ATP7A is a novel target of retinoic acid receptor ��2 in neuroblastoma cells

Increased retinoic acid receptor β (RARβ₂) gene expression is a hallmark of cancer cell responsiveness to retinoid anticancer effects. Moreover, low basal or induced RARβ₂ expression is a common feature of many human cancers, suggesting that RARβ₂ may act as a tumour suppressor gene in the absence of supplemented retinoid. We have previously shown that low RARβ₂ expression is a feature of advanced neuroblastoma. Here, we demonstrate that the ABC domain of the RARβ₂ protein alone was sufficient for the growth inhibitory effects of RARβ₂ on neuroblastoma cells. ATP7A, the copper efflux pump, is a retinoid-responsive gene, was upregulated by ectopic overexpression of RARβ₂. The ectopic overexpression of the RARβ₂ ABC domain was sufficient to induce ATP7A expression, whereas, RARβ₂ siRNA blocked the induction of ATP7A expression in retinoid-treated neuroblastoma cells. Forced downregulation of ATP7A reduced copper efflux and increased viability of retinoid-treated neuroblastoma cells. Copper supplementation enhanced cell growth and reduced retinoid-responsiveness, whereas copper chelation reduced the viability and proliferative capacity. Taken together, our data demonstrates ATP7A expression is regulated by retinoic acid receptor β and it has effects on intracellular copper levels, revealing a link between the anticancer action of retinoids and copper metabolism.     

Vasculogenic mimicry：a novel target for glioma therapy

Anti-angiogenic therapy has shown promising but insufficient efficacy on gliomas. Recent studies suggest that vasculogenic mimicry （VM）, or the formation of non-endothelial, tumor-cell-lined microvascular channels, occurs in aggressive tumors, including gliomas. There is also evidence of a physiological connection between the endothelial-lined vascuature and VM channels. Tumor cels, by virtue of their high plasticity, can form vessel-like structures themselves, which may function as blood supply networks. Our previous study on gliomas showed that microvessel density was comparably less in VM-positive tumors than in VM-negative tumors. Thus, VM may act as a complement to ensure tumor blood supply, especially in regions with less microvessel density. Patients with VM-positive gliomas survived a shorter period of time than did patients with VM-negative gliomas. Although the detailed molecular mechanisms for VM are not fully understood, glioma stem cells might play a key role, since they are involved in tumor tissue remodeling and contribute to neovasculadzation via transdifferentiation, In the future, successfu treatment of gliomas should involve targeting both VM and angiogenesis. In this review, we summarize the progress and challenges of VM in gliomas.     

ALCAM is a Novel Cytoplasmic Membrane Protein in TNF-α Stimulated Invasive Cholangiocarcinoma Cells

Background: Cholangiocarcinoma (CCA), or bile duct cancer, is incurable with a high mortality rate dueto a lack of effective early diagnosis and treatment. Identifying cytoplasmic membrane proteins of invasiveCCA that facilitate cancer progression would contribute toward the development of novel tumor markers andeffective chemotherapy. Materials and Methods: An invasive CCA cell line (KKU-100) was stimulated usingTNF-α and then biotinylated and purified for mass spectrometry analysis. Novel proteins expressed wereselected and their mRNAs expression levels were determined by real-time RT-PCR. In addition, the expressionof ALCAM was selected for further observation by Western blot analysis, immunofluorescent imaging, andantibody neutralization assay. Results: After comparing the proteomics profile of TNF-α induced invasive withnon-treated control cells, over-expression of seven novel proteins was observed in the cytoplasmic membraneof TNF-α stimulated CCA cells. Among these, ALCAM is a novel candidate which showed significant highermRNA- and protein levels. Immunofluorescent assay also supported that ALCAM was expressed on the cellmembrane of the cancer, with increasing intensity associated with TNF-α. Conclusions: This study indicatedthat ALCAM may be a novel protein candidate expressed on cytoplasmic membranes of invasive CCA cells thatcould be used as a biomarker for development of diagnosis, prognosis, and drug or antibody-based targetedtherapies in the future.     

