Cancer Research Center of Lyon, Inserm U1052, CNRS UMR 5286, Université de Lyon, Centre Léon Bérard, 69373, Lyon, France.
Equipe Labellisée La Ligue Contre Le Cancer, Lyon, France.
J Exp Clin Cancer Res. 2023 Nov 27;42(1):318. doi: 10.1186/s13046-023-02909-5.
TGFβ induces several cell phenotypes including senescence, a stable cell cycle arrest accompanied by a secretory program, and epithelial-mesenchymal transition (EMT) in normal epithelial cells. During carcinogenesis cells lose the ability to undergo senescence in response to TGFβ but they maintain an EMT, which can contribute to tumor progression. Our aim was to identify mechanisms promoting TGFβ-induced senescence escape.
In vitro experiments were performed with primary human mammary epithelial cells (HMEC) immortalized by hTert. For kinase library screen and modulation of gene expression retroviral transduction was used. To characterize gene expression, RNA microarray with GSEA analysis and RT-qPCR were used. For protein level and localization, Western blot and immunofluorescence were performed. For senescence characterization crystal violet assay, Senescence Associated-β-Galactosidase activity, EdU staining were conducted. To determine RSK3 partners FLAG-baited immunoprecipitation and mass spectrometry-based proteomic analyses were performed. Proteosome activity and proteasome enrichment assays were performed. To validate the role of RSK3 in human breast cancer, analysis of METABRIC database was performed. Murine intraductal xenografts using MCF10DCIS.com cells were carried out, with histological and immunofluorescence analysis of mouse tissue sections.
A screen with active kinases in HMECs upon TGFβ treatment identified that the serine threonine kinase RSK3, or RPS6KA2, a kinase mainly known to regulate cancer cell death including in breast cancer, reverted TGFβ-induced senescence. Interestingly, RSK3 expression decreased in response to TGFβ in a SMAD3-dependent manner, and its constitutive expression rescued SMAD3-induced senescence, indicating that a decrease in RSK3 itself contributes to TGFβ-induced senescence. Using transcriptomic analyses and affinity purification coupled to mass spectrometry-based proteomics, we unveiled that RSK3 regulates senescence by inhibiting the NF-κΒ pathway through the decrease in proteasome-mediated IκBα degradation. Strikingly, senescent TGFβ-treated HMECs display features of epithelial to mesenchymal transition (EMT) and during RSK3-induced senescence escaped HMECs conserve EMT features. Importantly, RSK3 expression is correlated with EMT and invasion, and inversely correlated with senescence and NF-κΒ in human claudin-low breast tumors and its expression enhances the formation of breast invasive tumors in the mouse mammary gland.
We conclude that RSK3 switches cell fate from senescence to malignancy in response to TGFβ signaling.
TGFβ可诱导多种细胞表型，包括衰老、伴随有分泌程序的稳定细胞周期停滞以及正常上皮细胞中的上皮-间充质转化（EMT）。在癌变过程中，细胞失去了对 TGFβ诱导的衰老的反应能力，但它们保持 EMT，这可能有助于肿瘤进展。我们的目的是确定促进 TGFβ诱导的衰老逃逸的机制。
使用 hTert 永生化的原代人乳腺上皮细胞（HMEC）进行体外实验。为了进行激酶文库筛选和基因表达的调节，使用了逆转录病毒转导。为了描述基因表达，使用 RNA 微阵列和 GSEA 分析以及 RT-qPCR。为了检测蛋白质水平和定位，进行了 Western blot 和免疫荧光。为了进行衰老特征鉴定，进行了结晶紫测定、衰老相关-β-半乳糖苷酶活性、EdU 染色。为了确定 RSK3 的伙伴，进行了 FLAG 诱饵免疫沉淀和基于质谱的蛋白质组学分析。进行了蛋白酶体活性和蛋白酶体富集测定。为了验证 RSK3 在人类乳腺癌中的作用，对 METABRIC 数据库进行了分析。使用 MCF10DCIS.com 细胞进行了小鼠乳腺内异种移植实验，并对小鼠组织切片进行了组织学和免疫荧光分析。
在 TGFβ处理的 HMEC 中进行的活性激酶筛选发现，丝氨酸/苏氨酸激酶 RSK3（也称为 RPS6KA2），一种主要用于调节包括乳腺癌在内的癌细胞死亡的激酶，逆转了 TGFβ诱导的衰老。有趣的是，RSK3 的表达在 TGFβ刺激下以 SMAD3 依赖性方式降低，其组成型表达挽救了 SMAD3 诱导的衰老，表明 RSK3 本身的减少有助于 TGFβ 诱导的衰老。使用转录组分析和亲和纯化结合基于质谱的蛋白质组学，我们揭示了 RSK3 通过抑制 NF-κB 途径来调节衰老，其通过减少蛋白酶体介导的 IκBα 降解来实现。引人注目的是，在 TGFβ 处理的衰老 HMEC 中显示出上皮到间充质转化（EMT）的特征，并且在 RSK3 诱导的衰老逃逸中，保留了 EMT 特征。重要的是，RSK3 的表达与 EMT 和侵袭相关，并且在 Claudin-low 型乳腺癌的人类肿瘤中与衰老和 NF-κB 呈负相关，其表达增强了在小鼠乳腺中形成乳腺浸润性肿瘤。
我们得出结论，RSK3 响应 TGFβ 信号转导将细胞命运从衰老转变为恶性。
