Study On The Mechanism Of SPOCK1/SIX1 Signal Axis In Regulating Breast Cancer Progression

Background:Worldwide,breast cancer,the most frequently diagnosed cancer and the most lethal gynecologic malignancy in women,is an increasing concern because of rising morbidity.Although major advances have been made in treatment for breast cancer,there is no cure for metastatic breast cancer at present.The 5-year survival rate of breast cancer patients is only 26%once spread metastasized occurs.Metastatic tumor,a main cause of death in most patients,is the major challenge of the clinical treatment for patients with breast cancer.The invasion and metastasis of breast cancer are multi-stage and complicated biological processes.They were triggered by multiple genes and intracellular signal transduction,which is also a key and difficult point for researchers to overcome.Thus,it is urgent to furtherly study the molecular mechanisms of breast cancer proliferation and metastasis,finding new diagnostic and therapeutic targets with high specificity and strong pertinence,providing new strategies for breast cancer treatment,and providing a theoretical basis for improving the prognosis of patients with breast cancer.Sparc/osteonectin,cwcv and kazal-like domains proteoglycan 1(SPOCK1)belongs to family member of multidomain testicular proteoglycan,which includes SPARC,TESTICAN-2 and TESTICAN-3,that involved in cell proliferation and metastasis.SPARC has been properly reported involving in cell proliferation,angiogenesis,apoptosis and epithelial-to-mesenchymal transition.Recently,researchers payed more and more attention to whether SPOCK1 was involved in the development of cancer.Some reports revealed that SPOCK1 related to distant metastasis of tumors,could activate Wnt/?-catenin,mTOR-S6K and NF-?B signaling pathway,and participated in processes of tumor proliferation,apoptosis and metastasis.It was confirmed that SPOCK1 was a novel oncogene involving in the occurrence and development of cancer.Meanwhile,some researchers reported that SPOCK1 was highly expressed in breast cancer,which might suggest the poor prognosis of patients.However,the SPOCK1-induced underlying mechanisms and functions,including cancer development and metastasis process are far from clear.Therefore,it is crucial for clarifying the potential mechanism and function of SPOCK1 in breast cancer evolution to provide a basis for improving the prognosis of breast cancer patients.SIX1 is highly expressed in a variety of tumor cells,such as liver cancer,breast cancer,colon cancer,etc.,which could suggest the poor prognosis of patients.Studies have confirmed that SIX1 participates in the process of epithelial-mesenchymal transition as a transcription factor and promotes tumor cell migration and invasion.However,it is unclear whether SIX1 and SPOCK 1 interact in the mechanism of breast cancer evolution.Objectives:The purpose was to clarify the impact of SPOCK1/SIX1 on breast cancer evolution and illustrate the potential molecular mechanism:1.To evaluate the role and clinical significance of SPOCK1/SIX1 in the early diagnosis and prognosis of breast cancer patients.2.To reveal the role and potential regulatory mechanisms of SPOCK1 played in breast cancer cell cycle,proliferation,metastasis and epithelial-mesenchymal transition.3.To explore the downstream target gene of SPOCK1 regulation,further explain their potential molecular mechanisms involved in breast cancer evolution.Materials and methods:1.Breast cancer database data and tissue specimen staining analysis:1)Oncomine,HPA(The Human Protein Atlas)and Ualcan databases were searched to analyze the expression of SPOCK1 and SIX1 in breast cancer tissues.2)Immunohistochemical staining was used to evaluate the expression of SPOCK1 in 80 breast cancer tissues,and the relationship between its high expression and the clinicopathological parameters of patients.3)The patient survival analysis curves were drawn by Kaplan Meier-plotter and SurvExpress databases,and evaluating the value of SPOCK1 and SIX1 in patients' prognostic assessment.2.In vitro experiments:1)Western blot assay to detect the expression of SPOCK1 in normal breast and breast cancer cell lines.2)Selecting MCF7 and SKBR3 cell lines with relatively high SPOCK1 expression and MDA-MB-231 and HS 578T cell lines with relatively low SPOCK1 expression constructed SPOCK1 