The catalytic phosphoinositol 3-kinase isoform p110δ is required for glioma cell migration and invasion

Glioblastoma multiforme (GBM) is a highly invasive and aggressive primary brain tumour in which loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a negative regulator of PI3K signalling, is a common feature. PTEN/PI3K/Akt signalling is involved in the regulation of proliferation, apoptosis and cell migration. Deregulation of PI3K signalling is considered an essential driver in gliomagenesis. However, the role of different PI3K isoforms in glioma is still largely unclear. Here we show that the catalytic PI3K isoform p110δ is consistently expressed at a high level in various glioma cell lines. We used small interfering RNA to selectively deplete p110δ and to determine its tumourigenic roles in PTEN-deficient cells. Interestingly, knockdown of p110δ decreased the cell migration and invasion ability of all GBM cell lines tested. Mechanistically, p110δ knockdown reduced the protein levels of focal adhesion kinase and cell division cycle 42, key regulators of cellular migration. In contrast, pharmacologic inhibition of p110δ by IC87114 or CAL-101 also clearly impaired glioma cell migration but had no obvious effect on the invasion capacity thus pinpointing to possible kinase-dependent and -independent roles of p110δ in glioma pathology. In summary, our data provide novel evidence that in glioma cells p110δ is a key regulator of cell movement and thus may contribute to the highly invasive phenotype of GBM. Isoform specific targeting of PI3Kδ may be beneficial in the treatment of glioblastoma multiforme by specifically inhibiting tumour cell migration capacity.     

Evidence of p38γ and p38δ involvement in cell transformation processes

The p38 mitogen-activated protein kinase (p38MAPK) signal transduction pathway is an important regulator of cell processes, whose deregulation leads to the development and progression of cancer. Defining the role of each p38MAPK family member in these processes has been difficult. To date, most studies of the p38MAPK pathways focused on function of the p38α isoform, which is widely considered to negatively regulate malignant transformation; nonetheless, few reports address the p38γ and p38δ isoforms. Here, we used embryonic fibroblasts derived from mice lacking p38γ or p38δ and show evidence that these isoforms participate in several processes involved in malignant transformation. We observed that lack of either p38γ or p38δ increased cell migration and metalloproteinase-2 secretion, whereas only p38δ deficiency impaired cell contact inhibition. In addition, lack of p38γ in K-Ras-transformed fibroblasts led to increased cell proliferation as well as tumorigenesis both in vitro and in vivo. Our results indicate that p38γ and p38δ have a role in the suppression of tumor development.     

PKCδ regulates Mdm2 independently of p53 in the apoptotic response to DNA damage

Apoptosis is the key process in which cells with defective genome can be eliminated. Dys-regulation of apoptosis causes accumulation of irreparable mutation arisen from DNA damage and is the underlying cause of carcinogenesis. PKCδ is a multifunctional kinase involved in signal transduction of genotoxic-induced apoptosis. Previous studies have demonstrated that PKCδ transactivates p53 in response to DNA damage. These findings led us to determine if Mdm2, a nuclear phospho-protein and negative regulator of p53, could also be a PKCδ-modulated substrate. We discovered that inhibition of PKCδ down-regulates Mdm2 protein expression regardless of p53 status. Given that Mdm2 mRNA change was detected in p53-proficient, but not deficient cells, PKCδ affected Mdm2 on the post-translational level. Interestingly, treatment of MG132 restored Mdm2 expression to the steady-state level. Further investigation showed that PKCδ inhibited Mdm2 ubiquitination in p53-deficient cells and loss of PKCδ resulted in an increase in Mdm2 proteosomal degradation. Moreover, P300/CBP-associated factor (PCAF), an ubiquitin ligase 3 for Mdm2, was observed to participate in Mdm2 ubiquitination by PKCδ inhibition and knock-down of PCAF rescued Mdm2 diminution. Finally, as shown for PKCδ, Mdm2 was also required to exert pro-apoptotic response caused by genotoxic agents in p53-null cells. In addition, overexpression of Mdm2 restored inhibitory effect of apoptosis in cells silenced for PKCδ. Taken together, we conclude that PKCδ regulates Mdm2 expression distinctively of p53 pathway by affecting Mdm2 ubiquitination and maintenance of Mdm2 expression by PKCδ is important to ensure normal genotoxic cell death response in human cancer cells.     

Δ133p53 is an independent prognostic marker in p53 mutant advanced serous ovarian cancer

Background:

We aimed to evaluate the clinical relevance of p53 and p73 isoforms that modulate the function of p53.

