Suppression of mTOR complex 2-dependent AKT phosphorylation in melanoma cells by combined treatment with rapamycin and LY294002

Background  Inhibition of mTOR complex 1 (mTORC1) with rapamycin leads to phosphorylation of AKT in some cancer cells, with unknown biological consequences. The role of this phosphorylation in melanoma is unknown, although preliminary clinical data indicate poor activity of rapalogues in melanoma.
Objectives  We aimed at elucidating the role of AKT phosphorylation after mTORC1 inhibition in melanoma cells.
Methods  Western blotting, apoptosis assays, cell cycle analyses and viability assays were performed to analyse the effects of rapamycin and LY294002 treatment on melanoma cells. For suppression of mTOR complex 2 (mTORC2) an siRNA directed against rictor was used.
Results  Rapamycin showed limited effects on cell viability but resulted in strong and lasting AKT phosphorylation in melanoma cells. Combined PI3K/mTOR inhibition with LY294002 had pronounced effects on viability but also led to increased AKT phosphorylation after prolonged treatment. In contrast, combination of rapamycin plus LY294002 suppressed AKT phosphorylation. Suppression of AKT phosphorylation did not correlate with decreases in cell viability. Inhibition of mTORC2 led to reduced levels of phosphorylated AKT.
Conclusions  mTORC1 inhibition with rapamycin and with LY294002 can lead to AKT phosphorylation in melanoma cells via mTORC2. Combination of rapamycin and LY294002 suppresses AKT phosphorylation but without significant effect on treatment efficacy.     

Targeting mTORC1/2 with OSI‐027 inhibits proliferation and migration of keloid keratinocytes

Keloid is a dermal proliferative disorder characterized by the excessive proliferation and migration of keratinocytes and fibroblasts. Over‐activation of the serine/threonine protein kinase, mammalian target of rapamycin (mTOR), plays a pivotal role in the process. Here, we show that both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) were hyper‐activated in keloid‐derived primary keratinocytes. Further, OSI‐027, an mTOR kinase inhibitor, potently inhibited proliferation and migration of keloid keratinocytes. At the molecular level, OSI‐027 disrupted the assembly of mTORC1 (mTOR–Raptor) and mTORC2 (mTOR–Rictor–mLST8). Further, OSI‐027 almost completely blocked the phosphorylation of the mTORC1 substrates, S6K1, S6 and 4EBP1, and the mTORC2 substrate, AKT, at Ser‐473. The OSI‐027 treatment of keloid keratinocytes showed more effectively inhibited cell proliferation and migration compared to the mTORC1 inhibitor, rapamycin. Moreover, restoring mTORC1 activation by the introduction of the constitutively active S6K1 only partly alleviated OSI‐027‐induced inhibition of keloid keratinocytes. Notably, mTOR2 inhibition by Rictor siRNAs also inhibited keloid keratinocyte proliferation and migration, but less efficiently than OSI‐027. Together, our results imply that concurrent targeting of mTORC1/2 by OSI‐027 potently inhibits the proliferation and the migration of keloid keratinocytes. Thus, OSI‐027 may have translational value for the treatment of keloid.     

Akt/mTOR活化抗UVB诱导HaCaT细胞凋亡的研究

目的 探讨Akt/mTOR信号通路活化抗中波紫外线（UVB）诱导的HaCaT细胞凋亡。方法 UVB照射角质形成细胞,Western印迹检测Akt/mTOR通路中相关信号分子的动态水平变化。免疫荧光 Hoechst 33342染色观察HaCaT细胞凋亡率。结果 UVB能活化Akt/mTOR信号通路,并在一定范围内（5 ～ 30 mJ/cm2）成剂量依赖性,在一定范围内（5 ～ 30 min）成时间依赖性。EGFR抑制剂PD 153035、PI3K抑制剂LY 294002和mTOR抑制剂雷帕霉素能显著抑制UVB对Akt/mTOR信号通路的活化作用。UVB照射前加入雷帕霉素、LY 294002预处理,HaCaT细胞凋亡率增加。结论 Akt/mTOR活化抗UVB诱导的HaCaT细胞凋亡。     

