RESUMEN

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Asunto(s)

Movimiento Celular , Técnicas Citológicas/métodos , Línea Celular Tumoral , Proliferación Celular , Impedancia Eléctrica , Glioblastoma/patología , Humanos , Invasividad Neoplásica , Transducción de Señal , Factores de Tiempo

RESUMEN

Sustained reliance on androgen receptor (AR) after failure of AR-targeting androgen deprivation therapy (ADT) prevents effective treatment of castration-recurrent (CR) prostate cancer (CaP). Interfering with the molecular machinery by which AR drives CaP progression may be an alternative therapeutic strategy but its feasibility remains to be tested. Here, we explore targeting the mechanism by which AR, via RhoA, conveys androgen-responsiveness to serum response factor (SRF), which controls aggressive CaP behavior and is maintained in CR-CaP. Following a siRNA screen and candidate gene approach, RNA-Seq studies confirmed that the RhoA effector Protein Kinase N1 (PKN1) transduces androgen-responsiveness to SRF. Androgen treatment induced SRF-PKN1 interaction, and PKN1 knockdown or overexpression severely impaired or stimulated, respectively, androgen regulation of SRF target genes. PKN1 overexpression occurred during clinical CR-CaP progression, and hastened CaP growth and shortened CR-CaP survival in orthotopic CaP xenografts. PKN1's effects on SRF relied on its kinase domain. The multikinase inhibitor lestaurtinib inhibited PKN1 action and preferentially affected androgen regulation of SRF over direct AR target genes. In a CR-CaP patient-derived xenograft, expression of SRF target genes was maintained while AR target gene expression declined and proliferative gene expression increased. PKN1 inhibition decreased viability of CaP cells before and after ADT. In patient-derived CaP explants, lestaurtinib increased AR target gene expression but did not significantly alter SRF target gene or proliferative gene expression. These results provide proof-of-principle for selective forms of ADT that preferentially target different fractions of AR's transcriptional output to inhibit CaP growth.

Asunto(s)

Andrógenos/metabolismo , Neoplasias de la Próstata/terapia , Proteína Quinasa C/metabolismo , Factor de Respuesta Sérica/metabolismo , Animales , Carbazoles/farmacología , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Progresión de la Enfermedad , Furanos , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias de la Próstata/metabolismo , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismo

Asunto(s)

Antineoplásicos/uso terapéutico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Chaperoninas/antagonistas & inhibidores , Expresión Génica , Linfoma Anaplásico de Células Grandes/tratamiento farmacológico , Linfoma Anaplásico de Células Grandes/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/genética , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Humanos , Linfoma Anaplásico de Células Grandes/metabolismo , Nucleofosmina , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/efectos adversos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Resultado del Tratamiento

RESUMEN

Epstein-Barr virus (EBV) is directly implicated in several B-cell lymphoid malignancies. EBV-associated lymphomas are characterized by prominent activation of the NF-κB pathway and targeting this pathway establishes a rationale for a therapeutic approach. The ubiquitin/proteasome signaling plays an essential role in the regulation of the NF-κB pathway. Ixazomib is an FDA-approved, orally bioavailable proteasome inhibitor. Here we report the first preclinical evaluation of ixazomib-mediated growth-inhibitory effects on EBV-infected B-lymphoblastoid cell lines Raji and Daudi. Ixazomib induced apoptosis in these cell lines in a dose-dependent manner. Cell-cycle analysis demonstrated ixazomib treatment induced cell-cycle arrest at the G2-M phase with a concomitant decrease in G0-G1 and S phases. The results further revealed an increase in p53, p21, and p27 levels and a decrease in survivin and c-Myc protein levels. Mechanistically, ixazomib treatment resulted in the accumulation of polyubiquitinated proteins, including phosphorylated IκBα with a significant reduction of p65 subunit nuclear translocation. Altogether, our preclinical data support the rationale for in vivo testing of ixazomib in EBV-associated B-cell neoplasms. IMPLICATIONS: This preclinical study supports the use of oral proteasome inhibitor ixazomib for targeting NF-κB signaling in the treatment of EBV-associated B-cell neoplasms.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/4/839/F1.large.jpg.

