RESUMO
Missense mutations in the DNA binding domain of p53 are observed frequently in esophageal squamous cell carcinoma (ESCC). Recent studies have revealed the potentially oncogenic transcriptional networks regulated by mutant p53 proteins. However, majority of these studies have focused on common "hotspot" p53 mutations while rarer mutations are poorly characterized. In this study, we report the characterization of rare, "non-hotspot" p53 mutations from ESCC. In vitro tumorigenic assays performed following ectopic-expression of certain "non-hotspot" mutant p53 proteins caused enhancement of oncogenic properties in squamous carcinoma cell lines. Genome-wide transcript profiling of ESCC tumor samples stratified for p53 status, revealed several genes exhibiting elevated transcript levels in tumors harboring mutant p53. Of these, ARF6, C1QBP, and TRIM23 were studied further. Reverse transcription-quantitative PCR (RT-qPCR) performed on RNA isolated from ESCC tumors revealed significant correlation of TP53 transcript levels with those of the three target genes. Ectopic expression of wild-type and several mutant p53 forms followed by RT-qPCR, chromatin affinity-purification (ChAP), and promoter-luciferase assays indicated the exclusive recruitment of p53 mutants-P190T and P278L, to the target genes leading to the activation of expression. Several functional assays following knockdown of the target genes revealed a significant suppression of tumorigenicity in squamous carcinoma cell lines. Rescue experiments confirmed the specificity of the knockdown. The tumorigenic effects of the genes were confirmed in nude mice xenograft assays. This study has therefore identified novel oncogenic targets of "non-hotspot" mutant p53 proteins relevant for ESCC besides validating the functional heterogeneity of the spectrum of tumor-specific p53 mutations.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Animais , Camundongos , Humanos , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias Esofágicas/patologia , Camundongos Nus , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Proteínas de Ligação ao GTP/genética , Proteínas de Transporte/genética , Proteínas Mitocondriais/genéticaRESUMO
Though primarily a tumor suppressor, TP53 harboring specific missense mutations located in the region encoding the DNA binding domain exhibits a gain of function by transcriptional activation of oncogenes. We performed microarray-based messenger RNA profiling of squamous cell carcinoma of the oral tongue (SCCOT) and identified significant elevation of SMARCD1 in samples exhibiting p53 nuclear stabilization. Activation of SMARCD1 by mutant p53 was confirmed by evaluation of additional tongue cancer samples as well as The Cancer Genome Atlas expression datasets. SMARCD1 knockdown in HNSCC cells resulted in a significant reduction in several tumorigenic characteristics including cell viability, ability to form colonies in liquid and solid media and cell migration. We identified significantly increased SMARCD1 transcript levels in tumor versus matched normal samples in SCCOT as well as in other cancer types. Increased SMARCD1 expression predicted poor survival in HNSCC tumors harboring missense p53 mutations. Our results suggest SMARCD1 to be a novel transcriptional target of mutant p53.
Assuntos
Proteínas Cromossômicas não Histona/genética , Mutação , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas Cromossômicas não Histona/metabolismo , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Rebaudioside-A is the second most abundant sweet diterpene glycoside (1-3%) present in the leaves of Stevia rebaudiana Bertoni, and is now being considered as a possible sucrose substitute due to its pleasant organoleptic properties and associated health benefits. In the present study, a novel in situ enzymatic transglycosylation of stevioside has been developed by pre-treating the stevia leaves with cellulase and adding soluble starch as the glucosyl donor. The results confirm that the transglycosylation of stevioside led to an enrichment in the rebaudioside-A content from 4% to 66%. This was further purified by multiple column chromatography to obtain 95% pure rebaudioside-A. The isolated rebaudioside-A showed concentration-dependent α-glucosidase inhibitory activity with IC50=35.01 µg/ml. Thus the study highlights the biotransformation of stevioside present in stevia leaves to rebaudioside-A by a simple, inexpensive and eco-friendly process that has commercial potential.
