RESUMEN

Ionizing radiation (IR) is frequently applied in the treatment of rectal adenocarcinoma, however, there is marked variance in the response to radiochemotherapy between individual tumors. In our previous investigations, it was shown that the overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K) confers radioresistance to malignant melanoma and colorectal carcinoma (CRC) in vitro, however, the underlying mechanism remains to be elucidated. As hnRNP K, a p53 binding partner and cofactor for the transcriptional activation of p53 target genes, is overexpressed in CRC, the present study investigated the possible radioprotective effect of the hnRNP K/p53­induced upregulation of p21 (also known as WAF1/CIP1) in rectal adenocarcinoma. Immunohistochemistry was performed for hnRNP K, p53 and p21 in a series of 68 consecutive cases of rectal adenocarcinoma with full molecular characterization following radiochemotherapy and 14 corresponding pre­therapeutic biopsies, and the results were correlated with clinicopathological characteristics and the percentage of vital tumor cells following therapy. In addition, pathway analyses, protein immunoprecipitation, western immunoblotting and immunofluorescence microscopy were performed to identify dysregulated kinase signaling and hnRNP K targets upon exposure of CRC cells to IR. Although the fraction of vital tumor cells upon neoadjuvant therapy was significantly higher in hnRNP K/p21­positive tumors (P=0.0047 and P=0.0223, Students' t­test), no significant association was found between the protein expression levels of hnRNP K, p53 and p21 (P>0.05, χ2 test). Irradiation enhanced apoptotic pathway activation via p53/CHK2 phosphorylation and poly (ADP­ribose) polymerase cleavage, and induced the overexpression and interaction of hnRNP K and p53. However, p53 Ser15­phosphorylation was independent of the presence of hnRNP K, and there was no measurable effect of hnRNP K on the expression of p21 in vitro. Taken together, the results of the present study support a radioprotective role for hnRNP K, which may be mediated through an interaction with p53, however, this effect appears to be independent of the hnRNP K/p53­induced upregulation of p21 in rectal adenocarcinoma.

Asunto(s)

Quimioradioterapia , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/radioterapia , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoprecipitación , Masculino , Persona de Mediana Edad , Proteína p53 Supresora de Tumor/genética

RESUMEN

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is overexpressed in malignant tumors and involved in DNA damage response upon ionizing radiation (IR). Here, we investigate its role in radioresistance of colorectal carcinoma (CRC) and evaluate a pharmacological approach to enhance CRC radiosensitivity via downregulation of hnRNP K. We show that hnRNP K is overexpressed in CRC tissue specimens and upregulated in response to IR in vitro, which occurs faster in KRAS-mutant CRC cells. HnRNP K knockdown impairs cell survival, cell cycle progression and KRAS-dependent radioresistance and increases apoptosis. Using the chicken chorioallantoic membrane assay, a decrease in xenograft tumor growth and radioresistance upon hnRNP K depletion could be verified in vivo, and comparable effects were achieved by suppression of hnRNP K expression using the MEK inhibitor MEK162 (Binimetinib). In summary, KRAS-mutant CRC shows intrinsic radioresistance along with rapid upregulation of hnRNP K in response to IR that can effectively be targeted by MEK inhibition. Our results point towards a possible use of MAPK pathway inhibitors to decrease radioresistance of KRAS-mutant CRC via downregulation of hnRNP K.

Asunto(s)

Biomarcadores de Tumor/genética , Neoplasias Colorrectales/radioterapia , Quinasas Quinasa Quinasa PAM/metabolismo , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genética , Tolerancia a Radiación/genética , Ribonucleoproteínas/metabolismo , Transducción de Señal/efectos de la radiación , Anciano , Anciano de 80 o más Años , Animales , Apoptosis/efectos de la radiación , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Relación Dosis-Respuesta en la Radiación , Femenino , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Ribonucleoproteína Heterogénea-Nuclear Grupo K , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Masculino , Persona de Mediana Edad , Fenotipo , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Tolerancia a Radiación/efectos de los fármacos , Ribonucleoproteínas/genética , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Regulación hacia Arriba