MCF7 Human Breast Cancer Cells Engineered to Stably Overexpress DEPTOR via CRISPR/Cas9 to Reduce Anabolic Capacity (2020)

As a potent cellular inhibitor of both mTOR complexes, DEPTOR protein has the potential to dramatically alter the anabolic potential of many different cell types and conditions. Manipulation of DEPTOR protein content means that cellular anabolism can be more aggressively regulated at a single bottleneck of key cellular signaling pathways. This approach could be particularly advantageous in a number of disease states where anabolically aggressive behavior gives rise to specific pathology. To evaluate this concept, we developed a novel subset of the immortalized MCF7 human epithelial breast cancer cell line. By utilizing CRISPR/Cas9 to facilitate a targeted insertion of a constitutively active DEPTOR gene into the human AAVS1 genomic safe harbor site, we examined direct and indirect measures of cellular anabolism to determine the efficacy of abating the anabolic potential of this type of human cancer. MCF7 cells overexpressing DEPTOR (DEP) were generated by genome integration of a constitutively active DEPTOR gene via a homologous directed repair (HDR) based template following a double stranded break (DSB) at the human AAVS1 safe harbor site by Cas9 nuclease. A second group of cells were used as a control by treating them with a vector containing a red fluorescent protein (RFP) gene in lieu of the DEPTOR gene. Both vectors contain a separate promoter for green fluorescence protein (GFP) gene, and N‐acetyltransferase. Both groups of cells were subjected to 14 days of puromycin selection, and appropriate fluorophore presence was confirmed. Rates of protein synthesis were measured using deuterium heavy water stable isotope methods, and cellular anabolic signaling was assessed via immunoblotting. Twenty‐four hour rates of protein synthesis were significantly reduced in DEP compared to RFP (23.390 ± 2.26 vs. 37.314 ± 0.21 %/day, respectively; p=0.029), representing a 37.6% reduction. DEPTOR protein content was found to be significantly higher in the DEP group compared to RFP controls (9.593 ± 3.209 vs. 1 ± 0.552, respectively; p&lt;0.001). Total (0.978 ± 0.202 vs 1 ± 0.162; p=0.841) and phosphorylated (0.65 ± 0.445 vs 1 ± 0.406; p=0.186) p70 S6 Kinase Thr389 were not found to be different between DEP and RFP. The phosphorylated to total ratio of p70 S6 Kinase was lower in DEP vs RFP (0.619 ± 0.315 vs 0.978 ± 0.255, respectively; p=0.05). Interestingly, both total 4E‐BP1 (1.340 ± 0.107 vs 1 ± 0.102, p&lt;0.001) and Thr 37/46 phosphorylation (2.106 ± 0.742 vs 1 ± 0.559, p=0.015) were significantly higher in the DEP vs RFP groups, respectively, but the phosphorylated to total ratios were not different between groups. These results indicate that DEPTOR overexpression dramatically impacts the anabolic activity of MCF7 human cancer cells, as shown by significant reductions in protein synthesis rates, as well as phosphorylation of the downstream mTORC1 target p70 S6 Kinase. Increases in phosphorylated and total 4E‐BP1 could be the result of long‐term inhibition of mTORC1 by DEPTOR protein, possibly indicating the need for evaluation of 4E‐BP1’s unique role in regulating transitions between Cap‐Dependent and Cap‐Independent mRNA translation. Support or Funding Information Funding provided by the Sydney &amp; JL Huffine’s Institute and the College of Education and Human Development.