Abstract 4723: Genetic ablation of pregnancy zone protein (PZP) promotes breast cancer progression by activating TGF-β/SMAD signalling

Abstract

Abstract A significant portion of breast cancer (BC) incidence is attributed to a hereditary predisposition to the disease. Germ-line variants in known BC-associated genes explain less than 30% of women with clinical signs of hereditary cancer syndrome. Using the whole-exome sequencing (WES) of genetically enriched BRCA-negative BC patients, we identified six novel candidate variants that are likely to contribute to BC predisposition. Particularly, the obtained data argued that pregnancy zone protein gene PZP might function as a tumor suppressor gene, likewise protein-truncating nonsense variant p.Arg680* could represent a moderate-risk breast cancer susceptibility allele. PZP is reported to bind with TGFβ1/β2 proteins, therefore, we hypothesize that the loss of PZP function may affect TGFβ signalling in breast cancer. We analysed the functional consequences of PZP nonsense mutation on TGFβ/SMAD signalling using CRISPR/Cas9 based genetic ablation of PZP in MCF7 and T47D breast cancer cells. GFP positive cells were sorted using FACS Aria III (BD biosciences) and genome editing events were identified using T7 endonuclease assay followed by DNA sequencing. PZP knockout cells were further verified by Anti-PZP Antibody (Abcam, UK) targeting loop region of PZP. The modulation of TGFβ signalling was quantified using qRT-PCR of TGFβ ligand/receptor mRNAs. The upregulation of TGFβ/SMAD signalling was evaluated by phospho-SMAD2, -SMAD3 protein levels. The wound healing and colony-forming assays were performed to compare cell migration and proliferation index in PZP wild-type [MCF7PZPwt, T47DPZPwt] versus PZP-deficient BC cells. DNA sequencing and Western blot confirmed the successful knockout using CRISPR/Cas9 approach [MCF7PZPsgRNA1, MCF7PZPsgRNA2, T47DPZPsgRNA1, and T47DPZPsgRNA2]. The PZP knockout cells have shown 2-2.8-fold increase in IC50 compared to wild-type cells following tamoxifen treatment (T47DPZPwt vs T47DPZPsgRNA1: 3.45µM vs 7.90µM; MCF7PZPwt vs MCF7PZPsgRNA1: 8.3µM vs 21µM). We documented the significant up-regulation of TGFβ1, β2 and βR2 transcripts in PZP-knockout clones. Of note, a very high up-regulation of TGFβ2 was observed in MCF7PZPsgRNA1 (197-fold), MCF7PZPsgRNA2 (498-fold) cells, whereas T47DPZPsgRNA1, T47DPZPsgRNA2 cells showed 2.8 and 5.5 fold up-regulation compared to wild-type cells (p=0.0001). Further, we reported an increase in SMAD2 and SMAD3 phosphorylation in PZP knockout cells. The relative wound width of PZP knockout cells significantly reduced compared to wildtype (p=0.0045). The clonogenic potential of PZP knockout cells was 3.5-fold higher compared to the wild-type counterpart. Our findings suggest that loss-of-function of PZP may favour breast cancer progression by up-regulating TGF-β/SMAD signalling. The research is supported by Indo Russia grant # INT/RUS/RSF/P-11 and RSF grant # 19-15-00207. Citation Format: Rohit Kumar, Ekaterina Kuligina, Anna Sokolenko, Quadir Siddiqui, Ashok K. Varma, Syed K. Hasan. Genetic ablation of pregnancy zone protein (PZP) promotes breast cancer progression by activating TGF-β/SMAD signalling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4723.