The Function And Mechanism Of Complement Regulatory ProteinCD59 In Regulating The Proliferation And Metastasis Of Breast Cancer

Breast cancer is so far the most frequent cancer among women with an estimated 232,670 new cases diagnosed this year(29%of all malignancies in women)and ranks second overall(15%)for estimated 40,000 deaths,after lung and bronchus carcinoma.Diagnosis at early stage is beneficial for patients.Although there are prognostic factors for long-term survival,breast cancer distant metastasis,especially lung metastasis,remains treacherous clinical challenge with a median survival of<2 years after diagnosis.Therefore,there's unmet need to discover prognostic biomarkers for breast cancer lung metastasis.Human CD59 is a membrane complement regulatory protein(mCRP)anchored to the cell membrane by glycanphosphatidylinositol(GPI),protecting innocent host cells from activated complement destruction.It has been shown that in most solid tumors,CD59 is overexpressed comparing with adjacent normal tissues and inhibition of CD59 function can facilitate breast tumor treatment.In addition,CD59 is also involved in tumor invasion and metastasis.To comprehend the role of CD5 9 in breast cancer cell proliferation,infiltration and the role of reveal CD59 on the prognosis of patients with pulmonary metastasis of breast cancer and provide potential therapeutic targets for clinical treatment,the mechanism of CD59 in breast cancer should be studied in deep,it has important theoretical and clinical value.PART1 MDA-MB-231-HM express higher level of CD59 but not of CD46 and CD55 than MDA-MB-231 probably through Akt/ERK signaling pathwayThe objective of this part were to identify the expression of CD59 in breast cancer cells MDA-MB-231-HM compared with its in parental cells MDA-MB-231 increased significantly,analysis the possible mechanism of high expression,continue to explore aspects of CD59 on breast cancer cell proliferation,invasion and lay the theoretical basis for the orthotopic xenograft model.Through comparing the high and low(or not)transfer potential cell lines,or compare the difference of primary tumors and metastases,by means of proteome research,compare protein expression in expressing quantity,location and modify status differences,to find proteins related to the pathological changes and disease specific proteins,are main ideas in the study of molecular mechanism of tumor metastasis.we chose MDA-MB-231-HM to reveal the roles of mCRPs that has been demonstrated prone to lung metastasis compared to its derived cell MDA-MB-231.we performed immunoblotting assay with total cell lysates.The results showed that CD59,but not other two mCRPs CD46 and CD55 was significantly up-regulated in MDA-MB-231-HM.The flow cytometry assay further confirmed that CD59 expression in cell membrane was significantly increased in MDA-MB-231-HM compared with its original cells.Recently we reported that CD59 constitutive expression is controlled by Spl,while inducible expression is regulated by NF-?B and enhancer-binding CREB.To investigate the underlying mechanisms by which MDA-MB-231-HM up-regulates CD59,we first examined the nuclear levels of Spl and phosphorylated Spl and the nucleus translocation of NF-?B.The results show that there are no significant differences between these two cell lines for Spl and NF-?B signalings.However,another transcriptional regulator CREB was found to be higher phosphorylated in MDA-MB-231-HM compared with MDA-MB-231.It has been demonstrated that CREB can be phosphorylated by phospho-Akt and phospho-ERK directly or indirectly,so we tested the phosphorylation levels of Akt and ERK.The result showed that phospho-Akt and phospho-ERK increased significantly in MDA-MB-231-HM compared with MDA-MB-231.We discovered that among three mCRPs,only CD59 was unregulated in MDA-MB-231-HM.CD59 transcriptional regulation involves many pathways.In our study,we show that maybe due to Akt and ERK pathway activation,CD59 is up regulated in MDA-MB-231-HM.Akt and ERK play vital roles in cancer,maybe one of the effect is up regulating CD59 through CREB thus promoting cell growth and metastasis in MDA-MB-321-HM.We hypothesized that probably via Akt and ERK pathways,CD59 is up-regulated in MDA-MB-231-HM compared with MDA-MB-231.PART2 Insufficiency of CD59 suppresses tumor growth and inhibits lung metastasis in MDA-MB-231-HMThe main purpose of this part were to explore the effects of CD59 on cell proliferation and metastasis in MDA-MB-231-HM,analysis the value of CD59 in the invasion and metastasis of breast cancer.Using specific siRNA against CD59 we induced the insufficient CD59 expression in MDA-MB-231-HM cells by two vectors,designated as 231-HM-shCD59 and 231-HM-shCD59,(the related were designated as 231-HM-Vector and 231-HM-Vector',respectively).As shown in Figure 2a,CD59 was remarkably knocked down in MDA-MB-231-HM,in which 231-HM-shCD59 appears more efficient,and was further chosen for future study.Next we used CCK8 assay to examine the effect of CD59 insufficiency on regulating cell proliferation.As shown in Figure 2b,231-HM-shCD59 cells with insufficient CD59 expression grew much slower than 231-HM-Vector cells starting from day 4 after cell plating(P<0.01).Then we implanted 231-HM-Vector and 231-HM-shCD59 cells into the left fourth mammary fat pads of female athymic nude mice,respectively.Primary tumors developed successfully in all mice,and the insufficient CD59 expression dramatically slowed down tumor growth as shown in tumor sizes(*P<0.02,**P<0.01).At 5 weeks all mice were sacrificed,tumors and lungs were further resected.Tumors were weighed and lungs were examined by hematoxylin and eosin(H&E)staining.The tumor sizes in 231-HM-shCD59-1 cells implanted mice are clearly demonstrated to be much smaller than those in control mice,and the related tumor weights showed the consistent results(representative images are