Genotype analysis of the human endostatin variant p.D104N in benign and malignant adrenocortical tumors

OBJECTIVE: Endostatin is a potent endogenous inhibitor of angiogenesis. It is derived from the proteolytic cleavage of collagen XVIII, which is encoded by the COL18A1 gene. A polymorphic COL18A1 allele encoding the functional polymorphism p.D104N impairs the activity of endostatin, resulting in a decreased ability to inhibit angiogenesis. This polymorphism has been previously analyzed in many types of cancer and has been considered a phenotype modulator in some benign and malignant tumors. However, these data are controversial, and different results have been reported for the same tumor types, such as prostate and breast cancer. The purpose of this study was to genotype the p.D104N variant in a cohort of pediatric and adult patients with adrenocortical tumors and to determine its possible association with the biological behavior of adrenocortical tumors. METHODS: DNA samples were obtained from 38 pediatric and 56 adult patients (0.6-75 yrs) with adrenocortical tumors. The DNA samples were obtained from peripheral blood, frozen tissue or paraffin-embedded tumor blocks when blood samples or fresh frozen tissue samples were unavailable. Restriction fragment length polymorphism analysis was used to genotype the patients and 150 controls. The potential associations of the p.D104N polymorphism with clinical and histopathological features and oncologic outcome (age of onset, tumor size, malignant tumor behavior, and clinical syndrome) were analyzed. RESULTS: Both the patient group and the control group were in Hardy-Weinberg equilibrium. The frequencies of the p.D104N polymorphism in the patient group were 81.9% (DD), 15.9% (DN) and 2.2% (NN). In the controls, these frequencies were 80.6%, 17.3% and 2.0%, respectively. We did not observe any association of this variant with clinical or histopathological features or oncologic outcome in our cohort of pediatric and adult patients with adrenocortical tumors

Analysis of copy number variations of IGF1R, SF1 and FGFR4 genes in adrenocortical tumors from children and adults

