RESUMEN
The oncogenic BRAFV600E kinase leads to abnormal activation of the MAPK signaling pathway and thus, uncontrolled cellular proliferation and cancer development. Based on our previous virtual screening studies which issued 2-acetamido-1,3 benzothiazole-6-carboxamide scaffold as active pharmacophore displaying selectivity against the mutated BRAF, eleven new substituted benzothiazole derivatives were designed and synthesized by coupling of 2-acetamidobenzo[d]thiazole-6-carboxylic acid with the appropriate amines in an effort to provide even more efficient inhibitors and tackle drug resistance often developed during cancer treatment. All derived compounds bore the benzothiazole scaffold substituted at position-2 by an acetamido moiety and at position-6 by a carboxamide functionality, the NH moiety of which was further linked through an alkylene linker to a sulfonamido (or amino) aryl (or alkyl) functionality or a phenylene linker to a sulfonamido aromatic (or non-aromatic) terminal pharmacophore in the order -C6 H4 -NHSO2 -R or reversely -C6 H4 -SO2 N(H)-R. These analogs were subsequently biologically evaluated as potential BRAFV600E inhibitors and antiproliferative agents in several colorectal cancer and melanoma cell lines. In all assays applied, one analog, namely 2-acetamido-N-[3-(pyridin-2-ylamino)propyl]benzo[d]thiazole-6-carboxamide (22), provided promising results in view of its use in drug development.
Asunto(s)
Antineoplásicos , Benzotiazoles , Línea Celular Tumoral , Benzotiazoles/farmacología , Antineoplásicos/farmacología , Proliferación Celular , Relación Estructura-Actividad , Ensayos de Selección de Medicamentos AntitumoralesRESUMEN
PLX7904 and PLX8394 are novel BRAFV600E inhibitors-BRAFi that are designed to evade the paradoxical MAPK activation, a trait for the name "paradox breakers"-PB. Current FDA approved inhibitors (Vemurafenib, Dabrafenib, Encorafenib) although improved progression-free survival of mtBRAF melanoma patients suffer from this treatment related side effect. mtBRAF Colorectal Cancer (CRC) is resistant to the approved BRAF inhibitors, although combinatorial treatment co-targeting BRAF and EGFR/MEK is offering a promising prospect. In an effort to explore the potential of the novel BRAF inhibitors-PB to impede CRC cell proliferation, they were tested on RKO, HT29 and Colo-205 cells, bearing the BRAFV600E mutation. This study shows that the BRAF paradox breakers PLX7904 and PLX8394 cause a more prolonged MAPK pathway inhibition and achieve a stronger blockage of proliferation and reduced viability than PLX4720, the sister compound of Vemurafenib. In some treatment conditions, cells can undergo apoptosis. Genomic analysis on the more resistant RKO cells treated with PLX7904, PLX8394 and PLX4720 showed similar gene expression pattern, but the alterations imposed by the PB were more intense. Bioinformatic analysis resulted in a short list of genes representing potential master regulators of the cellular response to BRAF inhibitors' treatments. From our results, it is clear that the BRAF paradox breakers present a notable differential regulation of major pathways, like MAPK signalling, apoptosis, cell cycle, or developmental signalling pathways. Combinatorial treatments of BRAFi with Mcl-1 and Notch modulators show a better effect than mono-treatments. Additional pathways could be further exploited in novel efficient combinatorial treatment protocols with BRAFi.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Compuestos Heterocíclicos con 2 Anillos/farmacología , Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Sulfonamidas/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mutación Puntual/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
BACKGROUND: NOXA and MCL1 are involved in the intrinsic pathway of apoptosis, where Noxa selectively binds to MCL1 and prevents it from inhibiting apoptosis. Both factors are considered as potential tumour biomarkers, while MCL1 has attracted interest as target in cancer. The purpose of this study was to investigate the expression of NOXA and MCL1 in 160 CRC tumour samples, to investigate their significance, also in combination with IAPs, DR5 expression and KRAS gene mutations in CRC. MATERIALS AND METHODS: Fresh frozen colorectal tissue was obtained from patients undergoing surgery for CRC. Real-time quantitative PCR was performed for the determination of mRNA expression levels. Protein expression was determined immunohistochemically. Differences in the mRNA expression profile were evaluated with the nonparametric Wilcoxon signed ranks test. Statistical analysis was performed with the use of Mann-Whitney U test and receiver-operating characteristic (ROC) curve. RESULTS: NOXA was found to be overexpressed in CRC tumours (P < .0001), even from early stage. Moreover, NOXA/MCL1 mRNA expression was significantly elevated in tumour samples compared to normal pairs (P < .0001). ROC curve analysis showed that both NOXA expression and its combination with Mcl1 expression have fair discriminatory value between CRC and normal colorectal tissue. Combinatorial ROC analysis revealed the most significant discriminatory value of NOXA, MCL1 with cIAP1 and cIAP2 (AUC = 0.834, P < .0001) as a 5-gene panel of markers. CONCLUSION: Noxa, Mcl1, DR5, cIAP1 and cIAP2 mRNA expressions are significantly deregulated in CRC and could provide a panel of markers with significant discriminatory value between CRC and normal colorectal tissue.