麦冬皂苷B通过抑制PI3K/Akt/mTOR通路诱导人黑色素瘤A375细胞凋亡

目的探讨麦冬皂苷B对人黑色素瘤(Melanoma)A375细胞增殖,凋亡和细胞周期的影响以及相关机制。方法麦冬皂苷B处理A375细胞后,采用CCK-8法检测细胞活力;流式细胞仪检测A375细胞周期变化及细胞凋亡水平;Hoechst33258荧光染色法观察细胞凋亡形态;免疫印迹法检测麦冬皂苷B对A375细胞PI3K,p-PI3K,AKT,p-AKT蛋白以及通路下游凋亡相关蛋白Bcl-2,Bax,Cleaved-caspase3表达的影响。结果麦冬皂苷B能抑制A375细胞增殖活力及PI3K/Akt/mTOR通路的活化;麦冬皂苷B促进了A375细胞内Bax和Cleaved-caspase 3的表达,抑制Bcl-2表达,诱导人黑色素瘤细胞发生凋亡;麦冬皂苷B阻滞了A375细胞细胞周期,G0/G1期细胞比例明显升高;PI3K/Akt/mTOR通路活化剂IGF-1处理A375细胞后抑制了麦冬皂苷B的上述作用。结论麦冬皂苷B能抑制A375细胞的增殖,诱导细胞发生凋亡,阻滞细胞周期于G0/G1期。     

Phytochemical modulation of the Akt/mTOR pathway and its potential use in cutaneous disease

The mechanistic target of rapamycin (mTOR) is involved in the regulation of cellular growth, proliferation, lipid synthesis, and protein translation. The mTOR pathway involves two complexes: the mechanistic target of rapamycin complex 1 (mTORC1) and the mechanistic target of rapamycin complex 2 (mTORC2). Both mTOR complexes have been implicated in the development and progression of various skin diseases including melanoma, psoriasis, and acne vulgaris. Here, we review the role of both mTORC1 and mTORC2 as well as their upstream modulators, phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt), and their downstream targets in various dermatologic diseases. Phytochemicals, plant-derived naturally occurring compounds, have been shown to regulate the mTOR pathway and may serve as novel therapeutic agents in dermatological disease. Here, we review phytochemicals in the context of the mTOR pathway and their potential use in cutaneous disease.     

黑素瘤相关通路及治疗的新进展

黑素瘤的发病机制与遗传、环境因素如紫外线长期照射有关.研究表明,黑素瘤的发生发展主要与细胞外调节蛋白激酶通路和磷酸酰肌醇3激酶/蛋白激酶/哺乳动物雷帕霉素靶蛋白信号通路改变相关.相关的基因改变有:BRAF基因、NRAS基因、GNAQ基因和GNA 11基因、C-kit基因、苏氨酸蛋白激酶基因等.黑素瘤是皮肤侵袭性肿瘤,对于放疗和化疗均不敏感.针对黑素瘤发病机制的分子靶向治疗发挥了重要的作用,包括免疫治疗、基因治疗以及针对肿瘤血供的治疗.生物治疗比传统治疗有更好的靶向性和特异性.     

Mammalian target of rapamycin complex 1 (mTORC1) may modulate the timing of anagen entry in mouse hair follicles

Mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and survival. There is limited evidence that mTOR influences hair follicles (HFs), which undergo cycles of quiescence (telogen), growth (anagen) and regression (catagen). We sought to investigate whether mTOR, in particular mTOR complex 1 (mTORC1), regulates the hair growth cycle by employing biochemical, immunohistochemical and functional approaches in vivo. Here, we demonstrate that quantitative analysis of mTORC1 kinase activity shows phase‐dependent changes, and phosphorylated mTOR at S2448 (p‐mTOR) was localized in certain sites of HFs in a phase‐dependent manner. These results were indicative of mTOR's role in hair growth initiation. Finally, in a pharmacological challenge in vivo using the specific mTORC1 inhibitor, rapamycin, hair cycle initiation was delayed, suggesting a functional relevance of mTORC1 in anagen entry. Based on our findings, we propose that mTORC1 may participate in hair cycle regulation, namely the timing of anagen initiation.     