Introdução: Uma elevada incidência de tumores adrenocorticais pediátricos e de adultos é observada nas regiões sul e sudeste do Brasil. Hiperexpressão dos genes IGF1R, SF1 e FGFR4 tem sido descrita em tumores adrenocorticais. Apesar de hiperexpressão ser um evento comum em diversas neoplasias, ainda não são claros os mecanismos moleculares que seriam responsáveis por essa falha na regulação da expressão. Objetivos: Determinar o número de cópias dos genes IGF1R, SF1 e FGFR4 em tumores adrenocorticais diagnosticados em crianças e adultos. Adicionalmente correlacionaremos os dados de expressão gênica e/ou protéica de IGF1R, SF1 e FGFR4 com o diagnóstico histológico e evolutivo dos tumores adrenocorticais. Pacientes e métodos: Sessenta e quatro pacientes com tumores adrenocorticais foram selecionados para o estudo. Todos os pacientes foram submetidos à avaliação clínica e tratamento cirúrgico. Oito glândulas adrenais normais obtidas em cirurgias renais ou autópsias foram utilizadas como controles. DNA genômico extraído dos tecidos normais e tumorais da glândula suprarrenal foram utilizados como substrato nas reações de multiplex ligation-dependent probe amplification (MLPA) com o intuito de se determinar o número de cópias dos genes IGF1R, SF1 e FGFR4. PCR em tempo real (SYBR Green) foi realizado para confirmar os dados de MLPA para os genes IGF1R e SF1. Resultados: Amplificação do gene IGF1R foi detectada por MLPA e confirmada por PCR em tempo real SYBR Green em apenas um carcinoma adrenocortical. Adicionalmente, amplificação gênica de outros loci (IGFBP3, FGFR4 e NSD1) bem como de sondas controles foi observada, sugerindo uma condição aneuplóide neste tumor maligno. Amplificação de SF1 foi detectada em 10 tumores adrenocorticais (8 pediátricos e 2 de adultos). Os valores de expressão gênica foram significantemente maiores em tumores associados com amplificação gênica quando comparados com tumores sem amplificação. Além disso, imunorreatividade para SF-1 foi detectada nos tumores com aumento no número de cópias. Doze amplificações do locus FGFR4 (3 pediátricos e 9 de adultos) foram demonstradas por MLPA. A amplificação do locus FGFR4 e hiperexpressão deste gene foram significantemente mais relacionados a carcinomas. Conclusões: Amplificação do gene IGF1R é um evento raro nos tumores adrenocorticais pediátricos e de adultos. A hiperexpressão de IGF1R em tumores adrenocorticais pediátricos não foi secundária à amplificação gênica. Amplificação do gene SF1 foi evidenciada predominantemente em tumores adrenocorticais pediátricos e se correlacionou com hiperexpressão gênica e protéica. Amplificação do locus FGFR4 foi demonstrada predominantemente em tumores adrenocorticais malignos de adultos. Amplificação de oncogenes representa um mecanismo molecular relevante na tumorigênese adrenocorticalIntroduction: A high incidence of adrenocortical tumors in children and adults has been observed in Southern and Southeastern regions of Brazil. Overexpression of IGF1R, SF1 and FGFR4 genes have been described in adrenocortical tumors. Despite of overexpression be a common event in several neoplasias, the molecular mechanism implicated in this upregulation remains unknown. Objectives: To determine the copy number of IGF1R, SF1 and FGFR4 genes in pediatric and adult adrenocortical tumors. Additionally, correlate with IGF1R, SF1 and FGFR4 gene and/or protein expression data as well as with the histological diagnosis and evolution of the adrenocortical tumors. Patients and methods: Sixty and four patients with adrenocortical tumors were selected for this study. All patients were submitted to clinical evaluation and surgical treatment. Eight normal adrenal glands obtained in renal surgery or autopsies were used as controls. The MLPA reactions were performed with the DNA extracted from adrenal gland tissues in order to determine the copy number of IGF1R, SF1 and FGFR4 genes. SYBR Green real-time PCR was carried out to confirm MLPA data for IGF1R and SF1 genes. Results: IGF1R amplification was detected by MLPA and confirmed by SYBR green real-time PCR in only one adrenocortical carcinoma. Additionally, other loci amplification was detected (IGFBP3, FGFR4 and NSD1) as well as for control probes, suggesting aneuploidy in this malignant tumor. SF1 amplifications were shown in 10 adrenocortical tumors (8 from children and 2 from adults). The SF1 mRNA levels were significantly higher in adrenocortical tumors associated with increased SF1 gene copies when compared with adrenocortical tumors without gene amplification. Moreover, all adrenocortical tumors with SF1 gene amplification showed a strong SF1 staining. Twelve FGFR4 locus amplifications (3 from children and 9 from adults) were demonstrated by MLPA. FGFR4 locus amplification and overexpression of this gene were significantly more related to carcinomas. Conclusions: IGF1R amplification is a rare event in adrenocortical tumors and it was not responsible for the IGF1R overexpression of pediatric and adult adrenocortical tumors. SF1 gene amplification was detected predominantly in pediatric adrenocortical tumors and was associated with gene and protein overexpression. FGFR4 locus amplification was demonstrated mainly in adult maligant adrenocortical tumors. FGFR4 amplification and upregulation were more associated to adrenocortical carcinomas. Oncogenes amplification represents an important molecular mechanism in adrenocortical tumorigenesi

Analysis of expression and silencing of insulin-like growth factor 1 receptor in human adrenocortical tumors