Asunto(s)
Neoplasias Colorrectales/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Biomarcadores de Tumor , Células CACO-2 , Neoplasias Colorrectales/metabolismo , Femenino , Células HCT116 , Células HT29 , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ubiquitina-Proteína Ligasas/genética , Regulación hacia ArribaRESUMEN
High expression of Inhibitor of apoptosis proteins (IAPs) has been related to colorectal cancer (CRC) progression, resistance to treatment and poor prognosis. TRAIL (TNF-related apoptosis-inducing ligand) through its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2) can selectively induce cancer cell apoptosis. The mRNA expression of DR4, DR5, c-IAP1, c-IAP2, XIAP and BIRC5/Survivin genes was examined in 100 paired (cancerous-normal) colorectal tissue specimens by real-time PCR, 50 of which were KRAS wild-type and 50 KRAS-mutant. DR5, XIAP and BIRC5/Survivin genes are significantly up-regulated (p < 0.0001, p = 0.012 and p = 0.0003, respectively), whereas c-IAP1 and c-IAP2 genes are significantly down-regulated at mRNA and protein levels in CRC (p < 0.0001 for both). ROC analyses showed that DR5, cIAP1 and cIAP2 expression has discriminatory value between CRC and normal tissue (AUC = 0.700, p < 0.0001 for DR5; AUC = 0.628, p = 0.011 for cIAP1; AUC = 0.673, p < 0.0001 for cIAP2). Combinatorial ROC analysis revealed the marginally fair discriminatory value of 5 genes as a panel (AUC = 0.685, p < 0.0001). Kaplan-Meier survival curves revealed significant association of cIAP2 down-regulation in CRC with lower overall survival probability of CRC patients (p = 0.0098). DR5, BIRC5/Survivin, XIAP, c-IAP1 and c-IAP2 mRNA expression are significantly deregulated in CRC and could provide a panel of markers with significant discriminatory value between CRC and normal colorectal tissue.
Asunto(s)
Apoptosis/genética , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/diagnóstico , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , ARN Mensajero/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Survivin , Regulación hacia Arriba , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
BACKGROUND: High expression levels of Inhibitors of Apoptosis Proteins (IAPs) have been correlated with poor cancer prognosis and block the cell death pathway by interfering with caspase activation. SMAC-mimetics are small-molecule inhibitors of IAPs that mimic the endogenous SMAC and promote the induction of cell death by neutralizing IAPs. METHODS: In this study, anti-tumour activity of new SMAC-mimetics Birinapant and AT-406 is evaluated against colorectal adenocarcinoma cells and IAP cross-talk with either oncogenic BRAF or BCL-2, or with the TRAIL are further exploited towards rational combined protocols. RESULTS: It is shown that pre-treatment of SMAC-mimetics followed by their combined treatment with BRAF inhibitors can decrease cell viability, migration and can very efficiently sensitize colorectal tumour cells to apoptosis. Moreover, co-treatment of TRAIL with SMAC-mimetics can efficiently sensitize resistant tumour cells to apoptosis synergistically, as shown by median effect analysis. Finally, Birinapant and AT-406 can synergise with BCL-2 inhibitor ABT-199 to reduce viability of adenocarcinoma cells with high BCL-2 expression. CONCLUSIONS: Proposed synergistic rational anticancer combined protocols of IAP antagonists Birinapant and AT-406 in 2D and 3D cultures can be later further exploited in vivo, from precision tumour biology to precision medical oncology.