PI3K/Akt/mTOR信号通路在皮肤鳞状细胞癌中的研究进展

【摘要】 皮肤鳞状细胞癌的发病机制复杂，晚期治疗手段缺乏。本文综述磷脂酰肌醇3-激酶/蛋白激酶B/哺乳类动物雷帕霉素靶蛋白（PI3K/Akt/mTOR）信号通路在皮肤鳞状细胞癌发病机制中的作用及针对其的治疗进展，为皮肤鳞状细胞癌的靶向治疗提供新思路。     

mTORC1信号通路在狼疮鼠体内的表达及意义

目的：检测mTORC1信号通路在系统性红斑狼疮鼠体内的表达,探讨其在系统性红斑狼疮发病中的作用。方法： 研究对象为6只B6.MRL/lpr狼疮小鼠和6只C57正常小鼠。RT-PCR和Western blot检测肾脏组织Akt、mTOR、p70S6K、IL-17 mRNA和蛋白的表达。细胞内染色流式细胞术检测脾脏Th17淋巴细胞亚群比例。结果： 狼疮鼠组Akt、mTOR、p70S6K、IL-17 mRNA、磷酸化蛋白表达水平高于对照组（P<0.05）;狼疮鼠组的Th17细胞占CD4+T细胞的比例高于正常对照组（P<0.05）。结论： mTORC1（Akt-mTOR-p70S6K）信号通路在SLE的发病中起着重要作用。     

丹参酮ⅡA体外促进黑素瘤A375细胞自噬及信号通路的实验研究

目的：探讨丹参酮ⅡA体外对黑素瘤细胞A375细胞自噬的影响及其信号通路研究。方法0.5、1、2、4 mg/L丹参酮ⅡA作用黑素瘤A375细胞24、48、72 h后,噻唑蓝（MTT）比色法检测A375细胞的增殖活性。1、2、4 mg/L丹参酮ⅡA作用黑素瘤A375细胞48 h,采用流式细胞仪检测细胞自噬小体的数量,Western印迹检测自噬相关蛋白Beclin?1和微管相关蛋白1轻链3（LC3）?Ⅱ蛋白及磷酸肌醇3激酶（PI3K）、蛋白激酶B（Akt）、雷帕霉素靶蛋白（mTOR）、p70S6激酶1（p70S6K1）蛋白的表达水平。结果 MTT分析显示,0.5、1、2和4 mg/L丹参酮ⅡA分别作用黑素瘤A375细胞24、48、72 h,均能抑制A375细胞的增殖能力,且抑制作用呈剂量和时间依赖性（F=2564.12、1235.25,均P<0.05）。流式细胞仪显示,1、2和4 mg/L丹参酮ⅡA作用A375细胞48 h后,细胞内自噬小体比例分别为6.91%±0.35%、13.11%±0.73%、25.51%±0.83%,均明显高于对照组（0.41%±0.02%）,各组间差异有统计学意义（均P<0.05）。Western印迹显示,1、2和4 mg/L丹参酮ⅡA作用A375细胞48 h后,细胞自噬相关蛋白Beclin?1和LC3?Ⅱ表达水平随丹参酮ⅡA浓度增加而升高,各丹参酮ⅡA组间差异有统计学意义,且高于对照组（均P<0.05）。而PI3K?Akt?mTOR?p70S6K1信号通路中PI3K、p?Akt、p?mTOR和p?p70S6K1蛋白表达随丹参酮ⅡA浓度增加而下降,各丹参酮ⅡA组间差异有统计学意义,且低于对照组（均P<0.05）。结论丹参酮ⅡA可通过抑制P13K?Akt?mTOR?p70S6K1信号通路,促进黑素瘤细胞发生自噬。     

p62通过调控PI3K/AKT通路促进黑素瘤增殖

目的探讨p62促进黑素瘤A375细胞生长增殖的作用及具体机制。方法取生长状态良好的黑素瘤A375细胞,采用干扰p62表达的siRNA片段转染A375细胞;EDU着色法检测干扰组和对照组的细胞分裂情况;采用Western blot检测干扰组和对照组PI3K/AKT/mTOR及下游因子的表达情况。结果干扰组的EDU着色数目较对照组明显减少;p62干扰后PI3K/AKT/mTOR及下游因子的表达明显受抑制。结论p62可能通过调控PI3K/AKT通路发挥促进黑素瘤增殖的作用。     

Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/AkT signaling pathways

Keloid, a chronic fibro-proliferative disease, exhibits distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells (MCs), and a milieu of enriched cytokines. Previous studies have demonstrated that co-culture with MCs stimulate type I collagen synthesis in fibroblasts, but the signaling mechanisms remain largely unknown. In this study, we investigated the signaling pathways involved in MC-stimulated type I collagen synthesis and the effects of green tea extract (GTE) and its major catechin, (-)-epigallocatechin-3-gallate (EGCG), on collagen homeostasis in keloid fibroblasts. Our results showed that MCs significantly stimulated type I collagen expression in keloid fibroblasts, and the upregulation of type I collagen was significantly attenuated by blockade of phosphatidylinositol-3-kinase (PI-3K), mammalian target of rapamycin (mTOR), and p38 MAPK signaling pathways, but not by blockade of ERK1/2 pathway. Furthermore, GTE and EGCG dramatically inhibited type I collagen production possibly by interfering with the PI-3K/Akt/mTOR signaling pathway. Our findings suggest that interaction between MCs and keloid fibroblasts may contribute to excessive collagen accumulation in keloids and imply a therapeutic potential of green tea for the intervention and prevention of keloids and other fibrotic diseases.     

PI3K/Akt/mTOR信号通路与皮肤肿瘤靶向治疗

P13K/AkdmTOR信号转导通路是促存活通路,在很多肿瘤中组成性激活.该通路激活的机制是肿瘤抑制基PTEN功能缺失、P13K扩增或突变、Akt扩增或突变.近年研究发现,该通路失常可促进肿瘤细胞的存活和生长,持续活化在皮肤肿瘤发病中起着重要的作用,已经发现抑制该通路中的信号分子可以治疗多种肿瘤,目前,针对该通路的抑制药物也在研究中,主要集中于mTOR抑制剂.     

依巴斯汀通过抑制AKT/mTOR通路诱导人黑素瘤细胞自噬

目的：研究依巴斯汀对人黑素瘤细胞自噬的影响及机制。方法：体外培养人黑素瘤细胞A375和M14,采用CCK-8增殖实验检测细胞活力,并计算IC50;利用mCherry-EGFP-LC3B双荧光指示系统检测自噬流;采用Western blot验证自噬相关蛋白LC3,Beclin1及信号通路蛋白的表达。结果：依巴斯汀可显著抑制人黑素瘤细胞的活力;依巴斯汀明显诱导人黑素瘤细胞中自噬小体和自噬溶酶体的产生;依巴斯汀显著上调人黑素瘤细胞中LC3-Ⅱ/Ⅰ的比值以及Beclin1的表达,同时抑制AKT/mTOR信号通路的活化,降低p-AKT/AKT和p-mTOR/mTOR。结论：依巴斯汀通过抑制AKT/mTOR通路诱导人黑素瘤细胞自噬的发生。     

五倍子瘢痕膏对瘢痕疙瘩miR-21/mTOR信号通路相关分子表达的影响

目的观察五倍子瘢痕膏对瘢痕疙瘩miR-21/mTOR信号通路关键分子miR-21、磷脂酰肌醇3-激酶(PI3K)、10号染色体张力缺失蛋白磷酸酶(PTEN)、蛋白激酶B(Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)表达的影响。方法将36只裸鼠瘢痕疙瘩模型随机分成治疗组和对照组,每组18只。治疗组涂抹五倍子瘢痕膏,对照组仅涂抹制作五倍子瘢痕膏的基质,3次/d,连续30 d。然后分别应用逆转录聚合酶链反应(RT-PCR)和免疫组化技术检测治疗组、对照组及正常皮肤组中miR-21和PI3K、PTEN、Akt、mTOR的表达。结果RT-PCR检测结果显示,治疗组和正常皮肤组miR-21相对表达量差异无统计学意义(P>0.05,t=1.24),但二者与对照组相比均明显降低,差异有统计学意义(P<0.05,t=2.76、2.81);免疫组化检测结果显示,PI3K、Akt、mTOR在对照组中高表达,而在治疗组和正常皮肤组中低表达;PTEN在对照组中低表达,而在治疗组和正常皮肤组中高表达。结论五倍子瘢痕膏抑制瘢痕疙瘩成纤维细胞增殖的机制可能与其通过抑制miR-21表达而上调PTEN表达,进而下调mTOR信号通路中PI3K、Akt、mTOR表达有关。其中PTEN是负反馈调节因子;下游的核糖体蛋白S6激酶(S6K)、真核细胞翻译起始因子4E结合蛋白(4EBP),二者均是蛋白翻译的关键调节因子。     

Dynamic interplay between autophagic flux and Akt during melanoma progression in vitro