Introdução O sistema dos fatores de crescimento semelhantes à insulina (IGF) desempenha importante papel no crescimento e desenvolvimento celular normal. Hiperexpressão do gene IGF1R tem sido demonstrada em diversos tumores, sugerindo que a expressão deste receptor represente um pré-requisito fundamental para transformação celular. Nosso grupo de pesquisa demonstrou o aumento de expressão de IGF1R em tumores adrenocorticais pediátricos. Objetivos: Induzir o silenciamento do gene IGF1R por siRNA na linhagem de tumor adrenocortical humano NCI H295R, bem como avaliar os efeitos in vitro por meio da análise de proliferação celular e apoptose desta linhagem celular. Adicionalmente, avaliar a expressão de IGF-1R e de microRNAs relacionados a sua transcrição em tumores adrenocorticais humanos. Pacientes e métodos: A linhagem celular de carcinoma adrenocortical humano NCI H295R foi cultivada e submetida ao tratamento com 2 siRNAs específicos para IGF-1R. Todos os experimentos foram realizados em quatro grupos: (1) células não tratadas com siRNA, (2) células tratadas com siRNA # 1, (3) células tratadas com siRNA # 2 e (4) células tratadas com o siRNA controle negativo. A expressão gênica e proteica de IGF-1R foram determinadas por meio das técnicas de PCR em tempo real e Western Blot, respectivamente. Os efeitos do silenciamento de IGF-1R in vitro foram avaliados por ensaios de proliferação celular e análise de atividade de caspases. Além disso, 202 pacientes com tumor adrenocortical foram selecionados para o estudo de expressão proteica de IGF-1R por imunohistoquímica. Para avaliação de expressão de microRNAs relacionados à expressão de IGF-1R (miR-100, 375, 145 e 126) por PCR em tempo real foram selecionados 32 pacientes dos 202 disponíveis. Resultados: A expressão de IGF-1R foi significantemente diminuída nas células tratadas com siRNA # 1 e siRNA # 2. Os valores relativos de RNA mensageiro de IGF1R diminuíram aproximadamente 50% e as análises de Western Blot revelaram uma redução de 30% na proteína de IGF-1R. A diminuição de expressão foi acompanhada por uma redução de 40% na taxa de crescimento celular in vitro e um aumento de 45% das taxas de apoptose. A análise de expressão dos microRNAs 100, 375, 145 e 126 demostrou que a expressão de IGF-1R não se correlaciona com a expressão destes RNAs pequenos. Adicionalmente, a análise de expressão proteica de IGF-1R em tumores adrenocorticais humanos revelou que expressão forte (20%) de IGF-1R foi mais comum em carcinomas de adultos. Além disso, a imunolocalização do IGF-1R nos carcinomas (19%) foi mais frequentemente nuclear em relação aos adenomas de adultos. Conclusões: Os dados obtidos reforçam a importância de IGF-1R nas vias tumorigênicas das neoplasias malignas do córtex da glândula suprarrenal. A inibição deste receptor foi capaz de inibir o crescimento tumoral in vitro por meio da redução das taxas de proliferação celular e aumento da apoptose em linhagem celular de carcinoma adrenocortical humano. Além disso, a expressão proteica nuclear de IGF-1R foi mais comum entre os carcinomas, sugerindo representar um marcador biológico desta neoplasiaIntroduction: The insulin-like growth factor (IGF) system plays a key role in normal cell growth and development. IGF1R overexpression has been demonstrated in several tumors suggesting that its expression is a prerequisite for cell transformation. We demonstrated IGF1R overexpression in pediatric adrenocortical tumors. Objectives: To induce IGF1R silencing by siRNA in a human adrenocortical cell line NCI H295R and evaluate its effects on cell proliferation and apoptosis. Additionally, evaluate the expression of IGF-1R protein and microRNAs related to its transcription in human adrenocortical tumors. Patients and methods: The human adrenocortical tumor cell line NCI H295R was cultured and treated with 2 specific IGF1R siRNA. All experiments were carried out in four groups: (1) untreated NCI H295R cells, (2) NCI H295R cells transfected with specific IGF1R siRNA # 1, (3) NCI H295R cells transfected with specific IGF1R siRNA # 2 and (4) NCI H295R cells transfected with a negative control. IGF-1R gene and protein expression was determined by the techniques of real-time PCR and Western blot, respectively. We assessed the effects of IGF-1R silencing on cell proliferation and apoptosis. Moreover, 202 patients with adrenocortical tumors were selected for the study of IGF-1R protein expression by immunohistochemistry. In the analysis of microRNAs that are related to IGF1R (miR-100, 375, 145 e 126) by real time PCR, 32 out 202 patients were selected. Results: IGF-1R levels were significantly decreased in cells that were treated with IGF-1R siRNA # 1 and siRNA # 2. The relative values of IGF1R mRNA decreased approximately 50% and Western blot analysis revealed a 30% of reduction in IGF-1R protein. Downregulation of this gene was accompanied by a reduction in 40% of cell growth in vitro and an increase in 45% of apoptosis. The analysis of microRNAs demonstrated that IGF1R expression is not correlated with the expression of these small RNAs. Additionally, the analysis of IGF-1R protein expression in human adrenocortical tumors revealed that strong expression (20%) of IGF-1R was more common in adult carcinomas. Moreover, the nuclear IGF-1R was more frequent in carcinomas diagnosed in adults (19%) when compared to adenomas. Conclusions: These data demonstrate the importance of IGF-1R in tumorigenic pathways of malignant neoplasms of the adrenocortical gland. IGF-1R silencing could inhibit tumor growth in vitro by reducing cell proliferation and increasing apoptosis in a cell line of human adrenocortical carcinoma. Furthermore, nuclear IGF-1R expression was more frequent in carcinomas diagnosed in adults, suggesting that IGF-1R may be a biological marker of this neoplasi