Asunto(s)
Antineoplásicos/farmacología , Azocinas/farmacología , Compuestos de Bencidrilo/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Neoplasias Colorrectales/genética , Dipéptidos/farmacología , Indoles/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Sulfonamidas/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Células CACO-2 , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Células HT29 , Humanos , MutaciónRESUMEN
Autophagy is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components. Autophagy has a controversial role in cancer--both in protecting against tumor progression by isolation of damaged organelles, or by potentially contributing to cancer growth. The impact of autophagy in RAS induced transformation still remains to be further analyzed based on the differential effect of RAS isoforms and tumor cell context. In the present study, the effect of KRAS/BRAF/PIK3CA oncogenic pathways on the autophagic cell properties and on main components of the autophagic machinery like p62 (SQSTM1), Beclin-1 (BECN1) and MAP1LC3 (LC3) in colon cancer cells was investigated. This study provides evidence that BRAF oncogene induces the expression of key autophagic markers, like LC3 and BECN1 in colorectal tumor cells. Herein, PI3K/AKT/MTOR inhibitors induce autophagic tumor properties, whereas RAF/MEK/ERK signalling inhibitors reduce expression of autophagic markers. Based on the ineffectiveness of BRAFV600E inhibitors in BRAFV600E bearing colorectal tumors, the BRAF related autophagic properties in colorectal cancer cells are further exploited, by novel combinatorial anti-cancer protocols. Strong evidence is provided here that pre-treatment of autophagy inhibitor 3-MA followed by its combination with BRAFV600E targeting drug PLX4720 can synergistically sensitize resistant colorectal tumors. Notably, colorectal cancer cells are very sensitive to mono-treatments of another autophagy inhibitor, Bafilomycin A1. The findings of this study are expected to provide novel efficient protocols for treatment of otherwise resistant colorectal tumors bearing BRAFV600E, by exploiting the autophagic properties induced by BRAF oncogene.
Asunto(s)
Antineoplásicos/farmacología , Autofagia/fisiología , Neoplasias Colorrectales/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Beclina-1/metabolismo , Células CACO-2 , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Neoplasias Colorrectales/patología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Células HCT116 , Células HT29 , Humanos , Indoles/farmacología , Macrólidos/farmacología , Proteínas Asociadas a Microtúbulos/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteína Sequestosoma-1/metabolismo , Sulfonamidas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
Reorganization of cytoskeleton via actin remodeling is a basic step of cell locomotion. Although cell migration of normal and cancer cells can be stimulated by a variety of intra- and extra-cellular factors, all paths ultimate on the regulation of cofilin activity. Cofilin is a small actin-binding protein able to bind both forms of actin, globular and filament, and is regulated by phosphorylation at Serine 3. Following phosphorylation at serine 3 cofilin is inactive, therefore cannot bind actin molecules and cytoskeleton remodeling is impaired. The histone methyltransferase EZH2 is frequently over expressed in many tumour types including colorectal cancer (CRC). EZH2 over activity, which results in epigenetic gene-silencing, has been associated with many tumour properties including invasion, angiogenesis and metastasis but little is known about the underneath molecular mechanisms. Herein, we report that EZH2 is able to control cofilin activity and consequently cell locomotion of CRC cell lines through a non-conventional novel axis that involves integrin signaling. Indeed, we show how genetic and pharmacological inhibition (DZNep and GSK343) of EZH2 function produces hyper phosphorylation of cofilin and reduces cell migration. We previously demonstrated by chromatin immuno-precipitation that Integrin alpha 2 (ITGα2) expression is regulated by EZH2. In the present study we provide evidence that in EZH2-silenced cells the signaling activity of the de-repressed ITGα2 is able to increase cofilin phosphorylation, which in turn reduces cell migration. This study also proposes novel mechanisms that might provide new anti-metastatic strategies for CRC treatment based on the inhibition of the epigenetic factor EZH2 and/or its target gene.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Movimiento Celular , Neoplasias del Colon/metabolismo , Integrina alfa2/metabolismo , Proteínas de Neoplasias/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Factores Despolimerizantes de la Actina/genética , Células CACO-2 , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Proteína Potenciadora del Homólogo Zeste 2 , Humanos , Integrina alfa2/genética , Proteínas de Neoplasias/genética , Complejo Represivo Polycomb 2/genéticaRESUMEN
As the increased knowledge of tumour heterogeneity and genetic alterations progresses, it exemplifies the need for further personalized medicine in modern cancer management. Here, the similarities but also the differential effects of RAS and BRAF oncogenic signalling are examined and further implications in personalized cancer diagnosis and therapy are discussed. Redundant mechanisms mediated by the two oncogenes as well as differential regulation of signalling pathways and gene expression by RAS as compared to BRAF are addressed. The implications of RAS vs BRAF differential functions, in relevant tumour types including colorectal cancer, melanoma, lung cancer are discussed. Current therapeutic findings and future viewpoints concerning the exploitation of RAS-BRAF-pathway alterations for the development of novel therapeutics and efficient rational combinations, as well as companion tests for relevant markers of response will be evaluated. The concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance posed a major therapy hindrance.
