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Study of Impacto of Mutational Allelic Expression Imbalance On Drug Treatment Response
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Atualmente, existem dois inibidores da proteína PI3K aprovados para tratar pacientes com o subtipo mais comum de cancro da mama diagnosticado em mulheres, cujos tumores contém mutações no gene PIK3CA e que foram previamente submetidas a terapia endócrina. Para eleger pacientes para esta terapia-alvo, a existência de mutações no gene PIK3CA, em tumores, é atualmente verificada por testes de DNA, ignorando a diferença de expressão entre os alelos normal e mutante – expressão alélica diferencial mutacional (EAD). Consequentemente, esta limitação pode levar à administração destes inibidores em doentes que não expressam o alelo mutante, necessário para produzir a proteína visada pela terapia. Nesses casos, não existem benefícios, mas os efeitos secundários podem aparecer, reforçando a necessidade de melhorar a seleção de pacientes e a identificação da dose mais adequada do fármaco para administrar a cada paciente. Para avaliar esta hipótese, o objetivo principal foi desenvolver um modelo de linha celular induzível com EAD variável entre ambos os alelos para avaliar a resposta aos inibidores de PI3K. O primeiro passo envolveu a transfeção de um plasmídeo contendo o alelo normal do gene PIK3CA em células cancerígenas, usando Lipofectamina 3000, um método mediado por lípidos. Diante dos desafios na transfecção deste plasmídeo de grande tamanho, as condições de transfecção foram otimizadas, mas o protocolo só teve sucesso com a transfecção de um plasmídeo mais pequeno. Posteriormente, a reação em cadeia da polimerase (PCR) seguida de sequenciamento por Sanger foram realizados para confirmar a integridade e corrigir a montagem dos plasmídeos utilizados para gerar os clones de expressão necessários para estabelecer o modelo baseado em células, dada a ausência de eficiência detetável na transfecção. Dado que tanto as integridades quanto a correta montagem foram confirmadas, concluímos que o mé todo de transfecção escolhido provavelmente não é adequado para a transfeção do plasmídeo pretendido e nos futuros trabalhos exploraremos métodos alternativos de transfecção.
Currently, there are two targeted PI3K inhibitors approved to treat patients with the most common subtype of breast cancer diagnosed in women, whose tumors harbor mutations in the PIK3CA gene, and who were previously submitted to endocrine therapy. To elect patients for this targeted therapy, the existence of mutations in the PIK3CA gene in tumors is currently verified by DNA testing overlooking the expression difference between the wild-type and mutant alleles – mutational differential allelic expression (DAE). Consequently, this limitation may lead to the administration of those inhibitors in patients who do not express the mutant allele, needed to produce the protein targeted by the therapy. In these cases, the benefits are absent, but the side effects can still appear, reinforcing the need to improve patient selection and the identification of the most adequate dose of the drug to administer to each patient. To evaluate this hypothesis, the main objective was to develop a cell based inducible cell line model of variant DAE between both alleles to assess the response to PI3K inhibitors. The first step was to transfect a plasmid having the wild type of PIK3CA into cancer cells using Lipofectamine 3000, a lipid-mediated method. In the face of challenges in transfecting this large size plasmid, the transfection conditions were optimized, but the protocol was only successful with the transfection of a smaller control plasmid. Subsequently, polymerase chain reaction (PCR) followed by Sanger sequencing was employed to confirm the integrity and correct the assembly of plasmids used to generate the expression clones required for establishing the cell-based model, given the absence of detectable transfection efficiency. Given that both integrity and good assembly were confirmed, we concluded that the chosen transfection method is most probably not suitable to transfect the intended plasmid and for future work we will explore alternative transfection methods.
Currently, there are two targeted PI3K inhibitors approved to treat patients with the most common subtype of breast cancer diagnosed in women, whose tumors harbor mutations in the PIK3CA gene, and who were previously submitted to endocrine therapy. To elect patients for this targeted therapy, the existence of mutations in the PIK3CA gene in tumors is currently verified by DNA testing overlooking the expression difference between the wild-type and mutant alleles – mutational differential allelic expression (DAE). Consequently, this limitation may lead to the administration of those inhibitors in patients who do not express the mutant allele, needed to produce the protein targeted by the therapy. In these cases, the benefits are absent, but the side effects can still appear, reinforcing the need to improve patient selection and the identification of the most adequate dose of the drug to administer to each patient. To evaluate this hypothesis, the main objective was to develop a cell based inducible cell line model of variant DAE between both alleles to assess the response to PI3K inhibitors. The first step was to transfect a plasmid having the wild type of PIK3CA into cancer cells using Lipofectamine 3000, a lipid-mediated method. In the face of challenges in transfecting this large size plasmid, the transfection conditions were optimized, but the protocol was only successful with the transfection of a smaller control plasmid. Subsequently, polymerase chain reaction (PCR) followed by Sanger sequencing was employed to confirm the integrity and correct the assembly of plasmids used to generate the expression clones required for establishing the cell-based model, given the absence of detectable transfection efficiency. Given that both integrity and good assembly were confirmed, we concluded that the chosen transfection method is most probably not suitable to transfect the intended plasmid and for future work we will explore alternative transfection methods.
Descrição
Palavras-chave
Breast cancer PIK3CA Targeted therapy Cell culture Transfection