RhoC promotes human melanoma invasion in a PI3K/Akt-dependent pathway

Overexpression of the small GTPase, RhoC, in various human cancers has been correlated with high metastatic ability and poor prognosis. Rho-kinase (ROCK) is an important effector of Rho GTPases. The oncogenic serine/threonine kinase Akt (also known as PKB) is a downstream effector of phosphatidylinositol-3 kinase (PI3K). Akt activation contributes to the neoplastic phenotype by promoting cell cycle progression, increasing antiapoptotic functions, and enhancing tumor cell invasion. Rho signaling via ROCK has been previously shown either to activate or to downregulate PI3K/Akt. Using a human radial growth phase melanoma cell line, WM35, we have established stable transfectants that overexpress RhoC (called WM35RhoC). We found that overexpression of RhoC increased phosphorylated-Akt (Ser473/474/472, pAkt) expression and promoted cell invasion. Inhibition of RhoC with C3 transferase downregulated pAkt expression and decreased cell invasion in these cells. In addition, inhibition of PI3K, Akt, or ROCK partially decreased invasion. Further, inhibition of PI3K but not ROCK decreased the pAkt level. These results suggest that RhoC promotes invasion in part via activation of a PI3K/Akt pathway, in a manner independent of ROCK signaling. We propose that RhoC promotes melanoma progression via separate mechanisms that regulate the PI3K/Akt pathway and the ROCK signaling pathway.     

Activation of the PI3K/AKT signalling pathway in non‐melanoma skin cancer is not mediated by oncogenic PIK3CA and AKT1 hotspot mutations

Please cite this paper as: Activation of the PI3K/AKT signalling pathway in non‐melanoma skin cancer is not mediated by oncogenic PIK3CA and AKT1 hotspot mutations. Experimental Dermatology 2010; 19 : e222–e227. Abstract: Non‐melanoma skin cancer represents the most frequent human cancer entity. Activation of the PI3K/AKT signalling pathway has been reported both in squamous cell carcinoma (SCC) of the skin and in basal cell carcinoma (BCC). In many cancers, including SCC of the head and neck, the oesophagus and the penis, activation of this pathway is mediated by oncogenic PIK3CA and AKT1 mutations. We therefore screened 61 non‐melanoma skin cancer samples (30 SCC and 31 BCC) for the presence of activating PIK3CA and AKT1 mutations. PIK3CA hotspot mutations were analysed using a highly sensitive SNaPshot assay, and exon 4 of AKT1 was sequenced directly. In addition, immunohistochemistry was performed for phosphorylated AKT protein. Immunohistochemical expression of pAkt was observed both in SCC and in BCC samples, indicating an activation of the PI3K/AKT pathway. Although SCC showed higher expression levels than BCC, this difference was not significant. However, none of the 61 non‐melanoma skin cancer samples revealed any PIK3CA and AKT1 hotspot mutations at the investigated loci. We conclude that PIK3CA and AKT1 hotspot mutations do not contribute to the activation of the PI3K/AKT signalling pathway in non‐melanoma skin cancer. The distinct PIK3CA mutation spectrum between SCC of the skin and SCC of other tissues may reflect the different carcinogens which are involved into the mutagenesis of these cancers. PIK3CA and AKT1 hotspot mutations are obviously not caused by UV light exposure, the main risk factor in non‐melanoma skin cancer.     

Overproduction of VEGF concomitantly expressed with its receptors promotes growth and survival of melanoma cells through MAPK and PI3K signaling

Vascular endothelial growth factor (VEGF) is an important mediator of tumor-associated angiogenesis, and consequently it has been associated with metastasis. We report here that the overexpression of VEGF(165) in melanoma xenografts promotes an acceleration of tumor growth and an increase in angiogenesis as well as the spontaneous metastasis formation. In addition, VEGF receptors (VEGFR)1, VEGFR2 and neurophilin-1 are expressed in A375 melanoma cells. Forced overexpression of VEGF in these cells induces cell growth and triggers survival activity in serum-starved cultures, by a mechanism dependent on the mitogen-activating protein kinase signaling pathway. Furthermore, these effects are dependent MEK 1/2 activity. Kinase domain region-specific tyrosine kinase inhibitors dramatically reduced DNA synthesis to 20% with respect to the controls, although they did not completely suppress either the p44 or p42-phosphorylated forms of extracellular signal-regulated protein kinase. These inhibitors also provoked a decrease in Akt phosphorylation. We observed a dramatic reduction in survival after treatment with phosphatidylinositol 3'-kinase (PI3K)-specific inhibitor in the presence of specific tyrosinase inhibitors. We suggest that the overproduction of VEGF(165) concomitantly expressed with its receptors favors cell growth and survival of melanoma cells through MAPK and PI3K signaling pathways. These data support the involvement in melanoma growth and survival of a VEGF-dependent internal autocrine loop mechanism, at least in vitro.     