Asunto(s)
Genes ras/genética , Mutación , Neoplasias/genética , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal/genética , Humanos , Neoplasias/tratamiento farmacológicoRESUMEN
Current clinical problems in colorectal cancer (CRC) diagnostics and therapeutics include the disease complexity, tumor heterogeneity, and resistance to targeted therapeutics. In the present study, we examined 171 CRC adenocarcinomas from Greek patients undergoing surgery for CRC to determine the frequency of KRAS, BRAF, and PIK3CA point mutations from different areas of tumors in heterogeneous specimens. Ninety two out of 171 (53.8%) patients were found to bear a KRAS mutation in codons 12/13. Of the 126 mutations found, 57.9% (73/126) were c.38G>A mutations (p.G13D) and 22.2% (28/126) were c.35G>T (p.G12V). Remarkably, RAS mutations in both codons 12 and 13 were recorded in the same tumor by pyrosequencing. Moreover, differences in KRAS mutations between tumor center and periphery revealed tumor heterogeneity in 50.7% of the specimens. BRAF c.1799T>A (V600E) mutations were moderately detected in 4/171 (2.3%) specimens, whereas most PIK3CA mutations were revealed by pyrosequencing 6/171 (3.5%). Remarkable tumor heterogeneity is revealed, where double mutations of KRAS in the same tumor and different KRAS mutation status between tumor core and margin are detected with high frequency. It is expected that these findings will have a major impact in cancer diagnosis and personalized therapies.
Asunto(s)
Neoplasias Colorrectales/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Fosfatidilinositol 3-Quinasa Clase I , Codón , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Mutación Puntual , Proteínas Proto-Oncogénicas p21(ras)RESUMEN
Previous studies have uncovered several transcription factors that determine biological alterations in tumor cells to execute the invasion-metastasis cascade, including the epithelial-mesenchymal transition (EMT). We sought to investigate the role of miR-21 in colorectal cancer regulation. For this purpose, miR-21 expression was quantified in a panel of colorectal cancer cell lines and clinical specimens. High expression was found in cell lines with EMT properties and in the vast majority of human tumor specimens. We demonstrate in a cell-specific manner the occupancy of MIR-21 gene promoter by AP-1 and ETS1 transcription factors and, for the first time, the pattern of histone posttranslational modifications necessary for miR-21 overexpression. We also show that Integrin-ß4 (ITGß4), exclusively expressed in polarized epithelial cells, is a novel miR-21 target gene and plays a role in the regulation of EMT, since it is remarkably de-repressed after transient miR-21 silencing and downregulated after miR-21 overexpression. miR-21-dependent change of ITGß4 expression significantly affects cell migration properties of colon cancer cells. Finally, in a subgroup of tumor specimens, ROC curve analysis performed on quantitative PCR data sets for miR-21, ITGß4, and PDCD4 shows that the combination of high miR-21 with low ITGß4 and PDCD4 expression is able to predict presence of metastasis. In conclusion, miR-21 is a key player in oncogenic EMT, its overexpression is controlled by the cooperation of genetic and epigenetic alterations, and its levels, along with ITGß4 and PDCD4 expression, could be exploited as a prognostic tool for CRC metastasis.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias Colorrectales/genética , Epigénesis Genética , Redes Reguladoras de Genes , Integrina beta4/genética , MicroARNs/genética , Proteínas de Unión al ARN/metabolismo , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Transición Epitelial-Mesenquimal , Histonas/metabolismo , Humanos , Integrina beta4/metabolismo , MicroARNs/metabolismo , Metástasis de la Neoplasia , Procesamiento Proteico-Postraduccional , Proteína Proto-Oncogénica c-ets-1/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
Despite the significant advances in clinical research, surgical resection, radiotherapy and chemotherapy are still used as the primary method for cancer treatment. As compared to conventional therapies that often induce systemic toxicity and eventually contribute to tumor resistance, the TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that selectively triggers apoptosis in various cancer cells by interacting with its proapoptotic receptors DR4 and KILLER/DR5, while sparing the normal surrounding tissue. The intensive studies of TRAIL signaling pathways over the past decade have provided clues for understanding the molecular mechanisms of TRAIL-induced apoptosis in carcinogenesis and identified an array of therapeutic responses elicited by TRAIL and its receptor agonists. Analysis of its activity at the molecular level has shown that TRAIL improves survival either as monotherapies or combinatorial therapies with other mediators of apoptosis or anticancer chemotherapy. Combinatorial treatments amplify the activities of anticancer agents and widen the therapeutic window by overcoming tumor resistance to apoptosis and driving cancer cells to self-destruction. Although TRAIL sensitivity varies widely depending on the cell type, nontransformed cells are largely resistant to death mediated by TRAIL Death Receptors (DRs). Genetic alterations in cancer can contribute in tumor progression and often play an important role in evasion of apoptosis by tumor cells. Remarkably, RAS, MYC and HER2 oncogenes have been shown to sensitise tumor cells to TRAIL induced cell death. Here, we summarise the cross-talk of oncogenic and apoptotic pathways and how they can be exploited toward efficient combinatorial therapeutic protocols.