The Mechanism Of SHOC2 To Promote Breast Cancer Progression And Regulate The Sensitivity Of MTOR Inhitor For Breast Cancer

BackgroudBreast cancer is the most common type of cancer in women worldwide,and it involves a multi-step process associated with the abnormal expression of several oncogenes and tumor suppressor genes and the activation of signal pathways.In addition to surgery,chemotherapy,endocrine therapy and targeted therapy are important methods for breast cancer treatment.In recent years,everolimus has shown many productive results,across a variety of clinical trials.As such,everolimus(marketed as Afinitor)was approved for use in combination with the steroidal aromatase inhibitor exemestane in breast cancer patients with advanced cancer that is hormone receptor positive,HER-2 negative,where the cancer was refractory to non-steroidal aromatase inhibitors(all patients had received prior treatment with either letrozole or anastrazole).Whilst it is clear that the use of everolimus use in breast cancer have wide potential in the clinic,they also are associated with key issues that may ultimately limit their application and range in terms of therapeutic use.The inhibition of mTOR with everolimus can stimulate the activation of MAPK feedback pathways.Numerous types of malignant tumors are associated with the abnormal activation of Ras-Raf-ERK.Active.Extracellular stimulation signals convert GDP-Ras to GTP-Ras,GTP-Ras binds to and promotes Raf activation,and activates Raf.However,it is unclear how the signal specificity is achieved within the wide biological responses controlled by the ERK pathway.Scaffolding proteins facilitate interactions and functions of core signaling partners of the ERK pathway and play indispensable roles in controlling the specific ERK1/2 activities.Example of these proteins is leucine rich repeat scaffold protein SHOC2,which was identified as a positive regulator of the Ras pathway.SHOC2 has been reported to regulate the Ras signaling cascade in a number of ways;in particular,it has essential roles in embryogenesis and normal biological processes.Furthermore,the role of SHOC2 in various types of malignant cells has been examined,which has highlighted its potential role in tumorigenesis.SHOC2 has also been shown to be a positive regulator for Ras-Raf-ERK that contributes to the malignant properties of different tumor cells by modulating the growth,transformation,migration,invasion of cancer cells,and facilitate drug resistance.Therefore,as the key regulator of Raf-ERK signal pathway,SHOC2 might play an important role in the mechanism of resistance to everolimus for breast cancer.In this study,the association between SHOC2 upregulation and the clinicopathological features of breast cancer was investigated,as well as the prognostic value of SHOC2.We preliminary study the role of Raf-ERK pathway mediated by SHOC2 in the mechanism of mTOR inhibitor everolimus resistance for breast cancer,which will provide a theoretical and experimental evidence for the further study of breast cancer targeted therapyPart I Correlation between SHOC2 expression and pathologicalparameters and prognosis of breast cancer patients,and itsregulations on breast cancer cell proliferation,apoptosis and RaspathwayObjective::To study the expression of SHOC2 protein in the breast cancer tissues and the paired para-tumor breast tissue,analyse the correlation between SHOC2 expression and clinicopathologic parameters and research the significance of the SHOC2 expression in breast cancer.We furthermore examine the effects of SHOC2 expression on cell proliferation,cell cycle,apoptosis,and Ras pathway in vitro by breast cancer cells.Methods:Use the technique of RT-PCR、Western Blotting and IHC to detect the expression of SHOC2 mRNA and protein in tissues of invasive breast cancer and their para-tumor counterparts,respectively.Analyse the association between SHOC2 expression and clinicopathologic parameters and the prognostic significance for breast cancer patients.We established breast cancer cell line of stable SHOC2 knockdown and overexpression and then examine the cell proliferation,cell cycle,apoptosis,and Ras pathway by MTT,flow cytometry and Western Blotting.Results:1.Compared with the para-tumor breast,breast cancer tissues show higher expression of SHOC2 on the level of mRNA and protein(P<0.01).2.IHC analysis reveal that the high expression of SHOC2 