RESUMEN

Conventional mitogen-activated protein kinase (MAPK) family members are among the most sought-after oncogenic effectors for the development of novel human cancer treatment strategies. MEK5/ERK5 has been the less-studied MAPK subfamily, despite its increasingly demonstrated relevance in the growth, survival, and differentiation of normal cells. MEK5/ERK5 signalling has already been proposed to have pivotal roles in several cancer hallmarks, and to mediate the effects of a range of oncogenes. Accumulating evidence indicates the contribution of MEK5/ERK5 signalling to therapy resistance and the benefits of using MEK5/ERK5 inhibitory strategies in the treatment of human cancer. Here, we explore the major known contributions of MEK5/ERK5 signalling to the onset and progression of several types of cancer, and highlight the potential clinical relevance of targeting MEK5/ERK5 pathways.

Asunto(s)

MAP Quinasa Quinasa 5/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Neoplasias/metabolismo , Animales , Humanos , Neoplasias/tratamiento farmacológico , Transducción de Señal

RESUMEN

The MEK5/ERK5 signaling pathway is emerging as an important contributor to colon cancer onset, progression and metastasis; however, its relevance to chemotherapy resistance remains unknown. Here, we evaluated the impact of the MEK5/ERK5 cascade in colon cancer cell sensitivity to 5-fluorouracil (5-FU). Increased ERK5 expression was correlated with poor overall survival in colon cancer patients. In colon cancer cells, 5-FU exposure impaired endogenous KRAS/MEK5/ERK5 expression and/or activation. In turn, MEK5 constitutive activation reduced 5-FU-induced cytotoxicity. Using genetic and pharmacological approaches, we showed that ERK5 inhibition increased caspase-3/7 activity and apoptosis following 5-FU exposure. Mechanistically, this was further associated with increased p53 transcriptional activation of p21 and PUMA. In addition, ERK5 inhibition increased the response of HCT116 p53+/+ cells to 5-FU, but failed to sensitize HCT116 p53-/- cells to the cytotoxic effects of this chemotherapeutic agent, suggesting a p53-dependent axis mediating 5-FU sensitization. Finally, ERK5 inhibition using XMD8-92 was shown to increase the antitumor effects of 5-FU in a murine subcutaneous xenograft model, enhancing apoptosis while markedly reducing tumor growth. Collectively, our results suggest that ERK5-targeted inhibition provides a promising therapeutic approach to overcome resistance to 5-FU-based chemotherapy and improve colon cancer treatment.

Asunto(s)

Antimetabolitos Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Fluorouracilo/farmacología , MAP Quinasa Quinasa 5/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Benzodiazepinonas/farmacología , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Proliferación Celular , Neoplasias del Colon/genética , Activación Enzimática/efectos de los fármacos , Células HCT116 , Humanos , MAP Quinasa Quinasa 5/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Proteína Quinasa 7 Activada por Mitógenos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

miR-143 and miR-145 are downregulated in colon cancer. Here, we tested the effect of restoring these miRNAs on sensitization to cetuximab in mutant KRAS (HCT116 and SW480) and wild-type KRAS (SW48) colon cancer cells. We evaluated cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and the modulation of signaling pathways involved in immune effector cell-mediated elimination of cancer cells. Stable miR-143 or miR-145 overexpression increased cell sensitivity to cetuximab, resulting in a significant increase of cetuximab-mediated ADCC independently of KRAS status. Importantly, HCT116 cells overexpressing these miRNAs triggered apoptosis in result of cetuximab-mediated ADCC, effected by peripheral blood mononuclear cells (p < 0.01). This was associated with increased apoptosis and caspase-3/7 activity, and reduced Bcl-2 protein expression (p < 0.01). In addition, caspase inhibition abrogated cetuximab-mediated ADCC in HCT116 cells overexpressing either miR-143 or miR-145 (p < 0.01). Furthermore, Bcl-2 silencing led to high level of cetuximab-mediated ADCC, compared to control siRNA (p < 0.05). Importantly, granzyme B inhibition, abrogated cetuximab-mediated ADCC, reducing caspase-3/7 activity (p < 0.01). Collectively, our data suggests that re-introduction of miR-143 or miR-145 may provide a new approach for development of therapeutic strategies to re-sensitize colon cancer cells to cetuximab by stimulating cetuximab-dependent ADCC to induce cell death.

