同时靶向 RSK 和 AKT 可有效抑制 YB-1 介导的乳腺癌细胞中电离辐射诱导的 DNA 双链断裂修复。

Simultaneous Targeting of RSK and AKT Efficiently Inhibits YB-1-Mediated Repair of Ionizing Radiation-Induced DNA Double-Strand Breaks in Breast Cancer Cells.

机构信息

Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tübingen, Tübingen, Germany; German Cancer Consortium (DKTK), partner site Tübingen, and German Cancer Research Center (DKFZ), Heidelberg, Germany.

出版信息

Int J Radiat Oncol Biol Phys. 2021 Feb 1;109(2):567-580. doi: 10.1016/j.ijrobp.2020.09.005. Epub 2020 Sep 12.

DOI:10.1016/j.ijrobp.2020.09.005

PMID:32931865

Abstract

PURPOSE

Y-box binding protein 1 (YB-1) overexpression is associated with chemotherapy- and radiation therapy resistance. Ionizing radiation (IR), receptor tyrosine kinase ligands, and mutation in KRAS gene stimulate activation of YB-1. YB-1 accelerates the repair of IR-induced DNA double-strand breaks (DSBs). Ribosomal S6 kinase (RSK) is the main kinase inducing YB-1 phosphorylation. We investigated the impact of RSK targeting on DSB repair and radiosensitivity.

MATERIALS AND METHODS

The triple negative breast cancer (TNBC) cell lines MDA-MB-231, MDA-MB-468, and Hs 578T, in addition to non-TNBC cell lines MCF7, HBL-100, and SKBR3, were used. MCF-10A cells were included as normal breast epithelial cells. The RSK inhibitor LJI308 was used to investigate the role of RSK activity in S102 phosphorylation of YB-1 and YB-1-associated signaling pathways. The activation status of the underlying pathways was investigated by Western blotting after treatment with pharmacologic inhibitors or transfection with siRNA. The impact of LJI308 on DSB repair and postirradiation cell survival was tested by the γH2AX foci and the standard clonogenic assays, respectively.

RESULTS

LJI308 inhibited the phosphorylation of RSK (T359/S363) and YB-1 (S102) after irradiation, treatment with EGF, and in cells expressing a KRAS mutation. LJI308 treatment slightly inhibited DSB repair only in some of the cell lines tested. This was shown to be due to PI3K-dependent stimulation of AKT or constitutive AKT activity mainly in cancer cells but not in normal breast epithelial MCF-10A cells. Simultaneous targeting of AKT and RSK strongly blocked DSB repair in all cancer cell lines, independent of TNBC status or KRAS mutation, with a minor effect in MCF-10A cells. Cotargeting of RSK- and AKT-induced radiation sensitivity in TNBC MDA-MB-231 and non-TNBC MCF7 cells but not in MCF-10A cells.

CONCLUSIONS

Simultaneous targeting of RSK and AKT might be an efficient approach to block the repair of DSBs after irradiation and to induce radiosensitization of breast cancer cells.

摘要

目的

Y 盒结合蛋白 1（YB-1）过表达与化疗和放疗抵抗有关。电离辐射（IR）、受体酪氨酸激酶配体和 KRAS 基因突变刺激 YB-1 的激活。YB-1 加速了 IR 诱导的 DNA 双链断裂（DSB）的修复。核糖体 S6 激酶（RSK）是诱导 YB-1 磷酸化的主要激酶。我们研究了 RSK 靶向对 DSB 修复和放射敏感性的影响。

材料和方法

使用三阴性乳腺癌（TNBC）细胞系 MDA-MB-231、MDA-MB-468 和 Hs 578T，以及非 TNBC 细胞系 MCF7、HBL-100 和 SKBR3，另外还包括正常乳腺上皮细胞 MCF-10A。使用 RSK 抑制剂 LJI308 研究 RSK 活性在 YB-1 的 S102 磷酸化和 YB-1 相关信号通路中的作用。用药物抑制剂处理或 siRNA 转染后，通过 Western 印迹法研究潜在通路的激活状态。通过 γH2AX 焦点和标准克隆形成测定法分别测试 LJI308 对 DSB 修复和照射后细胞存活的影响。

结果

LJI308 抑制了照射后、EGF 处理和表达 KRAS 突变的细胞中 RSK（T359/S363）和 YB-1（S102）的磷酸化。LJI308 处理仅在一些测试的细胞系中轻微抑制 DSB 修复。这是由于 PI3K 依赖性刺激 AKT 或 AKT 的组成性活性引起的，主要在癌细胞中，但不在正常乳腺上皮 MCF-10A 细胞中。在所有癌细胞系中，同时靶向 AKT 和 RSK 可强烈阻断 DSB 修复，与 TNBC 状态或 KRAS 突变无关，而在 MCF-10A 细胞中影响较小。在 TNBC MDA-MB-231 和非 TNBC MCF7 细胞中，同时靶向 RSK 和 AKT 诱导的放射敏感性，但在 MCF-10A 细胞中没有。