Serum-and Glucocorticoid-inducible Kinase 1 Is Essential For Osteoclastogenesis And Promotes Breast Cancer Bone Metastasis

Purpose1.To study the role of SGK1 on osteoclast formation and function and its underlying molecular mechanism;2.To observe the effect of SGK1 inhibitor on bone and other tissues of ovariectomized mice by intraperitoneal injection;3.To select breast cancer bone metastasis cell lines and establish bone metastasis animal models,and to observe the effect of SGK1 inhibitors on bone metastasis.4.To analyze the differential expression of SGK1 gene in breast cancer patients and its relationship with clinical results.Methods1.Use RT-q PCR and Western Blot to detect the changes of SGK1 gene expression in the process of osteoclast differentiation in RANKL-induced murine primary bone marrow mononuclear cells（BMM）and murine mononuclear cell line RAW 264.7.The osteoclast differentiation efficiency and function are detected by TRAP staining and bone plate resorption experiments.2.Use small molecule inhibitors of SGK1:GSK 650394,SGK1-sh RNA,SGK1-si RNA,to inhibit or silence SGK 1 protein,gene,or mRNA and interfere RANKL-induced osteoclast precursor cell differentiation process.3.Use RT-q PCR,Western Blot,or other methods to detect the expression changes of transcription factors and signaling pathways.Use Western Blot,immunofluorescence,Ca²⁺fluorescence probe to detect Orai1 channel’s protein expression and changes in functions.4.Establish ovariectomized（OVX）mouse model and intraperitoneally inject GSK 650394 in the mice.8 weeks later,collect mice tibia,femur,serum.Sample of femurs for Micro-CT scanning for bone mass analysis.Sections of the samples are for histological analysis.Serum are for detecting the CTX,OPG,and RANKL levels.5.Select breast cancer bone metastasis cell lines,establish mouse model of bone metastasis.Inject GSK650394 intraperitoneally to observe the incidence of bone metastasis and status of bone destruction.6.Use Oncomine and Onco Lnc database to analyze the differential expression of SGK 1 in different subtypes of breast cancer patients and its relationship with survival.Results1.During RANKL-induced osteoclast differentiation,SGK1 mRNA and protein levels were significantly increased.And SGK 1 inhibitors could effectively reduce the osteoclast precursor cell differentiation efficiency and osteoclast bone resorption function.2.After inhibiting the expression or function of SGK1,NF-k B and PI3K/AKT signaling pathways and osteoclast-related transcription factors NFATc1and c-Fos were significantly inhibited.3.In the process of RANKL-induced osteoclast differentiation,the expression of Orai1 increased,its calcium influx effect was enhanced,and the differentiation ability of osteoclast precursor cells became stronger after Orai1 overexpressed.And this phenomenon can be reversed by GSK 650394.4.In the OVX mouse model,intraperitoneal injection of SGK1 inhibitor can significantly reduce bone loss.In bone metastasis mouse models,intraperitoneal injection of SGK1 inhibitors can significantly reduce the incidence of bone metastases.5.In breast cancer patients,the mRNA level of SGK 1 is significantly higher than that of healthy people.KM plotter’s survival analysis based on a large scale of samples shows that SGK1 has no significant effect on the prognosis of breast cancer patients,but we found high expression of SGK1 can predict poor prognosis in other data sets GSE2603and GSE12276,suggesting that SGK1 has potential carcinogenicity.ConclusionAccording to the results of this study,increased expression of SGK 1 is an important activator in the process of osteoclast differentiation,bone resorption and bone metastasis.Through upregulation of NF-k B and PI3K/AKT pathway,SGK 1 can pro-mote the abundance and activity of osteoclast precursor cell membrane SOCE channel Orai1,and induce intracellular calcium oscillations(Ca²⁺Oscillations).Then transcription factors NFATc1 and c-Fos are activated,initiating osteoclast differentiation.SGK 1 inhibitors may reduce the incidence of bone metastasis by inhibiting osteoclastogenesis and its function,or direct cytotoxic effects on tumor cells.Although analysis through different database showed that SGK 1 overall prognosis for breast cancer patients is not yet clear（more precise conclusion requires further study in a particular cohort of adjustment for confounding factors）,SGK1 is still expected to be a therapeutic target for bone metastases.