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Neoplasias/tratamiento farmacológico , Oncogenes , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Animales , Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos , Humanos , Sistema de Señalización de MAP Quinasas , Terapia Molecular Dirigida , Neoplasias/genética , Neoplasias/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéuticoRESUMEN
Epithelial-Mesenchymal Transition is a good example of cell plasticity. In tumorigenesis, this process has been associated with metastasis. Overexpression of EZH2 has been detected in most malignant human tumors, including colorectal carcinomas. Herein, we provide evidence supporting the idea that oncogenic Epithelial-Mesenchymal Transition in colon cancer cell models is partially controlled by epigenetic factors such as the transcription regulator EZH2. Evaluation of EZH2 mRNA and protein levels revealed overexpression in cell lines with metastatic traits. Analysis of EZH2 mRNA expression was expanded in clinical samples of colon cancer, and high level of EZH2 correlates with appearance of metastasis. Furthermore, inhibition of ERK and AKT pathways in metastatic colon cancer cell lines attenuates EZH2 overexpression. EZH2 promoter analysis illustrates presence of putative AP-1 binding sites and occupancy of transcription factors such as FRA-1 and C-JUN is demonstrated here on EZH2 promoter. Abrogation of EZH2 expression impairs the ability of colon cancer cells to move associated with anoikis in three-dimensional environment. Integrin alpha2 was identified to be a novel EZH2 target by chromatin immunoprecipitation and short hairpin RNA analysis. This study proposes that activation of ERK/AKT pathways and FRA1/C-JUN induce EZH2 overexpression, which results in Integrin alpha2 silencing. Our results show how deregulation of epigenetic factors and mechanisms can affect cancer cell aggressiveness and propose EZH2 as a potential metastasis marker and/or therapeutic target for colorectal cancer treatment.
Asunto(s)
Anoicis , Transición Epitelial-Mesenquimal , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Integrina alfa2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células CACO-2 , Movimiento Celular , Supervivencia Celular , Transformación Celular Neoplásica , Colon/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Proteína Potenciadora del Homólogo Zeste 2 , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HCT116 , Humanos , Integrina alfa2/metabolismo , Mucosa Intestinal/metabolismo , Metástasis Linfática , Complejo Represivo Polycomb 2/genética , Regiones Promotoras Genéticas , Unión Proteica , Transporte de Proteínas , ARN Interferente Pequeño/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
The objective of the ONCODEATH consortium [EU Research Consortium "ONCODEATH" (2006-2010)] was to achieve sensitization of solid tumor cells to death receptor related therapies using rational mechanism-based drug combinations of targeted therapies. In this collaborative effort, during a period of 42 mo, cell and animal model systems of defined oncogenes were generated. Exploitation of generated knowledge and tools enabled the consortium to achieve the following research objectives: (1) elucidation of tumor components which confer sensitivity or resistance to TRAIL-induced cell death; (2) providing detailed knowledge on how small molecule Hsp90, Aurora, Choline kinase, BRAF inhibitors, DNA damaging agents, HDAC and DNMT inhibitors affect the intrinsic apoptotic amplification and execution machineries; (3) optimization of combined action of TRAIL with these therapeutics for optimum effects with minimum concentrations and toxicity in vivo. These findings provide mechanistic basis for a pharmacogenomic approach, which could be exploited further therapeutically, in order to reach novel personalized therapies for cancer patients.