Asunto(s)

Compuestos de Boro/farmacología , Infecciones por Virus de Epstein-Barr/patología , Glicina/análogos & derivados , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/virología , Antineoplásicos/farmacología , Línea Celular Tumoral , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Glicina/farmacología , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Terapia Molecular Dirigida , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/metabolismo , Fosforilación , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

The S100 calcium-binding protein A4 (S100A4) induces epithelial mesenchymal transition, migration, invasion, angiogenesis and metastasis. Its induced expression in several cancer types correlates with poor prognosis. Apart from the functional and transcriptional regulatory aspects of S100A4, its post-transcriptional regulation is not yet clearly elucidated. In this study, we show that microRNAs (miR) miR-505-5p and miR-520c-3p target the 3'-UTR of S100A4 and inhibits its expression and its mediated migration and invasion. 5-Aza treatment significantly increased miR-520c-3p expression and reduced the S100A4 protein amounts. The upstream promoter region of miR-520c is hypermethylated irrespective of the metastasis status of colorectal cancer (CRC) patient tissues and in all analyzed CRC cell lines. Moreover, in a cohort of CRC patient specimen (n = 59), miR-520c-3p was significantly downregulated. miR-520c-3p stably expressing HCT116 cells showed a reduced metastasis formation in livers after implanting in mice spleen. Taken together, our findings demonstrate that S100A4 is post-transcriptionally regulated by tumor suppressor miRs, miR-505c-5p and miR-520c-3p, and particularly miR-520c-3p expression is epigenetically silenced in CRC.

Asunto(s)

Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica/genética , MicroARNs/genética , Proteína de Unión al Calcio S100A4/biosíntesis , Animales , Neoplasias Colorrectales/patología , Metilación de ADN/genética , Progresión de la Enfermedad , Epigénesis Genética/genética , Silenciador del Gen , Células HCT116 , Xenoinjertos , Humanos , Ratones , Reacción en Cadena de la Polimerasa , Proteína de Unión al Calcio S100A4/genética

RESUMEN

Metastasis is a multistep molecular network process, which is lethal for more than 90% of the cancer patients. Understanding the regulatory functions of metastasis-inducing molecules is in high demand for improved therapeutic cancer approaches. Thus, we studied the post-transcriptional regulation of the crucial carcinogenic and metastasis-mediating molecule metastasis associated in colon cancer 1 (MACC1). In silico analysis revealed MACC1 as a potential target of miR-218, a tumor suppressor miRNA. Expression of these two molecules inversely correlated in colorectal cancer (CRC) cell lines. In a cohort of CRC patient tissues (n = 59), miR-218 is significantly downregulated and MACC1 is upregulated compared with normal mucosa. Luciferase reporter assays with a construct of the MACC1-3'-UTR harboring either the wild type or the mutated miR-218 seed sequence confirmed the specificity of the targeting. miR-218 inhibited significantly MACC1 protein expression, and consistently, MACC1-mediated migration, invasion and colony formation in CRC cells. Anti-miR-218 enhanced the MACC1-mediated migration, invasion and colony formation. Similar findings were observed in the gastric cancer cell line MKN-45. Further, we performed methylation-specific PCR of the SLIT2 and SLIT3 promoter, where miR-218 is encoded in intronic regions. The SLIT2 and SLIT3 promoters are hypermethylated in CRC cell lines. miR-218 and SLIT2 expressions correlated positively. Methyltransferase inhibitor 5-Azacytidine induced miR-218 expression and inhibited the expression of its target MACC1. We also determined that MACC1 has alternative polyadenylation (APA) sites, which results in different lengths of 3'-UTR variants in a CRC cell line. Taken together, miR-218 is post-transcriptionally inhibiting the MACC1 expression and its metastasis-inducing abilities.