Methods:

This prospective multicentre study included 154 patients with stage III and IV serous ovarian cancer. A functional yeast-based assay and subsequent sequencing were performed to analyse the p53 mutational status. Expression of p53 and p73 isoforms was determined using RT–qPCR.

Results:

Δ133p53 expression constituted an independent prognostic marker for recurrence-free (hazard ratio=0.571, P=0.016, 95% CI: 0.362–0.899) and overall survival (hazard ratio=0.365, P=0.004, 95% CI: 0.182–0.731) in patients with p53 mutant ovarian cancer (n=121). High Δ40p53 expression was associated with favourable tumour grading (P=0.037) and improved recurrence-free survival (33.4 vs 19.6 months, P=0.029), but not overall survival (43.1 vs 33.6 months, P=0.139), in patients with p53 wild-type cancer (n=33). Neither the p53 mutational status nor p73 isoform expression possessed prognostic significance in the examined ovarian cancer cases.

Conclusion:

Δ133p53 expression was associated with prognosis in the vast majority of ovarian cancer cases, that is, patients with p53 mutant advanced serous carcinomas. Thus, our findings underline the importance of considering the complex p53 regulatory network.     

氯化镉诱发16HBE细胞恶变过程中TEF-1δ p31异常表达的研究

背景与目的:探讨氯化镉(CdCl2)诱发人支气管上皮细胞(16HBE)恶性转化过程中不同阶段人翻译延伸因子TEF-1δ p31 mRNA表达的变化,以进一步阐明CdCl2致癌的分子机制.材料与方法:应用RT与PCR技术,以及Taqman荧光定量PCR方法,检测不同浓度CdCl2诱发16HBE恶性转化3个不同阶段细胞(转化期间细胞、转化细胞和成瘤细胞)的TEF-1δ p31 mRNA表达量的变化.结果:①相对于非转化16HBE对照细胞,CdCl2诱发恶性转化3个不同阶段细胞的TEF-1δ mRNA表达水平均显著升高(P＜0.01或P＜0.05),5 μmol/L CdCl2处理组各阶段细胞内的TEF-1δ平均表达量分别是对照细胞的3.4、5.2和6.9倍;10 μmol/L CdCl2处理组各阶段细胞内的TEF-1δ平均表达量分别是对照细胞的4.1、6.9和6.3倍;15 μmol/L CdCl2处理组各阶段转化细胞内的TEF-1δ 平均表达量分别是对照细胞的1.4、2.9和8.4倍.提示TEF-1δ的异常表达量与CdCl2诱发16HBE细胞恶变程度之间有正相关关系(r＞0.799.P＜0.01).②16HBE细胞恶变不同阶段TEF-1δ p31的异常表达水平与CdCl2的剂量相关(P=0.001).结论:氯化镉在诱发16HBE细胞恶变过程中,存在明显的人翻译延伸因子TEF-1δ p31异常表达的现象,其表达水平与细胞恶性转化程度和CdCl2的剂量密切相关,这可能是氯化镉诱发人细胞肿瘤的重要分子致癌机制之一.     

p120 Catenin Translocation is Involved in Enhancement of Hepatoma Cellular Malignant Features

OBJECTIVE To investigate the relationship between p 120^ctn translocation and hepatocellular carcinoma cell malignant features and the relationship between p 120^ctn and β-catenin translocation in cell signaling. METHODS Human hepatocellular carcinoma cells were over expressed with p120^ctn isoform 3A using a DNA transfection method. The effects of transfection and expression of p120^ctn and its binding capacity to E-cadherin were examined using immunoprecipitation and immunoblotting methods. p120^ctn subcellular localization and its relation with β-catenin were detected using immunofluorescent microscopy, p120^ctn phosphorylation was produced by EGF treatment. Cell adhesion, cell migration and cell proliferation were also examined in this study. RESULTS We found that p 120^ctn expression was increased after transfection and the binding capacity of p120^ctn to E-cadherin was enhanced. Tyrosine phosphorylation of p120^ctn increased after transfection and EGF treatment. p120^ctn and β-catenin cellular localization displayd a membrane and cytoplasmic expression pattern, but they translocated into the nucleus for relocalization after p120^ctn overexpression plus EGF stimulation. Cell adhesion ability was increased and migration ability reduced after transfection without EGF. Following transfection without EGF cellular proliferation was reduced, but increased after EGF treatment. CONCLUSION Our results suggest that p120^ctn plays an important role in hepatocellular carcinoma cell adhesion, migration and proliferation. In addition there is a relationship between p120^ctn and β-catenin subcellular localization and signaling.     