Overexpression of miR-370 and downregulation of its novel target TGFβ-RII contribute to the progression of gastric carcinoma

MicroRNAs (miRNAs) are endogenous non-coding RNAs that are known to be involved in the pathogenesis of tumors. Gastric carcinoma (GC) is a common malignancy worldwide. The aim of this study was the identification of the expression signature and functional roles of aberrant miRNAs in GC. Initial screening established a profile of aberrantly expressed miRNAs in tumors. miR-370 was confirmed to be overexpressed in GC tissues. Higher expression of miR-370 in GC tissues was associated with more advanced nodal metastasis and a higher clinical stage compared with controls. In addition, significantly higher level of miR-370 was noted in the plasma of GC patients compared with controls. Patients having more invasive or advanced tumors also exhibited a higher plasma level of miR-370. In vitro assays indicated that exogenous miR-370 expression enhanced the oncogenic potential of GC cells. The AGS-GFPM2 cells with exogenous miR-370 expression also exhibited enhanced abdominal metastatic dissemination in nude mice. Reporter assays confirmed that miR-370 targeted predicted sites in 3'UTR of transforming growth factor-β receptor II (TGFβ-RII) gene. The exogenous miR-370 expression decreased TGFβ-RII expression and the phosphorylation of Smad3 elicited by TGFβ1. The TGFβ1-mediated repression in cell migration was reverted by exogenous miR-370 expression. A reverse correlation between miR-370 and TGFβ-RII expression was noted in GC tissues. This study concludes that miR-370 is a miRNA that is associated with GC progression by downregulating TGFβ-RII. The miRNA expression profile described in this study should contribute to future studies on the role of miRNAs in GC.     

miR-19a和miR-19b在骨肉瘤中的表达及其临床意义

目的：探讨miR-19a和miR-19b在骨肉瘤组织及配对瘤旁组织中的表达及其与临床病理特征的相关性。方法：收集32对骨肉瘤和配对的瘤旁组织,运用实时荧光定量PCR（qPCR）检测骨肉瘤组织及其瘤旁组织中miR-19a和miR-19b的表达,分析其与骨肉瘤临床病理特征的相关性及其临床意义。结果：qPCR结果显示骨肉瘤组织中miR-19a和miR-19b的平均表达量较瘤旁组织中均明显上调（P均 < 0.05）。二者的表达水平呈正相关（r=0.685,P=0.000）。miR-19a和miR-19b的表达与骨肉瘤病理分级之间存在正相关（r=0.478,P=0.027）;miR-19a的表达与临床分期呈正相关（r=0.365,P=0.031）且与预后相关。Cox回归多因素分析发现miR-19a可作为骨肉瘤患者预后的影响因子（P=0.037）。结论：miR-19a和miR-19b在骨肉瘤中表达上调,在骨肉瘤的发生发展中可能发挥重要的作用,其中miR-19a可能作为骨肉瘤独立的预后标志物。     

miR-19a和miR-19b对抑癌基因SEPT7的调控

目的确定抑癌基因隔蛋白7(SEPT7)是微小RNA(miR)-19a和miR-19b的靶基因之一.方法脂质体介导转染miR-19a/19b寡聚核苷酸抑制物(miR-19aI和miR-19bI)于人脑胶质瘤细胞株SNB19细胞.采用荧光素酶实验检测miR-19a/19b对SEPT7的直接调控关系;实时荧光定量聚合酶链反应...     

TNF-α in promotion and progression of cancer

Tumour necrosis factor alpha is a member of the TNF/TNFR cytokine superfamily. In common with other family members, TNF-α is involved in maintenance and homeostasis of the immune system, inflammation and host defence. However, there is a ‘dark side’ to this powerful cytokine; it is now clear that, especially in middle and old age, TNF-α is involved in pathological processes such as chronic inflammation, autoimmunity and, in apparent contradiction to its name, malignant disease. This article will discuss the involvement of TNF-α in the inflammatory network that contributes to all stages of the malignant process, and consider the possibility that TNF-α may be a target for cancer therapy.     

Jab1 is a target of EGFR signaling in ERα-negative breast cancer

Introduction

c-Jun activation domain-binding protein-1 (Jab1) is a multifunctional signaling protein that previously has been shown to be a master regulator of a poor prognostic gene signature in invasive breast cancer and to mediate the action of S100A7. Since epidermal growth factor receptor (EGFR), like S100A7, is often expressed in estrogen receptor-alpha-negative (ERα^-) breast cancer, we set out to investigate the role of Jab1 in mediating EGFR signaling, another facet of the ERα^- phenotype.

Methods

MDA-MB-231 and MDA-MB-468 ERα^-/EGFR⁺ cell lines were assessed for localization of Jab1 and levels of downstream genes by immunofluorescence and nuclear protein extract assay following treatment with epidermal growth factor (EGF) and extracellular signal-regulated kinase (ERK) pathway inhibitor. A cohort of 424 human breast tumors was also assessed by immunohistochemistry.