Asunto(s)

Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica/genética , MicroARNs/genética , Factores de Transcripción/biosíntesis , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Humanos , Transactivadores , Factores de Transcripción/genética , Transcripción Genética

RESUMEN

The aberrant activity of Wnt signaling is an early step in the transformation of normal intestinal cells to malignant tissue, leading to more aggressive tumors, and eventually metastases. In colorectal cancer (CRC), metastasis accounts for about 90% of patient deaths, representing the most lethal event during the course of the disease and is directly linked to patient survival, critically limiting successful therapy. This review focuses on our studies of the metastasis-inducing gene S100A4, which we identified as transcriptional target of ß-catenin. S100A4 increased migration and invasion in vitro and metastasis in mice. In patient CRC samples, high S100A4 levels predict metastasis and reduced patient survival. Our results link pathways important for tumor progression and metastasis: the Wnt signaling pathway and S100A4, which regulates motility and invasiveness. S100A4 suppression by interdicting Wnt signaling has potential for therapeutic intervention. As proof of principle, we applied S100A4 shRNA systemically and prevented metastasis in mice. Furthermore, we identified small molecule inhibitors from high-throughput screens of pharmacologically active compounds employing an S100A4 promoter-driven reporter. Best hits act, as least in part, via intervening in the Wnt pathway and restricted metastasis in mouse models. We currently translate our findings on restricting S100A4-driven metastasis into clinical practice. The repositioned FDA-approved drug niclosamide, targeting Wnt signaling, is being tested in a prospective phase II clinical trial for treatment of CRC patients. Our assay for circulating S100A4 transcripts in patient blood is used to monitor treatment success.

RESUMEN

The increasing unraveling of the molecular basis of cancer offers manifold novel options for intervention strategies. However, the discovery and development of new drugs for potential clinical applications is a tremendously time-consuming and costly process. Translating a novel lead candidate compound into an approved clinical drug takes often more than a decade, and the success rate is very low due to versatile efforts including defining its pharmacokinetics, pharmacodynamics, side effects as well as lack of sufficient efficacy. Thus, strategies are needed to minimize time and costs, while maximizing success rates. A very attractive strategy for novel cancer therapeutic options is the repositioning of already approved drugs. These medicines, approved for the treatment of non-malignant disorders, have already passed some early costs and time, have been tested in humans and are ready for clinical trials as anti-cancer drugs. Here we discuss the repositioning of nonsteroidal anti-inflammatory drugs (NSAID), statins, anti-psychotic drugs, anti-helminthic drugs and vitamin D as anti-tumor agents. We focus on their novel actions and potential for inhibition of cancer growth and metastasis by interfering with target molecules and pathways, which drive these malignant processes. Furthermore, important pre-clinical and clinical data are reviewed herein, which elucidate their therapeutic mechanisms which enable their repositioning for cancer therapy and disruption of metastasis.

Asunto(s)

Antineoplásicos/farmacología , Reposicionamiento de Medicamentos , Neoplasias/tratamiento farmacológico , Animales , Progresión de la Enfermedad , Humanos , Terapia Molecular Dirigida , Metástasis de la Neoplasia , Neoplasias/patología

RESUMEN

The microRNA (miRNA) landscape changes during the progression of cancer. We defined a metastasis-associated miRNA landscape using a systematic approach. We profiled and validated miRNA and mRNA expression in a unique series of human colorectal metastasis tissues together with their matched primary tumors and corresponding normal tissues. We identified an exclusive miRNA signature that is differentially expressed in metastases. Three of these miRNAs were identified as key drivers of an EMT-regulating network acting though a number of novel targets. These targets include SIAH1, SETD2, ZEB2, and especially FOXN3, which we demonstrated for the first time as a direct transcriptional suppressor of N-cadherin. The modulation of N-cadherin expression had significant impact on migration, invasion, and metastasis in two different in vivo models. The significant deregulation of the miRNAs defining the network was confirmed in an independent patient set as well as in a database of diverse malignancies derived from more than 6,000 patients. Our data define a novel metastasis-orchestrating network based on systematic hypothesis generation from metastasis tissues.