lentiviral silencing and over-expressing cell lines,respectively.3)The effects of SPOCK1 on breast cancer cell cycle and proliferation were tested by thiazole blue test(MTT),EdU proliferation,clone formation and flow cytometry.At the same time,the changes of cell cycle related proteins were detected by Western blot.4)The effects of SPOCK1 on the invasion and metastasis ability of breast cancer cells were detected by wound healing,migration and invasion assays,and the changes of EMT markers were detected by Western blot.5)Using UCSC Xena and GEPIA2 databases to analyze the relationship between SPOCK1 and EMT-related markers,and further using immunofluorescence and Western blot assays to verify.6)Detecting the effect of different expressing SPOCK1 on AKT/mTOR pathway by Western blot.7)Detect the influence of SPOCK1-induced cell proliferation,metastasis and EMT progress after dealing with LY 294002(PI3K inhibitor)and Rapamycin(mTOR inhibitor).8)Immunofluorescence co-localization and co-immunoprecipitation experiments were used to verify the interaction between SPOCK1 and SIX1.9)Examining the effects on SPOCK1-induced cell cycle,proliferation,metastasis and EMT progress used si-RNA to knocking down SIX1 expression.3.In vivo experiments:1)Nude mice were inoculated breast cancer cells with different expression of SPOCK1 to compare the tumor formation.2)Injection breast cancer cells with different expression of SPOCK1 into nude mice tail vein to establishing tumor metastatic model compared the formation of lung metastases in nude mice,and further confirmed by hematoxylin-eosin staining.3)Immunohistochemical staining of nude mice tumor sections were used to comparing the effect of Ki67,E-cadherin and Vimentin by different expressing SPOCK1.Results:1.SPOCK1 was abnormally and strongly expressed and associated with poor prognostic in BC.SPOCK1 was significantly higher expressed in BC tissues compared to adjacent non-tumor ones.The positive rate(93.8%)and strongly positive rate(72.5%)of SPOCK1 in BC were both significantly higher than in adjacent non-tumor tissues(30.0%and 10%)(P<0.001).Notably,aberrant expression of SPOCK1 was associated with histological differentiation and LN metastasis.Moreover,high expression levels of SPOCK1 were markedly related to adverse outcomes and high-risk in breast cancer patients.2.SPOCK1 accelerated cell cycle progression and promoted cell proliferation in BC.Overexpression of SPOCK1 could enhance breast cancer cell cycle and proliferation in vitro and promote cell growth in vivo,while SPOCK1 knockdown displayed opposite results.3.SPOCK1 motivated BC metastasis via EMT process.High SPOCK1 expression motivated breast cancer cell motility and accelerated cell migration,invasion and EMT progress,and promoted lung metastasis in nude mice metastasis models.Conversely,SPOCK1 knockdown displayed the opposite effects.4.SPOCK1 promoted the progression of breast cancer by AKT/mTOR signaling pathway.The oncogenic activity of SPOCK1 was significantly correlated with AKT/mTOR pathway.LY 294002(PI3K inhibitor)and Rapamycin(mTOR inhibitor)could inverse SPOCK1-induced cell proliferation,metastasis and EMT progress in breast cancer.5.SIX1 was high expressed and interacted with SPOCK1 in breast cancer.SIX1 was high expressed in breast cancer,which was associated with poor prognosis of patients.The expression of SIX changed according to the alteration of SPOCK1,and co-immunoprecipitation assay further confirmed that SIX1 was interacted with SPOCK1 in breast cancer.6.The SPOCK1/SIX1 signal axis regulated breast cancer progression via AKT/mTOR signaling activity.Silencing the expression of SIX1 effectively restrained the SPOCK1-mediated cell proliferation,clone formation and cell cycle progression,and reversed SPOCK1-induced cell motility,migration and invasion.Conclusions:1.SPOCK1 and SIX1 were highly expressed in breast cancer,which could indicate the adverse outcomes for patients.2.High expression of SPOCK1 accelerated cell cycle progression and enhanced cell proliferation,and promoted cell metastasis via EMT progress in breast cancer.3.SPOCK1/SIX1 signal axis played a crucial role in breast cancer occurrence and development,and AKT/mTOR signaling pathway involved in breast cancer evolution SPOCK1/SIX1-mediated.4.SIX1 deficiency could reverse the promotion effect of SPOCK1 on breast cancer malignant behaviors.