PI3K/AKT信号转导通路和VEGF在子宫内膜异位症中的表达

目的:探讨子宫内膜异位症(EMs)患者中磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/AKT)和血管内皮生长因子(VEGF)表达水平。方法:采用EMs患者32例,取在位子宫内膜和异位内膜,无EMs患者34例,取子宫内膜,作为对照组。采用半定量RT-PCR法检测PI3K/AKT与VEGFmRNA表达,ELISA法检测PI3K/AKT蛋白和VEGF蛋白表达。结果:正常子宫内膜、异位子宫内膜和在位子宫内膜PI3K、AKT和VEGF mRNA和蛋白表达水平差异有统计学意义(P0.01)。异位内膜PI3K、AKT和VEGF的mRNA和蛋白的表达均高于在位内膜及正常内膜(P0.01)。EMs中,Ⅰ、Ⅱ期PI3K、AKT和VEGF mRNA和蛋白的表达与Ⅲ、Ⅳ期异位内膜相比,均无显著性差异(P0.05)。结论:PI3K/AKT信号转导通路和VEGF共同参与EMs的发生和发展。     

Vertical inhibition of the mTORC1/mTORC2/PI3K pathway shows synergistic effects against melanoma in vitro and in vivo

The phosphatidyl inositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway has been shown to be involved in the development of melanoma. PI-103 is a kinase inhibitor blocking PI3K class IA and mTOR complex 1 and 2. Here, we studied the effect of targeting the PI3K/mTORC1/mTORC2 pathway by PI-103 and rapamycin in melanoma cells and in a melanoma mouse model. Dual targeting of PI3K and mTOR by PI-103 induced apoptosis and cell-cycle arrest, and inhibited viability of melanoma cells in vitro. Combined treatment with PI-103 and the prototypic mTORC1 inhibitor rapamycin led to the synergistic suppression of AKT and ribosomal S6 protein phosphorylation and to the induction of apoptosis. In vivo, PI-103 and rapamycin displayed only modest single-agent activity, but the combination significantly reduced the tumor growth compared with both single agents. These data show that blocking the PI3K/mTORC1/mTORC2 pathway using the combination of two distinct small-molecule inhibitors ("vertical inhibition") leads to superior efficacy against malignant melanoma in vitro and in vivo.     

Trends in melanoma epidemiology suggest three different types of melanoma

BACKGROUND: It has been suggested that the incidence of thin melanomas but not of thick tumours is rising in fair-skinned populations, although the reason for this discrepancy is not understood. OBJECTIVES: To describe temporal trends in melanoma epidemiology in a limited part of France in order to confirm this observation and to provide an explanation. METHODS: This is a retrospective population- and academic centre-based study in which all melanomas diagnosed in the department of the Bas-Rhin, France between January 1980 and December 2004 were included. RESULTS: The study included 2094 melanomas diagnosed in 2020 patients. There was a steady increase in incidence of thin (< 1 mm) melanomas, mainly located on the trunk, and to a lesser extent in the head and neck region, in both sexes, and of intermediate (1-2 mm) melanomas in men. The incidence of intermediate melanomas in women and of thick (> 2 mm) melanomas, as well as mortality related to melanoma, remained stable. There was a steady decline of mean and median Breslow thickness. The 12 months median delay to diagnosis of thick tumours was significantly shorter than the 24 months delay to diagnosis of thin tumours. CONCLUSIONS: Temporal trends suggest the existence of three unrelated types of melanoma: type I, thick melanomas, with stable incidence; type II, thin melanoma with a steady and important increase in incidence, mainly located on the trunk; and type III, melanoma with a slower increase in incidence, mainly located on the head and neck region.     

