Mechanisms Underlying The Role Of RSK2 In Promoting The Proliferation And Migration In HER2 Positive Breast Cancer Cells

Background and objectives: Breast Cancer is the most common cancer in women,which seriously affects women’s health and life quality.HER2 positive breast cancer is a very dangerous subtype of breast cancer,accounting for about 15-20%.It has rapid tumor progression,is prone to lymph node or blood metastasis,and is not sensitive to endocrine therapy.There has been a variety of anti-tumor drugs targeting HER2,such as trastuzumab,lapatinib and so on,but due to the low sensitivity of tumor cells to drugs or drug resistance,eventually lead to recurrence.Therefore,developing novel targeted therapies for HER2 positive breast cancer is still an urgent problem to be solved.In recent years,a large number of studies have shown that RSK2 is highly expressed in a variety of tumor tissues,and promotes cell proliferation and metastasis,playing a key role in cancer progression.Therefore,targeting RSK2 represents a potential strategy for the treatment of many types of human cancer.It has been reported that RSK2 promotes cell metastasis in HER2 positive breast cancer,however,the related molecular mechanism is not clear.Therefore,the purpose of this study is to explore the role and underlying mechanism of RSK2 in HER2 positive breast cancer,which will provide new ideas and directions for targeted therapy and combined drug treatment of HER2 positive breast cancer.Methods: 1.The expressions of RSK2 in various subtypes of breast cancer and its relationship with prognosis were analyzed by database.2.The expression of RSK2 was detected by immunohistochemistry in clinical samples,and the relationship between the expression level of RSK2 and the prognosis of patients was analyzed.3.HER2 positive breast cancer cells were transfected with RSK2 si RNA or treated with RSK2 inhibitor LJH685,and cell survival and proliferation were detected by CCK-8 and Ed U assays.4.HER2 positive breast cancer cells were transfected with RSK2 si RNA or treated with RSK2 inhibitor LJH685,and the cell migration was detected by wound healing assay.5.HER2 positive breast cancer cells were transfected with RSK2 si RNA,followed by lapatinib treatment,and the cell survival and proliferation were detected by CCK-8 and Ed U assays.6.HER2 positive breast cancer cells were treated with Lapatinib and RSK2 inhibitor LJH685 simultaneously,and the cell survival and proliferation were detected by CCK-8 and Ed U assays.7.HER2 positive breast cancer cells were transfected with RSK2 si RNA,and the expression of HER2 protein was examined by Western blot,and the level of HER2 m RNA was detected by QPCR assay.8.HER2 positive breast cancer cells were transfected with RSK2 si RNA,followed by proteasome inhibitor MG132 or autophagy inhibitor chloroquine(CQ)treatment,and on the expression of HER2 protein was detected by Western blot.9.HER2 positive breast cancer cells with overexpressed RSK2 were treated with the protein synthesis inhibitor cycloheximide(CHX),and the expression of HER2 protein was detected by Western blot assay.10.HEK293 T cells with RSK2 overexpression or knockdown were transfected with the ubiquitin plasmid,and the effect of RSK2 on the ubiquitination of HER2 was detected by co-immunoprecipitation assay.11.The binding of RSK2 and HER2 was detected by co-immunoprecipitation and GST pull-down assay.12.The effect of RSK2 on HER2 phosphorylation was detected by in vitro kinase assay.Results: 1.Database analysis and immunohistochemical results of clinical samples showed that RSK2 was highly expressed in HER2 positive breast cancer cells,and is associated with poor prognosis of patients.2.Silencing RSK2 or RSK2 inhibitor LJH685 significantly inhibited the cell proliferation and migration of HER2 positive breast cancer cells.3.Silencing RSK2 or RSK2 inhibitor LJH685 can significantly enhance the sensitivity of HER2 positive breast cancer cells to lapatinib.4.Silencing of RSK2 down-regulated the protein expression of HER2,but did not affect the m RNA level of HER2.5.RSK2 inhibited the degradation of HER2 by proteasomal pathway.6.RSK2 decreased the ubiquitin level of HER2,inhibits its proteasomal degradation,thus up-regulating its protein expression.7.The interaction between RSK2 and HER2 was demonstrated with co-immunoprecipitation and GST pull-down assay.8.RSK2 phosphorylated HER2 at the serine/threonine site was demonstrated by the in vitro kinase assay.Conclusion: Silencing of RSK2 inhibits the cell proliferation and migration in HER2 positive breast cancer cells.Inhibition of RSK2 enhanced the sensitivity of HER2 positive breast cancer cells to lapatinib.RSK2 can directly bind to HER2,promote HER2 phosphorylation and inhibit its ubiquitination,block its degradation through proteasomal pathway,thus up-regulating HER2 protein expression.There are 22 figures,9 tables and 78 references in this paper.