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BACKGROUND: We recently demonstrated decreased tumor suppressor gene liver kinase B1 (LKB1) level in lung transplant recipients diagnosed with bronchiolitis obliterans syndrome. STE20-related adaptor alpha (STRADα) functions as a pseudokinase that binds and regulates LKB1 activity. METHODS: A murine model of chronic lung allograft rejection in which a single lung from a B6D2F1 mouse was orthotopically transplanted into a DBA/2J mouse was employed. We examined the effect of LKB1 knockdown using CRISPR-CAS9 in vitro culture system. RESULTS: Significant downregulation of LKB1 and STRADα expression was found in donor lung compared to recipient lung. STRADα knockdown significantly inhibited LKB1, pAMPK expression but induced phosphorylated mammalian target of rapamycin (mTOR), fibronectin, and Collagen-I, expression in BEAS-2B cells. LKB1 overexpression decreased fibronectin, Collagen-I, and phosphorylated mTOR expression in A549 cells. CONCLUSIONS: We demonstrated that downregulation of LKB1-STRADα pathway accompanied with increased fibrosis, results in development of chronic rejection following murine lung transplantation.
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Fibronectinas , Trasplante de Pulmón , Animales , Ratones , Fibronectinas/genética , Fibronectinas/metabolismo , Regulación hacia Abajo , Ratones Endogámicos DBA , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Pulmón/metabolismo , Biomarcadores , Genes Supresores de Tumor , Aloinjertos , Colágeno/genética , Colágeno/metabolismo , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
BACKGROUND: EpCAM (Epithelial cell adhesion molecule) is often dysregulated in epithelial cancers. Prior studies implicate EpCAM in the regulation of oncogenic signaling pathways and epithelial-to-mesenchymal transition. It was recently demonstrated that EpCAM contains a thyroglobulin type-1 (TY-1) domain. Multiple proteins with TY-1 domains are known to inhibit cathepsin-L (CTSL), a cysteine protease that promotes tumor cell invasion and metastasis. Analysis of human cancer sequencing studies reveals that somatic EpCAM mutations are present in up to 5.1% of tested tumors. METHODS: The Catalogue of Somatic Mutations in Cancer (COSMIC) database was queried to tabulate the position and amino acid changes of cancer associated EpCAM mutations. To determine how EpCAM mutations affect cancer biology we studied C66Y, a damaging TY-1 domain mutation identified in liver cancer, as well as 13 other cancer-associated EpCAM mutations. In vitro and in vivo models were used to determine the effect of wild type (WT) and mutant EpCAM on CTSL activity and invasion. Immunoprecipitation and localization studies tested EpCAM and CTSL protein binding and determined compartmental expression patterns of EpCAM mutants. RESULTS: We demonstrate that WT EpCAM, but not C66Y EpCAM, inhibits CTSL activity in vitro, and the TY-1 domain of EpCAM is responsible for this inhibition. WT EpCAM, but not C66Y EpCAM, inhibits tumor cell invasion in vitro and lung metastases in vivo. In an extended panel of human cancer cell lines, EpCAM expression is inversely correlated with CTSL activity. Previous studies have demonstrated that EpCAM germline mutations can prevent EpCAM from being expressed at the cell surface. We demonstrate that C66Y and multiple other EpCAM cancer-associated mutations prevent surface expression of EpCAM. Cancer-associated mutations that prevent EpCAM cell surface expression abrogate the ability of EpCAM to inhibit CTSL activity and tumor cell invasion. CONCLUSIONS: These studies reveal a novel role for EpCAM as a CTSL inhibitor, confirm the functional relevance of multiple cancer-associated EpCAM mutations, and suggest a therapeutic vulnerability in cancers harboring EpCAM mutations.
