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1.
Biomed Res Int ; 2014: 936031, 2014.
Article in English | MEDLINE | ID: mdl-25110710

ABSTRACT

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.


Subject(s)
Adenocarcinoma/metabolism , Adrenal Cortex Neoplasms/metabolism , Gene Amplification , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/biosynthesis , Receptor, IGF Type 1/biosynthesis , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adolescent , Adrenal Cortex Neoplasms/genetics , Adrenal Cortex Neoplasms/pathology , Adult , Aged , Child , Child, Preschool , Female , Humans , Infant , Male , MicroRNAs/biosynthesis , MicroRNAs/genetics , Middle Aged , Neoplasm Proteins/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , Receptor, IGF Type 1/genetics
2.
São Paulo; s.n; 2014. [121] p. ilus, tab, graf.
Thesis in Portuguese | LILACS | ID: biblio-870827

ABSTRACT

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...


Introduction: 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...


Subject(s)
Humans , Male , Female , Adrenal Cortex Neoplasms , Immunohistochemistry , MicroRNAs , Proteins , Receptor, IGF Type 1 , RNA, Small Interfering
3.
Clinics (Sao Paulo) ; 67(2): 95-8, 2012.
Article in English | MEDLINE | ID: mdl-22358232

ABSTRACT

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.


Subject(s)
Adenoma/genetics , Adrenal Cortex Neoplasms/genetics , Adrenocortical Carcinoma/genetics , Endostatins/genetics , Gene Frequency/genetics , Genotype , Polymorphism, Genetic/genetics , Adolescent , Adult , Aged , Child , Child, Preschool , DNA Mutational Analysis , Epidemiologic Methods , Female , Genotyping Techniques , Humans , Infant , Male , Middle Aged , Young Adult
5.
Mol Cell Endocrinol ; 351(1): 96-100, 2012 Mar 31.
Article in English | MEDLINE | ID: mdl-22019903

ABSTRACT

The insulin-like growth factor (IGF) signaling pathway has many important roles in normal cell growth and development. Remarkably, all of the components of this system (IGFs, receptors, and binding proteins) are expressed in human fetal adrenals. Beckwith-Wiedemann syndrome, a congenital overgrowth disorder characterized by a high risk of development of childhood tumors, is also distinguished by a high incidence of adrenocortical carcinomas. This disease has been associated with structural abnormalities at the 11p15 locus, which harbors the IGF2 gene as well as the genes coding for insulin, H19, and p57kip2. Notably, rearrangements at the 11p15 locus and overexpression of IGF2 were also described in sporadic adrenocortical tumors. In addition, the IGF2 overexpression was exclusively demonstrated in adults with adrenocortical tumors as a frequent feature of the malignant state. More recent studies demonstrated that the interaction of IGF-2 with IGF receptor type 1 (IGF-1R) plays also a pivotal role in adrenocortical tumorigenesis. IGF1R expression levels were significantly higher in pediatric adrenocortical carcinomas, suggesting that IGF1R expression represents a potential prognostic marker in this group of patients. These findings indicate that the IGF system is an important pathway for autonomous growth of adrenocortical cells and potential inhibitors of this system could be a rational therapeutic target for adrenocortical tumors.


Subject(s)
Adrenal Cortex Neoplasms , Beckwith-Wiedemann Syndrome , Biomarkers, Tumor , Cell Transformation, Neoplastic , Insulin-Like Growth Factor II , Receptor, IGF Type 1 , Signal Transduction/genetics , Adolescent , Adrenal Cortex Neoplasms/genetics , Adrenal Cortex Neoplasms/mortality , Adrenal Cortex Neoplasms/pathology , Adrenal Cortex Neoplasms/therapy , Adult , Beckwith-Wiedemann Syndrome/genetics , Beckwith-Wiedemann Syndrome/metabolism , Beckwith-Wiedemann Syndrome/pathology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Child , Child, Preschool , Cyclin-Dependent Kinase Inhibitor p57/genetics , Cyclin-Dependent Kinase Inhibitor p57/metabolism , Gene Expression Regulation, Neoplastic , Genetic Loci , Humans , Insulin-Like Growth Factor II/genetics , Insulin-Like Growth Factor II/metabolism , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism
6.
Clinics ; 67(2): 95-98, 2012. ilus, tab
Article in English | LILACS | ID: lil-614631

ABSTRACT

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 percent (DD), 15.9 percent (DN) and 2.2 percent (NN). In the controls, these frequencies were 80.6 percent, 17.3 percent and 2.0 percent, 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.


Subject(s)
Adolescent , Adult , Aged , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Young Adult , Adenoma/genetics , Adrenal Cortex Neoplasms/genetics , Adrenocortical Carcinoma/genetics , Endostatins/genetics , Genotype , Gene Frequency/genetics , Polymorphism, Genetic/genetics , DNA Mutational Analysis , Epidemiologic Methods , Genotyping Techniques
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