Stage 1 Generation of genetically modified triple negative breast cancer cell lines with stable overexpression (ELK3-OE) or stable inhibition (ELK3-KD) of the ELK3 transcription factor by lentiviral transduction
Activity 1.1 Design of the overall experimental procedure and genetic constructs to be used in the generation of the genetically modified triple negative breast cancer cell lines.
Activity 1.2 Lentiviral-mediated transduction of triple-negative breast cancer cell lines and generation of 4 genetically modified cell lines
Activity 1.3 Validation of the genetic modification for the 4 triple negative breast cancer cell lines, in terms of their phenotype (GFP expression) and at the level of genomic DNA, messenger RNA and ELK3 protein.
Stage 2 Evaluation of the cellular effects determined by the overexpression and inhibition of the transcription factor ELK3 in triple negative breast cancer cells, in terms of the cells’ migration capacity in 3D microfluidic devices, their phenotype on the epithelial-mesenchymal continuum by flow cytometry and their stemness potential in mammosphere formation assays
Activity 2.1 Assessment of the impact of the transcription factor ELK3 in the migration of triple-negative breast cancer cells in 3D microfluidic devices
Activity 2.2 Assessment of the impact of the transcription factor ELK3 in the epithelial-mesenchymal transition of triple-negative breast cancer cells by flow cytometry
Activity 2.3 Assessment of the impact of the transcription factor ELK3 on the stemness capacity of triple negative breast cancer cells in mammosphere formation assays
Stage 3 Identification of the molecular signaling pathways modulated by the transcription factor ELK3 that could be involved in the metastasis of triple negative breast cancer, by using microarray technology and RT-qPCR.
Activity 3.1 Identification of all gene expression changes triggered by the overexpression and inhibition of the transcription factor ELK3 in triple-negative breast cancer cells, by microarray technology
Activity 3.2 Validation of gene expression changes determined by the overexpression and inhibition of the transcription factor ELK3 in triple negative breast cancer cells that could be involved in metastasis, by RT-qPCR
Activity 3.3 Identification of the molecular mechanisms that could explain the impact of the ELK3 transcription factor in the metastasis of triple negative breast cancer, through the functional analysis of gene expression differences