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Catepsina L/antagonistas & inhibidores , Molécula de Adhesión Celular Epitelial/genética , Mutación , Neoplasias/genética , Animales , Catepsina L/fisiología , Molécula de Adhesión Celular Epitelial/fisiología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Invasividad NeoplásicaRESUMEN
In the realm of biomedical advancement, extracellular vesicles (EVs) are revolutionizing our capacity to diagnose, monitor, and predict disease progression. However, the comprehensive exploration and clinical application of EVs face significant limitations due to the current isolation techniques. The size exclusion chromatography, commercial precipitation reagents, and ultracentrifugation are frequently employed, necessitating skilled operators and entailing challenges related to consistency, reproducibility, quality, and yields. Notably, the formidable challenge of extracellular vesicle isolation persists when dealing with clinical samples of limited availability. This study addresses these challenges by aiming to devise a rapid, user-friendly, and high-recovery EVs isolation technique tailored for blood samples. The NTI-EXO precipitation method demonstrated a 5-fold increase in the recovery of serum EVs compared to current methodologies. Importantly, we illustrate that a mere two drops of blood (â¼100 µL) suffice for the recovery of enriched EVs. The integrity and quality of these isolated EVs were rigorously assessed for the size, purity, and contaminants. This method was validated through the successful isolation of EVs from organ transplant recipients to detect disease-specific exosomal markers, including LKB1, SARS-CoV-2 spike protein, and PD-L1. In conclusion, NTI-EXO method can be used for small clinical samples, thereby advancing discoveries in the EV-centric domain and propelling the frontiers of biomedical research and clinical applications.
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Over the years, scientists have studied the behavior and anatomy of many animals to understand the own species. However, despite the continuous efforts, it is often difficult to know for certain how the brain works due to the differences between the brains of animals and the human brain. While the use of animal models for research continues, the origin of human cognition and neurological disorders needs further elucidation. To that end, in vitro organoids that exhibit in vivo characteristics of the human brain have been recently developed. These brain-like organoids enable researchers to dive deeper into understanding the human brain, its neurological structures, and the causes of neurological pathologies. This paper reviews the recent developments in the regeneration of brain-like organoids using Matrigel and other alternatives. Further, gel-free methods that may enhance the regeneration process of organoids are discussed. Finally, the vascularized brain organoid growth and development in both in vitro and in vivo conditions are detailed.
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Encéfalo , Organoides , Animales , Humanos , Encéfalo/patología , Modelos AnimalesRESUMEN
Next generation sequencing of human cancer mutations has identified novel therapeutic targets. Activating Ras oncogene mutations play a central role in oncogenesis, and Ras-driven tumorigenesis upregulates an array of genes and signaling cascades that can transform normal cells into tumor cells. In this study, we investigated the role of altered localization of epithelial cell adhesion molecule (EpCAM) in Ras-expressing cells. Analysis of microarray data demonstrated that Ras expression induced EpCAM expression in normal breast epithelial cells. Fluorescent and confocal microscopy showed that H-Ras mediated transformation also promoted epithelial-to-mesenchymal transition (EMT) together with EpCAM. To consistently localize EpCAM in the cytosol, we generated a cancer-associated EpCAM mutant (EpCAM-L240A) that is retained in the cytosol compartment. Normal MCF-10A cells were transduced with H-Ras together with EpCAM wild-type (WT) or EpCAM-L240A. WT-EpCAM marginally effected invasion, proliferation, and soft agar growth. EpCAM-L240A, however, markedly altered cells and transformed to mesenchymal phenotype. Ras-EpCAM-L240A expression also promoted expression of EMT factors FRA1, ZEB1 with inflammatory cytokines IL-6, IL-8, and IL1. This altered morphology was reversed using MEK-specific inhibitors and to some extent JNK inhibition. Furthermore, these transformed cells were sensitized to apoptosis using paclitaxel and quercetin, but not other therapies. For the first time, we have demonstrated that EpCAM mutations can cooperate with H-Ras and promote EMT. Collectively, our results highlight future therapeutic opportunities in EpCAM and Ras mutated cancers.
