Biological Function And Clinical Significance Of RDGN Members In Breast Cancer

Part Ⅰ Dachshund 1(DACH1)suppresses breast tumor growth,and DACH1 expression is inversely correlated with the level of cluster of differentiation 44(CD44).Purpose: DACH1 is an important member of the retinal determination gene network(RDGN),and acts as a key cell fate determination factor.It has been reported that the dysregulation of DACH1 contributes to tumorigenesis,invasion and metastasis of human breast neoplasm.However,the exact molecular mechanisms for the anti-tumor roles of DACH1 in breast carcinoma are still lack of comprehensive and extensive understanding.CD44 is a transmembrane glycoprotein that serves as the receptor for the extracellular matrix component hyaluronic acid.Previous studies have demonstrated that CD44 is associated with the initiation and development of breast cancer,but the association between CD44 and the clinico-pathological characteristics as well as the prognosis in breast cancer remains controversial.In this study,we comprehensively evaluated the correlation between the expression of DACH1 and CD44 and the clinico-pathological features as well as clinical outcomes of breast cancer patients,and analyzed whether there was any association between the m RNA levels of DACH1 and CD44 and epithelial-mesenchymal transition(EMT)markers,cancer stem cells(CSCs)markers as well as proliferation-related genes.In addition,we also tested whether DACH1 inhibited breast tumor growth,and further explored whether DACH1 affected CD44 expression in breast cancer.Methods: We conducted a meta-analysis on breast cancer Gene Expression Omnibus(GEO)datasets and performed statistical analysis on the Cancer Genome Atlas(TCGA)as well as immunohistochemistry(IHC)analysis on the breast cancer tissue microarray,comprehensively assessing the correlation between the expression of DACH1 and CD44 and clinico-pathological characteristics as well as the prognosis of breast cancer patients.We exploited GSE20685 and breast cancer cell line data to evaluate the association between the m RNA levels of DACH1 and CD44 and the m RNA expression of EMT markers,CSCs markers and proliferation-related genes.Besides,we also constructed breast cancer cells with DACH1 over-expression and mice transplanted tumor models to test the anti-growth role of DACH1 and further explore the impact of DACH1 on CD44 expression in breast cancer.Results: DACH1 expression was much lower in human breast tumors and poorly-differentiated tumors than normal breast and well-differentiated tumors,respectively.DACH1 expression was much higher in Luminal-type breast cancer than triple-negative breast cancer(TNBC),while CD44 expression was remarkably lower in Luminal-type tumors in comparison with TNBC.Correlation analysis showed that the m RNA expression of DACH1 was inversely associated with the markers of EMT and CSCs as well as proliferation-related genes,while CD44 m RNA level was positively correlated with these genes.At both m RNA and protein levels,the expression of CD44,Fibronectin and Vimentin in breast cancer cells with DACH1 over-expression was much lower than the controls.IHC analysis on mice transplanted tumor tissues displayed that the levels of CD44,Myc,Sox2,Fibronectin,Vimentin,epidermal growth factor receptor(EGFR)and Ki-67 in tumors with DACH1 over-expression were significantly lower than the controls.In addition,DACH1 over-expression remarkably suppressed breast tumor growth in vivo.Survival analysis showed that higher m RNA expression of DACH1 was associated with better overall survival(OS),relapse-free survival(RFS)and metastasis-free survival(MFS),while higher m RNA level of CD44 was correlated with worse OS and MFS in breast cancer.Conclusions: Our study indicated that DACH1 and CD44 were both closely correlated with the clinico-pathological features of breast cancer.DACH1 level was negatively correlated with CD44 expression in breast cancer,and DACH1 inhibited breast tumor growth.In addition,low expression of DACH1 and high level of CD44 were both unfavorable elements for the prognosis of breast cancer patients.Part Ⅱ Sine oculis(SIX)expression is correlated with clinical stages,molecular subtypes and prognosis in breast cancer.Purpose: SIX is another key RDGN component.Members of SIX family include SIX1,SIX2,SIX3,SIX4,SIX5 and SIX6.It has been reported that SIX1 increases CSCs through promoting extracellular signal-regulated kinase and transforming growth factor-β pathways,and high SIX1 expression is