The “other” mTOR complex: New insights into mTORC2 immunobiology and their implications

A central role of the mechanistic target of rapamycin (mTOR) in regulation of fundamental cell processes is well recognized. mTOR functions in two distinct complexes: rapamycin‐sensitive mTOR complex (C) 1 and rapamycin‐insensitive mTORC2. While the role of mTORC1 in shaping immune responses, including transplant rejection, and the influence of its antagonism in promoting allograft tolerance have been studied extensively using rapamycin, lack of selective small molecule inhibitors has limited understanding of mTORC2 biology. Within the past few years, however, intracellular localization of mTORC2, its contribution to mitochondrial fitness, cell metabolism, cytoskeletal modeling and cell migration, and its role in differentiation and function of immune cells have been described. Studies in mTORC2 knockdown/knockout mouse models and a new class of dual mTORC1/2 inhibitors, have shed light on the immune regulatory functions of mTORC2. These include regulation of antigen‐presenting cell, NK cell, T cell subset, and B cell differentiation and function. mTORC2 has been implicated in regulation of ischemia/reperfusion injury and graft rejection. Potential therapeutic benefits of antagonizing mTORC2 to inhibit chronic rejection have also been described, while selective in vivo targeting strategies using nanotechnology have been developed. We briefly review and discuss these developments and their implications.     

雷帕霉素对糖尿病大鼠肾脏病变和VEGF及其受体表达的影响

目的 研究雷帕霉素对糖尿病大鼠早期肾脏病变的影响,探讨血管内皮生长因子（VEGF）在糖尿病肾病发病中的作用。方法 SD大鼠经链脲佐菌素（STZ,65 mg/kg）一次性腹腔注射建立糖尿病模型。建模成功后分为雷帕霉素治疗组（建模后12周开始用雷帕霉素治疗4周,n=9）、血管紧张素Ⅱ(ATⅡ)受体拮抗剂L-158809治疗组（阳性对照组,建模后12周开始用L-158809治疗4周,n=5）和糖尿病肾病组（建模12周后用等量蒸馏水灌胃4周,n=9）,以未建模SD大鼠作为正常对照组（n=9）。采集各组大鼠血、尿样本和肾脏组织标本,观察和分析各组大鼠肾小球结构和功能的改变;Western blotting和免疫组织化学法检测肾脏组织VEGF及其受体VEGFR2的表达。结果 与正常对照组比较,糖尿病肾病组大鼠24 h尿白蛋白增加、内生肌酐清除率升高、肾小球体积增大、肾小球基膜增厚,两组间比较差异均有统计学意义（P<0.01）。与糖尿病肾病组比较,雷帕霉素治疗组和阳性对照组大鼠24 h尿白蛋白较少,且肾小球体积增大和基膜增厚均不明显。大鼠肾脏组织VEGF和VEGFR2表达,糖尿病肾病组明显高于正常对照组（P<0.01）,雷帕霉素治疗组和阳性对照组显著低于糖尿病肾病组（P<001）。结论 雷帕霉素具有减少糖尿病肾病大鼠蛋白尿和缓解肾小球肥大和基膜增厚的作用。抑制肾脏组织VEGF和VEGFR2蛋白表达可能是雷帕霉素延缓糖尿病肾病发病的可能机制之一。     

Okadaic acid increases autophagosomes in rat neurons: implications for Alzheimer's disease

Autophagosomes are accumulated in Alzheimer's disease (AD), but the regulatory pathway of autophagy in AD remains largely unknown. By using electron microscopy, Western blotting, and immunocytochemistry, here we show that autophagosomes are accumulated in rat neurons by okadaic acid (OA), a protein phosphatase-2A inhibitor known to enhance tau phosphorylation, beta-amyloid (Abeta) deposition, and neuronal death, which are the pathological hallmarks of AD. Autophagy can be generally induced via several distinct pathways, such as inhibition of mTOR or activation of beclin-1. Interestingly, OA increased both mTOR and beclin-1 pathways simultaneously, which suggests that autophagy in OA-treated neurons is induced mainly via the beclin-1 pathway, and less so via mTOR inhibition. Finally, inhibition of autophagy by 3MA reduced cytotoxicity in OA-treated neurons. Our novel findings provide new insights into the pathology of and therapeutic intervention for AD.     