What is the Best Frontline Therapy for Patients with CLL and 17p Deletion?

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease with significant variation in disease progression, response to therapy, and survival outcome. Deletions of 17p or mutations of TP53 have been identified as one of the poorest prognostic factors, being predictive of short time for disease progression, lack of response to therapy, short response duration, and short overall survival. The treatment of patients with CLL has improved significantly with the development of chemoimmunotherapy, but this benefit was not pronounced in patients with 17p deletion. We compare various treatment strategies used in these patients, including FCR-like chemoimmunotherapy, alemtuzumab, other antibody combinations, or novel targeted therapies with promising results. Allogeneic stem cell transplantation offers the possibility for long-term disease control in these patients and should be considered early in younger, transplant-eligible patients. The current state of therapy is far from optimal and resources should be applied to studying therapeutic options for patients who have CLL with loss of p53 function.     

p53家族新成员p63和p73

抑癌基因p53由于其在大多数人肿瘤细胞中的频繁缺失和突变而倍受重视.近年来,随着对p53基因的深入研究,p53家族的其它成员也不断被发现.     

p53基因家族的新成员 p73和 p63

p53作为广泛存在的肿瘤抑制基因,最近发现了其家族成员：p73和p63。它们在结构和功能上具有相似性,均能触发细胞周期停滞,诱导凋亡,但它们在肿瘤抑制和组织发育中起着截然不同的作用,深入了解它们彼此之间的相似性和差异将对理解肿瘤发生的机制产生重要的影响。本文总结了最近几年来此领域内的最新进展。另外,p73和p63在C端的SAM结构说明它们可能参与组织的发育过程。     

HPV DNA,E6I-mRNA expression and p16 immunohistochemistry in head and neck cancer – How valid is p16 as surrogate marker?

It has been proposed that p16(INK4A) qualifies as a surrogate marker for viral oncogene activity in head and neck cancer (HNSCC). By analyzing 78 HNSCC we sought to validate the accuracy of p16(INK4A) as a reliable marker of active HPV infections in HNSCC. To this end we determined HPV DNA (HPVD) and E6*I mRNA (HPVR) expression status and correlated these results with p16(INK4A) staining. In tonsillar SCC 12/20 were HPVD+ and 12/12 of these showed active HPV infections whereas in non-tonsillar SCC 10/58 were HPVD+ and 5/10 showed active HPV infections. Thus, we prove about 8% of non-tonsillar SCC to be also correlated with HPV-associated carcinogenesis. Strikingly, 3/14 (21.4%) of tonsillar and non-tonsillar HPVD+/HPVR+ cases did not show p16(INK4A) overexpression and these cases would have been missed when applying initial p16(INK4A) staining only. However, in 13 cases negative for HPV, DNA p16(INK4A) was overexpressed. In conclusion, our data confirm tonsillar SCC to be predominantly but not only associated with active HPV infections. Furthermore, our data show that p16(INK4A) overexpression is not evident in a subgroup of HNSCC with active HPV infection. Definitive HPV data should therefore be utilized in diagnostics and treatment modalities of HPV positive and HPV negative HNSCC patients, resulting in a paradigm shift regarding these obviously different tumor entities.     

p53家族新成员p63基因

p63是近几年发现的p53家族新成员,它编码结构和功能不同的异构体,全长表达的p63蛋白能激活p53的相关靶基因,阻遏细胞周期和诱导细胞凋亡;而N端被截短的p63蛋白却拮抗p53和全长表达的p63的功能.而且p63编码的活性产物对调节胚胎形成时外胚层的分化、各种上皮组织的正常发生与分化及维持细胞形态具有重要作用,其缺乏会引起一系列的发育畸形.p63基因在肿瘤发生、细胞凋亡和组织发育的研究中备受关注.     

Induction of apoptosis by quercetin is mediated through AMPKα1/ASK1/p38 pathway

Effective strategies for cancer prevention and treatment can be identified by understanding the mechanism of apoptotic pathways. In this study, we investigated the regulatory mechanism of quercetin-induced apoptosis through apoptosis signal-regulating kinase (ASK)-1 and mitogen-activated protein kinase pathways. Our results showed that quercetin increased apoptotic cell death through reactive oxygen species (ROS) generation and was responsible for ASK1 activation. Increasing ASK1 activity was accompanied by p38 activation. Interestingly, AMP-activated protein kinase (AMPK) seemed to be a critical controller of quercetin-regulated ASK1/p38 activation. Blocking AMPKα1 activity using Compound C, a synthetic inhibitor or siRNA showed that quercetin-activated ASK1 could not stimulate p38 activity. Thus, we suggested that quercetin-exerted apoptotic effects involve ROS/AMPKα1/ASK1/p38 signaling pathway, and AMPKα1 is a necessary element for apoptotic event induced by ASK1.     