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Transición Epitelial-Mesenquimal , Transducción de Señal , Humanos , Línea Celular Tumoral , Citosol/metabolismo , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Transición Epitelial-Mesenquimal/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
An important mechanism by which pancreatic cancer avoids antitumor immunity is by recruiting regulatory T cells (Tregs) to the tumor microenvironment. Recent studies suggest that suppressor Tregs and effector Th17 cells share a common lineage and differentiate based on the presence of certain cytokines in the microenvironment. Because IL-6 in the presence of TGF-ß has been shown to inhibit Treg development and induce Th17 cells, we hypothesized that altering the tumor cytokine environment could induce Th17 and reverse tumor-associated immune suppression. Pan02 murine pancreatic tumor cells that secrete TGF-ß were transduced with the gene encoding IL-6. C57BL/6 mice were injected s.c. with wild-type (WT), empty vector (EV), or IL-6-transduced Pan02 cells (IL-6 Pan02) to investigate the impact of IL-6 secretion in the tumor microenvironment. Mice bearing IL-6 Pan02 tumors demonstrated significant delay in tumor growth and better overall median survival compared with mice bearing WT or EV Pan02 tumors. Immunohistochemical analysis demonstrated an increase in Th17 cells (CD4(+)IL-23R(+) cells and CD4(+)IL-17(+) cells) in tumors of the IL-6 Pan02 group compared with WT or EV Pan02 tumors. The upregulation of IL-17-secreting CD4(+) tumor-infiltrating lymphocytes was substantiated at the cellular level by flow cytometry and ELISPOT assay and mRNA level for retinoic acid-related orphan receptor γt and IL-23R by RT-PCR. Thus, the addition of IL-6 to the tumor microenvironment skews the balance toward Th17 cells in a murine model of pancreatic cancer. The delayed tumor growth and improved survival suggests that induction of Th17 in the tumor microenvironment produces an antitumor effect.
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Linfocitos T CD4-Positivos/inmunología , Interleucina-17/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Pancreáticas/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Línea Celular Tumoral , Separación Celular , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunohistoquímica , Interleucina-17/biosíntesis , Interleucina-6/inmunología , Interleucina-6/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/metabolismo , Transducción GenéticaRESUMEN
INTRODUCTION: EpCAM is a cell-surface glycoprotein that is overexpressed in the majority of epithelial carcinomas. However, the functional role of EpCAM in regulating cancer invasion remains controversial, and the mechanism(s) underlying EpCAM-mediated regulation of breast cancer invasion remain to be defined. METHODS: EpCAM expression was manipulated in breast cancer cell lines using RNA interference and cDNA expression constructs. Recombinant EpCAM was used to rescue EpCAM signaling following specific ablation of EpCAM. Protein and gene expression, invasion, transcription factor activity, and protein phosphorylation were measured using standard molecular biology techniques. RESULTS: In loss-of-function, and gain-of-function experiments we demonstrate that EpCAM expression is associated with increased breast cancer invasion in vitro and in vivo. We demonstrate further that specific ablation of EpCAM expression is associated with decreased activator protein-1 (AP-1) transcription factor activity. Phosphoprotein analyses confirm that specific ablation of EpCAM is associated with decreased phosphorylation of the AP-1 subunit c-Jun. Recombinant soluble extracellular EpCAM (rEpCAM) is able to rescue invasion, AP-1 transcription factor activity, and c-Jun phosphorylation in a dose-dependent fashion. Pharmacologic inhibitors, and constitutively active constructs of the c-Jun N-terminal kinase (JNK) signal transduction pathway, suggest that the impact of EpCAM expression on AP-1 transcription factor activity is mediated through the JNK pathway. In functional rescue experiments, forced expression of c-Jun rescues invasion in breast cancer cells following specific ablation of EpCAM. CONCLUSIONS: These data demonstrate for the first time that EpCAM expression can influence the JNK/AP-1 signal transduction pathway, and suggest that modulation of AP-1 transcription factor activity contributes to EpCAM-dependent breast cancer invasion. These data have important implications for the design and application of molecular therapies targeting EpCAM.