Results

EGF treatment of cell lines resulted in increased Jab1 nuclear expression. This effect was inhibited by the ERK pathway inhibitor, PD98059. EGF treatment was also associated with colocalization of pERK (phosphorylated ERK) and Jab1 as well as regulation of the Jab1 downstream target gene, p27. When Jab1 activity was knocked down, p27 levels were restored to pre-EGF treatment level. Analysis of EGFR and Jab1 expression in a cohort of invasive breast tumors by tissue microarray and immunohistochemistry confirmed a relationship between EGFR and increased nuclear Jab1 within the ERα^- subset (n = 154, P = 0.019). The same association was also confirmed for S100A7 and Jab1 (P = 0.036), and high Jab1 nuclear expression was most frequent in tumors that were positive for both EGFR and S100A7 (P = 0.004).

Conclusion

Jab1 is a target of EGFR signaling in ERα^- cell lines and breast tumors and therefore may be a common central factor and potential therapeutic target for important cell signaling pathways in ERα^- breast cancer.     

Identification of odontogenic ameloblast associated as a novel target gene of the Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway plays a key role in development and carcinogenesis. Although some target genes of this signaling have been identified in various tissues and neoplasms, the comprehensive understanding of the target genes and their roles in the development of human cancer, including hepatoma and colorectal cancer remain to be fully elucidated. In this study, we searched for genes regulated by the Wnt signaling in liver cancer using HuH-7 hepatoma cells. A comparison of the expression profiles between cells expressing an active form of mutant β-catenin and cells expressing enhanced green fluorescent protein (EGFP) identified seven genes upregulated by the mutant β-catenin gene (CTNNB1). Among the seven genes, we focused in this study on ODAM, odontogenic, ameloblast associated, as a novel target gene. Interestingly, its expression was frequently upregulated in hepatocellular carcinoma, colorectal adenocarcinoma, and hepatoblastoma. We additionally identified a distant enhancer region that was associated with the β-catenin/TCF7L2 complex. Further analyses revealed that ODAM plays an important role in the regulation of the cell cycle, DNA synthesis, and cell proliferation. These data may be useful for clarification of the main molecular mechanism(s) underlying these cancers.     

ADAM-9 is a novel mediator of tenascin-C-stimulated invasiveness of brain tumor–initiating cells

Background

Tenascin-C (TNC), an extracellular matrix protein overexpressed in malignant gliomas, stimulates invasion of conventional glioma cell lines (U251, U87). However, there is a dearth of such information on glioma stemlike cells. Here, we have addressed whether and how TNC may regulate the invasiveness of brain tumor–initiating cells (BTICs) that give rise to glioma progenies.

Methods

Transwell inserts coated with extracellular matrix proteins were used to determine the role of TNC in BTIC invasion. Microarray analysis, lentiviral constructs, RNA interference-mediated knockdown, and activity assay ascertained the role of proteases in TNC-stimulated BTIC invasion in culture. Involvement of proteases was validated using orthotopic brain xenografts in mice.

Results

TNC stimulated BTIC invasiveness in a metalloproteinase-dependent manner. A global gene expression screen identified the metalloproteinase ADAM-9 as a potential regulator of TNC-stimulated BTIC invasiveness, and this was corroborated by an increase of ADAM-9 protein in 4 glioma patient–derived BTIC lines. Notably, RNA interference to ADAM-9, as well as inhibition of mitogen-activated protein kinase 8 (c-Jun NH2-terminal kinase), attenuated TNC-stimulated ADAM-9 expression, proteolytic activity, and BTIC invasiveness. The relevance of ADAM-9 to tumor invasiveness was validated using resected human glioblastoma specimens and orthotopic xenografts where elevation of ADAM-9 and TNC expression was prominent at the invasive front of the tumor.

Conclusions

This study has identified TNC as a promoter of the invasiveness of BTICs through a mechanism involving ADAM-9 proteolysis via the c-Jun NH2-terminal kinase pathway.     

非小细胞肺癌中miR-19a和miR-19b的表达及临床意义

目的:分析miR-19a、miR-19b在非小细胞肺癌中的表达及其与临床病理特征的关系。方法:非小细胞肺癌手术标本61例及其癌旁正常组织,提取总RNA,采用实时定量PCR方法检测miR-19a、miR-19b在非小细胞肺癌及其癌旁正常组织中的表达,并分析其与临床病理特征的关系。结果:miR-19a、miR-19b在非小细胞肺癌组织中的表达高于对应的癌旁正常肺组织,其表达与临床分期、病理类型及淋巴结有无转移相关（P<0.05）。而在不同年龄、性别和吸烟史患者间差异无统计学意义（P>0.05）。结论:miR-19a、miR-19b高表达与非小细胞肺癌的临床分期、病理分型及有无淋巴结转移密切相关,miR-19a、miR-19b有可能成为非小细胞肺癌的重要肿瘤标志物之一。     

Combined treatment of IL-1α and TNF-α potentiates the antitumour effect of hyperthermia

