Validação da variante c.3140A>G e de suas potenciais proteínas alvo relacionadas a via PIK3CA/AKT/mTOR em neoplasias mamárias de cadelas
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Data
2022-03-07
Autores
Perossi, Isabela Fernanda Spinelli
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O câncer de mama (CM) é um relevante problema de saúde pública, pois representa a segunda principal causa de morte relacionada ao câncer em mulheres em todo o mundo. Os cães são utilizados como modelo para estudos desses tumores, pois além da grande ocorrência, possuem semelhanças biológicas e fisiopatológicas. A via PIK3CA/AKT/mTOR está fortemente desregulada no câncer de mama e desempenha papel central na homeostase celular. A literatura recente retrata que todas as mutações somáticas identificadas a partir do gene PIK3CA alteraram as sequências de aminoácidos, dentre eles a variante c.3140A> G. Esse gene participa da regulação de uma ampla gama de atividades celulares relevantes englobando várias proteínas importantes. Na medicina veterinária o estudo desta via é incipiente. O objetivo deste trabalho foi analisar a variante c.3140A>G do gene PI3KCA e a expressão das proteínas alvo PIK3CA, PTEN, HIF, VHL, ZEB1, ZEB 2, Caspase-3 e PARP1 como marcadores prognósticos em um estudo prospectivo, verificando a expressão gênica dessa variante e, em um estudo retrospectivo, avaliando a expressão proteica e gênica de alvos moleculares à jusante da via correlacionando com o prognóstico nas cadelas. No estudo retrospectivo, o DNA de 24 fragmentos de tumores mamários caninos e 20 fragmentos normais de mama de cadelas foi extraído e a mutação verificada usando TaqMan® Mutation Detection Assay. A imunohistoquímica foi realizada com o kit Reveal HRP Conjugate e sua análise pelo método Histoscore. Duas pacientes (8,3%) apresentaram a mutação c.3140A> G, sendo que uma paciente apresentou metástase pulmonar e morreu em 30 dias após o diagnóstico e a outra paciente permanece viva. Os dados de imunohistoquímica revelaram que a expressão das proteínas PIK3CA, HIF, ZEB2 e PARP1 foi maior na paciente que veio a óbito em comparação com a que permanece viva (p<0,05). No estudo prospectivo, para a análise proteica as amostras foram provenientes de 58 cadelas com neoplasia mamária, previamente identificadas por análise histopatológica. A imunohistoquímica foi realizada com o kit Reveal HRP Conjugate e sua análise pelo método Histoscore. Para a análise genica as amostras são provenientes de 13 pacientes caninas acometidas por neoplasia mamária primária, o DNA foi extraído segundo o método de Sambrook & Russel e realizada a análise para expressão quantitativa utilizando o sistema Step One Plus. Através da imunohistoquimica foi observado que a positividade de PIK3CA foi significativamente associada a linfonodo regional8 acometido, à metástase a distância, às pacientes com fenótipos HER2+, Triplo Negativo e Luminal B e também à menor sobrevida. Por meio da expressão gênica observamos, entre as pacientes vivas e com óbito, expressão gênica mais elevada de ZEB2 e PARP1 comparado aqueles sem metástase, o que não foi verdadeiro para as expressões de PIK3CA e HIF1 α. Em conclusão, os dados observados neste trabalho são promissores no estudo de novos marcadores moleculares de prognostico adequados para o desenvolvimento de novas terapias, ainda assim mais pesquisas destinadas a esclarecer e validar as relações entre a via PI3K/AKT/mTOR.