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Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Antígenos de Neoplasias/genética , Neoplasias de la Mama/genética , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Molécula de Adhesión Celular Epitelial , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Desnudos , Invasividad Neoplásica , Fosforilación , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein expressed in epithelial tissues. EpCAM forms intercellular, homophilic adhesions, modulates epithelial junctional protein complex formation, and promotes epithelial tissue homeostasis. EpCAM is a target of molecular therapies and plays a prominent role in tumor biology. In this review, we focus on the dynamic regulation of EpCAM expression during epithelial-to-mesenchymal transition (EMT) and the functional implications of EpCAM expression on the regulation of EMT. EpCAM is frequently and highly expressed in epithelial cancers, while silenced in mesenchymal cancers. During EMT, EpCAM expression is downregulated by extracellular signal-regulated kinases (ERK) and EMT transcription factors, as well as by regulated intramembrane proteolysis (RIP). The functional impact of EpCAM expression on tumor biology is frequently dependent on the cancer type and predominant oncogenic signaling pathways, suggesting that the role of EpCAM in tumor biology and EMT is multifunctional. Membrane EpCAM is cleaved in cancers and its intracellular domain (EpICD) is transported into the nucleus and binds ß-catenin, FHL2, and LEF1. This stimulates gene transcription that promotes growth, cancer stem cell properties, and EMT. EpCAM is also regulated by epidermal growth factor receptor (EGFR) signaling and the EpCAM ectoderm (EpEX) is an EGFR ligand that affects EMT. EpCAM is expressed on circulating tumor and cancer stem cells undergoing EMT and modulates metastases and cancer treatment responses. Future research exploring EpCAM's role in EMT may reveal additional therapeutic opportunities.
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Molécula de Adhesión Celular Epitelial/fisiología , Transición Epitelial-Mesenquimal/fisiología , Línea Celular Tumoral , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Humanos , Proteínas con Homeodominio LIM/metabolismo , Proteínas Musculares/metabolismo , Neoplasias/metabolismo , Neoplasias/fisiopatología , Células Neoplásicas Circulantes/metabolismo , Células Madre Neoplásicas/fisiología , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , beta Catenina/metabolismoRESUMEN
RNA interference (RNAi) is a powerful new tool for the selective ablation of gene expression, facilitating loss-of-function studies. However, appropriate controls are considered essential to confirm the specificity of RNAi experiments. The most stringent control is rescue of the target gene in a form that is refractory to RNAi. To facilitate rescue of the target gene, we have created improved dual expression lentiviral vectors with the ability to simultaneously drive expression of a shRNA for RNA interference and a rescue transgene in a single vector system. In proof-of-principle experiments, we ablated more than 90% of target gene expression by targeting either the open reading frame, or the 3' UTR region. Target gene expression was successfully rescued with a cDNA containing silent third-codon point mutations in the targeted region or with native cDNA when the 3' UTR was targeted. Finally, expression of the rescue transgene can be manipulated by positional cloning and appropriate promoter selection. The dual expression lentiviral vectors described here represent a versatile strategy for confirming the integrity of RNAi experiments and may facilitate functional analyses even in the absence of an established gain-of-function model system.
