Inhibition of monocarboxylate transporter-4 depletes stem-like glioblastoma cells and inhibits HIF transcriptional response in a lactate-independent manner.
Inhibition of monocarboxylate transporter-4 depletes stem-like glioblastoma cells and inhibits HIF transcriptional response in a lactate-independent manner.
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胶质母细胞瘤(GBM)是最常见的成人恶性脑肿瘤，常有缺氧区存在，肿瘤缺氧区的增加与临床预后不良有关。为了揭示低氧中的重要基因，我们在低氧条件下处理GBM神经球，并确定单羧酸转运体4(MCT4)是最上调的基因之一。为了研究MCT4在GBM中的临床重要性，我们检查了临床结果，发现MCT4的过度表达与患者较短的生存期有关。与此一致的是，MCT4上调与GBM侵袭性间充质亚群相关，而MCT4下调与GBM侵袭性较弱的G-CIMP(胶质瘤CpG甲基化表型)亚型相关。组织芯片免疫组织化学分析证实，与低级别星形细胞瘤相比，高级别星形细胞瘤MCT4蛋白水平升高，进一步表明MCT4是一个临床相关的靶点。为了在体外测试对MCT4的需求，我们用慢病毒转导含有针对MCT4的短发夹RNA(ShRNAs)的神经球，导致两个株系在低氧条件下生长抑制50-80%。MCT4基因敲除与干细胞标记物CD133表达的细胞百分比降低和细胞凋亡率增加有关。我们还发现，流式分选的CD133阳性细胞的MCT4水平几乎是CD133阴性细胞的6倍，这表明干细胞群体可能对MCT4有更高的需求。最重要的是，MCT4沉默也减缓了体内GBM异种移植的生长。有趣的是，尽管MCT4是一种很好的乳酸输出物，但我们发现细胞内和细胞外的乳酸水平在MCT4沉默后都没有改变，这表明了一种新的乳酸输出不依赖于乳酸输出物的机制来抑制基底膜的生长。为了确定这一可能的机制，我们对对照和shMCT4表达的神经球进行了微阵列分析，发现MCT4被敲除后，多个缺氧诱导因子(HIF)调节基因的表达显著减少。使用依赖于缺氧反应元件(HRE)的绿色荧光蛋白(GFP)报告线进一步验证了HIF转录反应的总体减少。
Hypoxic regions are frequent in glioblastoma (GBM), the most common type of malignant adult brain tumor, and increased levels of tumor hypoxia have been associated with worse clinical outcomes. To unmask genes important in hypoxia, we treated GBM neurospheres in hypoxia and identified monocarboxylate transporter-4 (MCT4) as one of the most upregulated genes. To investigate the clinical importance of MCT4 in GBM, we examined clinical outcomes and found that MCT4 overexpression is associated with shorter patient survival. Consistent with this, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM, and MCT4 downregulation correlated with the less aggressive G-CIMP (Glioma CpG Methylator Phenotype) subset of GBM. Immunohistochemical analysis of tissue microarrays confirmed that MCT4 protein levels were increased in high-grade as compared with lower-grade astrocytomas, further suggesting that MCT4 is a clinically relevant target. To test the requirement for MCT4 in vitro, we transduced neurospheres with lentiviruses encoding short-hairpin RNAs (shRNAs) against MCT4, resulting in growth inhibition of 50–80% under hypoxia in two lines. MCT4 knockdown was associated with a decreased percentage of cells expressing the stem-cell marker CD133 and increased apoptotic fraction. We also found that flow-sorted CD133-positive cells had almost sixfold higher MCT4 levels than CD133-negative cells, suggesting that the stem-like population might have a greater requirement for MCT4. Most importantly, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo. Interestingly, whereas MCT4 is a well-characterized lactate exporter, we found that both intracellular and extracellular lactate levels did not change following MCT4 silencing, suggesting a novel lactate export-independent mechanism for growth inhibition in GBMs. To identify this potential mechanism, we performed microarray analysis on control and shMCT4-expressing neurospheres and found a dramatic reduction in the expression of multiple Hypoxia-Inducible Factor (HIF)-regulated genes following MCT4 knockdown. The overall reduction in HIF transcriptional response was further validated using a hypoxia response element (HRE)-dependent green-fluorescent protein (GFP) reporter line.
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DOI：
10.4161/cc.8.20.9701
发表时间：
2009-10-15
期刊：
Cell cycle (Georgetown, Tex.)
影响因子：
--
作者：
Heddleston JM;Li Z;McLendon RE;Hjelmeland AB;Rich JN
通讯作者：
Rich JN
影响因子：
5.2
作者：
Bar, Eli E.;Chaudhry, Aneeka;Eberharta, Charles G.
通讯作者：
Eberharta, Charles G.
影响因子：
3.3
作者：
Li, Kay Ka Wai;Pang, Jesse Chung-sean;Ng, Ho-keung
通讯作者：
Ng, Ho-keung
影响因子：
6.6
作者：
Cheng, Chang;Edin, Nina F. Jeppesen;Bergersen, Linda H.
通讯作者：
Bergersen, Linda H.
影响因子：
4.1
作者：
Dimmer, KS;Friedrich, B;Bröer, S
通讯作者：
Bröer, S