SUMOylation modulates FOXK2-mediated paclitaxel sensitivity in breast cancer cells.
SUMOylation modulates FOXK2-mediated paclitaxel sensitivity in breast cancer cells.
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DOI：
10.1038/s41389-018-0038-6
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发表时间：
2018-03-13
期刊：
影响因子：
6.2
通讯作者：
Lam EW
中科院分区：
文献类型：
--
作者：
Nestal de Moraes G;Ji Z;Fan LY;Yao S;Zona S;Sharrocks AD;Lam EW
叉头转录因子FOXK2在抑制乳腺癌肿瘤发生和介导细胞毒性药物作用中起关键作用。然而，调控FOXK2生物学功能的机制仍然知之甚少。在这里，我们研究了SUMOylation在调节foxk2介导的药物敏感性中的作用。我们在FOXK2序列中确定了SUMOylation共识基序，并通过将赖氨酸527和633分别转化为精氨酸，将谷氨酸529和635分别转化为丙氨酸，构建了两个SUMOylation缺陷双突变体。我们发现，与野生型（WT） FOXK2相比，FOXK2 summoyl-缺陷（K527/633 R）和（E529/635 A）突变体在介导紫杉醇的细胞毒性功能方面都无效。当过表达时，与野生型（WT） FOXK2不同，K527/633 R突变体对MCF-7和MDA-MB-231细胞对紫杉醇的敏感性几乎没有影响，通过细胞活力和克隆实验检测。我们的研究结果还表明，与野生型FOXK2相比，过表达K527/633 R突变形式FOXK2或空表达载体的MCF-7细胞具有较低的肿瘤抑制转录靶点FOXO3的蛋白质和mRNA水平。ChIP实验一致显示，与野生型FOXK2不同，sumoylylation -defective （K527/633 R）突变体不能与FOXO3启动子结合，尽管在MCF-7细胞中表达了与野生型FOXK2相当水平的蛋白质，并且具有相同的亚细胞定位。有趣的是，野生型和K527/633 R突变体FOXK2的表达对MCF-7 TaxR紫杉醇耐药细胞的增殖和紫杉醇敏感性都没有影响。与此一致的是，野生型和（K527/633 R）突变体FOXK2在这些细胞中都不能与内源性FOXO3启动子结合。总之，我们的研究结果表明，SUMOylation正调节FOXK2的转录活性，并通过肿瘤抑制因子FOXO3介导紫杉醇的细胞毒性反应。
The forkhead transcription factor FOXK2 plays a critical role in suppressing tumorigenesis and mediating cytotoxic drug action in breast cancer. However, the mechanism by which the biological function of FOXK2 is regulated remains poorly understood. Here, we investigated the role of SUMOylation in modulating FOXK2-mediated drug sensitivity. We identified SUMOylation consensus motifs within the FOXK2 sequence and constructed two SUMOylation-defective double mutants by converting lysine 527 and 633 to arginines and glutamic acid 529 and 635 to alanines, respectively. We found that both the FOXK2 SUMOylation-deficient (K527/633 R) and (E529/635 A) mutants were ineffective in mediating the cytotoxic function of paclitaxel when compared to the wild-type (WT) FOXK2. When overexpressed, unlike the wild-type (WT) FOXK2, the K527/633 R mutant had little effect on the sensitivity of MCF-7 and MDA-MB-231 cells to paclitaxel, as examined by cell viability and clonogenic assays. Our results also showed that MCF-7 cells overexpressing the K527/633 R mutant form of FOXK2 or the empty expression vector have lower protein and mRNA levels of its tumour suppressive transcriptional target FOXO3 compared to the wild-type FOXK2. Consistently, ChIP assays revealed that unlike wild-type FOXK2, the SUMOylation-defective (K527/633 R) mutant is unable to bind to the FOXO3 promoter, despite expressing comparable levels of protein and having the same subcellular localization as the wild-type FOXK2 in MCF-7 cells. Interestingly, expression of neither the wild-type nor the K527/633 R mutant FOXK2 had any effect on the proliferation and paclitaxel sensitivity of the MCF-7 TaxR paclitaxel-resistant cells. In agreement, both the wild-type and the (K527/633 R) mutant FOXK2 failed to bind to the endogenous FOXO3 promoter in these cells. Collectively, our results suggest that SUMOylation positively regulates FOXK2 transcriptional activity and has a role in mediating the cytotoxic response to paclitaxel through the tumour suppressor FOXO3.
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