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Vectores Genéticos , Lentivirus , Interferencia de ARN , Línea Celular , Expresión Génica , Humanos , TransgenesRESUMEN
BACKGROUND: Pancreatic cancer is a lethal malignancy that frequently acquires resistance to conventional chemotherapies often associated with overexpression of inhibitors of apoptosis proteins (IAPs). We have recently described a novel means to deliver second mitochondria-derived activator of caspases (SMAC) mimetics selectively to cancer cells employing the sigma-2 ligand/receptor interaction. The intrinsic death pathway agonist SMAC offers an excellent opportunity to counteract the anti-apoptotic activity of IAPs. SMAC mimetics have been used to sensitize several cancer types to chemotherapeutic agents but cancer-selective delivery and appropriate cellular localization have not yet been considered. In our current study, we tested the ability of the sigma-2/SMAC drug conjugate SW IV-134 to sensitize pancreatic cancer cells to gemcitabine. METHODS: Using the targeted SMAC mimetic SW IV-134, inhibition of the X-linked inhibitor of apoptosis proteins (XIAP) was induced pharmacologically and its impact on cell viability was studied alone and in combination with gemcitabine. Pathway analyses were performed by assessing caspase activation, PARP cleavage and membrane blebbing (Annexin-V), key components of apoptotic cell death. Single-agent treatment regimens were compared with combination therapy in a preclinical mouse model of pancreatic cancer. RESULTS: The sensitizing effect of XIAP interference toward gemcitabine was confirmed via pharmacological intervention using our recently designed, targeted SMAC mimetic SW IV-134 across a wide range of commonly used pancreatic cancer cell lines at concentrations where the individual drugs showed only minimal activity. On a mechanistic level, we identified involvement of key components of the apoptosis machinery during cell death execution. Furthermore, combination therapy proved superior in decreasing the tumor burden and extending the lives of the animals in a preclinical mouse model of pancreatic cancer. CONCLUSION: We believe that the strong sensitizing capacity of SW IV-134 in combination with clinically relevant doses of gemcitabine represents a promising treatment option that warrants clinical evaluation.
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Antimetabolitos Antineoplásicos/administración & dosificación , Antineoplásicos/administración & dosificación , Compuestos de Azabiciclo/administración & dosificación , Desoxicitidina/análogos & derivados , Oligopéptidos/administración & dosificación , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Compuestos de Azabiciclo/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/administración & dosificación , Desoxicitidina/farmacología , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Oligopéptidos/farmacología , Neoplasias Pancreáticas/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
TFF1 is a member of a unique family of gastrointestinal peptides. Loss of TFF1 expression has been observed in the majority of human gastric cancers and the biological significance of this loss has been demonstrated in a Tff1 knockout mouse model. However, few TFF1 gene mutations or allelic loss have also been documented. To understand the molecular mechanism repressing the TFF1 gene expression, the 5'-flanking region of the human TFF1 gene was characterized. We found a repressor region (-241 to -84), which is active in MKN45 and IMGE5 cells expressing endogenous TFF1 gene. A consensus binding site for C/EBPbeta was identified and EMSA analysis demonstrated specific binding of CEBPbeta. Mutation of this C/EBPbeta element potentiated the transactivation of TFF1 by 50% and 145% for MKN45 and IMGE5 cells respectively. Furthermore, co-transfection of C/EBPbeta isoforms specifically decreased TFF1 promoter activity. These findings suggest that C/EBPbeta is involved in the down-regulating of TFF1 gene expression and this mode of repression may account at least in part for the loss of TFF1 gene expression in transformed human and mice gastric epithelial cells.
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Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/genética , Proteínas/metabolismo , Factores de Transcripción/metabolismo , Región de Flanqueo 5'/genética , Animales , Secuencia de Bases , Proteína beta Potenciadora de Unión a CCAAT/genética , Ensayo de Cambio de Movilidad Electroforética , Humanos , Luciferasas , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligonucleótidos , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Proteínas/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Factor Trefoil-1 , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
This article presents two hybrid strategies for the modeling and optimization of the glucose to gluconic acid batch bioprocess. In the hybrid approaches, first a novel artificial intelligence formalism, namely, genetic programming (GP), is used to develop a process model solely from the historic process input-output data. In the next step, the input space of the GP-based model, representing process operating conditions, is optimized using two stochastic optimization (SO) formalisms, viz., genetic algorithms (GAs) and simultaneous perturbation stochastic approximation (SPSA). These SO formalisms possess certain unique advantages over the commonly used gradient-based optimization techniques. The principal advantage of the GP-GA and GP-SPSA hybrid techniques is that process modeling and optimization can be performed exclusively from the process input-output data without invoking the detailed knowledge of the process phenomenology. The GP-GA and GP-SPSA techniques have been employed for modeling and optimization of the glucose to gluconic acid bioprocess, and the optimized process operating conditions obtained thereby have been compared with those obtained using two other hybrid modeling-optimization paradigms integrating artificial neural networks (ANNs) and GA/SPSA formalisms. Finally, the overall optimized operating conditions given by the GP-GA method, when verified experimentally resulted in a significant improvement in the gluconic acid yield. The hybrid strategies presented here are generic in nature and can be employed for modeling and optimization of a wide variety of batch and continuous bioprocesses.