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Terapia Molecular Dirigida , Receptores de Muerte Celular/antagonistas & inhibidores , Animales , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias del Colon/metabolismo , Inhibidores Enzimáticos/administración & dosificación , Humanos , Receptores de Muerte Celular/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Colorectal cancer is a common disease that involves genetic alterations, such as inactivation of tumour suppressor genes and activation of oncogenes. Among them are RAS and BRAF mutations, which rarely coexist in the same tumour. Individual members of the Rho (Ras homology) GTPases contribute with distinct roles in tumour cell morphology, invasion and metastasis. The aim of this study is to dissect cell migration and invasion pathways that are utilised by BRAFV600E as compared to KRASG12V and HRASG12V oncoproteins. In particular, the role of RhoA (Ras homolog gene family, member A), Rac1 (Ras-related C3 botulinum toxin substrate 1) and Cdc42 (cell division cycle 42) in cancer progression induced by each of the three oncogenes is described. METHODS: Colon adenocarcinoma cells with endogenous as well as ectopically expressed or silenced oncogenic mutations of BRAFV600E, KRASG12V and HRASG12V were employed. Signalling pathways and Rho GTPases were inhibited with specific kinase inhibitors and siRNAs. Cell motility and invasion properties were correlated with cytoskeletal properties and Rho GTPase activities. RESULTS: Evidence presented here indicate that BRAFV600E significantly induces cell migration and invasion properties in vitro in colon cancer cells, at least in part through activation of RhoA GTPase. The relationship established between BRAFV600E and RhoA activation is mediated by the MEK-ERK pathway. In parallel, KRASG12V enhances the ability of colon adenocarcinoma cells Caco-2 to migrate and invade through filopodia formation and PI3K-dependent Cdc42 activation. Ultimately increased cell migration and invasion, mediated by Rac1, along with the mesenchymal morphology obtained through the Epithelial-Mesenchymal Transition (EMT) were the main characteristics rendered by HRASG12V in Caco-2 cells. Moreover, BRAF and KRAS oncogenes are shown to cooperate with the TGFß-1 pathway to provide cells with additional transforming properties. CONCLUSION: This study discriminates oncogene-specific cell migration and invasion pathways mediated by Rho GTPases in colon cancer cells and reveals potential new oncogene-specific characteristics for targeted therapeutics.
Asunto(s)
Movimiento Celular , Proteína Oncogénica p21(ras)/genética , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal/efectos de los fármacos , Proteínas de Unión al GTP rho/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Forma de la Célula , Neoplasias del Colon , Regulación hacia Abajo , Activación Enzimática , Transición Epitelial-Mesenquimal , Técnicas de Silenciamiento del Gen , Humanos , Mutación Missense , Invasividad Neoplásica , Proteína Oncogénica p21(ras)/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Seudópodos/metabolismo , Transcripción Genética , Factor de Crecimiento Transformador beta1/farmacología , Factor de Crecimiento Transformador beta1/fisiología , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
Documented sensitivity of melanoma cells to PLX4720, a selective BRAFV600E inhibitor, is based on the presence of mutant BRAF(V600E) alone, while wt-BRAF or mutated KRAS result in cell proliferation. In colon cancer appearance of oncogenic alterations is complex , since BRAF, like KRAS mutations, tend to co-exist with those in PIK3CA and mutated PI3K has been shown to interfere with the successful application of MEK inhibitors. When PLX4720 was used to treat colon tumours, results were not encouraging and herein we attempt to understand the cause of this recorded resistance and discover rational therapeutic combinations to resensitize oncogene driven tumours to apoptosis. Treatment of two genetically different BRAF(V600E) mutant colon cancer cell lines with PLX4720 conferred complete resistance to cell death. Even though p-MAPK/ ERK kinase (MEK) suppression was achieved, TRAIL, an apoptosis inducing agent, was used synergistically in order to achieve cell death by apoptosis in RKO(BRAFV600E/PIK3CAH1047) cells. In contrast, for the same level of apoptosis in HT29(BRAFV600E/PIK3CAP449T) cells, TRAIL was combined with 17-AAG, an Hsp90 inhibitor. For cells where PLX4720 was completely ineffective, 17-AAG was alternatively used to target mutant BRAF(V600E). TRAIL dependence on the constitutive activation of BRAF(V600E) is emphasised through the overexpression of BRAF(V600E) in the permissive genetic background of colon adenocarcinoma Caco-2 cells. Pharmacological suppression of the PI3K pathway further enhances the synergistic effect between TRAIL and PLX4720 in RKO cells, indicating the presence of PIK3CA(MT) as the inhibitory factor. Another rational combination includes 17-AAG synergism with TRAIL in a BRAF(V600E) mutant dependent manner to commit cells to apoptosis, through DR5 and the amplification of the apoptotic pathway. We have successfully utilised combinations of two chemically unrelated BRAF(V600E) inhibitors in combination with TRAIL in a BRAF(V600E) mutated background and provided insight for new anti-cancer strategies where the activated PI3KCA mutation oncogene should be suppressed.