associated with poor prognosis of Luminal-type breast cancer patients.Through regulating the expression of SOX2 and E-cadherin,SIX2 drives tumor metastasis.SIX3 acts as an anti-tumor factor in multiple cancer types.Currently,the roles of SIX4,SIX5 and SIX6 in breast cancer have been rarely reported.In this study,we explored whether there was any correlation between SIX expression and clinico-pathological characteristics and assessed the prognostic value of SIX members in breast cancer.Methods: We performed statistical analysis on TCGA breast cancer data to evaluate the association between SIX expression and clinical stages,the status of hormone receptors and molecular subtypes.Besides,we also employed public breast cancer data platform to perform the survival analysis of SIX in breast cancer.Results: The m RNA levels of SIX1-4 in breast tumors were significantly higher than para-carcinoma tissues.The m RNA levels of SIX1 and SIX4 in stage III-IV breast tumors were slightly higher than stage I-II breast cancer,while the m RNA expression of SIX3 in stage III-IV breast tumors was lower than stage I-II breast cancer.The m RNA levels of SIX1 and SIX4 in estrogen receptor(ER)-positive breast cancer tissues were higher than ER-negative breast tumors,while the m RNA expression of SIX2,SIX3 and SIX6 in ER-positive breast cancer was significantly lower than ER-negative tumors.The m RNA levels of SIX2,SIX3 and SIX6 in progesterone receptor(PR)-positive breast cancer were remarkably lower than PR-negative tumors.In addition,the m RNA expression of SIX family members was correlated with molecular subtypes and clinical outcomes in breast cancer.Conclusions: Our analysis indicated that the levels of SIX family members were associated with tumor stages,the status of hormone receptors,molecular subtypes and prognosis in breast cancer.Part Ⅲ Eyes absent 2(EYA2)is correlated with molecular subtypes,predicts poor survival,and promotes the proliferation of breast cancer cells.Purpose: EYA2,another important RDGN member,is a transcriptional activator.EYA2 is pivotal to organ development,but aberrant regulation of EYA2 has been reported in multiple human tumors.However,the role of EYA2 in breast cancer is still lack of full understanding.In this study,we comprehensively evaluated whether EYA2 was associated with clinico-pathological features and survival performance in breast cancer,and explored whether EYA2 had an impact on the proliferation of breast cancer cells.Methods: We conducted a meta-analysis on breast cancer GEO datasets,expression analysis of TCGA data and IHC analysis on the breast cancer tissue microarray,comprehensively evaluating whether EYA2 expression was correlated with tumor differentiation,status of hormone receptors and molecular subtypes of breast cancer.We exploited GSE25066 to perform correlation analysis,exploring whether there was any association between EYA2 m RNA level and mesenchymal markers,CSCs markers as well as proliferation-related genes.We constructed breast cancer cell lines with EYA2 over-expression to explore whether EYA2 affected cell proliferation through clone-forming assay and Ed U experiment and whether EYA2 affected the expression of proliferative markers through western blot and immunofluorescence assay.In addition,public data platform was used to analyze the prognostic value of EYA2 in breast cancer.Results: The mRNA level of EYA2 in breast cancer was lower than normal breast tissues.EYA2 m RNA expression in poorly-differentiated breast cancer was higher than well-differentiated tumors.At m RNA and protein levels,EYA2 expression in ER-positive and PR-positive breast cancer was lower than ER-negative and PR-negative breast tumors,respectively.EYA2 level in TNBC was significantly higher in comparison with Luminal-type breast cancer.Correlation analysis showed that EYA2 m RNA level was positively associated with markers of TNBC,mesenchyme and CSCs as well as the proliferation-related gene.Over-expression of EYA2 enhanced the proliferation of breast tumor cells,accompanied by the up-regulation of EGFR,proliferating cell nuclear antigen,Cyclin E and Y-box binding protein 1.Besides,high m RNA level of EYA2 was an unfavorable prognostic marker for breast cancer patients.Conclusions: Our study indicated that EYA2 was enriched in TNBC,and high EYA2 expression was correlated with poor clinical outcomes of breast cancer patients.In addition,EYA2 enhanced the proliferation of breast cancer cells with the up-regulation of proliferation-related proteins.