Asunto(s)

Neoplasias Colorrectales/genética , Neoplasias Colorrectales/secundario , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Animales , Antígenos CD/genética , Cadherinas/genética , Proteínas de Ciclo Celular/genética , Bases de Datos Factuales , Transición Epitelial-Mesenquimal/genética , Factores de Transcripción Forkhead , N-Metiltransferasa de Histona-Lisina/genética , Proteínas de Homeodominio/genética , Humanos , Ratones Desnudos , Metástasis de la Neoplasia/genética , Proteínas Nucleares/genética , Valores de Referencia , Proteínas Represoras/genética , Reproducibilidad de los Resultados , Ubiquitina-Proteína Ligasas/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc

RESUMEN

MicroRNAs (miRNAs) have been widely studied in order to elucidate their biological functions. MicroRNA microarrays or miRNA overexpression libraries generated by synthesis and cloning of individual miRNAs have been used to study their different roles. In this work, we have developed a novel methodology to express mature miRNAs and other small RNAs from a double convergent RNA polymerase III promoter. We show that the generated miRNAs function similarly to those processed from primary transcripts or pri-miRNAs. This system allowed us to produce a lentiviral library expressing the whole population of small RNAs present in a metastatic cell line. A functional screening using this library led to the identification of hsa-miR-30b and hsa-miR-30c as negative regulators of cell death induced by loss of attachment (anoikis). Importantly, we demonstrated that the acquisition of anoikis resistance via these miRNAs is achieved through down-regulation of caspase 3 expression. Moreover, overexpression of these miRNAs resulted in a decrease of other types of caspase 3-dependent cell death and enhanced the survival of MCF10A acinar cells in morphogenesis assays, suggesting a putative role as oncomirs. In summary, this novel methodology provides a powerful and effective way for identifying novel small RNAs involved in a particular biological process.

Asunto(s)

Anoicis/genética , Caspasa 3/genética , MicroARNs/genética , Regiones no Traducidas 3' , Secuencia de Bases , Sitios de Unión , Caspasa 3/metabolismo , Técnicas de Cultivo de Célula , Forma de la Célula , Represión Enzimática , Femenino , Expresión Génica , Biblioteca de Genes , Células HCT116 , Células HEK293 , Humanos , Glándulas Mamarias Humanas/citología , MicroARNs/metabolismo , Morfogénesis , Interferencia de ARN

RESUMEN

UNLABELLED: Malignant cell transformation, invasion, and metastasis are dependent on the coordinated rewiring of gene expression. A major component in the scaffold of these reprogramming events is one in which epithelial cells lose intercellular connections and polarity to adopt a more motile mesenchymal phenotype, which is largely supported by a robust transcriptional machinery consisting mostly of developmental transcription factors. This study demonstrates that the winged helix transcription factor, FOXQ1, contributes to this rewiring process, in part by directly modulating the transcription of TWIST1, itself a key mediator of metastasis that transcriptionally regulates the expression of important molecules involved in epithelial-to-mesenchymal transition. Forced expression and RNA-mediated silencing of FOXQ1 led to enhanced and suppressed mRNA and protein levels of TWIST1, respectively. Mechanistically, FOXQ1 enhanced the reporter activity of TWIST1 and directly interacted with its promoter. Furthermore, enhanced expression of FOXQ1 resulted in increased migration and invasion in colorectal cancer cell lines, whereas knockdown studies showed the opposite effect. Moreover, using the in vivo chicken chorioallantoic membrane metastasis assay model, FOXQ1 significantly enhanced distant metastasis with minimal effects on tumor growth. IMPLICATIONS: These findings reveal FOXQ1 as a modulator of TWIST1-mediated metastatic phenotypes and support its potential as a biomarker of metastasis.

Asunto(s)

Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Transición Epitelial-Mesenquimal , Factores de Transcripción Forkhead/metabolismo , Metástasis de la Neoplasia , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/genética , Perros , Células Epiteliales/metabolismo , Células Epiteliales/patología , Factores de Transcripción Forkhead/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Células de Riñón Canino Madin Darby , Datos de Secuencia Molecular , Invasividad Neoplásica , Neoplasias Experimentales , Proteínas Nucleares/genética , Alineación de Secuencia , Proteína 1 Relacionada con Twist/genética