Despite advances in cancer diagnosis and therapy, metastatic melanoma remains untreatable, due to its notorious resistance to apoptosis, deeming traditional therapies obsolete. Deregulated PI3K/Akt signalling is a common oncogenic event enabling melanocyte transformation and represents a significant and ‘druggable’ pathway in melanoma. Emerging data show that the ability of cancer cells to survive is also facilitated by alteration of vital homoeostatic mechanisms, such as autophagy. Although the role of autophagy in melanoma is still controversial, recent studies suggest that basal autophagy is down‐modulated in primary melanomas. However, the dynamic connection between pro‐tumorigenic PI3K/Akt and autophagy during melanoma progression has not been systematically studied. By using human primary melanocytes, incipient melanoma and metastatic melanoma cell lines, we show that early in melanomagenesis, increased Akt activity is associated with a low baseline autophagic flux. However, during melanoma progression, metastatic melanoma cells regain the ability to stimulate autophagic flux, supporting survival. Heightened autophagy is associated with an attenuated Akt activation status and can be suppressed by overexpressing a constitutive active mutant of Akt. On the other hand, blocking the higher Akt activity of primary melanoma is sufficient to incite autophagy. Interestingly, we found that although Akt supports survival of melanocytes and all melanoma cell lines, autophagy inhibition specifically targeted the metastatic melanoma cells, thus indicating a stage‐specific requirement for Akt and autophagic flux, throughout melanoma progression. Therefore, this study highlights a dynamic interplay between Akt signalling and autophagic rescue in melanoma, which should be considered in the design of therapeutic strategies targeting these pathways.     

TNF-alpha impairs the S-G2/M cell cycle checkpoint and cyclobutane pyrimidine dimer repair in premalignant skin cells: role of the PI3K-Akt pathway

Tumor necrosis factor-alpha (TNF-alpha) is induced by UVB radiation and has been implicated in the early stages of skin carcinogenesis. Here, we show that in normal keratinocytes and the transformed keratinocyte cell lines, HaCaT and A431, TNF-alpha stimulates protein kinase B/Akt, which results in activation of the survival complex mTORC1 (mammalian target of rapamycin complex 1) and inhibition of the proapoptotic proteins Bad and FoxO3a. In UVB-irradiated HaCaT cells (10-20 mJ cm(-2)), TNF-alpha increased the proportion of cycling cells and enhanced the rate of apoptosis. A significantly higher proportion of UVB-treated HaCaT cells containing unrepaired cyclobutane pyrimidine dimers (CPDs) escaped the G2/M cell cycle checkpoint in the presence of TNF-alpha (9.5+/-3.3 vs 4.8+/-2.2%). After treatment with the PI3K inhibitor LY294002, only 1.2+/-0.7% of CPD-containing HaCaT cells were actively cycling. TNF-alpha enhanced apoptosis less potently and did not increase the level of CPD or stimulate cell cycle progression in normal keratinocytes. Our data suggest that TNF-alpha overrides the G2/M checkpoint in premalignant skin cells and allows for some cells containing unrepaired CPD to enter the cell cycle. The effect of TNF-alpha seems to be dependent on Akt activation and may constitute a relevant mechanism enhancing mutagenesis and tumor development.     

mTOR is activated in the majority of malignant melanomas

The objective of this study was to determine whether activation of the kinase mammalian target of rapamycin (mTOR) is associated with human melanoma. We found moderate or strong hyperphosphorylation of ribosomal protein S6 in 78/107 melanomas (73%). In contrast, only 3/67 benign nevi (4%) were moderately positive, and none were strongly positive. These data indicate that mTOR activation is very strongly associated with malignant, compared to benign, melanocytic lesions. Next, we tested six melanoma-derived cell lines for evidence of mTOR dysregulation. Five of the six lines showed persistent phosphorylation of S6 after 18 hours of serum deprivation, and four had S6 phosphorylation after 30 minutes of amino-acid withdrawal, indicating inappropriate mTOR activation. The proliferation of three melanoma-derived lines was blocked by the mTOR inhibitor rapamycin, indicating that mTOR activation is a growth-promoting factor in melanoma-derived cells. mTOR is directly activated by the small guanosine triphosphatase Ras homolog enriched in brain (Rheb), in a farnesylation-dependent manner. Therefore, to investigate the mechanism of mTOR activation, we used the farnesyl transferase inhibitor FTI-277, which partially blocked the growth of three of the six melanoma cell lines. Together, these data implicate activation of mTOR in the pathogenesis of melanoma, and suggest that Rheb and mTOR may be targets for melanoma therapy.