Department of medical sciences, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
Cell Commun Signal. 2018 Oct 1;16(1):64. doi: 10.1186/s12964-018-0278-2.
Mammalian target of rapamycin (mTOR) is a master regulator of various cellular responses by forming two functional complexes, mTORC1 and mTORC2. mTOR signaling is frequently dysregulated in pancreatic neuroendocrine tumors (PNETs). mTOR inhibitors have been used in attempts to treat these lesions, and prolonged progression free survival has been recorded. If this holds true also for the multiple endocrine neoplasia type 1 (MEN1) associated PNETs is yet unclear. We investigated the relationship between expression of the MEN1 protein menin and mTOR signaling in the presence or absence of the mTOR inhibitor rapamycin.
In addition to use of menin wild type and menin-null mouse embryonic fibroblasts (MEFs), menin was silenced by siRNA in pancreatic neuroendocrine tumor cell line BON-1. Panels of protein phosphorylation, as activation markers downstream of PI3k-mTOR-Akt pathways, as well as menin expression were evaluated by immunoblotting. The impact of menin expression in the presence and absence of rapamycin was determinate upon Wound healing, migration and proliferation in MEFs and BON1 cells.
PDGF-BB markedly increased phosphorylation of mTORC2 substrate Akt, at serine 473 (S473) and threonine 450 (T450) in menin MEFs but did not alter phosphorylation of mTORC1 substrates ribosomal protein S6 or eIF4B. Acute rapamycin treatment by mTORC1-S6 inhibition caused a greater enhancement of Akt phosphorylation on S473 in menin cells as compared to menin MEFs (116% vs 38%). Chronic rapamycin treatment, which inhibits both mTORC1and 2, reduced Akt phosphorylation of S473 to a lesser extent in menin MEFs than menin MEFs (25% vs 75%). Silencing of menin expression in human PNET cell line (BON1) also enhanced Akt phosphorylation at S473, but not activation of mTORC1. Interestingly, silencing menin in BON1 cells elevated S473 phosphorylation of Akt in both acute and chronic treatments with rapamycin. Finally, we show that the inhibitory effect of rapamycin on serum mediated wound healing and cell migration is impaired in menin MEFs, as well as in menin-silenced BON1 cells.
Menin is involved in regulatory mechanism between the two mTOR complexes, and its reduced expression is accompanied with increased mTORC2-Akt signaling, which consequently impairs anti-migratory effect of rapamycin.
哺乳动物雷帕霉素靶蛋白（mTOR）通过形成两个功能复合物 mTORC1 和 mTORC2，成为各种细胞反应的主调控因子。胰腺神经内分泌肿瘤（PNET）中 mTOR 信号经常失调。已经尝试使用 mTOR 抑制剂来治疗这些病变，并记录到无进展生存期延长。但是对于多发性内分泌肿瘤 1 型（MEN1）相关的 PNET 是否也是如此还不清楚。我们研究了 MEN1 蛋白 menin 的表达与存在或不存在 mTOR 抑制剂 rapamycin 时的 mTOR 信号之间的关系。
除了使用 menin 野生型和 menin 缺失型小鼠胚胎成纤维细胞（MEFs）外，还通过 siRNA 沉默胰腺神经内分泌肿瘤细胞系 BON-1 中的 menin。通过免疫印迹评估磷酸化蛋白组作为 PI3k-mTOR-Akt 通路下游的激活标志物，以及 menin 的表达。在 MEFs 和 BON1 细胞中，测定有和没有 rapamycin 时 menin 表达对伤口愈合、迁移和增殖的影响。
PDGF-BB 明显增加了 menin MEFs 中 mTORC2 底物 Akt 的丝氨酸 473（S473）和苏氨酸 450（T450）的磷酸化，但没有改变 mTORC1 底物核糖体蛋白 S6 或 eIF4B 的磷酸化。通过 mTORC1-S6 抑制的急性 rapamycin 处理导致 menin 细胞中 Akt 的 S473 磷酸化增强程度大于 menin MEFs（116%比 38%）。慢性 rapamycin 处理（同时抑制 mTORC1 和 2）导致 menin MEFs 中 Akt 的 S473 磷酸化比 menin MEFs 减少（25%比 75%）。在人 PNET 细胞系（BON1）中沉默 menin 表达也增强了 Akt 的 S473 磷酸化，但不激活 mTORC1。有趣的是，在 rapamycin 的急性和慢性处理中，沉默 BON1 细胞中的 menin 也增加了 Akt 的 S473 磷酸化。最后，我们表明 rapamycin 对血清介导的伤口愈合和细胞迁移的抑制作用在 menin MEFs 以及沉默 menin 的 BON1 细胞中受损。
menin 参与了两个 mTOR 复合物之间的调节机制，其表达减少伴随着 mTORC2-Akt 信号的增加，这反过来又损害了 rapamycin 的抗迁移作用。
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