Asunto(s)
Indoles/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Sulfonamidas/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Células CACO-2 , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Citometría de Flujo , Furanos/farmacología , Células HT29 , Humanos , Immunoblotting , Inmunoprecipitación , Microscopía Fluorescente , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridinas/farmacología , Pirimidinas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The TAF4b subunit of the transcription factor IID, which has a central role in transcription by polymerase II, is involved in promoter recognition by selective recruitment of activators. The activating protein-1 (AP-1) family members participate in oncogenic transformation via gene regulation. Utilizing immunoprecipitation of endogenous protein complexes, we documented specific interactions between Jun family members and TATA box binding protein-associated factors (TAF) in colon HT29 adenocarcinoma cells. Particularly, TAF4b and c-Jun were found to colocalize and interact in the nucleus of advanced carcinoma cells and in cells with epithelial-to-mesenchymal transition (EMT) characteristics. TAF4b was found to specifically regulate the AP-1 target gene involved in EMT integrin alpha6, thus altering related cellular properties such as migration potential. Using a chromatin immunoprecipitation approach in colon adenocarcinoma cell lines, we further identified a synergistic role for TAF4b and c-Jun and other AP-1 family members on the promoter of integrin alpha6, underlining the existence of a specific mechanism related to gene expression control. We show evidence for the first time of an interdependence of TAF4b and AP-1 family members in cell type-specific promoter recognition and initiation of transcription in the context of cancer progression and EMT.
Asunto(s)
Integrina alfa6/genética , Invasividad Neoplásica/genética , Neoplasias/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción AP-1/genética , Factor de Transcripción TFIID/genética , Activación Transcripcional/genética , Células CACO-2 , Desdiferenciación Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Integrina alfa6/metabolismo , Neoplasias/metabolismo , Regiones Promotoras Genéticas/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción TFIID/metabolismoRESUMEN
ELK transcription factors are known to be expressed in a number of regions in the nervous system. We show by RT-PCR that the previously described Elk1, Elk3/Elk3b/Elk3c and Elk4 mRNAs are expressed in adult dorsal root ganglia (DRG), together with the novel alternatively spliced isoforms Elk1b, Elk3d and Elk4c/Elk4d/Elk4e. These isoforms are also expressed in brain, heart, kidney and testis. In contrast to Elk3 protein, the novel Elk3d isoform is cytoplasmic, fails to bind ETS binding sites and yet can activate transcription by an indirect mechanism. The Elk3 and Elk4 genes are overlapped by co-expressed Pctk2 (Cdk17) and Mfsd4 genes, respectively, with the potential formation of Elk3/Pctaire2 and Elk4/Mfsd4 sense-antisense mRNA heteroduplexes. After peripheral nerve injury the Elk3 mRNA isoforms are each upregulated approximately 2.3-fold in DRG (P<0.005), whereas the natural antisense Pctaire2 isoforms show only a small increase (21%, P<0.01) and Elk1 and Elk4 mRNAs are unchanged.
