RESUMEN
Tumor relapse after therapy typifies hepatocellular carcinoma (HCC) and is believed to be attributable to residual cancer stem cells (CSCs) that survive treatment. We have previously identified a CSC population derived from HCC that is characterized by CD133. Despite our growing knowledge of the importance of this subset of cells in driving HCC, the regulatory mechanism of CD133 is not known. Epigenetic changes are believed to be essential in the control of cancer and stem cells. Here, we report the epigenetic regulation of CD133 by miR-142-3p. The interaction between CD133 and miR-142-3p was identified by in silico prediction and substantiated by luciferase reporter analysis. Expression of CD133 was found to be inversely correlated with miR-142-3p in HCC clinical samples as well as in cell lines. Importantly, lower miR-142-3p expression in HCC was significantly associated with worst survival. Functional studies with miR-142-3p stably transduced in HCC cells demonstrated a diminished ability to self-renew, initiate tumor growth, invade, migrate, induce angiogenesis and resist chemotherapy. Rescue experiments whereby CD133 and miR-142-3p is simultaneously overexpressed compensated the deregulated ability of the cells to confer these features. Thus, miR-142-3p directly targets CD133 to regulate its ability to confer cancer and stem cell-like features in HCC.
Asunto(s)
Antígenos CD/metabolismo , Carcinoma Hepatocelular/metabolismo , Glicoproteínas/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , MicroARNs/metabolismo , Células Madre Neoplásicas/metabolismo , Péptidos/metabolismo , Antígeno AC133 , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Células Hep G2 , Xenoinjertos , Humanos , Neoplasias Hepáticas/genética , Ratones , Ratones Desnudos , MicroARNs/genética , Células Madre Neoplásicas/patología , TransfecciónRESUMEN
Tumor-initiating cells (TIC), also known as cancer stem cells, are regarded widely as a specific subpopulation of cells needed for cancer initiation and progression. TICs have yet to be identified in esophageal tumors that have an increasing incidence in developed countries. Here, we report a CD90(+) cell population found in esophageal squamous cell carcinoma (ESCC), which is endowed with stem cell-like properties and high tumorigenic and metastatic potential. mRNA profiling of these cells suggested pathways through which they drive tumor growth and metastasis, with deregulation of an Ets-1/MMP signaling pathway and epithelial-mesenchymal transition figuring prominently. These cells possessed higher self-renewal activity and were sufficient for tumor growth, differentiation, metastasis, and chemotherapeutic resistance. CD90(+) TICs were isolated and characterized from ESCC clinical specimens as well as ESCC cell lines. In freshly resected clinical specimens, they represented a rare cell population, the levels of which correlated with strong family histories and lymph node metastasis. Our results prompt further study of this CD90(+) population of esophageal TICs as potential therapeutic targets.
Asunto(s)
Carcinoma de Células Escamosas/secundario , Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Neoplasias Esofágicas/patología , Células Madre Neoplásicas/patología , Antígenos Thy-1/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Adhesión Celular , Diferenciación Celular , Progresión de la Enfermedad , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/secundario , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Metástasis Linfática , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Estadificación de Neoplasias , Células Madre Neoplásicas/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Antígenos Thy-1/química , Antígenos Thy-1/genéticaRESUMEN
Mitotic progression of mammalian cells is tightly regulated by the E3 ubiquitin ligase anaphase promoting complex (APC)/C. Deregulation of APC/C is frequently observed in cancer cells and is suggested to contribute to chromosome instability and cancer predisposition. In this study, we identified Daxx as a novel APC/C inhibitor frequently overexpressed in prostate cancer. Daxx interacts with the APC/C coactivators Cdc20 and Cdh1 in vivo, with the binding of Cdc20 dependent on the consensus destruction boxes near the N-terminal of the Daxx protein. Ectopic expression of Daxx, but not the D-box deleted mutant (DaxxΔD-box), inhibited the degradation of APC/Cdc20 and APC/Cdh1 substrates, leading to a transient delay in mitotic progression. Daxx is frequently upregulated in prostate cancer tissues; the expression level positively correlated with the Gleason score and disease metastasis (P = 0.027 and 0.032, respectively). Furthermore, ectopic expression of Daxx in a non-malignant prostate epithelial cell line induced polyploidy under mitotic stress. Our data suggest that Daxx may function as a novel APC/C inhibitor, which promotes chromosome instability during prostate cancer development.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/antagonistas & inhibidores , Cadherinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Inestabilidad Cromosómica , Mitosis , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Antígenos CD , Proteínas Cdc20 , Ciclo Celular , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proteínas Co-Represoras , Células HEK293 , Células HeLa , Humanos , Masculino , Chaperonas Moleculares , Mutación , Clasificación del Tumor , Metástasis de la Neoplasia , Proteínas Nucleares/genética , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
Esophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is a devastating disease characterized by distinctly high incidences and mortality rates. However, there remains limited understanding of molecular events leading to development and progression of the disease, which are of paramount importance to defining biomarkers for diagnosis, prognosis, and personalized treatment. By high-throughout transcriptome sequence profiling of nontumor and ESCC clinical samples, we identified a subset of significantly differentially expressed genes involved in integrin signaling. The Rab25 gene implicated in endocytic recycling of integrins was the only gene in this group significantly downregulated, and its downregulation was confirmed as a frequent event in a second larger cohort of ESCC tumor specimens by quantitative real-time PCR and immunohistochemical analyses. Reduced expression of Rab25 correlated with decreased overall survival and was also documented in ESCC cell lines compared with pooled normal tissues. Demethylation treatment and bisulfite genomic sequencing analyses revealed that downregulation of Rab25 expression in both ESCC cell lines and clinical samples was associated with promoter hypermethylation. Functional studies using lentiviral-based overexpression and suppression systems lent direct support of Rab25 to function as an important tumor suppressor with both anti-invasive and -angiogenic abilities, through a deregulated FAK-Raf-MEK1/2-ERK signaling pathway. Further characterization of Rab25 may provide a prognostic biomarker for ESCC outcome prediction and a novel therapeutic target in ESCC treatment.
