A novel tumor-promoting function residing in the 5′ non-coding region of vascular endothelial growth factor mRNA
A novel tumor-promoting function residing in the 5′ non-coding region of vascular endothelial growth factor mRNA
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DOI：
10.1371/journal.pmed.0050094
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发表时间：
2008-05-01
期刊：
影响因子：
15.8
通讯作者：
Rokutan, Kazuhito
中科院分区：
文献类型：
--
作者：
Masuda, Kiyoshi;Teshima-Kondo, Shigetada;Rokutan, Kazuhito
背景血管内皮生长因子-A（Vascular endothelial growth factor-A，VEGF）是肿瘤发生、发展的重要调节因子之一，因此被认为是肿瘤治疗的重要靶点。然而，临床试验表明，抗VEGF单药治疗不如标准化疗有效。在证据的基础上，我们假设，VEGF mRNA可能有未识别的功能（S）在癌cells.Methods和FindingsKnockdown的VEGF与VEGF靶向小干扰（si）RNA的人结肠癌细胞系（HCT 116）的易感性增加5-氟尿嘧啶，依托泊苷，或阿霉素引起的细胞凋亡。重组人VEGF（165）不能完全抑制这种凋亡。相反，VEGF（165）的过度表达增加了对抗癌药物诱导的细胞凋亡的抵抗，而抗VEGF（165）中和抗体不能完全阻断这种抵抗。我们制备了编码信号序列突变的全长vegf mRNA、缺乏非翻译区（UTR）的vegf mRNA或突变的5 'UTR的质粒。利用这些质粒，我们发现vegfmRNA的5 ' UTR具有抗凋亡活性。5 'UTR介导的活性不受蛋白质合成抑制剂放线菌酮的影响。我们建立了稳定表达vegf 5 ' UTR或突变的5 ' UTR的HCT 116克隆。表达5 ' UTR的克隆，而不是突变的克隆，在体外显示出增加的锚定非依赖性生长，并且当植入无胸腺裸鼠中时形成进行性肿瘤。基因芯片和实时荧光定量PCR分析表明，表达vegf 5 'UTR的肿瘤具有上调的抗凋亡基因、多药耐药基因和促生长基因，而促凋亡基因下调。值得注意的是，在表达5 'UTR的肿瘤中，信号转导和转录激活因子1（STAT 1）的表达被显著抑制，导致STAT 1应答基因簇（43个基因）的下调。结果，肿瘤对干扰素（IFN）α治疗完全没有反应。我们发现，稳定沉默的内源性血管内皮生长因子mRNA在HCT 116细胞增强STAT 1的表达和IFN α respons.ConclusionsThese研究结果表明，癌细胞有一个生存系统，是由血管内皮生长因子mRNA的调节，并意味着，血管内皮生长因子mRNA及其蛋白质可能协同促进肿瘤细胞的恶性。因此，抗VEGF转录物策略（例如基于siRNA的基因沉默）与抗VEGF抗体治疗的组合可以改善靶向VEGF的抗癌疗法。
BackgroundVascular endothelial growth factor-A (VEGF) is one of the key regulators of tumor development, hence it is considered to be an important therapeutic target for cancer treatment. However, clinical trials have suggested that anti-VEGF monotherapy was less effective than standard chemotherapy. On the basis of the evidence, we hypothesized that vegf mRNA may have unrecognized function(s)in cancer cells.Methods and FindingsKnockdown of VEGF with vegf-targeting small-interfering (si) RNAs increased susceptibility of human colon cancer cell line (HCT116) to apoptosis caused with 5-fluorouracil, etoposide, or doxorubicin. Recombinant human VEGF(165) did not completely inhibit this apoptosis. Conversely, overexpression of VEGF(165) increased resistance to anti-cancer drug-induced apoptosis, while an anti-VEGF(165)-neutralizing antibody did not completely block the resistance. We prepared plasmids encoding full-length vegf mRNA with mutation of signal sequence, vegf mRNAs lacking untranslated regions (UTRs), or mutated 5 ' UTRs. Using these plasmids, we revealed that the 5 ' UTR of vegf mRNA possessed anti-apoptotic activity. The 5 ' UTR-mediated activity was not affected by a protein synthesis inhibitor, cycloheximide. We established HCT116 clones stably expressing either the vegf 5 ' UTR or the mutated 5 ' UTR. The clones expressing the 5 ' UTR, but not the mutated one, showed increased anchorage-independent growth in vitro and formed progressive tumors when implanted in athymic nude mice. Microarray and quantitative real-time PCR analyses indicated that the vegf 5 ' UTR-expressing tumors had up-regulated anti-apoptotic genes, multidrug-resistant genes, and growth-promoting genes, while pro-apoptotic genes were down-regulated. Notably, expression of signal transducers and activators of transcription 1 (STAT1) was markedly repressed in the 5 ' UTR-expressing tumors, resulting in down-regulation of a STAT1-responsive cluster of genes (43 genes). As a result, the tumors did not respond to interferon (IFN)alpha therapy at all. We showed that stable silencing of endogenous vegf mRNA in HCT116 cells enhanced both STAT1 expression and IFN alpha responses.ConclusionsThese findings suggest that cancer cells have a survival system that is regulated by vegf mRNA and imply that both vegf mRNA and its protein may synergistically promote the malignancy of tumor cells. Therefore, combination of anti-vegf transcript strategies, such as siRNA-based gene silencing, with anti-VEGF antibody treatment may improve anti-cancer therapies that target VEGF.