Amplification of the Insulin-Like Growth Factor 1 Receptor Gene Is a Rare Event in Adrenocortical Adenocarcinomas: Searching for Potential Mechanisms of Overexpression

Context . IGF1R overexpression appears to be a prognostic biomarker of metastatic pediatric adrenocortical tumors. However, the molecular mechanisms that are implicated in its upregulation remain unknown. Aim. To investigate the potential mechanisms involved in IGF1R overexpression. Patients and Methods. We studied 64 adrenocortical tumors. IGF1R copy number variation was determined in all patients using MLPA and confirmed using real time PCR. In a subgroup of 32 patients, automatic sequencing was used to identify IGF1R allelic variants and the expression of microRNAs involved in IGF1R regulation by real time PCR. Results. IGF1R amplification was detected in an adrenocortical carcinoma that was diagnosed in a 46-year-old woman with Cushing's syndrome and virilization. IGF1R overexpression was demonstrated in this case. In addition, gene amplification of other loci was identified in this adrenocortical malignant tumor, but no IGF1R copy number variation was evidenced in the remaining cases. Automatic sequencing revealed three known polymorphisms but they did not correlate with its expression. Expression of miR-100, miR-145, miR-375, and miR-126 did not correlate with IGF1R expression. Conclusion. We demonstrated amplification and overexpression of IGF1R gene in only one adrenocortical carcinoma, suggesting that these combined events are uncommon. In addition, IGF1R polymorphisms and abnormal microRNA expression did not correlate with IGF1R upregulation in adrenocortical tumors

Amplification of the Insulin-Like Growth Factor 1 Receptor Gene Is a Rare Event in Adrenocortical Adenocarcinomas: Searching for Potential Mechanisms of Overexpression

Context. IGF1R overexpression appears to be a prognostic biomarker of metastatic pediatric adrenocortical tumors. However, the molecular mechanisms that are implicated in its upregulation remain unknown. Aim. To investigate the potential mechanisms involved in IGF1R overexpression. Patients and Methods. We studied 64 adrenocortical tumors. IGF1R copy number variation was determined in all patients using MLPA and confirmed using real time PCR. In a subgroup of 32 patients, automatic sequencing was used to identify IGF1R allelic variants and the expression of microRNAs involved in IGF1R regulation by real time PCR. Results. IGF1R amplification was detected in an adrenocortical carcinoma that was diagnosed in a 46-year-old woman with Cushing’s syndrome and virilization. IGF1R overexpression was demonstrated in this case. In addition, gene amplification of other loci was identified in this adrenocortical malignant tumor, but no IGF1R copy number variation was evidenced in the remaining cases. Automatic sequencing revealed three known polymorphisms but they did not correlate with its expression. Expression of miR-100, miR-145, miR-375, and miR-126 did not correlate with IGF1R expression. Conclusion. We demonstrated amplification and overexpression of IGF1R gene in only one adrenocortical carcinoma, suggesting that these combined events are uncommon. In addition, IGF1R polymorphisms and abnormal microRNA expression did not correlate with IGF1R upregulation in adrenocortical tumors