Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, USA.
Mol Cancer Ther. 2010 Jun;9(6):1831-41. doi: 10.1158/1535-7163.MCT-09-0880. Epub 2010 May 25.
Clusterin (CLU), in its cytoplasmic form, is abundant in many advanced cancers and has been established to be cytoprotective against chemotherapeutic agents including docetaxel. However, little is known of the mechanism of its induction. Here, we provide evidence that AKT plays a critical role in upregulating cytoplasmic/secretory sCLU, which is responsible for docetaxel resistance. Western blot analysis indicated that docetaxel-resistant sublines derived from DU145 and PC3 prostate tumor cell lines displayed a markedly increased phospho-AKT level closely accompanied by heightened sCLU expression when compared with parental cells. To examine if AKT has a role in sCLU expression, AKT blockade was done by treatment with a specific inhibitor, API-2, or dominant-negative AKT transduction before analysis of sCLU gene expression. Loss of AKT function resulted in loss of sCLU and was accompanied by chemosensitization to docetaxel and increased cell death via a caspase-3-dependent pathway. To confirm that AKT affected resistance to docetaxel through sCLU and not through other mediators, tumor cells were first transfected with full-length CLU for overexpression and then treated with the AKT inhibitor API-2. We found that once sCLU was overexpressed, API-2 could not chemosensitize the tumor cells to docetaxel. Thus, the chemoresistance to docetaxel is mediated by sCLU and it can be induced by AKT. Lastly, AKT was found to mediate sCLU induction via signal transducer and activator of transcription 1 activation, which we have earlier shown to drive sCLU gene expression. These results identify a previously unrecognized pathway linking AKT to cytoprotection by sCLU in tumor cells.
簇集蛋白（CLU）在其细胞质形式中在许多晚期癌症中含量丰富，并已被证实对包括多西紫杉醇在内的化疗药物具有细胞保护作用。然而，其诱导机制知之甚少。在这里，我们提供的证据表明 AKT 在上调细胞质/分泌型 sCLU 中起关键作用，sCLU 负责多西紫杉醇耐药。Western blot 分析表明，与亲本细胞相比，源自 DU145 和 PC3 前列腺肿瘤细胞系的多西紫杉醇耐药亚系显示出明显增加的磷酸化 AKT 水平，同时伴随着 sCLU 表达的升高。为了研究 AKT 是否在 sCLU 表达中起作用，在用特异性抑制剂 API-2 处理或转导显性失活 AKT 后进行 sCLU 基因表达分析之前，对 AKT 进行了阻断。AKT 功能的丧失导致 sCLU 的丧失，并伴有对多西紫杉醇的化疗增敏作用，通过 caspase-3 依赖性途径导致细胞死亡增加。为了确认 AKT 通过 sCLU 而不是通过其他介质影响多西紫杉醇的耐药性，首先用全长 CLU 转染肿瘤细胞以过表达，然后用 AKT 抑制剂 API-2 处理。我们发现，一旦 sCLU 过表达，API-2 就不能使肿瘤细胞对多西紫杉醇产生化疗增敏作用。因此，多西紫杉醇的耐药性是由 sCLU 介导的，它可以被 AKT 诱导。最后，发现 AKT 通过信号转导和转录激活因子 1 的激活来介导 sCLU 的诱导，我们之前已经证明该激活因子可驱动 sCLU 基因表达。这些结果确定了一种以前未被识别的途径，将 AKT 与肿瘤细胞中 sCLU 的细胞保护作用联系起来。