检测子宫内膜异位症中PI3K/Akt通路和VEGF的临床意义

目的:探讨子宫内膜异位症患者中血管内皮生长因子(VEGF)、磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/Akt)表达水平及其与血管生成的关系,并且探讨VEGF与PI3K在调控血管生成过程中的相互关系。方法:利用S-P方法对32例EM手术标本和30例子宫肌瘤中VEGF、Akt及微血管密度(MVD)的表达进行检测。结果:EM中正常内膜、在位内膜和异位内膜Akt、VEGF阳性表达率分别为20.0%(6/30)、37.5%(12/32)、84.4%(27/32)、60.0%(18/30)、75.0%(24/32)、87.5%(28/32);EM中正常内膜、在位内膜和异位内膜MVD分别是(10.37±5.24)、(19.14±7.10)、(48.24±10.54)。Akt、VEGF和血管密度在EM中异位内膜较正常内膜组高表达,在调控血管生成过程中,Akt与VEGF呈正相关。结论:PI3K/Akt、VEGF均参与EM的发生发展,在EM血管生成中发挥关键作用,共同参与EM过程。     

CDKN2A germline mutations in individuals with cutaneous malignant melanoma

Cyclin-dependent kinase inhibitor type 2A (CDKN2A) has been identified as a major melanoma susceptibility gene based on the presence of germline mutations in high-risk melanoma families. In this study, we sought to identify and characterize the spectrum of CDKN2A mutations affecting p16 inhibitor of cyclin-dependent kinase type 4 (INK4a) in individuals with melanoma using a population-based study design. DNA samples from 1189 individuals with incident multiple primary melanoma (MPM) and 2424 with incident single primary melanoma unselected for family history of melanoma were available for screening of CDKN2A (p16INK4a) mutations. Variants were classified for functional impact based on intragenic position, existing functional data, sequence, and structural analysis. The impact of individual mutations and functional groupings was assessed by comparing frequencies in cases of MPM versus cases with a single first primary melanoma, and by comparing the reported incidence rates in first-degree relatives. Our results show that mutations occur infrequently in these high-risk groups, and that they occur mainly in exons 1alpha and 2. Rare coding variants with putative functional impact are observed to increase substantially the risk of melanoma. With the exception of the variant in position -34 of CDKN2A of known functional consequence, the remaining rare variants in the non-coding region have no apparent impact on risk.     

Interleukin-18 prevents apoptosis via PI3K/Akt pathway in normal human keratinocytes

Interleukin-18 (IL-18) is a pleiotropic cytokine expressed in both immune and non-immune cells. In the present study, we demonstrate an anti-apoptotic role of IL-18 in normal human neonatal foreskin epidermal keratinocytes (NHEK-F). Cultured NHEK-F spontaneously produced the active form of IL-18. Treatment of NHEK-F cells with anti-IL-18 receptor alpha-chain neutralizing antibody increased apoptosis and caspase-3 activity. Exogenous IL-18 augmented phosphorylation of Akt and activation of NF-kappaB. The promotion of Akt phosphorylation by IL-18 was abolished by LY294002, a PI3K inhibitor, but not SN50, an NF-kappaB inhibitor, indicating that IL-18 functions via the PI3K/Akt pathway and independently of NF-kappaB. In addition, IL-18 was found to augment expression of anti-apoptotic proteins, Bcl-2, XIAP and glucose regulated protein78/BiP, while anti-IL-18 receptor alpha-chain neutralizing antibody suppressed expression of Bcl-2, XIAP, glucose regulated protein94 and protein disulfide isomerase. Taken together, these results indicate that IL-18 plays an important role in keratinocyte survival.     

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.     

Proliferation arrest in B-Raf mutant melanoma cell lines upon MAPK pathway activation

Due to elaborate control mechanisms, in benign tumors the activation of oncogenes primarily induces senescence, associated with cessation of cellular proliferation; for example, melanocytic nevi expressing mutant B-Raf. These mechanisms include the RB and/or the p53 pathway. The current model of melanomagenesis postulates that progression to immortal melanoma cells requires inactivating aberrations in signaling cascades controlling senescence. Thus, melanoma cells carrying mutant B-Raf should be resistant to mitogen-activated protein kinase (MAPK) pathway-induced senescence. Here, we demonstrate that hyperactivation of the MAPK pathway following activation of an inducible form of oncogenic C-Raf induces a senescence-like proliferation arrest in B-Raf mutant melanoma cells. This Raf-induced senescence is initially strictly dependent on MEK signaling, but seems to be independent of MAPK signaling after prolonged continuance. It is associated with reduced levels of RB phosphorylation and an increase in p21 expression, but is independent of p16(Ink4a) and p53. These data argue against the existence of fundamental changes in melanoma cells completely precluding senescence.     

