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

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Acknowledgement

The research is supported by Indo Russia grant # INT/RUS/RSF/P-11 and Russian Science Federation (RSF) grant # 19-15-00207.

Funding

The research was supported by public funding agencies under Indo Russia grant # INT/RUS/RSF/P-11 by Department of Science and Technology, Government of India and Russian Science Federation (RSF) grant # 19-15-00207.

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Authors

Contributions

RK and QS conducted experiments, data acquisition, and data analyses. EK and AS provided PZP mutation data. NG and SG contributed in silico data analyses and interpretation. SKH and AV designed the study, analyzed the data, and wrote the paper.

Corresponding authors

Correspondence to Ashok K. Varma or Syed K. Hasan.

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Conflict of interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

The study design was approved by the local Ethical Committee of N.N. Petrov Institute of Oncology. All procedures performed in the study were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consents were obtained from all individual participants included in the study.

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Kumar, R., Kuligina, E., Sokolenko, A. et al. Genetic ablation of pregnancy zone protein promotes breast cancer progression by activating TGF-β/SMAD signaling. Breast Cancer Res Treat 185, 317–330 (2021). https://doi.org/10.1007/s10549-020-05958-y

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