RESUMEN

Cancer is a complex disease process that evolves as a consequence of multiple malfunctions in key regulatory molecular networks. Understanding these networks will be essential to combat cancer. In this study, we focussed on central players in such networks. In a series of colon and breast cancer cell lines, we found that CD24 activates Src, and induces the activation of c-Jun and expression of c-Jun and c-Fos. Thereby CD24 increases the promoter activity and expression of miR-21, which in turn suppresses expression of Pdcd4 and PTEN. Co-transfection of a CD24 expression construct and an siRNA that silences Src showed that CD24-dependent upregulation of miR-21 is mediated by Src. Additionally, we found that miR-34a post-transcriptionally downregulates CD24 and Src expression, leading to the deactivation of c-Jun, reduced expression of c-Jun and c-Fos, inhibition of miR-21, and upregulation of Pdcd4 and PTEN. Furthermore, miR-34a-mediated inhibition of Src expression reduced migration and invasion of colorectal cancer cells. Resected tumor tissues from 26 colorectal patients showed significantly lower expression of Pdcd4 and miR-34a, and higher expression of CD24, Src and miR-21 compared to the corresponding normal tissues. Moreover, CD24 positively correlated with the amount of Src protein in tumor tissues, and a trend towards an inverse correlation between miR-34a and Src protein levels was also observed. Our results reveal essential players in the complex networks that regulate the progression of solid tumors such as colorectal cancer. These findings therefore identify novel therapeutic approaches for combating tumor growth and progression.

Asunto(s)

Antígeno CD24/metabolismo , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Antígeno CD24/genética , Línea Celular Tumoral , Células HCT116 , Células HT29 , Humanos , MicroARNs/genética , Proteínas Proto-Oncogénicas pp60(c-src)/genética

RESUMEN

BACKGROUND: Tri- and tetra-nucleotide repeats in mammalian genomes can induce formation of alternative non-B DNA structures such as triplexes and guanine (G)-quadruplexes. These structures can induce mutagenesis, chromosomal translocations and genomic instability. We wanted to determine if proteins that bind triplex DNA structures are quantitatively or qualitatively different between colorectal tumor and adjacent normal tissue and if this binding activity correlates with patient clinical characteristics. METHODS: Extracts from 63 human colorectal tumor and adjacent normal tissues were examined by gel shifts (EMSA) for triplex DNA-binding proteins, which were correlated with clinicopathological tumor characteristics using the Mann-Whitney U, Spearman's rho, Kaplan-Meier and Mantel-Cox log-rank tests. Biotinylated triplex DNA and streptavidin agarose affinity binding were used to purify triplex-binding proteins in RKO cells. Western blotting and reverse-phase protein array were used to measure protein expression in tissue extracts. RESULTS: Increased triplex DNA-binding activity in tumor extracts correlated significantly with lymphatic disease, metastasis, and reduced overall survival. We identified three multifunctional splicing factors with biotinylated triplex DNA affinity: U2AF65 in cytoplasmic extracts, and PSF and p54nrb in nuclear extracts. Super-shift EMSA with anti-U2AF65 antibodies produced a shifted band of the major EMSA H3 complex, identifying U2AF65 as the protein present in the major EMSA band. U2AF65 expression correlated significantly with EMSA H3 values in all extracts and was higher in extracts from Stage III/IV vs. Stage I/II colon tumors (p=0.024). EMSA H3 values and U2AF65 expression also correlated significantly with GSK3 beta, beta-catenin, and NF- B p65 expression, whereas p54nrb and PSF expression correlated with c-Myc, cyclin D1, and CDK4. EMSA values and expression of all three splicing factors correlated with ErbB1, mTOR, PTEN, and Stat5. Western blots confirmed that full-length and truncated beta-catenin expression correlated with U2AF65 expression in tumor extracts. CONCLUSIONS: Increased triplex DNA-binding activity in vitro correlates with lymph node disease, metastasis, and reduced overall survival in colorectal cancer, and increased U2AF65 expression is associated with total and truncated beta-catenin expression in high-stage colorectal tumors.