Breast cancer (BC) is a relevant public health problem, as it represents the second leading cause of cancer-related death in women worldwide. Dogs are used as a model for studies of these tumors, because in addition to their high occurrence, they have biological and pathophysiological similarities. The PIK3CA/AKT/mTOR pathway is strongly dysregulated in breast cancer and plays a central role in cellular homeostasis. Recent literature shows that all somatic mutations identified from the PIK3CA gene altered the amino acid sequences, including the variant c.3140A> G. This gene participates in the regulation of a wide range of relevant cellular activities encompassing several important proteins. In veterinary medicine, the study of this pathway is incipient. The objective of this work was to analyze the c.3140A>G variant of the PI3KCA gene and the expression of the target proteins PIK3CA, PTEN, HIF, VHL, ZEB1, ZEB 2, Caspase-3 and PARP1 as prognostic markers in a prospective study, verifying the gene expression of this variant and, in a retrospective study, evaluating the protein and gene expression of molecular targets downstream of the pathway correlating with the prognosis in bitches. In the retrospective study, DNA from 24 canine mammary tumor fragments and 20 canine normal mammary fragments was extracted and mutation verified using the TaqMan® Mutation Detection Assay. Immunohistochemistry was performed with the Reveal HRP Conjugate kit (Spring®) and its analysis was performed using the Histoscore method. Two patients (8.3%) had the c.3140A>G mutation, one patient had lung metastasis and died within 30 days of diagnosis and the other patient remains. Immunohistochemistry data revealed that the expression of PIK3CA, HIF, ZEB2 and PARP1 proteins was higher in the patient who died compared to the one who remained alive (p<0.05). In the prospective study, for protein analysis, the samples came from 58 bitches with mammary neoplasia, previously identified by histopathological analysis, immunohistochemistry was performed with the Reveal HRP Conjugate kit (Spring®) and its analysis by the Histoscore method. For the genetic analysis, the samples come from 13 canine patients affected by primary mammary neoplasia, the DNA was extracted according to the method of Sambrook & Russell and the analysis for quantitative expression was performed using the Step One Plus system (Applied Biosystems®, Foster City, CA, USA). Through immunohistochemistry, PIK3CA positivity was significantly associated with affected regional lymph node, distant metastasis, patients with HER2+, Triple 10 Negative and Luminal B phenotypes and also the lowest survival in univariate analysis. Through gene expression, we observed higher gene expression of ZEB2 and PARP1 among patients who were alive and with death compared to those without metastasis, which was not true for the expressions of PIK3CA and HIF1 α. In conclusion, the data observed in this work are promising in the study of new molecular markers of prognosis suitable for the development of new therapies, yet more research is aimed at clarifying and validating the relationships between the PI3K/AKT/mTOR pathway
Breast cancer (BC) is a relevant public health problem, as it represents the second leading cause of cancer-related death in women worldwide. Dogs are used as a model for studies of these tumors, because in addition to their high occurrence, they have biological and pathophysiological similarities. The PIK3CA/AKT/mTOR pathway is strongly dysregulated in breast cancer and plays a central role in cellular homeostasis. Recent literature shows that all somatic mutations identified from the PIK3CA gene altered the amino acid sequences, including the variant c.3140A> G. This gene participates in the regulation of a wide range of relevant cellular activities encompassing several important proteins. In veterinary medicine, the study of this pathway is incipient. The objective of this work was to analyze the c.3140A>G variant of the PI3KCA gene and the expression of the target proteins PIK3CA, PTEN, HIF, VHL, ZEB1, ZEB 2, Caspase-3 and PARP1 as prognostic markers in a prospective study, verifying the gene expression of this variant and, in a retrospective study, evaluating the protein and gene expression of molecular targets downstream of the pathway correlating with the prognosis in bitches. In the retrospective study, DNA from 24 canine mammary tumor fragments and 20 canine normal mammary fragments was extracted and mutation verified using the TaqMan® Mutation Detection Assay. Immunohistochemistry was performed with the Reveal HRP Conjugate kit (Spring®) and its analysis was performed using the Histoscore method. Two patients (8.3%) had the c.3140A>G mutation, one patient had lung metastasis and died within 30 days of diagnosis and the other patient remains. Immunohistochemistry data revealed that the expression of PIK3CA, HIF, ZEB2 and PARP1 proteins was higher in the patient who died compared to the one who remained alive (p<0.05). In the prospective study, for protein analysis, the samples came from 58 bitches with mammary neoplasia, previously identified by histopathological analysis, immunohistochemistry was performed with the Reveal HRP Conjugate kit (Spring®) and its analysis by the Histoscore method. For the genetic analysis, the samples come from 13 canine patients affected by primary mammary neoplasia, the DNA was extracted according to the method of Sambrook & Russell and the analysis for quantitative expression was performed using the Step One Plus system (Applied Biosystems®, Foster City, CA, USA). Through immunohistochemistry, PIK3CA positivity was significantly associated with affected regional lymph node, distant metastasis, patients with HER2+, Triple 10 Negative and Luminal B phenotypes and also the lowest survival in univariate analysis. Through gene expression, we observed higher gene expression of ZEB2 and PARP1 among patients who were alive and with death compared to those without metastasis, which was not true for the expressions of PIK3CA and HIF1 α. In conclusion, the data observed in this work are promising in the study of new molecular markers of prognosis suitable for the development of new therapies, yet more research is aimed at clarifying and validating the relationships between the PI3K/AKT/mTOR pathway
Descrição
Palavras-chave
Neoplasia mamária, Via pi3k/akt/mtor, Expressão proteica, Expressão gênica, Prognóstico, Metástase, Breast cancer, PI3K/AKT/mTOR pathway, Protein expression, Gene expression, Prognosis, Metastasis