RESUMO
BACKGROUND: To obtain a deep understanding of the mechanism by which breast cancer develops, the genes involved in tumorigenesis should be analyzed in vivo. Mouse mammary gland can regenerate completely from a mammary stem cell (MaSC), which enables us to analyze the effect of gene expression and repression on tumorigenesis in mammary gland regenerated from genetically manipulated MaSCs. Although lentiviral and retroviral systems have usually been applied for gene transduction into MaSCs, they are associated with difficulty in introducing long, repeated, or transcriptional termination sequences. There is thus a need for an easier and quicker gene delivery system. METHODS: We devised a new system for gene delivery into MaSCs using the piggyBac transposon vectors and electroporation. Compared with viral systems, this system enables easier and quicker transfection of even long, repeated, or transcriptional termination DNA sequences. We designed gene expression vectors of the transposon system, equipped with a luciferase (Luc) expression cassette for monitoring gene transduction into regenerative mammary gland in mice by in-vivo imaging. A doxycycline (Dox)-inducible system was also integrated for expressing the target gene after mammary regeneration to mimic the actual mechanism of tumorigenesis. RESULTS: With this new gene delivery system, genetically manipulated mammary glands were successfully reconstituted even though the vector size was > 200 kb and even in the presence of DNA elements such as promoters and transcription termination sequences, which are major obstacles to viral vector packaging. They differentiated correctly into both basal and luminal cells, and showed normal morphological change and milk production after pregnancy, as well as self-renewal capacity. Using the Tet-On system, gene expression can be controlled by the addition of Dox after mammary reconstitution. In a case study using polyoma-virus middle T antigen (PyMT), oncogene-induced tumorigenesis was achieved. The histological appearance of the tumor was highly similar to that of the mouse mammary tumor virus-PyMT transgenic mouse model. CONCLUSIONS: With this system, gene transduction in the mammary gland can be easily and quickly achieved, and gene expression can be controlled by Dox administration. This system for genetic manipulation could be useful for analyzing genes involved in breast cancer.
Assuntos
Diferenciação Celular/genética , Engenharia Genética/métodos , Glândulas Mamárias Animais/fisiologia , Neoplasias Mamárias Experimentais/genética , Células-Tronco/fisiologia , Animais , Linhagem Celular , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Doxiciclina/administração & dosagem , Feminino , Fibroblastos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/transplante , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células/métodos , Transfecção/métodosRESUMO
BACKGROUND: Chromosomal aberrations involving the anaplastic lymphoma kinase (ALK) gene have been observed in approximately 4% of patients with non-small cell lung cancer (NSCLC). Although these patients clinically benefit from treatment with various ALK tyrosine kinase inhibitors (ALK-TKIs), none of these can inhibit the development of resistance mutations. Considering inevitable drug resistance and the variety of available ALK-TKIs, it is necessary to predict the pattern of drug-resistance mutations to determine the optimal treatment strategy. OBJECTIVE: We aimed to establish a polymerase chain reaction (PCR)-based system to predict the development of resistance mutations against ALK-TKIs and identify therapeutic strategies using the upcoming ALK-TKIs repotrectinib (TPX-0005) and ensartinib (X-396) following recurrence on first-line alectinib treatment for ALK-positive NSCLC. METHODS: An error-prone PCR-based method for predicting drug resistance mutations was established and the half-maximal inhibitory concentration (IC50) values of the predicted ALK mutations were evaluated in a Ba/F3 cell-based assay. RESULTS: We predicted several resistance mutations against repotrectinib and ensartinib, and demonstrated that the next-generation ALK-TKI TPX-0131, was active against repotrectinib-resistant mutations and that the FLT3 inhibitor gilteritinib was active against ensartinib-resistant mutations. CONCLUSIONS: We developed a PCR-based system for predicting drug resistance mutations. When this system was applied to repotrectinib and ensartinib, the results suggested that these drugs can be used for the second-line treatment of ALK-positive NSCLC. Predicting resistance mutations against TKIs will provide useful information to aid in the development of effective therapeutic strategies.