Asunto(s)

Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Cetuximab/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , MicroARNs/genética , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Genes bcl-2/genética , Granzimas/antagonistas & inhibidores , Células HCT116 , Humanos , MicroARNs/biosíntesis , Proteínas Proto-Oncogénicas p21(ras)/genética , Interferencia de ARN , ARN Interferente Pequeño/genética

RESUMEN

BACKGROUND: Simultaneous isolation of nucleic acids and proteins from a single biological sample facilitates meaningful data interpretation and reduces time, cost and sampling errors. This is particularly relevant for rare human and animal specimens, often scarce, and/or irreplaceable. TRIzol(®) and TRIzol(®)LS are suitable for simultaneous isolation of RNA, DNA and proteins from the same biological sample. These reagents are widely used for RNA and/or DNA isolation, while reports on their use for protein extraction are limited, attributable to technical difficulties in protein solubilisation. RESULTS: TRIzol(®)LS was used for RNA isolation from 284 human colon cancer samples, including normal colon mucosa, tubulovillous adenomas, and colon carcinomas with proficient and deficient mismatch repair system. TRIzol(®) was used for RNA isolation from human colon cancer cells, from brains of transgenic Alzheimer's disease mice model, and from cultured mouse cortical neurons. Following RNA extraction, the TRIzol(®)-chloroform fractions from human colon cancer samples and from mouse hippocampus and frontal cortex were stored for 2 years and 3 months, respectively, at -80°C until used for protein isolation.Simple modifications to the TRIzol(®) manufacturer's protocol, including Urea:SDS solubilization and sonication, allowed improved protein recovery yield compared to the TRIzol(®) manufacturer's protocol. Following SDS-PAGE and Ponceau and Coomassie staining, recovered proteins displayed wide molecular weight range and staining pattern comparable to those obtainable with commonly used protein extraction protocols. We also show that nuclear and cytosolic proteins can be easily extracted and detected by immunoblotting, and that posttranslational modifications, such as protein phosphorylation, are detectable in proteins recovered from TRIzol(®)-chloroform fractions stored for up to 2 years at -80°C. CONCLUSIONS: We provide a novel approach to improve protein recovery from samples processed for nucleic acid extraction with TRIzol(®) and TRIzol(®)LS compared to the manufacturer`s protocol, allowing downstream immunoblotting and evaluation of steady-state relative protein expression levels. The method was validated in large sets of samples from multiple sources, including human colon cancer and brains of transgenic Alzheimer's disease mice model, stored in TRIzol(®)-chloroform for up to two years. Collectively, we provide a faster and cheaper alternative to the TRIzol(®) manufacturer`s protein extraction protocol, illustrating the high relevance, and wide applicability, of the present protein isolation method for the immunoblot evaluation of steady-state relative protein expression levels in samples from multiple sources, and following prolonged storage.

Asunto(s)

Proteínas/metabolismo , ARN/metabolismo , Adenoma Velloso/metabolismo , Adenoma Velloso/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patología , Células Cultivadas , Cloroformo/química , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Células HCT116 , Hipocampo/metabolismo , Humanos , Ratones , Neuronas/citología , Neuronas/metabolismo , Fosforilación , Estabilidad Proteica , Proteínas/química , Proteínas/aislamiento & purificación , ARN/aislamiento & purificación , Estabilidad del ARN , Juego de Reactivos para Diagnóstico , Urea/química

RESUMEN

BACKGROUND: MicroRNAs (miRNAs) are aberrantly expressed in human cancer and involved in the (dys)regulation of cell survival, proliferation, differentiation and death. Specifically, miRNA-143 (miR-143) is down-regulated in human colon cancer. In the present study, we evaluated the role of miR-143 overexpression on the growth of human colon carcinoma cells xenografted in nude mice (immunodeficient mouse strain: N: NIH(s) II-nu/nu). METHODOLOGY/PRINCIPAL FINDINGS: HCT116 cells with stable miR-143 overexpression (Over-143) and control (Empty) cells were subcutaneously injected into the flanks of nude mice, and tumor growth was evaluated over time. Tumors arose ∼ 14 days after tumor cell implantation, and the experiment was ended at 40 days after implantation. miR-143 was confirmed to be significantly overexpressed in Over-143 versus Empty xenografts, by TaqMan® Real-time PCR (p<0.05). Importantly, Over-143 xenografts displayed slower tumor growth compared to Empty xenografts from 23 until 40 days in vivo (p<0.05), with final volumes of 928±338 and 2512±387 mm(3), respectively. Evaluation of apoptotic proteins showed that Over-143 versus Empty xenografts displayed reduced Bcl-2 levels, and increased caspase-3 activation and PARP cleavage (p<0.05). In addition, the incidence of apoptotic tumor cells, assessed by TUNEL, was increased in Over-143 versus Empty xenografts (p<0.01). Finally, Over-143 versus Empty xenografts displayed significantly reduced NF-κB activation and ERK5 levels and activation (p<0.05), as well as reduced proliferative index, evaluated by Ki-67 immunohistochemistry (p<0.01). CONCLUSIONS: Our results suggest that reduced tumor volume in Over-143 versus Empty xenografts may result from increased apoptosis and decreased proliferation induced by miR-143. This reinforces the relevance of miR-143 in colon cancer, indicating an important role in the control of in vivo tumor progression, and suggesting that miR-143 may constitute a putative novel therapeutic tool for colon cancer treatment that warrants further investigation.

Asunto(s)

Apoptosis/fisiología , Neoplasias del Colon/metabolismo , MicroARNs/metabolismo , Animales , Apoptosis/genética , Proliferación Celular , Neoplasias del Colon/genética , Células HCT116 , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Ratones Desnudos , MicroARNs/genética , Ensayos Antitumor por Modelo de Xenoinjerto