Apoptosis Induced by Knockdown of uPAR and MMP-9 is Mediated by Inactivation of EGFR/STAT3 Signaling in Medulloblastoma

BackgroundMedulloblastoma is a highly invasive cancer of central nervous system diagnosed mainly in children. Matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator receptor (uPAR) are over expressed in several cancers and well established for their roles in tumor progression. The present study is aimed to determine the consequences of targeting these molecules on medulloblastoma progression. Methodology/Principal FindingsRadiation is one of the foremost methods applied for treating cancer and considerable evidence showed that radiation elevated uPAR and MMP-9 expression in medulloblastoma cell. Therefore efforts are made to target these molecules in non-irradiated and irradiated medulloblastoma cells. Our results showed that siRNA-mediated knockdown of uPAR and MMP-9, either alone or in combination with radiation modulated a series of events leading to apoptosis. Down regulation of uPAR and MMP-9 inhibited the expression of anti-apoptotic molecules like Bcl-2, Bcl-xL, survivin, XIAP and cIAPI; activated BID cleavage, enhanced the expression of Bak and translocated cyctochrome C to cytosol. Capsase-3 and -9 activities were also increased in uPAR- and MMP-9-downregulated cells. The apoptosis induced by targeting MMP-9 and uPAR was initiated by inhibiting epidermal growth factor receptor (EGFR) mediated activation of STAT3 and NF-κB related signaling molecules. Silencing uPAR and MMP-9 inhibited DNA binding activity of STAT3 and also reduced the recruitment of STAT3 protein at the promoter region of Bcl-2 and survivin genes. Our results suggest that inhibiting uPAR and MMP-9 reduced the expression of anti-apoptotic molecules by inactivating the transcriptional activity of STAT3. In addition, treating pre-established medulloblastoma with siRNAs against uPAR and MMP-9 both alone or in combination with radiation suppressed uPAR, MMP-9, EGFR, STAT3 expression and induced Bak activation leading to apoptosis. Conclusion/SignificanceTaken together, our results illustrated that RNAi mediated targeting of uPAR and MMP-9 might have therapeutic potential against medulloblastoma.

背景：髓母细胞瘤（Medulloblastoma）是一类主要发病人群为儿童的高侵袭性中枢神经系统恶性肿瘤。基质金属蛋白酶-9（Matrix metalloproteinase-9, MMP-9）与尿激酶型纤溶酶原激活物受体（urokinase plasminogen activator receptor, uPAR）在多种癌症中呈过表达状态，且已被证实其在肿瘤进展过程中发挥关键作用。本研究旨在明确靶向这两种分子对髓母细胞瘤进展的影响。

方法与主要结果：放疗是临床治疗癌症的核心手段之一，已有大量研究证实，放疗可上调髓母细胞瘤细胞中uPAR与MMP-9的表达。因此，本研究尝试在未接受放疗及接受放疗的髓母细胞瘤细胞中靶向干预这两种分子。结果显示，无论是单独使用小干扰RNA（small interfering RNA, siRNA）介导uPAR与MMP-9敲低，还是联合放疗，均可调控一系列细胞事件，最终诱导细胞凋亡。下调uPAR与MMP-9的表达可抑制抗凋亡分子Bcl-2、Bcl-xL、survivin、XIAP及cIAP1的转录与表达；激活BID蛋白切割，增强Bak蛋白的表达，并促进细胞色素C（cytochrome C）转位至胞浆。在uPAR与MMP-9表达下调的细胞中，半胱天冬酶-3（Caspase-3）与半胱天冬酶-9（Caspase-9）的活性亦显著升高。靶向MMP-9与uPAR所诱导的细胞凋亡，是通过抑制表皮生长因子受体（epidermal growth factor receptor, EGFR）介导的STAT3及核因子κB（nuclear factor kappa B, NF-κB）相关信号分子活化而启动的。沉默uPAR与MMP-9可抑制STAT3的DNA结合活性，同时减少STAT3蛋白在Bcl-2及survivin基因启动子区域的募集。本研究结果表明，抑制uPAR与MMP-9的表达可通过灭活STAT3的转录活性，下调抗凋亡分子的表达水平。此外，针对已建立的髓母细胞瘤模型，单独使用或联合放疗使用靶向uPAR与MMP-9的siRNA，均可抑制uPAR、MMP-9、EGFR及STAT3的表达，并激活Bak蛋白，进而诱导细胞凋亡。

结论与意义：综上，本研究结果证实，RNA干扰（RNA interference, RNAi）介导的uPAR与MMP-9靶向治疗或许具备治疗髓母细胞瘤的临床应用潜力。