PI3K/Akt/mTOR通路在SLE发病机制中的研究进展

PI3K/Akt/mTOR通路高度保守,广泛参与细胞增殖、分化及蛋白合成等活动,近年研究发现该通路的激活在系统性红斑狼疮的发病中起到重要作用,影响T、B淋巴细胞、巨噬细胞及间充质细胞等免疫功能,参与自噬调节。因此,该通路抑制剂的药物研发为SLE治疗提供新思路。本文从PI3K/Akt/mTOR通路组成和作用机制、与SLE发病的密切关系及该通路抑制剂的最新研究进展进行概述。     

Frequencies of BRAF and NRAS mutations are different in histological types and sites of origin of cutaneous melanoma: a meta-analysis

Background There have been conflicting data regarding the prevalence and clinicopathological characteristics of BRAF and NRAS mutations in primary cutaneous melanoma. Objectives To solve this controversy, this study used a meta‐analysis to evaluate the frequencies of BRAF and NRAS mutations, and the relationship between these mutations and clinicopathological parameters of cutaneous melanoma. Methods Data from studies published between 1989 and 2010 were combined. The BRAF and NRAS mutations were reported in 36 and 31 studies involving 2521 and 1972 patients, respectively. The effect sizes of outcome parameters were calculated by odds ratios (OR). Results BRAF and NRAS mutations were reported in 41% and 18% of cutaneous melanomas, respectively. The mutations were associated with histological subtype and tumour site, but not with age and sex. The BRAF mutation was frequently detected in patients with superficial spreading melanoma (OR = 2·021; P < 0·001) and in melanomas arising in nonchronic sun‐damaged skin (OR = 2·043; P = 0·001). In contrast, the NRAS mutation was frequently evident in patients with nodular melanoma (OR = 1·894; P < 0·001) and in melanomas arising in chronic sun‐damaged skin (OR = 1·887; P = 0·018). Conclusions This pooled analysis shows that the incidences of BRAF and NRAS mutations in cutaneous melanomas differ according to histological type and tumour location based on the degree of sun exposure.     

MAPK信号通路调控长波紫外线诱导的皮肤成纤维细胞组织蛋白酶K表达

【摘要】 目的 研究MAPK信号通路调控长波紫外线（UVA）诱导皮肤成纤维细胞组织蛋白酶K（CatK）的表达。 方法 原代培养的皮肤成纤维细胞取自儿童包皮。Western印迹检测10 J/cm2 UVA照射前及照射后0.75、1.5、3和6 h皮肤成纤维细胞中磷酸化JNK（p-JNK）、JNK、磷酸化P38（p-P38）、P38蛋白的表达。800 nmol/L SP600125（SP）、10 μmol/L SB203580（SB）分别孵育皮肤成纤维细胞,实验分为无UVA照射的对照组、SP组、SB组及10 J/cm2 UVA照射的对照（UVA）组、UVA-SP组、UVA-SB组。先以Western印迹检测各组UVA照射后1.5 h磷酸化c-Jun（p-c-Jun） 和磷酸化MAPKAPK2（p-MAPKAPK2）表达,再以RT-PCR 和Western印迹检测各组照射后48 h CatK mRNA、蛋白的表达。 结果 皮肤成纤维细胞在UVA照射后0.75、1.5 h,p-JNK表达的灰度值分别为4.77 ± 0.19和4.68 ± 0.09,p-P38分别为2.44 ± 0.13、2.30 ± 0.04,均较照射前（p-JNK为3.2 ± 0.27,p-P38为1.61 ± 0.08）显著升高（均P ＜ 0.05）;而在照射后3、6 h的表达与照射前相比差异无统计学意义（P ＞ 0.05）。p-c-Jun在UVA-SP组表达（2.55 ± 0.48）、p-MAPKAPK2在UVA-SB组表达（1.16 ± 0.12）均显著低于UVA组（分别为4.85 ± 0.96和2.46 ± 0.09）,均P ＜ 0.05。UVA-SP组、UVA-SB组CatK mRNA、蛋白的表达分别降为UVA组的38.9%、28.7%和55.7%、49.6%（P ＜ 0.05）,UVA-SP组CatK mRNA、蛋白的表达也均显著低于UVA-SB组（P ＜ 0.05）。 结论 JNK和P38信号通路在调控UVA上调的皮肤成纤维细胞CatK表达中起重要作用。     

恶性黑素瘤与磷脂酰肌醇3-激酶-Akt信号通路

磷脂酰肌醇3-激酶信号参与增殖、分化、凋亡和葡萄糖转运等多种细胞功能的调节.近年来发现,IA型磷脂酰肌醇3-激酶和其下游分子蛋白激酶B所组成的信号通路与人类恶性黑素瘤的发生发展密切相关.该通路调节黑素瘤细胞的增殖和存活,其活性异常不但能引起细胞恶性转化,而且与黑素瘤细胞的迁移、黏附、血管生成以及细胞外基质的降解等相关.目前以磷脂酰肌醇3-激酶-Akt信号通路关键分子为靶点的恶性黑素瘤治疗策略正在研究中.