Análise dos genes CYP1A1, CYP2D6 e GSTP1 em portadores de câncer de mama esporádico

Orientadora: Enilze Maria de Souza Fonseca RibeiroCoorientador: Iglenir João CavalliMonografia (Bacharelado) - Universidade Federal do Paraná. Setor de Ciencias Biológicas. Curso de Graduaçao em Ciencias BiológicasResumo : O câncer de mama é o segundo tipo de câncer mais freqüente no mundo, sendo o mais comum entre as mulheres (INCA/MS, 2006). Admite-se que a exposição prolongada a níveis aumentados de estrogênio seja o fator central na etiologia do câncer de mama (BERNSTEIN, 2002). O objetivo deste trabalho foi investigar a associação entre o câncer de mama e polimorfismos em três genes atuantes na via metabólica do estrogênio (CYP1A1, CYP2D6 e GSTP1). A amostra foi composta de 85 pacientes e 85 controles do sexo feminino, pareadas por idade (±5 anos) e etnia. Foram coletadas amostras de sangue periférico para extração de DNA pelo método de salting out, e em seguida realizou-se a análise molecular por PCR-RFLP, seguida de visualização em gel de agarose 3,0%. Nossos resultados não demonstraram associação entre a ocorrência de câncer de mama e os genótipos considerados de risco: OR= 1,57 (IC 95%= 0,77 - 3,20) para o polimorfismo CYP1A1 – Msp1, OR= 1,30 (IC 95%= 0,69 - 2,47) para o polimorfismo CYP2D6*4 e OR= 1,69 (IC 95%=0,92 – 3,11) para o polimorfismo GSTP1 – BsmA1. Ao combinar os genótipos considerados de risco dos três genes, não observamos associações com um genótipo de risco (OR= 1,28; IC 95%=0,61 – 2,70), mas encontramos associação com dois ou três genótipos (OR= 2,94; IC 95%=1,21 – 7,16). Na análise dos parâmetros clínicos (idade da menarca, idade da menopausa, tempo entre menarca e menopausa, tempo de uso de pílulas anticoncepcionais, número de filhos e idade da primeira gravidez), encontramos diferenças entre pacientes e controles quanto à idade média da menarca (t= 6,43; P0,01 para o gene GSTP1) e média do tempo entre menarca e menopausa (t= 2,18; P>0,02 para o gene CYP1A1 e t= 2,16; P>0,02 para o gene GSTP1). A análise dos parâmetros histopatológicos (grau histológico do tumor, envolvimento de linfonodos e tamanho do tumor) não apresentou diferenças estatisticamente significantes entre os subgrupos considerados de risco e as pacientes homozigotas selvagens para cada um dos genes estudados. Portanto, uma interação entre os genes em estudo pode estar influenciando o risco ao câncer de mama, assim como alguns dos parâmetros clínicos analisados. Para corroborar estes e os outros resultados do trabalho, torna-se importante a ampliação da amostra utilizada

Population analysis of xenobiotic metabolizing genes in South Brazilian Euro and Afro-descendants

Individual variability in xenobiotic metabolism has been associated with susceptibility to developing complex diseases. Genes involved in xenobiotic metabolism have been evaluated in association studies; the difficulty of obtaining accurate gene frequencies in mixed populations makes interpretation of the results difficult. We sought to estimate population parameters for the cytochrome P450 and glutathione S-transferase gene families, thus contributing to studies using these genes as markers. We describe the frequencies of six genes (CYP1A1, CYP2D6, CYP2E1, GSTM1, GSTT1, and GSTP1) and estimate population parameters in 115 Euro-descendants and 196 Afro-descendants from Curitiba, South of Brazil. PCR-based methods were used for genotyping, and statistical analysis were performed by AMOVA with ARLEQUIN software. The mutant allele frequencies in the Afro-descendants and Euro-descendants, respectively, were: CYP1A1*2A = 30.1% and 15.2%; CYP2D6*4 = 14.5% and 21.5%; CYP2E1*5B = 7.9% and 5%; GSTP1*B = 37.8% and 28.3%. The null genotype frequencies were: GSTM1*0 = 36.8% and 46.1%; GSTT1*0 = 24.2% and 17.4%

FANCD2 and BRCA1 have differential expression among the FA-BRCA genes in primary breast cancer/ Expressão diferencial de FANCD2 e BRCA1 no câncer de mama primário

The molecular pathways of DNA repair in tumors may play a role in tailoring patient therapy. The Fanconi anemia DNA repair pathway operates in the repair of DNA interstrand crosslink induced by several chemotherapeutic drugs. In this study we evaluated the expression of Fanconi anemia DNA repair genes (FANCA, C, D2, F, BRCA1 and PALB2) in 46 primary breast tumors and ten non-compromised breast samples, by Real-Time Quantitative Reverse Transcription PCR, and to correlated gene expression with breast cancer subtypes and clinico-pathological parameters. Tumor samples were classified in subtypes based on immunohistochemistry markers, and clinico-pathological parameters were obtained from the medical reports. FANCD2 was twice more expressed in tumors than in the non-compromised group (p= 0.02). BRCA1 showed a differential expression in the luminal group, three times less expressed in Luminal-B than in Luminal-A group (p= 0.01). In conclusion, the higher level of expression of FANCD2 in tumors may indicate activation of the Fanconi anemia DNA repair pathway, which has been implicated in breast carcinogenesis and in chemotherapeutic resistance. The loss of BRCA1 expression in the Luminal-B group may indicate that the use of cisplatin-based neo/adjuvant therapies is preferable, and that the use of taxol-based therapies should be avoided due to the risk of drug resistance

