Effect of CCAR2 depletion on the gene expression profile of BJ-hTERT cells. Homo sapiens

CCAR2 is a nuclear protein recently emerged as a pivotal player of the DNA damage response since it has been found involved in both apoptosis induction and DNA repair. Differently, its role in tumorigenesis and cancer progression is still elusive. In our studies we found that CCAR2 depletion impairs the proliferation of human cancer cell lines, but leaves unaffected the growth of normal immortalized cells. To better investigate this point we performed a genome wide gene expression analyses in U2OS and BJ-hTERT depleted of CCAR2 and we found that loss of this protein causes the deregulation of genes implicated in the AKT pathway specifically in U2OS cells, but not in BJ-hTERT. In accordance with these results we found a reduction in AKT activation in all the tested cancer cell lines depleted of CCAR2, but not in the normal ones. The defective activation of AKT is caused by the upregulation of TRB3 gene in cancer cells depleted of CCAR2 and finally results in the reduction of GSK3β phosphorylation, prevention of G1/S transition and inhibition of cancer cell growth. Overall design: The human normal fibroblast cell line BJ-hTERT was cultured in Dulbecco's Modified Eagle Medium (DMEM)/Medium 199 (4:1) supplemented with 10% of fetal calf serum and 10µg/ml hygromycin B as selection for hTERT transgene. To obtain a population of CCAR2-knock-out (KO) cells we used the CRISPR/Cas9 system as described in Magni et al, Oncotarget 2015. Cells were transfected with the Amaxa Nucleofector Device using the Basic Fibroblast Kit (VPI-1002) and U23 program. A population of 40% cells negative for CCAR2 was initially obtained. Then, after a second round of transfection, a mass culture with the 80% of cells KO for CCAR2 was produced. These cells were then collected in triplicates for RNA extraction and gene expression analyses.