Vasoactive cytokines, such as IL-1α and TNF-α, modulate the homeostatic state at the endothelial surface and cause various types of pathological damage in vascular systems. We investigated the potential therapeutic effects of IL-1α and TNF-α in combination with hyperthermia on SCK tumours grown in the legs of A/J mice. We first determined the effect of cytokines on tumour blood perfusion with the ⁸⁶Rb uptake method. When the host mice were given an i.p. injection of 25 μg/kg IL-1α or 50 μg/kg TNF-α, the tumour blood perfusion markedly declined to 46 and 82% of control, respectively. The combination of IL-1α and TNF-α reduced the ⁸⁶Rb uptake to 41% of control. Hyperthermia at 42·5°C for 1 h reduced the tumour blood flow to 71% of control. The tumour blood perfusion decreased further to 20% of control when the tumours were heated for 1 h at 42.5°C starting 4h after the injection of both IL-1α and TNF-α. The changes in clonogenic cell numbers in SCK tumours, as determined by the in vivo-in vitro assay, following various treatments was also investigated. At 4h after an i.p. injection of 25 μg/kg IL-1α or 50 μg/kg TNF-α, the clonogenicity of SCK tumours significantly decreased to 29 or 37% of control, respectively. Heating at 42.5°C for 1h caused a decline in the clonogenic cell number to 30% of control. When both IL-1α and TNF-α were given and tumours were heated 4h later at 42·5°C for 1h, the clonogenic cell number markedly declined to 0.4% of control. The time needed for control tumours to reach 4x their initial volume was about 3 days, and treatment with IL-1α or hyperthermia alone induced a tumour delay growth by about 1 day. The combined injection of IL-1α and TNF-α followed by a heating at 42·5°C for 1h delayed the tumour growth by 6 days. The results in this study suggest that prior impairment of blood circulation by the combined treatment of IL-1α and TNF-α potentiates hyperthermic damage in tumours.     

αB-crystallin is a novel predictor of resistance to neoadjuvant chemotherapy in breast cancer

Aims αB-crystallin is an anti-apoptotic protein commonly expressed in poor prognosis basal-like breast tumors, which are largely triple (estrogen receptor (ER), progesterone receptor (PR), and HER2) negative. We examined whether αB-crystallin expression in breast cancer was associated with a poor response to neoadjuvant (preoperative) chemotherapy. Methods One hundred and twelve breast cancer patients who received neoadjuvant chemotherapy and who had post-chemotherapy tumor specimens available for analysis were included in the study. Forty-nine percent of patients were treated with doxorubicin and cyclophosphamide (AC), 37% received AC in combination with a taxane, and 14% received other regimens. Paired pre- and post-chemotherapy tumor specimens were available for 33 patients. αB-crystallin expression was determined by immunohistochemistry in tissue microarrays. Results Seventeen percent of tumors were αB-crystallin positive. αB-crystallin expression was identical in 32 of 33 cases for which both pre- and post-chemotherapy tumor tissue was available. αB-crystallin expression was associated with ER-negative (P = 0.0024) and triple negative status (P = 0.005). Overall response rates (ORR) defined as ≥50% reduction in tumor size after treatment were 53% (clinical ORR) and 61% (pathological ORR). Although tumor grade, size, ER, PR, HER2 or triple negative status was not associated with response, αB-crystallin-positive tumors had poorer overall response rates than αB-crystallin-negative tumors (clinical ORR, 21% vs. 59%, respectively, P = 0.0045; pathological ORR, 16% vs. 70%, respectively, P < 0.0001). Conclusion αB-crystallin is a novel biomarker expressed predominantly in triple negative breast tumors that identifies a subset of chemotherapy-resistant tumors, which may contribute to their poor prognosis.     

Ca/calmodulin-dependent protein kinase IIγ, a critical mediator of the NF-κB network,is a novel therapeutic target in non-small cell lung cancer

The molecular mechanism that triggers constitutive activation of nuclear factor-kappa B (NF-κB) in non-small cell lung cancer (NSCLC) remains elusive. In this present study, we demonstrated that Ca²⁺/calmodulin-dependent protein kinase IIγ (CaMKIIγ) is a critical molecular switch of continuous activation of NF-κB in NSCLC. We found that CaMKIIγ was aberrantly expressed in human NSCLC tissues and correlated well with the degree of malignancy. Functionally, CaMKIIγ promoted the growth and survival of NSCLC cells via direct activation of NF-κB and multiple oncogenic signaling pathways in NSCLC. Taken together, our findings described a previously uncharacterized role of CaMKIIγ in NSCLC, and suggest a novel potential therapeutic target for NSCLC treatment.