LAUSR

Germline mutations in known breast cancer (BC) predisposing genes account for only 20 - 25% of hereditary breast cancer susceptibility. In this study, we anticipated that application of exome analysis to a series of genetically homogeneous Slavic BC patients will allow to identify novel recurrent BC predisposing mutations. 49 BRCA1/2-negative BC patients with evident clinical signs of the hereditary disease (family history and/or BC bilaterality and/or young onset) were subjected to Illumina-based whole exome sequencing. Two patients were found to carry a rare nonsense mutation in pregnancy zone protein (PZP) gene (p.Arg680*, rs145240281). This mutation was further analyzed in a case-control study involving 1046 high-risk BRCA1/2-negative BC cases, 1608 consecutive BC and 1082 middle-aged tumor-free women. Distribution of PZP p.Arg680* alleles was in agreement with its putative BC-predisposing role: 4/1046 (0.38%) and 9/1608 (0.56%) BC cases vs. 1/1082 (0.09%) controls, producing risk estimates in consecutive BC group equal to 6.08 (95% CI 0.770 to 48.096, P = 0.087]. 5/15 heterozygous for PZP p.Arg680* variant patients were diagnosed with multiple primary tumors (2 bilateral BCs, BC + colorectal cancer, bilateral BC + gastric cancer, BC + basal-cell like skin cancer). To the best of our knowledge, no reports associating gene PZP to cancer pathogenesis are available. In order to evaluate the functional effects of PZP p.Arg680*, we obtained two heterozygous mutant clones of CRISPR/Cas9-modified MCF7 cells containing 10-bp and 4-bp deletions adjacent to the desired targeted location. The real-time quantitative PCR confirmed significant down-regulation of PZP transcript in the clones carrying CRISPR/Cas9 induced deletions. Since this mutation results in a truncated protein, we also performed shRNA mediated knock-down of PZP. To study the effects of PZP mutation on cell proliferation and apoptosis, CRISPR/Cas9- and shRNA-modified clones were treated with tamoxifen and etoposide for 72 hours. The results have shown a significantly higher inhibitory concentration (IC50) for both drugs and higher clonogenic potential in CRISPR/Cas9-modified PZP clones compared to control MCF7 cells. Apoptosis induction was quantified using AnnexinV/PI staining; the reduced apoptosis in PZP-defective clones was documented. Taken together, obtained data argue that PZP may function as a tumor suppressor gene, and truncating variant p.Arg680* can represent a moderate-risk breast cancer susceptibility allele. This work has been supported by the Russian Science Foundation (grant 17-15-01384) and DST/INT/RUS/RSF/P-11 Citation Format: Ekaterina Sh Kuligina, Anna P. Sokolenko, Rohit Kumar, Ilya V. Bizin, Aglaya G. Iyevleva, Kirill Zagorodnev, Syed K. Hasan, Aleksandr A. Romanko, Ashok K. Varma, Evgeny N. Imyanitov. PZP as a new gene associated with increased breast cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4160.