Asunto(s)

Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/mortalidad , Proteínas de Unión al ADN/metabolismo , Proteómica , Neoplasias Colorrectales/patología , ADN/metabolismo , Proteínas de Unión al ADN/genética , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ganglios Linfáticos/patología , Masculino , Metástasis de la Neoplasia , Estadificación de Neoplasias , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción de Octámeros/genética , Factores de Transcripción de Octámeros/metabolismo , Factor de Empalme Asociado a PTB , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Factor de Empalme U2AF , Helicasa del Síndrome de Werner , beta Catenina/genética , beta Catenina/metabolismo

RESUMEN

MicroRNAs have come to represent a significant mechanism of post transcriptional gene regulation affecting processes as varied as cellular differentiation, proliferation, metabolism, apoptosis, and cancer. As more miRNAs are unravelled and their roles dissected, it has become evident that the involvement of these molecules in cancer is much more extensive than initially thought. Several miRNA expression analyses in both haematological malignancies and solid tumors have shown that, aside significant differences in expression between tumor and normal states, distinct tumor specific miRNA signatures exist. Additionally, the ability of miRNAs to mediate both oncogenic and tumor suppressor functions further broadens their functional significance. In recent years, efforts have intensified to utilize miRNAs therapeutically, especially in the context of oncomirs. As far as the impact and the success of this approach are concerned, it is still early days, but the potential is enormous. This review focuses on the important miRNAs that have been found to impact the tumorigenic process, how far we have come in terms of utilizing these molecules for therapy and the outlook for the near future.

Asunto(s)

Terapia Genética/métodos , MicroARNs/genética , Neoplasias/genética , Neoplasias/terapia , Oligonucleótidos Antisentido/uso terapéutico , Animales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , MicroARNs/metabolismo , Neoplasias/metabolismo

RESUMEN

Expression of the glycosylphosphatidylinositol-anchored membrane protein CD24 correlates with a poor prognosis for many human cancers, and in experimental tumors can promote metastasis. However, the mechanism by which CD24 contributes to tumor progression remains unclear. Here we report that in MTLy breast cancer cells CD24 interacts with and augments the kinase activity of c-src, a protein strongly implicated in promoting invasion and metastasis. This occurs within and is dependent upon intact lipid rafts. CD24-augmented c-src kinase activity increased formation of focal adhesion complexes, accelerated phosphorylation of FAK and paxillin and consequently enhanced integrin-mediated adhesion. Loss and gain of function approaches showed that c-src activity is necessary and sufficient to mediate the effects of CD24 on integrin-dependent adhesion and cell spreading, as well as on invasion. Together these results indicate that c-src is a CD24-activated mediator that promotes integrin-mediated adhesion and invasion, and suggest a mechanism by which CD24 might contribute to tumor progression through stimulating the activity of c-src or another member of the Src family.

Asunto(s)

Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Antígeno CD24/metabolismo , Integrinas/metabolismo , Microdominios de Membrana/enzimología , Microdominios de Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Antibacterianos/farmacología , Neoplasias de la Mama/metabolismo , Proteína Tirosina Quinasa CSK , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Doxiciclina/farmacología , Femenino , Fibronectinas/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Paxillin/metabolismo , Fosforilación , Unión Proteica , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Familia-src Quinasas

RESUMEN

MicroRNAs (miRNAs) are small non-coding RNAs which regulate gene expression by base-pairing to the 3'-UTR of the target mRNA. Recently, miRNAs have been shown to regulate cancer metastasis, however, central molecular mechanisms of this ability still need to be investigated. Epithelial to mesenchymal transition (EMT), which is characterized especially by repression of E-cadherin expression and increased cell motility, is an essential component of cancer metastasis and progression. In the present study, we found that Snai1, a known transcriptional repressor of E-cadherin and modulator of EMT, is post-transcriptionally targeted by miRNA-30a in non-small cell lung cancer (NSCLC). Consistent with this, microRNA-30a expression was found inversely proportional to the invasive potential of various NSCLC cell lines, correlating positively with E-cadherin (epithelial marker) and negatively with N-cadherin (mesenchymal marker) expression. Forced re-introduction of miR-30a significantly altered cell morphology, in vitro invasion and migration of invasive cell lines, this being paralleled by a downregulation of Snai1 and upregulation of E-cadherin expression. Using a chicken embryonic metastasis assay, we found that miR-30a suppresses in vivo distant metastasis to the lungs and liver. Finally, we screened the expression of miR-30a in 64 consecutively resected NSCLC patients and found that, in 81% of the patients, expression of miR-30a was downregulated significantly (p < 0.0001) in tumors compared to corresponding normal tissues. These results suggest that miR-30a targets Snai1, inhibits invasion and metastasis, and is downregulated in NSCLC.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Transición Epitelial-Mesenquimal/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , MicroARNs/genética , MicroARNs/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Cadherinas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Movimiento Celular , Embrión de Pollo , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos , Humanos , Neoplasias Pulmonares/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Factores de Transcripción de la Familia Snail , Transfección