Asunto(s)
Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Genes Supresores de Tumor , Proteínas de Unión al GTP rab/genética , Animales , Secuencia de Bases , Carcinoma de Células Escamosas/irrigación sanguínea , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Metilación de ADN , Regulación hacia Abajo , Neoplasias Esofágicas/irrigación sanguínea , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Perfilación de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Invasividad Neoplásica , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Regiones Promotoras Genéticas , Proteínas de Unión al GTP rab/biosíntesisRESUMEN
BACKGROUND & AIMS: Esophageal squamous cell carcinoma (ESCC) is the most commonly observed histologic subtype of esophageal cancer. ESCC is believed to develop via accumulation of numerous genetic alterations, including inactivation of tumor suppressor genes and activation of oncogenes. We searched for transcripts that were altered in human ESCC samples compared with nontumor tissues. METHODS: We performed integrative transcriptome sequencing (RNA-Seq) analysis using ESCC samples from 3 patients and adjacent nontumor tissues to identify transcripts that were altered in ESCC tissue. We performed molecular and functional studies of the transcripts identified and investigated the mechanisms of alteration. RESULTS: We identified protein tyrosine kinase 6 (PTK6) as a transcript that was significantly down-regulated in ESCC tissues and cell lines compared with nontumor tissues or immortalized normal esophageal cell lines. The promoter of the PTK6 gene was inactivated in ESCC tissues at least in part via hypermethylation and histone deacetylation. Knockdown of PTK6 in KYSE30 ESCC cells using small hairpin RNAs increased their ability to form foci, migrate, and invade extracellular matrix in culture and form tumors in nude mice. Overexpression of PTK6 in these cells reduced their proliferation in culture and tumor formation in mice. PTK6 reduced phosphorylation of Akt and glycogen synthase kinase (GSK)3ß, leading to activation of ß-catenin. CONCLUSIONS: PTK6 was identified as a transcript that is down-regulated in human ESCC tissues via epigenetic modification at the PTK6 locus. Its product appears to regulate cell proliferation by reducing phosphorylation of Akt and GSK3ß, leading to activation of ß-catenin. Reduced levels of PTK6 promote growth of xenograft tumors in mice; it might be developed as a marker of ESCC.
Asunto(s)
Carcinoma de Células Escamosas/enzimología , Neoplasias Esofágicas/enzimología , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Acetilación , Adulto , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Metilación de ADN , Epigénesis Genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Matriz Extracelular/metabolismo , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Histonas/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Fosforilación , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal , Transcripción Genética , Transfección , Proteínas Supresoras de Tumor/genética , beta Catenina/metabolismoRESUMEN
UNLABELLED: A novel theory in the field of tumor biology postulates that cancer growth is driven by a population of stem-like cells, called tumor-initiating cells (TICs). We previously identified a TIC population derived from hepatocellular carcinoma (HCC) that is characterized by membrane expression of CD133. Here, we describe a novel mechanism by which these cells mediate tumor growth and angiogenesis by systematic comparison of the gene expression profiles between sorted CD133 liver subpopulations through genome-wide microarray analysis. A significantly dysregulated interleukin-8 (IL-8) signaling network was identified in CD133(+) liver TICs obtained from HCC clinical samples and cell lines. IL-8 was found to be overexpressed at both the genomic and proteomic levels in CD133(+) cells isolated from HCC cell lines or clinical samples. Functional studies found enhanced IL-8 secretion in CD133(+) liver TICs to exhibit a greater ability to self-renew, induce tumor angiogenesis, and initiate tumors. In further support of these observations, IL-8 repression in CD133(+) liver TICs by knockdown or neutralizing antibody abolished these effects. Subsequent studies of the IL-8 functional network identified neurotensin (NTS) and CXCL1 to be preferentially expressed in CD133(+) liver TICs. Addition of exogenous NTS resulted in concomitant up-regulation of IL-8 and CXCL1 with simultaneous activation of p-ERK1/2 and RAF-1, both key components of the mitogen-activated protein kinase (MAPK) pathway. Enhanced IL-8 secretion by CD133(+) liver TICs can in turn activate an IL-8-dependent feedback loop that signals through the MAPK pathway. Further, in its role as a liver TIC marker CD133 also plays a functional part in regulating tumorigenesis of liver TICs by way of regulating NTS, IL-8, CXCL1, and MAPK signaling. CONCLUSION: CD133(+) liver TICs promote angiogenesis, tumorigenesis, and self-renewal through NTS-induced activation of the IL-8 signaling cascade.
