Rohit Kumar1, Ekaterina Kuligina2, Anna Sokolenko2, Quadir Siddiqui1, Nilesh Gardi3,4, Sudeep Gupta3,4, Ashok K Varma5,6, Syed K Hasan7,8. 1. Cell and Tumor Biology Group, Advanced Centre for Treatment, Research and Education in Cancer, Sector 22, Kharghar, Navi Mumbai, 410210, Maharashtra, India. 2. Laboratory of Molecular Oncology, N.N. Petrov Institute of Oncology, Pesochny-2, 197758, St.-Petersburg, Russia. 3. Department of Medical Oncology, Tata Memorial Centre, Mumbai, 400012, Maharashtra, India. 4. Homi Bhabha National Institute (HBNI), Anushaktinagar, Mumbai, 400094, India. 5. Varma Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, 410210, India. avarma@actrec.gov.in. 6. Homi Bhabha National Institute (HBNI), Anushaktinagar, Mumbai, 400094, India. avarma@actrec.gov.in. 7. Cell and Tumor Biology Group, Advanced Centre for Treatment, Research and Education in Cancer, Sector 22, Kharghar, Navi Mumbai, 410210, Maharashtra, India. shasan@actrec.gov.in. 8. Homi Bhabha National Institute (HBNI), Anushaktinagar, Mumbai, 400094, India. shasan@actrec.gov.in.
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-β 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-β2 ligand, TGF-β- receptor 2, and upregulation of phosphorylated regulatory-SMADs (pSMAD2 and pSMAD3) activating the pro-survival function of TGF-β/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.
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-β 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 cancerpatients (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-β2 ligand, TGF-β- receptor 2, and upregulation of phosphorylated regulatory-SMADs (pSMAD2 and pSMAD3) activating the pro-survival function of TGF-β/SMAD signaling in PZP knockout clones. Meta-analyses of data records of breast cancerpatients 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 cancerpatients.