Asunto(s)
Empalme Alternativo/genética , Ganglios Espinales/metabolismo , ARN sin Sentido/genética , ARN Mensajero/genética , Células Receptoras Sensoriales/metabolismo , Factores Complejos Ternarios/metabolismo , Animales , Axotomía/efectos adversos , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Modelos Animales de Enfermedad , Ganglios Espinales/citología , Regulación de la Expresión Génica/genética , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Regeneración Nerviosa/genética , Técnicas de Cultivo de Órganos , Traumatismos de los Nervios Periféricos , Nervios Periféricos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Células Receptoras Sensoriales/citología , Factores Complejos Ternarios/genética , Regulación hacia Arriba/genética , Proteína Elk-1 con Dominio ets/genética , Proteína Elk-1 con Dominio ets/metabolismo , Proteína Elk-4 del Dominio ets/genética , Proteína Elk-4 del Dominio ets/metabolismoRESUMEN
The presence of different forms of histone covalent modifications, such as phosphorylation, acetylation and methylation in localized promoter regions are markers for chromatin packing and transcription. Activation of RAS signalling pathways through oncogenic RAS mutations is a hallmark of colorectal cancer. Overexpression of Harvey-Ras oncogene induces epithelial-mesenchymal transition (EMT) in Caco-2 cells. We focused on the role of epigenetic modifications of histone H3 and its dependence on RAS signal transduction pathways and oncogenic transformation. Using cell lines stably overexpressing oncogenic Harvey-RAS with EMT phenotype, we studied the acquired changes in the H3 histone modification patterns. Two genes show inverse protein expression patterns after Ha-RAS overexpression: Cyclin D1, a cell cycle-related gene, and the EMT marker-gene E-cadherin. We report that these two genes demonstrate matching inverse histone repression patterns on their promoter, while histone markers associated with an active state of genes were affected by the RAS-activated signalling pathway MEK-ERK-MSK1. Furthermore, we show that though the level of methyltransferases enzymes was increased, the status of H3 three-methylation at lysine 27 (H3K27me(3)), associated with gene repression on the promoter of Cyclin D1, was lower. Together, these results suggest that histone covalent modifications can be affected by oncogenic RAS pathways to regulate the expression of target genes like Cyclin D1 or E-cadherin and that the dynamic balance of opposing histone-modifying enzymes is critical for the regulation of cell proliferation.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Células Epiteliales/metabolismo , Histonas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proteínas ras/metabolismo , Células CACO-2 , Cadherinas/biosíntesis , Cadherinas/genética , Transformación Celular Neoplásica , Neoplasias Colorrectales/patología , Ciclina D1/biosíntesis , Ciclina D1/genética , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Células Madre Mesenquimatosas/patología , Procesamiento Proteico-Postraduccional/genética , Transducción de Señal , Transgenes , Proteínas ras/genéticaRESUMEN
We developed inducible and constitutive expression systems of Ha-RasV12 in HEK 293 cells to examine early oncogenic RasV12 signaling. Inducible expression of oncogenic Ras-triggered growth arrest, early senescence, and later apoptosis. Gene expression profile analysis revealed early Ras proliferation and cell cycle genes like c-fos, cyclin E, cdk2, cell-cell contact, and signaling like integrin a6, MEK5, and free radical signaling genes, like proline oxidase. Therefore, Ras-mediated signaling is a fine regulated process both positively and negatively influencing cell cycle, senescence, and apoptosis pathways. Novel early RAS-target genes could be potentially exploited in cancer diagnostics and therapeutics.
Asunto(s)
Apoptosis/genética , Transformación Celular Neoplásica/genética , Senescencia Celular/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes ras , Proteínas ras/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Activación Enzimática , Perfilación de la Expresión Génica/métodos , Humanos , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Factores de Tiempo , Transfección , Proteínas ras/metabolismoRESUMEN
In colorectal cancer, BRAF and KRAS oncogenes are mutated in about 15% and 35% respectively at approximately the same stage of the adenoma-carcinoma sequence. Since these two mutations rarely coexist, further analysis to dissect their function of transformation in colon cancer is required. Caco-2 human colon adenocarcinoma cells were stably transfected with BRAF(V600E) (Caco-BR cells) or KRAS(G12V) (Caco-K cells) oncogenes. BRAF(V600E) is more efficient in transforming Caco-2 cells and altering their morphology. The dominant nature of BRAF(V600E) is evident by its ability to render Caco-2 cells tumorigenic in vivo all be it through selective extracellular signal-related kinase (ERK) 2 phosphorylation and high levels of cyclin D1. As a consequence, the cell cycle distribution of parental cells is altered and microsatellite instability is introduced. Attenuated ERK activation observed correlated with KSR downregulation by BRAF(V600E) without further implications to signaling. Highly activated ERK in case of KRAS(G12V) (Caco-K cells) leads to mild transformation causing Caco-K cells to express premature senescence-related markers and acquire growth factor-dependent viability. Interestingly, BRAF(WT)gets equally activated by upstream KRAS mutations present in colon adenocarcinoma cells such as DLD-1 and SW620. Taken together, these results suggest that the two oncogenes have different transforming capability in colon cancer, although they both use the mitogen-activated protein (MAP) kinase pathway to carry out their effect. In general, BRAF(V600E) presents greater potential in mediating tumorigenic effect as compared to KRAS(G12V) both in vivo and in vitro. These findings may have implications in personalised diagnosis and targeted therapeutics.