correlates with the advanced histologic grade(P<0.001),large tumor size(P=0.023),and negative status of ER(P=0.028).Survival analysis suggest that high expression of SHOC2 can obviously decrease the overall survival of the patients,espacially for the patients of ER negative.3.The results of experiments in vitro show that knockdown of SHOC2 protein can significantly inhibit the proliferation of breast cancer(P<0.001),as well as the S phase entry(P<0.001),and induce the apotosis of breast cancer(P<0.001).Knockdown of SHOC2 also can inhibit the activation of Ras signal pathway,while overexpression of SHOC2 can activate the Ras pathway.Conclusion:1.SHOC2 can play an important role in tumorigenesis of breast cancer and has significant prognostic value for breast cancer patients.2.The expression of SHOC2 can affect the proliferation and apotosis of breast cancer cells and associates with the activation of Ras signal pathway.Part II The mechanism of Ras-Raf-ERK pathway activation medicated by SHOC2 in the everolimus resistance of breast cancer cellsObjective:To study the role of SHOC2 protein in the everolimus resisitance of breast cancer,and make clear the mechanism regulated by SHOC2..Methods:Use CCK-8 asssy to analyze sensitivity to everolimus of different breast cancer cell lines and preliminary screening everolimus-resistant cell lines.Use flow cytometry to examine the effect of everolimus to the cell cycle of MDA-MB-231 and Hs578T breast cancer cells.Westen blot was adopted to examine the alteration of mTOR pathway and Raf-ERK pathway after everolimus,as well as SHOC2 knockdown.Co-IP assays was used to analyze whether SHOC2 protein can interact with Raf-1 in vitro and the change after everolimus.Raf-ERK pathway was inhibited by MEK-1 inhibitor PD98059 and the change of signal pathway,cell proliferation and cell cycle was examined by Western Blotting,CCK-8 asssy and flow cytometry.Results:1.MDA-MB-231 and Hs578T Triple-negative breast cancer cells were relatively resisitant to mTOR inhibitor everolimus2.Co-IP assay shows the binding of SHOC2 and Raf-1 was increased,which promoted the phosphorylation activation of Raf-1 after everolimus.Then Raf-1-ERK feedback pathway was actived in the MDA-MB-231and Hs578T Triple-negative breast cancer cells.3.SHOC2 knockdown could significantly inhibit Raf-1-ERK feedback pathway actived by everolimus.4.Inhibition of Raf-1-ERK feedback pathway could enhance the inhibition of mTOR inhibitor everolimus to the proliferation of MDA-MB-231and Hs578T triple-negative breast cells(P<0.001),and could block the cell cycle in G0/G1 phase,and significantly reduce the proportion of cells in S phase(P<0.001).Conclusion:1.SHOC2 could bind to Raf-1 and promote its phosphorylation activation,which was involved in the activation of the Raf-1-ERK feedback pathway in everolimus-resistant breast cancer cells.2.Inhibiting Raf-1-ERK feedback pathway could increased sensitivity of breast cancer cells to everolimus.Part Ⅲ Knockdown of SHOC2 could improve breast cancer cell sensitivity to mTOR inhibitor everolimus in vitro and vivoObjective:To verify the function of SHOC2 for the sensitivity to everolimus in breast cancer cells and research the sensitivity of nude mouse tumor to everolimus after SHOC2 knock down.Methods:Use the technique of RNAi to inhibit SHOC2 protein expression.After that,CCK-8 assay,clony formation experiment and flow cytometryranswell assay were done to compare the ability of proliferation,cell cycle and apotosis.Use the nude mouse transplantation tumor experiment to display the sensitivity to everolimus after SHOC2 knockdownResults:1.SHOC2 knockdown could significant enhance the inhibition of everolimus to the proliferation(P<0.001)and clony formation(P<0.001)of breast cancer cells.2.SHOC2 knockdown could significant enhance the inhibition of everolimus to S phase entry(P<0.001)and induction of apotosis(P<0.001)of breast cancer cells.3.The tumors of nude mouse in the SHOC2 knockdown combined everolimus group were significantly inhibited than the other groups(P<0.001).SHOC2 knockdown could enhance the sensitivity of nude mouse tumor to everolimu in vivo.Conclusion:1.SHOC2 knockdown could enhance the inhibition of everolimus to the breast cancer proliferation,as well as the effects of cell cycle arrest and induction of apotosis in vitro.2.SHOC2 knockdown could enhance the sensitivity of nude mouse tumor to everolimus in vivo.