaje. Se recomienda la estimulación temprana y los patrones de crianza para mejorar el desarrollo del lenguaje y el desempeño de los niños en las demás áreas.

Abstract: The goal of this article is to describe psychomotor development scores in children who attend the children's center through Form 028, corresponding to the Internationally Standardized Denver II Test. This is a study with a quantitative approach of descriptive-transversal character. A total of 42 children aged 18-36 months were recruited to attend the Denver II test in Santo Domingo de los Tsáchilas Children's Center in Ecuador. Children with severe pathologies with impediment to answer the form due to deficiency in their physical and mental capacity were excluded. Through the scores obtained by the application of the Denver test, it was observed that the deficit in the Personal-Social Area occurs more in boys (20%) than in girls (5%). In the Fine Motor Skill Area, the deficit occurs in both boys (10%) and girls (9%). In the Language Area it was observed that there is a significant retardation: it was more present in boys (35%) than in girls (18%). It was also observed that the deficit in the Gross Motor Area is higher in boys (15%) than in girls (5%). It is significant the high frequency of deficits shown in psychomotor development in the population evaluated, especially in the language area. Early stimulation and parenting patterns are recommended to improve language development and the performance of children in other areas.

Resumo: O presente estudo buscou descrever escores de desenvolvimento psicomotor em crianças que frequentam o Centro das Crianças usando o formulário de 028 correspondente ao Teste de Denver II, padronizado internacionalmente. Pesquisa quantitativa com abordagem transversal descritivo. 42 crianças com idades entre 18-36 meses que frequentam o centro infantil em Santo Domingo de los Tsáchilas, Ecuador, para a implementação do teste de Denver II foram recrutados. Crianças com doenças graves foram excluídos devido a um impedimento para atender o déficit de instrumento em sua capacidade física e mental. Usando os escores obtidos pela aplicação do teste de Denver foi observado que o déficit na área pessoal-social é mais comum em crianças (20%) e mulheres (5%). Na Área Motriz fina o déficit ocorre em crianças (10%) e mulheres (9%). Na área da linguagem foi observado que há um atraso acentuado, parecia mais filhos (35%) do que em meninas (18%). Como observou-se que o déficit na área Motriz Grande é mais comum em crianças (15%) do que meninas (5%). Destaca a alta freqüência de déficits no desenvolvimento neuropsicomotor na população estudada, especialmente em crianças. Onde a área mais significativa foi Idioma. estimulação precoce e padrões parentais é recomendado para melhorar o desenvolvimento da linguagem e desempenho das crianças em outras áreas.

Extracellular matrix-specific Caveolin-1 phosphorylation on tyrosine 14 is linked to augmented melanoma metastasis but not tumorigenesis.

Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells. CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading. Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation. Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM. Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation. In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development. Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung. Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1.

Bronchial hyperreactivity to methacholine in atopic versus nonatopic asthmatic schoolchildren and preschoolers.

BACKGROUND: Atopy and bronchial hyperreactivity are factors related to severe and unremitting asthma of childhood; however, the prevalence of these factors could be different according to age of the child. OBJECTIVE: To determine if methacholine bronchial hyperreactivity (BHR) differs between atopic and nonatopic preschoolers and schoolchildren with mild-moderate asthma. METHODS: Data obtained from 340 children with diagnosis of asthma or recurrent wheezing, matched by atopic conditions (positive or negative skin prick test) and age, and who underwent a methacholine bronchial challenge test (by spirometry in schoolchildren and by transcutaneous oxygen pressure [TcP(O2)] in preschoolers) were reviewed. RESULTS: Among 136 schoolchildren (9.07 ± 2.5 years), the prevalence of positive BHR was significantly higher among atopics than nonatopics (75% versus 48.5%, p = .001, respectively), even after controlling for gender and nutritional status (adjusted odds ratio [aOR] = 3.2129, 95% confidence interval [CI]: 1.5-6.8; p = .002). In addition, atopic schoolchildren had lower PC(20) and required a lower threshold dose of methacholine to induce a reaction (0.53 versus 0.82 mg/ml, p = .055 and .5 versus 1 mg/ml, p = .02, respectively) than nonatopics. Nevertheless, basal and predicted forced expiratory volume in one second (FEV1) were similar between groups. In contrast, among 204 preschoolers (4.74 ± 1.1 years), there were no differences in the prevalence of positive BHR between atopics and nonatopics (74.5% versus 72.5%, p = .75, respectively). Furthermore, basal TcP(O2), a higher fall of TcP(O2) and lower threshold doses of methacholine required for induction as measured by TcP(O2) were similar between the atopic and nonatopic preschoolers. CONCLUSIONS: Atopic asthmatic schoolchildren have greater hyperresponsiveness to methacholine than nonatopics (only among those with normal nutritional status). However, atopic and nonatopic asthmatic preschoolers have similar hyperresponsiveness to methacholine. Therefore, factors different from atopy may be responsible for wheeze in younger children.