Inhibition of calcium-independent phospholipase A2 activates p38 MAPK signaling pathways during cytostasis in prostate cancer cells

The p38 mitogen-activated protein kinase (MAPK) signaling pathways activated during cytostasis induced by Ca²⁺-independent phospholipase A₂ (iPLA₂) inhibition in prostate cancer cells were investigated. iPLA₂ inhibition using siRNA, or the selective inhibitor bromoenol lactone (BEL) and it's enantiomers, decreased growth in LNCaP (p53 positive) and PC-3 (p53 negative) human prostate cancer cells. Decreased cell growth correlated to time- and concentration-dependent activation of the mitogen-activated protein kinase p38 in both cell lines. Inhibition of cytosolic iPLA₂β using S-BEL, induced significantly higher levels of P-p53, p53, p21 and P-p38 expression than inhibition of microsomal iPLA₂γ using R-BEL. Inhibition of p38 using SB202190 or SB203580 inhibited BEL-induced increases in P-p53 (ser15), p53 and p21, and altered the number of cells in G1 in LNCaP cells, and S-phase in PC-3 cells. BEL treatment also induced reactive species in PC-3 and LNCaP cells, which was partially reversed by pretreatment with N-acetyl-cysteine (NAC). NAC subsequently inhibited BEL-induced activation of p38 and p53 in LNCaP cells. In addition, treatment of cells with NAC partially reversed the effect of BEL on cell growth and preserved cell morphology. Collectively, these data demonstrate the novel findings that iPLA₂ inhibition activates p38 by inducing reactive species, and further suggest that this signaling kinase is involved in p53 activation, cell cycle arrest and cytostasis.     

Treatment with sirolimus results in complete responses in patients with autoimmune lymphoproliferative syndrome

We hypothesized that sirolimus, an mTOR inhibitor, may be effective in patients with autoimmune lymphoproliferative syndrome (ALPS) and treated patients who were intolerant to or failed other therapies. Four patients were treated for autoimmune cytopenias; all had a rapid complete or near complete response. Two patients were treated for autoimmune arthritis and colitis, demonstrating marked improvement. Three patients had complete resolution of lymphadenopathy and splenomegaly and all patients had a reduction in double negative T cells, a population hallmark of the disease. Based on these significant responses, we recommend that sirolimus be considered as second-line therapy for patients with steroid-refractory disease.     

PI3K/Akt/mTOR通路抑制剂对白血病细胞株增殖及其PHLPP表达的影响

目的:观察PI3K/Akt/mTOR通路抑制剂wortmannin或rapamycin对白血病细胞株增殖及其PHLPP(PH domainleucine-rich repeat protein phosphatase)蛋白表达的影响。方法:以不同浓度的wortmannin或rapamycin分别作用于人类髓细胞白血病细胞系K562、HL-60,采用WST-1法检测细胞的增殖活性,AnnexinⅤ-FITC双染流式细胞术检测细胞凋亡,Westernblotting法检测细胞中p-Akt、Akt、PHLPP蛋白的表达。结果:Wortmannin以时间以及剂量依赖方式抑制K562、HL-60细胞的增殖(P<0.05),48 h的IC50值分别为(187.6±48.4)、(185.5±48.1)nmol/L。100 nmol/L wortmannin作用于K562细胞、50nmol/L wortmannin作用于HL-60细胞12和24 h后,细胞凋亡率均较对照细胞显著升高[(12.4±0.7)%、(17.6±2.3)%vs(5.0±0.6)%,P<0.05;(11.0±0.2)%、(17.9±1.6)%vs(6.8±0.4)%,P<0.05]。Wortmannin分别作用于K562、HL-60细胞12、24、36 h后,p-Akt、PHLPP的蛋白表达水平明显降低;rapamycin同样可使K562、HL-60细胞中PHLPP蛋白的表达水平降低。结论:PI3K/Akt/mTOR信号通路抑制剂抑制白血病细胞株增殖的同时降低其PHLPP蛋白的表达。     

Autophagy limits the cytotoxic effects of the AKT inhibitor AZ7328 in human bladder cancer cells

Background: Mutations that activate the PI3K/AKT/mTOR pathway are relatively common in urothelial (bladder) cancers, but how these pathway mutations affect AKT dependency is not known. We characterized the relationship between AKT pathway mutational status and sensitivity to the effects of the selective AKT kinase inhibitor AZ7328 using a panel of 12 well-characterized human bladder cancer cell lines.Methods: Sequenome DNA sequencing was performed to identify mutations in a panel of 12 urothelial cancer cell lines. Drug-induced proliferative inhibition and apoptosis were quantified using MTT assays and propidium iodide staining with FACS analyses. Protein activation via phosphorylation was measured by immunoblotting. Autophagy was measured by LC3 immunofluorescence and immunoblotting.Results: AZ7328 inhibited proliferation and AKT substrate phosphorylation in a concentration-dependent manner but had minimal effects on apoptosis. Proliferative inhibition correlated loosely with the presence of activating PIK3CA mutations and was strengthened in combination with the mTOR inhibitor rapamycin. AZ7328 induced autophagy in some of the lines, and in the cells exposed to a combination of AZ7328 and chemical autophagy inhibitors apoptosis was induced.Conclusions: The cytostatic effects of AZ7328 correlate with PIK3CA mutations and are greatly enhanced by dual pathway inhibition using an mTOR inhibitor. Furthermore, AZ7328 can interact with autophagy inhibitors to induce apoptosis in some cell lines. Overall, our results support the further evaluation of combinations of PI3K/AKT/mTOR pathway and autophagy inhibitors in pre-clinical in vivo models and ultimately in patients with PIK3CA mutant bladder cancers.