Genetic ablation of pregnancy zone protein promotes breast cancer progression by activating TGF-β/SMAD signaling
Genetic ablation of pregnancy zone protein promotes breast cancer progression by activating TGF-β/SMAD signaling
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DOI：
10.1007/s10549-020-05958-y
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发表时间：
2020-10-15
影响因子：
3.8
通讯作者：
Hasan, Syed K.
中科院分区：
文献类型：
--
作者：
Kumar, Rohit;Kuligina, Ekaterina;Hasan, Syed K.
妊娠带蛋白（Pregnancy zone protein，PZP）是一种蛋白酶抑制剂，在妊娠期血浆中的浓度显著升高。最近的研究揭示了PZPin激活生殖系突变在乳腺癌易感性中的作用，因此我们设计了一项研究来评估这种蛋白在肿瘤发病机制中的功能参与。方法利用CRISPR-Cas9方法在MCF 7和T47 D（乳腺癌）细胞系中产生PZP敲除细胞，并进行集落形成、细胞增殖和迁移测定。使用qRT-PCR和蛋白质印迹进行TGF-β和SMAD表达研究。使用TCGA联盟登记处中包括的乳腺癌患者（n = 1211）以及激素受体阳性（n = 118）和三阴性乳腺癌（TNBC）患者（n = 116）的独立队列的数据记录的荟萃分析，比较肿瘤与正常组织中的PZP表达。结果我们证明，基因切除PZP有效地抑制了他莫昔芬诱导的细胞凋亡，并增强了细胞增殖、迁移和集落形成能力。我们发现在他莫昔芬治疗后，与野生型对应物相比，CRISPR/Cas9介导的PZP敲除克隆的存活分数显著增加（p < 0.05）。PZP敲除显著促进乳腺癌细胞的体外迁移（p < 0.01）。我们观察到TGF-β 2配体、TGF-β受体2的高表达，以及磷酸化调节SMAD（pSMAD 2和pSMAD 3）的上调，激活PZP敲除克隆中TGF-β/SMAD信号传导的促存活功能。对乳腺癌患者数据记录的荟萃分析表明，PZP 6低表达与6年总生存率低相关（低表达者51.7% vs高表达者62.9%;p = 0.026）。我们还观察到，与激素受体阳性肿瘤相比，TNBC中PZPmRNA表达显著降低（p = 0.019）。结论PZP基因敲除可导致乳腺癌的进展，PZP基因表达降低与乳腺癌患者的生存率相关。
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.