The Role and Mechanism of CRT0066101 as an Effective Drug for Treatment of Triple-Negative Breast Cancer.
The Role and Mechanism of CRT0066101 as an Effective Drug for Treatment of Triple-Negative Breast Cancer.
复制标题
CRT0066101作为治疗三阴性乳腺癌的有效药物的作用和机制。
DOI：
10.33594/000000027
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发表时间：
2019-01-01
期刊：
影响因子：
--
通讯作者：
Chen, Liming
中科院分区：
文献类型：
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作者：
Liu, Yan;Wang, Yuzhi;Chen, Liming
背景/目的
乳腺癌在临床上分为三种主要亚型：雌激素受体阳性（ER+）乳腺癌、人表皮生长因子受体2阳性（HER 2+）乳腺癌和三阴性乳腺癌（TNBC）。在没有特异性靶向治疗的情况下，与ER+和HER 2+乳腺癌患者相比，TNBC患者的预后较差。蛋白激酶D（PRKD）家族成员在癌症进展中起着至关重要的作用。CRT 0066101是一种PRKD抑制剂，已报告在许多癌症类型中具有抗癌活性。然而，CRT 0066101在TNBC中的作用和机制尚未得到很好的研究。
方法
在乳腺癌样品和乳腺癌细胞系中分析PRKD的表达水平。分别通过细胞计数试剂盒8试验、细胞周期试验、碘化丙啶/膜联蛋白-V试验和异种移植小鼠模型研究了CRT 0066101抑制PRKD活性对TNBC细胞增殖、细胞周期、凋亡和肿瘤生长的影响。为了揭示CRT 0066101在TNBC中的分子机制，使用iTRAQ进行比较磷酸蛋白质组学分析。
结果
我们发现PRKD 2和PRKD 3在乳腺癌中优先表达。免疫组化证实PRKD 2和PRKD 3在TNBC中过表达。CRT 0066101可抑制PRKD的活性，在体外显著抑制增殖，增加TNBC细胞的凋亡和G1期细胞群，并在体内减少乳腺肿瘤体积。在接受和不接受CRT 0066101治疗的乳腺癌细胞之间进行的比较磷酸化蛋白质组学分析显示，抗乳腺癌作用涉及对复杂网络的调节，该复杂网络包含多种富集途径和促进多种癌症相关过程的几个枢纽节点，从而解释了CRT 0066101在体外和体内对TNBC的所述作用。最后，我们通过用CRT 0066101和针对PRKD 2和PRKD 3的小干扰RNA（siPRKD 2和siPRKD 3）处理来验证PRKD抑制的几个靶点，包括p-MYC（T58/ S62）、p-MAPK 1/3（T202/Y204）、p-AKT（S473）、p-YAP（S127）和p-CDC 2（T14）。
结论
PRKD抑制剂CRT 0066101通过调节磷酸化信号网络和抑制许多癌症驱动因子（包括MYC、MAPK 1/3、AKT、雅普和CDC 2）的磷酸化来发挥抗TNBC作用，从而深入了解CRT 0066101作为TNBC有效药物的重要作用及其分子机制。
BACKGROUND/AIMS
Breast cancer is clinically classified into three main subtypes: estrogen receptor-positive (ER+) breast cancer, human epidermal growth factor receptor 2-positive (HER2+) breast cancer, and triple-negative breast cancer (TNBC). Without specific targeted therapies, patients with TNBC have poorer prognosis compared with those with ER+ and HER2+ breast cancer. Protein kinase D (PRKD) family members play crucial roles in cancer progression. CRT0066101, a PRKD inhibitor, has been reported to have anticancer activity in many cancer types. Nevertheless, the role and mechanism of CRT0066101 in TNBC have not been well investigated.
METHODS
The expression level of PRKDs was analyzed in breast cancer samples and breast cancer cell lines. The effects of inhibiting PRKD activity with CRT0066101 on TNBC cell proliferation, cell cycle, apoptosis, and tumor growth were studied by Cell Counting Kit8 assay, cell cycle assay, propidium iodide/annexin-V assay, and a xenograft mouse model, respectively. To uncover the molecular mechanism of CRT0066101 in TNBC, comparative phosphoproteomic analysis using iTRAQ was employed.
RESULTS
We found that PRKD2 and PRKD3 were preferentially expressed in breast cancers. Immunohistochemistry confirmed the overexpression of PRKD2 and PRKD3 in TNBC. CRT0066101, which inhibited the activity of PRKDs, dramatically inhibited proliferation, increased apoptosis and the G1-phase population of TNBC cells in vitro, and reduced breast tumor volume in vivo. Comparative phosphoproteomic analysis between breast cancer cells with and without CRT0066101 treatment revealed that the anti-breast cancer effects involved regulation of a complex network containing multiple enriched pathways and several hub-nodes contributing to multiple cancer-related processes, thus explaining the described effects of CRT0066101 on TNBC in vitro and in vivo. Finally, we validated several targets of PRKD inhibition by treatment with CRT0066101 and small interfering RNAs against PRKD2 and PRKD3 (siPRKD2 and siPRKD3), including p-MYC(T58/ S62), p-MAPK1/3(T202/Y204), p-AKT(S473), p-YAP(S127), and p-CDC2(T14).
CONCLUSION
PRKD inhibitor CRT0066101 exhibits anti-TNBC effects via modulating a phosphor-signaling network and inhibiting the phosphorylation of many cancer-driving factors, including MYC, MAPK1/3, AKT, YAP, and CDC2, providing insight into the important roles as well as the molecular mechanism of CRT0066101 as an effective drug for TNBC.