The Relationship Between SHP-1 And Breast Cancer And The Underlying Mechanism Of SHP-1 In Suppressing Malignant Biological Behaviors In Breast Cancer

BackgroundBreast cancer has become one of the world’s most important causes that threaten human health seriously.Despite early diagnosis and treatment of breast cancer have made progress,about half a million women worldwide die from breast cancer each year.The incidence of breast cancer has increased 30 percent in the latest ten years in our country,which has already ranked the first in female malignant tumors.Breast cancer has a bad impact on the physical and mental health of patients.Therefore,how to make the pathogenesis clear,find a suitable target and treat patients effectively have become the current focus of the breast cancer research.The growth of body and stability of internal environment are maintained by the balance of cell survival and apoptosis.Various growth factors mediated cell signal transduction pathways participate in the interaction process.With the further study of malignant biological behaviors,scientists have recognized that the phosphorylation and dephosphorylation modification of pathway proteins are important in regulating cell signal transduction.The intricate equilibrium of phosphotyrosine residues on proteins is controlled by the opposing actions of protein tyrosine kinases(PTK)and protein tyrosine phosphatases(PTP).PTPs which serve as antagonists of tyrosine kinases can play prominent roles in cell proliferation,differentiation,apoptosis and so on.The increased PTK activity or decreased PTP activity has been regarded as an important cause in breast cancer development.Epidermal growth factor receptor(EGFR)is an essential component of PTK family including four members:HER1(EGFR),HER2,HER3 and HER4.When EGFR is combined with its specific ligand,its tyrosine residues in intracellular are phosphorylated,which play important roles in the cell proliferation,differentiation and signal transductionand.HER2,which is associated with the pathogenesis and progression of breast cancer,is greatly overexpressed in approximately 20%to 25%of breast cancer.Overexpression of HER2 often leads to distant metastasis in breast cancers,and its targeted drugs—transtuzumab,often developed resistance during one year.What’s more,the existing tyrosine kinase inhibitor(TKI)treatments in breast cancer have shown limited benefit.We have paid much attention to targeted therapy of PTK for years,but ignored the importance of PTP.Is it possible to reduce excessive intracellular tyrosine phosphorylation by PTP,and restore the original phosphorylation balance,thus maintaining the stability of cells?The SHP-1 gene,a family member of PTPs,is regarded as a negative regulator in signal transduction.SHP-1 can dephosphorylate some growth factor receptors,thus inhibiting cell proliferation and tumor growth.Our previous work showed that the proliferation of MDA-MB-231 cells was inhibited after the cells were transfected with SHP-1 gene.And the migration ability of MCF-7 cells was enhanced after SHP-1 reduction by RNA interference.However,the function of SHP-1 protein in breast cancer as well as its specific signaling pathway has not been completely clear so far.Previous researches have found that the MAPK signaling pathway is usually activated in breast cancer.MAPK signaling pathway mainly has three members,including Erkl/2,JNK and p38MAPK.A variety of small molecules can regulate breast cancer cell proliferation,invasion and metastasis through MAPK signaling pathway.We will investigate if MAPK signaling pathway is involved in the the regulation of breast cancer in our study.In this study,we first identified SHP-1 expression in human breast cancer tissues and studied the relationship between SHP-1 and a series of clinicopathological characteristics.Then we discussed the role of SHP-1 in breast cancer cell proliferation,migration and invasion through lentivirus infection or RNA interference.At last,we studied the molecular mechanism of the functional changes by western blot analysis.ObjectiveTo identify the expression of SHP-1 in human breast cancer tissues and the correlation between SHP-1 and some clinicopathological characteristics.To study the effect between SHP-1 and the prognosis of patients with breast cancer.To make sure the role of SHP-1 in regulation of breast cancer cell proliferation,migration and invasion and then to verify the molecular mechanism of the functional changes.Contents and methods1.To identify the expression of SHP-1 in human breast cancer tissues and the correlation between SHP-1 and clinicopathological characteristics The expression levels of SHP-1 in human breast cancer tissues were deteced by immunohistochemical analysis compared to adjacent non-cancerous breast tissues.The relationships between SHP-1 and clinicopathological characteristics,overall survival were analyzed by statistical methods.2.To make sure the role of SHP-1 in breast cancer cell proliferation in vitro and in vivo and verify the molecular mechanisms1)Establish MCF-7-SHP1 and MDA-MB-231-SHP1 cell lines with stable overexpression of SHP-1 by using packaging lentivirus.Establish MCF-7-SHP1-siSHP1 and MDA-MB-231-SHP1-siSHP1 cell lines with stable reduction of SHP-1 by RNA interference.Western blot analysis was used to detect the efficiency of transfection.2)Capacities of proliferation in breast cancer cell lines were detected by MTT assay after overexpressing and reducing expression of SHP-1.3)Colony formation abilities in breast cancer cell lines were detected by tablet colony formation assay after overexpression of SHP-1.4)Percentage of cells in S phase of the cell cycle in MCF-7 and MDA-MB-231 cells after overexpressing or reducing expression of SHP-1 was detected by EDU incorporation assay.5)The distribution of cell cycle in MCF-7 and MDA-MB-231 cells after stable overexpression of SHP-1 was detected by cell cycle analysis.6)The change of some cell cycle related proteins in MCF-7 and MDA-MB-231 after overexpression of SHP-1 was detected