Human BRCA2 Knockout Cell Line-LNCaP

Previous sequencing studies have shown that alterations in DNA damage response (DDR)-related genes are enriched in patients with metastatic castration-resistant prostate cancer (mCRPC). BRCA2 is a DDR and cancer susceptibility gene that is frequently deleted (homozygous and heterozygous) in men with aggressive prostate cancer. Here, researchers investigated the potential molecular mechanisms and genomic consequences of co-loss of BRCA2 and RB1 in prostate cancer. Results show that loss of BRCA2 leads to a castration-resistant phenotype in human prostate cancer cell lines (LNCaP and LAPC4). Co-loss of BRCA2-RB1 in human prostate cancer cells induces epithelial-mesenchymal transition, which is associated with an aggressive and more invasive disease phenotype. Importantly, PARP inhibitors attenuated cell growth in human mCRPC-derived organoids and in human CRPC cells carrying a single copy deletion of both genes.

Here, the researchers observed downregulation of BRCA2 protein and mRNA in RB1 knockdown LNCaP cells (Figure 1A). Interestingly, they also observed that loss of BRCA2 attenuated RB1 protein expression in all BRCA2 knockout LNCaP cells (Figure 1A). In addition, BRCA2-RB1 knockout/knockdown LNCaP cells showed relative resistance to the CDK4/6 inhibitor palbociclib as measured by MTT (Figure 1B). These data suggest that depletion of RB1 and/or BRCA2 in LNCaP cells is sufficient to induce inhibition of canonical downstream pathways by RB1.

Immunofluorescence staining using phalloidin revealed actin filament remodeling in LNCaP-BRCA2-RB1 cells, further supporting the changes in cell morphology upon simultaneous loss of BRCA2 and RB1 (Figure 1C). Knockdown/knockout of RB1 or BRCA2 alone induced a moderately invasive phenotype (Figure 1C). Phosphorylation of γH2AX was increased in LNCaP-BRCA2-RB1 cells compared with BRCA2 or RB1 knockout/knockdown LNCaP cells (Figure 1D). In addition, S2056 autophosphorylation of DNA-PKcs was slightly increased in LNCaP-BRCA2-RB1 cells compared with BRCA2 knockout LNCaP cells (Figure 1D). Loss of RB1 alone resulted in only a slight increase in γH2AX phosphorylation, but not in S2056 autophosphorylation of DNA-PKcs, compared with control LNCaP cells (Figure 1D). Treatment of LNCaP-BRCA2-RB1 cells with the PARPi olaparib and talazoparib resulted in greater cell growth inhibition than BRCA2-null LNCaP cells (Figure 1E). These data suggest that co-loss of BRCA2 and RB1 increases the sensitivity of prostate cancer cells to PARPi compared with loss of BRCA2 alone. In contrast, loss of RB1 alone was not associated with sensitivity of prostate cancer cells to PARPi (Figure 1E).

Figure 1. Co-loss of BRCA2 and RB1 induces invasive phenotype in LNCaP cells. (Chakraborty G, et al., 2020)