PO.MCB06.03 · 分子与细胞生物学

MPP8 通过与 SIRT1 和 ZEB1 相互作用抑制 PRKN 信号，从而促进膀胱癌的增殖和转移

MPP8 inhibits PRKN signaling through interactions with SIRT1 and ZEB1 to promote bladder cancer proliferation and metastasis

编号 3196 展板 6 时间 4/20 02:00–05:00 区域 Section 20 主讲 Stephany Gonzalez Tineo, BS

分会场 Epigenetic Changes as Molecular Markers of Cancer

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作者与单位 Authors & Affiliations

Stephany Gonzalez Tineo, Ryan M. Kemper, Surya K. Tripathi, Daniel J. Crona

Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina, North Carolina, NC

摘要 Abstract

中文摘要

背景：转移性膀胱癌（mBC）仍无法治愈，表观遗传失调与疾病进展相关。我们假设 H3K9me3 阅读蛋白 MPP8 与 III 类组蛋白去乙酰化酶 SIRT1 和转录因子 ZEB1 形成复合物以驱动增殖和转移。在此，我们探讨 MPP8 敲低（MPP8 KD）如何破坏 MPP8-SIRT1-ZEB1 相互作用，从而改变差异表达基因（DEGs）和基因网络，并消除增殖和转移。方法：使用 UM-UC-3 mBC 细胞比较 MPP8 KD 和未转染（MPP8 WT）细胞。RNA-seq 对差异表达进行定量。DEGs 在 R v4.1.1 中使用 DESeq2.3 进行分析。基因富集使用 GSEA v4.2.3（Hallmark 基因集）和 IPA 通路分析（Qiagen）进行评估。Co-IP WB 评估 MPP8-SIRT1-ZEB1 蛋白复合物。CellTiter-Glo™ 评估活力差异。克隆形成实验评估增殖，而迁移/侵袭实验评估转移潜能。将 MPP8 KD 和 MPP8 WT 细胞注射到 NSG 小鼠胁腹，测量肿瘤体积和 TBW 的变化，直至肿瘤 ≥ 2 cm³。结果：DEGs 的过度代表分析表明，MPP8 KD 减少了 mBC 细胞生长和 DNA 复制，并增强了对细胞迁移的抑制。GSEA 鉴定出下调的 E2F（NES=-1.9，q=0.02）以及 TNFalpha 经 NFKB 信号基因集的富集（NES=1.71，q=0.01）。IPA 揭示了帕金森病信号通路的激活（z-score=1.54，P=1.1E-03），且与 GSEA 分析一致，预测增殖减少和细胞应激反应。一个由顶级 DEGs 构成的自定义 IPA 基因网络包含帕金森病信号通路基因（如 PRKN），并表明 MPP8、SIRT1、ZEB1 均作用于通路基因的上游。此外，预测 SIRT1 和 PRKN 通过蛋白-蛋白相互作用发生互作，而 MPP8 KD 直接增加 PRKN 表达（LFC=1.658，q=2.39E-03）。在功能上，我们证明 MPP8 在 MPP8 WT 细胞中与 SIRT1 和 ZEB1 形成复合物，而在 MPP8 KD 细胞中 MPP8-SIRT1-ZEB1 相互作用丧失。MPP8 KD 降低了活力（48 小时后 44% 对比 84%；P=0.0001）、集落生长（10 天时集落覆盖平板 13% 对比 36%；P=0.0001）、迁移细胞数（18 小时后 97 对比 470 个细胞；P<0.0001）和侵袭细胞数（18 小时后 44 对比 130 个细胞；P=0.02）。注射后第 32 天，所有携带 MPP8 WT 细胞的小鼠均被处死（平均体积 2,859 ± 761 mm³；n=8）。第 32 天时，MPP8 KD1（平均体积 234 ± 447 mm³，P<0.0001；n=8）和 MPP8 KD2 小鼠（平均体积 1,341 ± 785 mm³，P=0.002；n=8）的肿瘤体积显著更小。第 32 天时，未观察到 TBW 的变化。结论：我们的数据提示 MPP8-SIRT1-ZEB1 轴是 mBC 的关键驱动因素，而 MPP8 KD 导致 PRKN 激活，这可能阻止 mBC 的增殖和转移。

查看英文原文 English abstract

Background: Metastatic bladder cancer (mBC) remains incurable, and epigenetic dysregulation is associated with disease progression. We hypothesize that the H3K9me3 reader protein MPP8 complexes with class III histone deacetylase SIRT1 and transcription factor ZEB1 to drive proliferation and metastasis. Here, we explore how MPP8 knockdown (MPP8 KD ) disrupts MPP8-SIRT1-ZEB1 interactions, thereby altering differentially expressed genes (DEGs) and gene networks and abrogating proliferation and metastasis. Methods: UM-UC-3 mBC cells were used to compare MPP8 KD and untransfected (MPP8 WT ) cells. RNA-seq quantified differential expression. DEGs were analyzed in R v4.1.1 using DESeq2.3. Gene enrichment was assessed with GSEA v4.2.3 (Hallmark sets) and IPA pathway analyses (Qiagen). Co-IP WBs evaluated MPP8-SIRT1-ZEB1 protein complexes. CellTiter-Glo™ evaluated differences in viability. Clonogenic assays assessed proliferation while migration/invasion assays evaluated metastatic potential. MPP8 KD and MPP8 WT cells were injected into NSG mouse flanks, and changes to both tumor volume and TBW were measured until tumors were ≥ 2 cm 3 . Results: Over-representation analysis of DEGs indicated MPP8 KD reduced mBC cell growth and DNA replication, as well as increased inhibition of cell migration. GSEA identified downregulated E2F (NES=-1.9, q=0.02) and enrichment of TNFalpha signaling via NFKB gene sets (NES=1.71, q=0.01). IPA revealed activation of the Parkinson's signaling pathway (z-score=1.54, P=1.1E-03), and consistent with GSEA analyses, predicted reduced proliferation and cellular stress responses. A custom IPA gene network of top DEGs included Parkinson's signaling pathway genes (e.g., PRKN) and demonstrated that MPP8, SIRT1, ZEB1 all act upstream of pathway genes. Moreover, SIRT1 and PRKN were predicted to interact through protein-protein interactions, while MPP8 KD directly increased PRKN expression (LFC=1.658, q=2.39E-03). Functionally, we demonstrated MPP8 forms a complex with SIRT1 and ZEB1 in MPP8 WT cells, while the MPP8-SIRT1-ZEB1 interactions were lost in MPP8 KD cells. MPP8 KD reduced viability (44% vs. 84% after 48 h; P=0.0001), colony growth (13% vs. 36% plate covered with colonies at 10 d; P=0.0001), number of migrated cells (97 vs. 470 cells after 18 h; P<0.0001), and number of invading cells (44 vs. 130 cells after 18 h; P=0.02). By D+32 post-injection, all mice with MPP8 WT cells were sacrificed (mean vol. 2,859 ± 761 mm 3 ; n=8). At D+32, tumor volume was significantly smaller in MPP8 KD1 (mean vol. 234 ± 447 mm 3 , P<0.0001; n=8) and MPP8 KD2 mice (mean vol. 1,341 ± 785 mm 3 , P=0.002; n=8). At D+32, changes in TBW were not observed. Conclusions: Our data suggest the MPP8-SIRT1-ZEB1 axis is a key driver of mBC, and MPP8 KD results in PRKN activation that could halt mBC proliferation and metastasis.

利益披露 Disclosure

S. Gonzalez Tineo, None.. R. M. Kemper, None.. S. K. Tripathi, None.. D. J. Crona, None.

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