by Western blot analysis.Then we explored a signaling pathway of SHP-1 in regulating cell proliferation in breast cancer.7)The change of capacity of tumor-forming was detected by in vivo proliferation assay.Then tumor tissues were excised from the nude mice and observed the levels of above proteins by immunohistochemical analysis.3.To study the effect of SHP-1 on breast cancer cell migration and invasion and identify the related molecular mechanism1)The changes of capacity of migration and invasion in breast cancer cell lines were detected by transwell and boyden assay after overexpression or reduction of SHP-1.2)The change of ability of cell migration was detected by would scratch assay.3)The change of some EMT related proteins in MCF-7 and MDA-MB-231 after overexpression of SHP-1 was detected by Western blot analysis.Then we explored a signaling pathway of SHP-1 in regulating cell migration and invasion in breast cancer.4)Tumor tissues excised from nude mice were used to observe the levels of above proteins by immunohistochemical analysis.4.Statistical analysisAll quantified data represented an average of at least three samples.SPSS 16.0 and GraphPad Prism 5.0 software were used for statistical analysis.All data are expressed as mean 士 SD.Significance was established by two-tailed Student’s t-test as appropriate.Significant associations between SHP-1 expression and some cl inicopathologic factors were assessed using Chi square test.Kaplan-Meier and log-rank tests were used to compare patient survival and to create survival curves based on SHP-1 scores.Multivariate survival analysis was performed for all clinicopathologic characteristics that were significant in univariate analysis using the Cox regression model.P<0.05 was considered statistically significant.Results1.The levels of SHP-1 protein were higher in adjacent non-cancerous breast tissue specimens than breast cancer tissue samples using immunohistochemical staining.The lower expression level of SHP-1 was significantly related to larger tumor size,negative ER and PR status.The expression levels of SHP-1 was significantly associated with overall survival,and decreased SHP-1 expression was a poor independent prognostic factor for breast cancer patients.1)SHP-1 was highly expressed in 74.4%(29/39)of adjacent non-cancerous breast tissues,while in only 50.8%(96/189)of breast cancer tissues.2)There were no any significant associations between SHP-1 and patient’s age,histological grade or lymph node metastasis.However,the lower expression level of SHP-1 was significantly related to larger tumor size(P=0.009),negative ER status(P=0.031)and negative PR status(P=0.011).3)The expression levels of SHP-1 was significantly correlated with overall survival,as patients with lower SHP-1 had worse survival(133.8 months vs.111.2 months,P=0.004).Decreased SHP-1 expression was a poor independent prognostic factor for breast cancer patients(P=0.036)using Cox proportional-hazards model.2.SHP-1 inhibit cell proliferation in vitro and in vivo in breast cancer and its underlying mechanism1)The overexpression and interference efficiency were detected by Western blot analysis.2)SHP-1 overexpression inhibited proliferation in stable MCF-7 and MDA-MB-231 cell lines,whereas SHP-1 knockdown promoted proliferation in transiently transfected MCF-7-SHP1 and MDA-MB-231-SHP1 cells by MTT assay.The EdU incorporation assay revealed that the percentage of cells in S phase reduced after upregulation of SHP-1,but elevated after downregulation of SHP-1.3)Overexpressed SHP-1 was observed to block cell cycle transition from G1 to S and G2 phase by cell cycle analysis;and the colony formation assay showed that SHP-1 upregulation significantly reduced the number of MCF-7 and MDA-MB-231 colonies compared with the controls.4)Western blot analysis showed that SHP-1 overexpression was associated with decreased k-ras,h-ras,phos-Erk1/2,p-GSK3β,β-catenin,cyclin D1 and c-Myc levels.5)The average volume and weight of subcutaneous tumors resulting from MCF-7-SHP1 and MDA-MB-231-SHP1 cell injection were significantly lower than those from MCF-7-NC and MDA-MB-231-NC cell injection.Immunohistochemical staining of tumor nodules originating from the in vivo demonstrated that MCF-7-SHP1 and MDA-MB-231-SHP1 cells had decreased p-ERK1/2,β-catenin,cyclin D1 and c-Myc levels compared with that from control groups.3.SHP-1 suppress cell migration and invation in breast cancer and its underlying mechanism1)After incubation for 8h and 12h respectively,the percentage of cells that migrated in both the MDA-MB-231-SHP1 and MCF-7-SHP1 cell groups was dramatically lower than in their control cells.In addition,increased SHP-1 expression led to a significant decrease in the invasion activity of MDA-MB-231 and MCF-7 cells on a matrigel.Conversely,the migration and invasion capacity was enhanced after siRNA knockdown of SHP-1 expression in MDA-MB-231-SHP1 and MCF-7-SHP1 cells.2)As evidenced by the wound-healing assay,SHP-1 overexpression decreased the migration capacity of breast cancer cells.3)SHP-1 overexpression resulted in downregulation of N-cadherin and Snail,upregulation of E-cadherin.Immunohistochemical staining of the tumor nodules had the similar results.Conclusion1.The levels of SHP-1 protein were higher in adjacent non-cancerous breast tissue specimens than breast cancer tissue samples using immunohistochemical staining.The lower expression level of SHP-1 was significantly correlated with larger tumor size,negative ER and PR status.The expression levels of SHP-1 was significantly associated with overall survival,as patients with lower SHP-1 had worse survival.Decreased SHP-1 expression was a poor independent prognostic factor for breast cancer patients.2.SHP-1 upregulation inhibited breast cancer cell proliferation in vitro and in vivo.And it suggested that SHP-1 could suppress tumor growth through Ras/Erk/GSK3β/β-catenin signaling pathway.3.SHP-1 could suppress breast cancer cell migration and invasion,and it might be achieved by Ras/Erk/GSK3β/Snail signaling pathway.