Asunto(s)
Antígenos CD/metabolismo , Proliferación Celular , Quimiocina CXCL1/fisiología , Glicoproteínas/metabolismo , Interleucina-8/fisiología , Neoplasias Hepáticas/patología , Células Madre Neoplásicas/fisiología , Neovascularización Patológica/fisiopatología , Neurotensina/fisiología , Péptidos/metabolismo , Transducción de Señal/fisiología , Antígeno AC133 , Animales , Antígenos CD/genética , Carcinoma Hepatocelular/irrigación sanguínea , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/cirugía , Línea Celular Tumoral , Células Cultivadas , Retroalimentación Fisiológica , Glicoproteínas/deficiencia , Glicoproteínas/genética , Hepatectomía , Humanos , Interleucina-8/deficiencia , Interleucina-8/genética , Hígado/irrigación sanguínea , Hígado/patología , Hígado/cirugía , Neoplasias Hepáticas/irrigación sanguínea , Neoplasias Hepáticas/cirugía , Ratones , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Células Madre Neoplásicas/inmunología , Neurotensina/farmacología , Péptidos/deficiencia , Péptidos/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Expression of microRNA genes is profoundly altered in cancer but their role in the development of androgen-independent prostate cancer has received limited attention as yet. In this study, we report a functional impact in prostate cancer cells for overexpression of the microRNA miR-616, which occurred consistently in cells that were androgen-independent (AI) versus androgen-dependent (AD). miR-616 overexpression was confirmed in malignant prostate tissues as opposed to benign prostate specimens. Stable miR-616 overexpression in LNCaP cells by a lentiviral-based approach stimulated AI prostate cancer cell proliferation in vitro whereas concomitantly reducing androgen-induced cell growth. More importantly, miR-616 overexpressing LNCaP cells overcame castration resistance as shown by an enhanced ability to proliferate in vivo after bilateral orchiectomy. Conversely, antagonizing miR-616 in AI prostate cancer cells yielded opposite effects. Microarray profiling and bioinformatics analysis identified the tissue factor pathway inhibitor TFPI-2 mRNA as a candidate downstream target of miR-616. In support of this candidacy, we documented interactions between miR-616 and the 3'UTR of TFPI-2 and determined TFPI-2 expression to be inversely correlated to miR-616 in a series of prostate cell lines and clinical specimens. Notably, reexpression of TFPI-2 in LNCaP cells with stable miR-616 overexpression rescued the AD phenotype, as shown by a restoration of androgen dependence and cell growth inhibition. Taken together, our findings define a functional involvement for miR-616 and TFPI-2 in the development and maintenance of androgen-independent prostate cancer.
Asunto(s)
Glicoproteínas/biosíntesis , MicroARNs/biosíntesis , Neoplasias de la Próstata/genética , Andrógenos/fisiología , Animales , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Desnudos , MicroARNs/genética , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Orquiectomía , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patologíaRESUMEN
A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs) or cancer stem cells (CSCs). Here we describe the identification and characterization of such cells from hepatocellular carcinoma (HCC) using the marker CD133. CD133 accounts for approximately 1.3%-13.6% of the cells in the bulk tumor of human primary HCC samples. When compared with their CD133â» counterparts, CD133(+) cells not only possess the preferential ability to form undifferentiated tumor spheroids in vitro but also express an enhanced level of stem cell-associated genes, have a greater ability to form tumors when implanted orthotopically in immunodeficient mice, and can be serially passaged into secondary animal recipients. Xenografts resemble the original human tumor and maintain a similar percentage of tumorigenic CD133(+) cells. Quantitative PCR analysis of 41 separate HCC tissue specimens with follow-up data found that CD133(+) tumor cells were frequently detected at low quantities in HCC, and their presence was also associated with worse overall survival and higher recurrence rates. Subsequent differential microRNA expression profiling of CD133(+) and CD133â» cells from human HCC clinical specimens and cell lines identified an overexpression of miR-130b in CD133(+) TICs. Functional studies on miR-130b lentiviral-transduced CD133â» cells demonstrated superior resistance to chemotherapeutic agents, enhanced tumorigenicity in vivo, and a greater potential for self renewal. Conversely, antagonizing miR-130b in CD133(+) TICs yielded an opposing effect. The increased miR-130b paralleled the reduced TP53INP1, a known miR-130b target. Silencing TP53INP1 in CD133â» cells enhanced both self renewal and tumorigenicity in vivo. Collectively, miR-130b regulates CD133(+) liver TICs, in part, via silencing TP53INP1.