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

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 ofPZPinactivating 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-beta and SMAD expression studies were performed using qRT-PCR and Western blot.PZPexpression 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 ofPZPefficiently 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-beta 2 ligand, TGF-beta- receptor 2, and upregulation of phosphorylated regulatory-SMADs (pSMAD2 and pSMAD3) activating the pro-survival function of TGF-beta/SMAD signaling in PZP knockout clones. Meta-analyses of data records of breast cancer patients indicated that lowPZPexpression 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 lowerPZPmRNA expression in TNBC as compared with hormone receptor-positive tumors (p = 0.019). Conclusion Taken together, our results suggest that genetic ablation ofPZPresults in tumor progression and low expression ofPZPis associated with poor survival of breast cancer patients.

Automated summary

The study revealed that genetic ablation of PZP results in tumor progression, and low expression of PZP is associated with poor survival of breast cancer patients.