Generation of STK3 knockout mammary epithelial cells using CRISPR/Cas9 technology

Abstract

Hippo is a tumor suppressor pathway and is related to the regulation of cell proliferation, apoptosis, organ size and tumorigenesis. In mammals, the Hippo pathway core is composed of 4 serine-threonine kinases: Mammalian Ste-20-like Kinase 1 and 2 (MST1 and MST2) and large suppressive tumor 1 and 2 (LATS1 and LATS2). When Hippo is active, the transcription co-activators YAP and TAZ are phosphorylated and retained in the cytoplasm. However, when the pathway is inactive, YAP and TAZ are not phosphorylated and enter the cell nucleus, where they activate transcription factors, increasing proliferation and inducing malignant phenotype in epithelial cells. Preliminary results from our laboratory showed that in malignant cells (T4-2), STK3 mRNA (gene encoding MST2) is shorter, a result of an exon skipping splicing that excludes exon 7 from the transcript (STK3”exon7). Analysis of RNAseq data from The Cancer Genome Atlas (TCGA) revealed that the variant STK3”exon7 is also found in samples from patients with breast cancer. With the exclusion of exon 7, the transcript is translated into a protein that is more susceptible to degradation. In this project, we propose to knockout (KO) the STK3 gene in non-tumoral epithelial cells of the human mammary gland using CRISPR/Cas9. This cellular model will serve as a basis for the development of experiments to elucidate the phenotypic effects of expressing d MST2”exon7 and reestablishing the expression of MST2 in different concentrations.