shown,P<0.01).In the H&E stained slices,we found metastasis in 3/10 lungs in the 231-HM-Vector group,while none of 10 lungs from the 231-HM-shCD59 group showed metastatic lesions,in which the representative images are shown.These data indicate that the insufficient CD5 9 expression would suppress cell proliferation both in vitro and in vivo,and further impair lung metastasis in vivo.By knocking down CD59 with specific shRNA,surprisingly,we found that the growth of MDA-MB-231-HM was significantly suppressed both in vitro and in vivo.As we expected,CD5 9 insufficiency in MDA-MB-231-HM ablated its metastatic lesions in the orthotopic xenograft model.CD59 transcriptional regulation involves many pathways.In this study,we showed that CD59 up-regulation in MDA-MB-231 cells may result from Akt and ERK pathway activation.Akt and ERK play critical roles in cancer;one of their functions is to up-regulate CD59 through CREB,thus promoting cell growth and metastasis in MDA-MB-321-HM cells.The protective roles of mCRPs,especially CD59,may facilitate tumor cell evasion from complement attack;the mCRPs are therefore obstacles to antibody-based immunotherapy for cancer.In addition,CD59 overexpression protects MCF-7 breast cancer cells from complement-mediated cytolysis and promotes MCF-7 cell proliferation by inhibiting BCL-2 expression;conversely,CD59 knockdown up-regulates Fas and caspase-3 to induce apoptosis.PART 3 High CD59 expression level is associated with lung metastasis and poor prognosis in breast cancer patientsThe goal of this part were to explore the clinical significance of CD59 expression in lung metastasis,discusses the possible influence on patients and the connection between the expression of CD59 and the clinical features of breast cancer patients.We performed IHC staining for CD59 expression,retrospectively collected the complete clinical data including age,tumor size,pathological assessment and follow-up visit.The Chi-squared test was used to evaluate the relationship between patient characteristics and Body Mass Index(BMI)category and to compare groups based on CD59.A multivariate logistic regression model for predicting metastasis was used considering both categorical(CD59 expression,tumor size,lymph node status,cancer embolus,ER status,PR status,and HER-2 status)and continuous variables(age)evaluated at diagnosis.we investigated a cohort of 120 breast cancer patients composed of 58 breast cancer patients with lung metastasis and 62 patients without metastasis.We performed IHC staining for CD59 expression,and the expression scores were blind ranked by two independent pathologists.The representative images of CD59 expression in each 3 patients with or without metastasis were shown.We retrospectively collected the complete clinical data including age,tumor size,pathological assessment and follow-up visit.Our study shows the patient characteristics according to CD59 category.(P=0.001).A higher percentage of HER-2 deletion patients were observed in patients with high expression of CD59.However,age,tumor size,node status,tumor histology,ER status,PR status and HER-2 status were not significantly correlated with CD59 expression.Among the 120 patients,the lung metastasis rate was 48.3%(58/120).Table 2 shows the univariate and multivariate analysis of the lung metastasis rate predictors using a logistic regression model.In the univariate model,there was no significant relationship between lung metastasis rate and tumor size,tumor histology,ER status,PR status and HER-2 status(P>0.05).However,lung metastasis was significantly associated with age,lymph node status,cancer embolus and CD59(P<0.05).Patients with age?35,positive lymph node status,cancer embolus and high expression CD59 were more likely to suffer lung metastasis.In the multivariate model,CD59 expression,lymph node status and cancer embolus were independent predictors of lung metastasis,with P value of 0.041,0.032 and 0.008,respectively.Notably,we applied Kaplan-Meier survival analysis and turned out that CD59 expression levels were negatively correlated with relapse free survival of breast cancer patients(P<0.01).The analysis indicates that patients with higher CD59 expression would have a poorer prognosis compared with patients with lower expression of CD59.These clinical data suggest that CD5 9 expression might be considered as a prognostic biomarker for lung metastasis and outcome of breast cancer patients.Inspiring different works by Madjd et al.and Mikkel et al.demonstrate that loss of CD59 expression in breast tumors correlates with poor survival,and CD59 exhibited an inverse correlation with metastatic capability.Here we show that CD59 overexpression is associated with MDA-MB-231-HM growth,metastasis and poor outcome of breast cancer patients.In the grand study involving 520 patients of Madjd et al.,lung metastasis was not considered as a parameter,while we focus on lung metastasis and established the cohort of 120 patients with selection of lung metastasis and non-metastasis.Besides,we noticed that in our study,in HR-and Her-2 negative patients,a larger portion of patients is with high expression of CD59.These differences may lead to the different conclusions we drawn.Mikkel et al.showed that CD59 was "associated with the aggressiveness of metastasis rather than metastasis colonization per se.May be due to the inhibition of cell growth,during the time of our study,CD59 knockdown resulted in better "outcome" in mice.However,it has been reported that CD59 can mediate signal transmission through Src family member Lck in T cells.We cannot rule out that in MDA-MB-231-HM,complex signals maybe transmitted by CD59 to promote cell proliferation and metastasis.As a biomarker in breast cancer,CD59 may play versatile roles.Depending on our study,we revealed that CD59 overexpression may be prognostic for lung metastasis and poor survival of breast cancer patients.