RESUMEN

Pdcd4 (programmed cell death protein 4) is an important novel tumour suppressor inhibiting transformation, translation, invasion and intravasation, and its expression is down-regulated in several cancers. However, little is known about the transcriptional regulation and the promoter of this important tumour suppressor. So far the following is the first comprehensive study to describe the regulation of Pdcd4 transcription by ZBP-89 (zinc-finger-binding protein 89), besides characterizing the gene promoter. We identified the transcriptional start sites of the human pdcd4 promoter, a functional CCAAT-box, and the basal promoter region. Within this basal region, computer-based analysis revealed several potential binding sites for ZBPs, especially for Sp (specificity protein) family members and ZBP-89. We identified four Sp1/Sp3/Sp4-binding elements to be indispensable for basal promoter activity. However, overexpression of Sp1 and Sp3 was not sufficient to enhance Pdcd4 protein expression. Analysis in different solid cancer cell lines showed a significant correlation between pdcd4 and zbp-89 mRNA amounts. In contrast with Sp transcription factors, overexpression of ZBP-89 led to an enhanced expression of Pdcd4 mRNA and protein. Additionally, specific knockdown of ZBP-89 resulted in a decreased pdcd4 gene expression. Reporter gene analysis showed a significant up-regulation of basal promoter activity by co-transfection with ZBP-89, which could be abolished by mithramycin treatment. Predicted binding of ZBP-89 to the basal promoter was confirmed by EMSA (electrophoretic mobility-shift assay) data and supershift analysis for ZBP-89. Taken together, data for the first time implicate ZBP-89 as a regulator of Pdcd4 by binding to the basal promoter either alone or by interacting with Sp family members.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/genética , Factores de Transcripción Sp/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Factor de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Plicamicina/farmacología , Regiones Promotoras Genéticas , Unión Proteica , Inhibidores de la Síntesis de la Proteína/farmacología , Análisis de Secuencia de ADN , Sitio de Iniciación de la Transcripción , Transcripción Genética

RESUMEN

Curcumin has promising potential in cancer prevention and therapy by interacting with proteins and modifying their expression and activity, which includes transcription factors, inflammatory cytokines and factors of cell survival, proliferation and angiogenesis. miR-21 is overexpressed in many tumours, promoting progression and metastasis. In the present study, we examined the potential of curcumin to regulate miR-21, tumour growth, invasion and in vivo metastasis in colorectal cancer. In Rko and HCT116 cells, we identified two new transcriptional start sites of the miR-21 gene and delineated its promoter region. PMA stimulation induced miR-21 expression via motifs bound with AP-1 (activator protein 1) transcription factors. Curcumin treatment reduced miR-21 promoter activity and expression in a dose-dependent manner by inhibiting AP-1 binding to the promoter, and induced the expression of the tumour suppressor Pdcd4 (programmed cell death protein 4), which is a target of miR-21. Curcumin-treated Rko and HCT116 cells were arrested in the G2/M phase with increasing concentrations. Furthermore, curcumin inhibited tumour growth, invasion and in vivo metastasis in the chicken-embryo-metastasis assay [CAM (chorionallantoic membrane) assay]. Additionally, curcumin significantly inhibited miR-21 expression in primary tumours generated in vivo in the CAM assay by Rko and HCT116 cells (P<0.00006 and P<0.035 respectively). Taken together, this is the first paper to show that curcumin inhibits the transcriptional regulation of miR-21 via AP-1, suppresses cell proliferation, tumour growth, invasion and in vivo metastasis, and stabilizes the expression of the tumour suppressor Pdcd4 in colorectal cancer.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/secundario , Curcumina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , MicroARNs/genética , Invasividad Neoplásica/prevención & control , Animales , Antineoplásicos/uso terapéutico , Proteínas Reguladoras de la Apoptosis/metabolismo , Ciclo Celular/efectos de los fármacos , Embrión de Pollo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Curcumina/uso terapéutico , Células HCT116 , Humanos , MicroARNs/antagonistas & inhibidores , Metástasis de la Neoplasia/tratamiento farmacológico , Proteínas de Unión al ARN/metabolismo , Activación Transcripcional/efectos de los fármacos

