Genetic ablation of pregnancy zone protein promotes breast cancer progression by activating TGF-ß/SMAD signaling.

ABSTRACT

PURPOSE: Pregnancy zone protein (PZP) is best known as protease inhibitor and its concentration in human blood plasma increases dramatically during pregnancy. Recent investigation revealed a role of PZP inactivating germ-line mutation in breast cancer predisposition, and therefore we designed a study to evaluate functional involvement of this protein in tumor pathogenesis. METHODS: PZP knockout cells were generated utilizing the CRISPR-Cas9 approach in MCF7 and T47D (breast cancer) cell lines, and colony formation, cell proliferation, and migration assays carried out. TGF-ß and SMAD expression studies were performed using qRT-PCR and Western blot. PZP expression in tumor vs normal tissue was compared using meta-analyses of data records of breast cancer patients (n = 1211) included in the TCGA consortium registry as well as in independent cohorts of hormone receptor-positive (n = 118) and triple-negative breast cancer (TNBC) patients (n = 116). RESULTS: We demonstrated that genetic ablation of PZP efficiently inhibits tamoxifen-induced apoptosis and enhances cell proliferation, migration, and colony-forming capacity. We found a significant increase in survival fraction of CRISPR/Cas9-mediated PZP knockout clones compared to wild-type counterpart after tamoxifen treatment (p < 0.05). The PZP knockout significantly promoted breast cancer cell migration (p < 0.01) in vitro. We observed high expression of TGF-ß2 ligand, TGF-ß- receptor 2, and upregulation of phosphorylated regulatory-SMADs (pSMAD2 and pSMAD3) activating the pro-survival function of TGF-ß/SMAD signaling in PZP knockout clones. Meta-analyses of data records of breast cancer patients indicated that low PZP expression is associated with poor overall survival at 6 years (51.7% vs 62.9% in low vs high expressers, respectively; p = 0.026). We also observed a significantly lower PZP mRNA expression in TNBC as compared with hormone receptor-positive tumors (p = 0.019). CONCLUSION: Taken together, our results suggest that genetic ablation of PZP results in tumor progression and low expression of PZP is associated with poor survival of breast cancer patients.