PIK3R1 (p85α) is somatically mutated at high frequency in primary endometrial cancer

Phosphoinositide 3-kinase (PI3K) is an important therapeutic target. Mutations in PIK3CA, which encodes p110α, the catalytic subunit of PI3K, occur in endometrioid endometrial cancers (EEC) and nonendometrioid endometrial cancers (NEEC). The goal of this study was to determine whether PIK3R1, which encodes p85α, the inhibitory subunit of PI3K, is mutated in endometrial carcinoma. We carried out exonic sequencing of PIK3R1 from 42 EECs and 66 NEECs. The pattern of PIK3R1 mutations was compared with the patterns of PIK3CA, PTEN, and KRAS mutations. The biochemical effect of seven PIK3R1 mutations was examined by stable expression in U2OS cells, followed by coimmunoprecipitation analysis of p110α, and Western blotting of phospho-AKT(Ser473) (p-AKT(Ser473)). We found that PIK3R1 was somatically mutated in 43% of EECs and 12% of NEECs. The majority of mutations (93.3%) were localized to the p85α-nSH2 and -iSH2 domains. Several mutations were recurrent. PIK3R1 mutations were significantly (P = 0.0015) more frequent in PIK3CA-wild type EECs (70%) than in PIK3CA mutant EECs (18%). Introduction of wild-type p85α into U2OS cells reduced the level of p-AKT(Ser473) compared with the vector control. Five p85α mutants, p85αdelH450-E451, p85αdelK459, p85αdelY463-L466, p85αdelR574-T576, and the p85αN564D positive control, were shown to bind p110α and led to increased levels of p-AKT(Ser473). The p85αR348X and p85αK511VfsX2 mutants did not bind p110α and showed no appreciable change in p-AKT(Ser473) levels. In conclusion, our study has revealed a new mode of PI3K alteration in primary endometrial tumors and warrants future studies to determine whether PIK3R1 mutations correlate with clinical outcome to targeted therapies directed against the PI3K pathway in EEC and NEEC.     

p53、p16、p27和PTEN基因与卵巢癌的关系

卵巢癌是一种严重威胁妇女生命健康的妇科常见恶性肿瘤，极易在早期发生浸润和转移，由于其具有起病隐匿、早期临床诊断率低、复发率高等特点，严重影响远期疗效。5年生存率只有25％～30％，死亡率居妇科恶性肿瘤首位。随着肿瘤研究的不断深人和分子生物学的飞速发展，已经发现了多种癌基因和抑癌基因，人们已经认识到恶性肿瘤除了与癌基因的表达与恶性病变有关以外，抑癌基因的失活也起到非常重要的作用。因此如何利用新方法、新技术     

胃癌中p14、p15、p16基因微卫星不稳定性的研究

 目的 对照检测伴有正常及高级别上皮内瘤变的胃腺癌组织中定位于染色体9p21区D9S166、D9S171、D9S941、D9S942和IFNA的微卫星不稳定性（MSI），探讨p14、p15和p16基因与胃癌发生发展的关系。方法 选择55例伴有正常及高级别上皮内瘤变的胃腺癌标本，应用手工显微切割、聚合酶链反应、高压凝胶电泳、硝酸银染色等技术, 对照分析正常、高级别上皮内瘤变和胃腺癌组织中9p21上的5个微卫星位点的MSI变化。结果 胃癌组织MSI总发生率为27 %（64／233），高级别上皮内瘤变MSI总发生率为18 %（42／233），正常组织中未发生MSI。胃癌组织中的MSI发生率显著高于高级别上皮内瘤变（P＜0.05）。在胃癌组织中，5个微卫星位点的MSI发生率依次为D9S171（45 %）、IFNA（41 %）、D9S941（22 %）、D9S166（16 %）、D9S942（15 %）；高级别上皮内瘤变组织中，依次为IFNA（22 %）、 D9S171（21 %）、D9S941（18 %）、D9S166（14 %）、D9S942（11 %）。在胃癌组织中D9S171位点的MSI发生率为45 %，在高级别上皮内瘤变的MSI发生率为21 %。两者差异有统计学意义（P＜0.05）。结论 胃癌中染色体9p21区发生有高频率的MSI，MSI 可能为胃癌发生过程中的早期分子事件，并且在胃癌发展过程中持续发挥作用。p14、p15和p16基因可能与胃癌的发生发展相关。