RESUMEN

MicroRNAs (miRNAs) are an emerging class of non-coding endogenous RNAs involved in multiple cellular processes, including cell differentiation. Treatment with retinoic acid (RA) results in neural differentiation of neuroblastoma cells. We wanted to elucidate whether miRNAs contribute to the gene expression changes induced by RA in neuroblastoma cells and whether miRNA regulation is involved in the transduction of the RA signal. We show here that RA treatment of SH-SY5Y neuroblastoma cells results in profound changes in the expression pattern of miRNAs. Up to 42 different miRNA species significantly changed their expression (26 up-regulated and 16 down-regulated). Among them, the closely related miR-10a and -10b showed the most prominent expression changes. Induction of miR-10a and -10b by RA also could be detected in LA-N-1 neuroblastoma cells. Loss of function experiments demonstrated that miR-10a and -10b are essential mediators of RA-induced neuroblastoma differentiation and of the associated changes in migration, invasion, and in vivo metastasis. In addition, we found that the SR-family splicing factor SFRS1 (SF2/ASF) is a target for miR-10a -and -10b in HeLa and SH-SY5Y neuroblastoma cells. We show here that changes in miR-10a and -10b expression levels may regulate SFRS1-dependent alternative splicing and translational functions. Taken together, our results give support to the idea that miRNA regulation plays a key role in RA-induced neuroblastoma cell differentiation. The discovery of SFRS1 as direct target of miR-10a and -10b supports the emerging functional interaction between two post-transcriptional mechanisms, microRNAs and splicing, in the neuronal differentiation context.

Asunto(s)

Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Receptores Inmunológicos/metabolismo , Tretinoina/farmacología , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Diferenciación Celular/genética , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Embrión de Pollo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Células HeLa , Humanos , Glicoproteínas de Membrana/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Neuroblastoma/genética , Neuroblastoma/patología , Proteínas Nucleares/genética , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Proteínas de Unión al ARN , Receptores Inmunológicos/genética , Factores de Empalme Serina-Arginina

RESUMEN

BACKGROUND: Axl is a receptor tyrosine kinase promoting anti-apoptosis, invasion and mitogenesis, and is highly expressed in different solid cancers. Axl basal transcriptional activity is driven by Sp1/Sp3, and overexpression of MZF-1 (myeloid zinc-finger 1) induces Axl transcription and gene expression. Furthermore, Axl expression is epigenetically controlled by CpG hypermethylation; however, little is known about inducible Axl gene expression and Axl regulation in haematopoetic malignancies. RESULTS: In the present study, we studied Axl transcriptional regulation under PMA-stimulated conditions in leukaemia cells. Luciferase analysis with sequential 5'-deletion constructs revealed that the -660/-580 region of the Axl promoter is indispensable for induced promoter activity under PMA stimulation. This region includes AP-1 (activator protein 1)/CREB [CRE (cAMP-response-element)-binding protein] motifs, five times partially overlapping TGCGTG repeats and multiple GT repeats. Mutational, supershift and ChIP (chromatin immunoprecipitation) analysis determined that AP-1 family members bind to AP-1 motifs and to the 5 × TGCGTG overlapping repeats, thus transactivating Axl promoter activity. Furthermore, specific inhibitors of PKC (protein kinase C), ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 reduced Axl expression. Additionally, mithramycin treatment abolished constitutive and PMA-induced Axl expression. CONCLUSIONS: Taken together the results of the present study suggest that PMA-induced Axl gene expression in leukaemia cells is mediated by AP-1 motifs and 5 × TGCGTG repeats within the promoter region -660/-580, and through the PKC/ERK1/2/AP-1 or PKC/p-38/AP-1 signalling axis.

Asunto(s)

Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Elementos de Respuesta , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Secuencia de Bases , Línea Celular Tumoral , Humanos , Leucemia/tratamiento farmacológico , Leucemia/enzimología , Leucemia/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Factor de Transcripción AP-1/genética , Tirosina Quinasa del Receptor Axl