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Inteligencia Artificial , Aspergillus niger/metabolismo , Gluconatos/metabolismo , Glucosa/metabolismo , Aspergillus niger/crecimiento & desarrollo , Biomasa , Reactores Biológicos , Simulación por Computador , Fermentación , Procesos EstocásticosRESUMEN
The epithelial cell adhesion molecule (EpCAM) is a 40-kD type I transmembrane protein that is overexpressed in human epithelial cancers and is currently the target of molecular therapy based on its overexpression at the cell surface. Recently, we and others have shown a role for EpCAM in cell signaling and carcinogenesis, and EpCAM expression seems to promote breast cancer invasion. Interleukin-8 (IL-8/CXCL-8) is an inflammatory cytokine that has recently been shown to modulate breast cancer invasion and angiogenesis. In preliminary experiments, we identified a correlation between EpCAM and IL-8 expression in primary human breast cancers. Specific ablation of EpCAM in breast cancer cell lines results in decreased IL-8 expression, and IL-8 contributes to EpCAM-dependent breast cancer invasion. Specific ablation of EpCAM is also associated with decreased NF-κB transcription factor activity, decreased phosphorylation of the NF-κB family member RELA, and increased IκBα protein expression. EpCAM modulates IL-8 expression at baseline, and following IL-1ß stimulation, which is known to be a potent inducer of NF-κB in breast cancer. In functional rescue experiments, specific ablation of RELA or forced expression of the NF-κB inhibitor protein IκBα prevented EpCAM-dependent rescue of IL-8 promoter activity. These studies show for the first time that EpCAM can modulate NF-κB transcription factor activity and IL-8 expression in breast cancer and confirm the role of EpCAM signaling in modulating breast cancer invasion. Further study is required to define the molecular mechanism(s) of EpCAM signaling in breast cancer and to direct the rational development of molecular therapies targeting EpCAM.
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Antígenos de Neoplasias/biosíntesis , Neoplasias de la Mama/metabolismo , Moléculas de Adhesión Celular/biosíntesis , Interleucina-8/biosíntesis , FN-kappa B/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Molécula de Adhesión Celular Epitelial , Femenino , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Células MCF-7 , FN-kappa B/genética , Invasividad Neoplásica , Transducción de SeñalRESUMEN
p53 is a tumor suppressor gene with well-characterized roles in cell cycle regulation, apoptosis, and maintenance of genome stability. Recent evidence suggests that p53 may also contribute to the regulation of migration and invasion. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is overexpressed in the majority of human epithelial carcinomas, including breast and colorectal carcinomas. We show by chromatin immunoprecipitation assays that p53 interacts with a candidate p53 binding site within the EpCAM gene. p53-mediated transcriptional repression of EpCAM was confirmed in gain-of-function and loss-of-function experimental systems. Induction of wild-type p53 was associated with a significant dose-dependent decrease in EpCAM expression; conversely, specific ablation of p53 was associated with a significant increase in EpCAM expression. At the functional level, specific ablation of p53 expression is associated with increased breast cancer invasion, and this effect is abrogated by concomitant specific ablation of EpCAM expression. Taken together, these biochemical and functional data are the first demonstration that (a) wild-type p53 protein binds to a response element within the EpCAM gene and negatively regulates EpCAM expression, and (b) transcriptional repression of EpCAM contributes to p53 control of breast cancer invasion.