Characterization of MTAP gene expression in breast cancer patients and cell lines

MTAP is a ubiquitously expressed gene important for adenine and methionine salvage. The gene is located at 9p21, a chromosome region often deleted in breast carcinomas, similar to CDKN2A, a recognized tumor suppressor gene. Several research groups have shown that MTAP acts as a tumor suppressor, and some therapeutic approaches were proposed based on a tumors\ub4 MTAP status. We analyzed MTAP and CDKN2A gene (RT-qPCR) and protein (western-blotting) expression in seven breast cancer cell lines and evaluated their promoter methylation patterns to better characterize the contribution of these genes to breast cancer. Cytotoxicity assays with inhibitors of de novo adenine synthesis (5-FU, AZA and MTX) after MTAP gene knockdown showed an increased sensitivity, mainly to 5-FU. MTAP expression was also evaluated in two groups of samples from breast cancer patients, fresh tumors and paired normal breast tissue, and from formalin-fixed paraffin embedded (FFPE) core breast cancer samples diagnosed as Luminal-A tumors and triple negative breast tumors (TNBC). The difference of MTAP expression between fresh tumors and normal tissues was not statistically significant. However, MTAP expression was significantly higher in Luminal-A breast tumors than in TNBC, suggesting the lack of expression in more aggressive breast tumors and the possibility of using the new approaches based on MTAP status in TNB

Lnc-uc.147 Is Associated with Disease Stage of Liver, Gastric, and Renal Cancer

Lnc-uc.147, a long non-coding RNA derived from a transcribed ultraconserved region (T-UCR), was previously evidenced in breast cancer. However, the role of this region in other tumor types was not previously investigated. The present study aimed to investigate lnc-uc.147 in different types of cancer, as well as to suggest lnc-uc.147 functional and regulation aspects. From solid tumor datasets analysis of The Cancer Genome Atlas (TCGA), deregulated lnc-uc.147 expression was associated with the histologic grade of hepatocellular carcinoma, and with the tumor stage of clear cell renal and gastric adenocarcinoma. Considering the epidemiologic relevance of liver cancer, silencing lnc-uc.147 reduced the viability and clonogenic capacity of HepG2 cell lines. Additionally, we suggest a relation between the transcription factor TEAD4 and lnc-uc.147 in liver and breast cancer cells

The Potential of NORAD–PUMILIO–RALGAPB Regulatory Axis as a Biomarker in Breast Cancer

Introduction: Long non-coding RNAs (LncRNA) represent a heterogeneous family of RNAs that have emerged as regulators of various biological processes through their association with proteins in ribonucleoproteins complexes. The dynamic of these interactions can affect cell metabolism, including cancer development. Annually, breast cancer causes thousands of deaths worldwide, and searching for new biomarkers is pivotal for better diagnosis and treatment. Methods: Based on in silico prediction analysis, we focus on LncRNAs that have binding sites for PUMILIO, an RBP family involved in post-transcriptional regulation and associated with cancer progression. We compared the expression levels of these LncRNAs in breast cancer and non-tumor samples from the TCGA database. We analyzed the impact of overall and disease-free survival associated with the expression of the LncRNAs and co-expressed genes and targets of PUMILIO proteins. Results: Our results found NORAD as the most relevant LncRNA with a PUMILIO binding site in breast cancer, differently expressed between Luminal A and Basal subtypes. Additionally, NORAD was co-expressed in a Basal-like subtype (0.55) with the RALGAPB gene, a target gene of PUMILIO related to chromosome stability during cell division. Conclusion: These data suggest that this molecular axis may provide insights for developing novel therapeutic strategies for breast cancer

Association of FOSL1 copy number alteration and triple negative breast tumors

Abstract Copy number alterations (CNAs) are a frequent feature in human breast cancer, and one of the hallmarks of genomic instability. The FOSL1, GSTP1 and CCND1 genes are located at 11q13, a cytoband commonly affected by CNA in breast cancer, with relevant function in progression and invasion. Our main goal was to analyze CNAs of these genes and determine their association with breast cancer subtypes. Seventy-three cases of invasive breast tumors [52 Luminal, 7 HER2+ and 14 triple negative (TNBC) subtypes] were analyzed by TaqMan assays. CNAs were observed for all genes, with gains more frequently observed. Gains of the FOSL1 gene were observed in 71% of the cases. This gene was the only one with a statistically significant difference (p<0.001) among tumor subtypes, with increased copy number in TNBC compared to luminal and HER2+. No significant association of CNA and clinical and histopathological parameters from the patients was observed. Additional studies in larger breast cancer patient cohorts based on more refined molecular subtypes are necessary to confirm the observed association of FOSL1 gain with aggressive breast tumors phenotypes