The Study Of TGFβ1 Function On Hepatic Metastasis And Stemness Of Triple Negative Breast Cancer

Partl The study of TGFβ1 function on TNBC liver metastasisBackground:Previous studies showed that TGFβ1 was highly expressed in triple negative breast cancer(TNBC)cell lines,however whether it involed in TNBC regulation was unclear.In our study,we studied TGFβ1 function on TNBC cell proliferation,invasion,liver metastasis in nude mice.Methods:MDA cells were either treated with or without TGFβ1 repeatedly,then injected into 3-4 weeks old nude mice.After 4-5 weeks,mice were sacrificed;tumor and liver were taken out for HE and immunohistochemical staining.Results:We found TGFβ1 pretreatment increased MDA cells proliferation,local invasion and metastasis to liver,immunohistochemical staining showed liver lesion resouced to primary tumor.Conclusions:TGFβ1 pretreatment may increase TNBC cells proliferation,local invasion and metastasis to liver.Part 2 The study of TGFβ1 function on the stemness of TNBCBackground:TGFβ play an important role in regulating cell proliferation,differentiation and apoptosis in autocrine or paracrine manners.Accumulating studied showed it may play an extremely important role in regulation of human breast cancer stem cells.We found that TGFβ1 is high expressed in TNBC cell lines,suggesting it may involved in breast cancer stem cell regulation.Methods:First,we used flow cytometry to analyze TGFβ1 function on TNBC CD44+/CD24-population.Then,sphere forming assay was to enrich breast cancer stem cell and sphere forming efficiency was used to accessing stem cell self-renewal ability.Further,PT-PCR was further applied to detect gene expression of stem cell marker OCT4、SOX2、NANOG,luminal epithelial marker(cytokeratin8,cytokeratinl8)and mesenchymal marker alpha smooth muscle actin(ACTA2)and cytokeratinl4.Finally,3D cultere was used to analyze TGFβ1 function on TNBC invasion.Results:Our results showed that TGFβ1 increased TNBC cells CD44+/CD24-population,increased their stem-like mesenchymal phenotype,increased stemness markers(OCT4、SOX2、NANOG)expression,promoted their self-renewal ability and increased TNBC stem cell invasion.Conclusions:Our results suggested TGFβ1 plays an important role in TNBC sternness.Part 3 CDK4 is essential for TGFβ1’s function on TNBC stemnessBackground:The cell cycle regulator,CDK4 and its binding partner cyclin D1 are the ultimate targets of many oncogenic signals,suggesting a central role for these proteins in cancer development and progression.Interestingly,CDK4 was also shown to promote normal stem cell expansion and inhibit differentiation of embryonic progenitors.As cancer stem cells are key to tumor development and tumor propagation,CDK4 may play an important role in regulating BCSC stemnessrs.We also found TGFβ1 to increase cyclin D1 expression and to induce its association with the kinase CDK4.In this part,we studied CDK4 function on mediating the effects of TGF(31 on TNBC stem cell regulation.Methods:Sphere forming assay and sphere forming efficiency was to accessing TNBC stem cell self-renewal ability.Expression level of lumiinal epithelial marker(cytokeratin8,cytokeratinl8)and mesenchymal marker alpha smooth muscle actin(ACTA2)and cytokeratinl4 was detected by RT-PCR.Results:We found that blocking CDK4 expression or kinase activity prevented TNBC stem cell self-renewal and stem-like mesenchymal phenotype.We also found TGFβ1 to increase cyclin D1 expression and to induce its association with the kinase CDK4.Modulation of this pathway either through knockdown of CDK4 expression with RNA interference or pharmacological inhibition of CDK4 completely blocked the effect of TGFβ1 on tumorsphere formation and stem-like mesenchymal phenotype,implicating CDK4 as a critical player in TGFβ1 TNBC cancer stem cell regulation.Conclusions:These results defined an essential role of cyclin D1/CDK4 complex in mediating the effects of TGFβ1 on TNBC stem cell regulation and highlight CDK4 as primary targets for novel therapies in basal-like triple negative breast cancers.Part 4 Association analysis between CSC and Triple negative breast cancerBackground:Some clinical datasets provide us more tools to analyze specific gene expression and cancers.Methods:We first performed a bioinformatic analysis of cancer stem cell surface marker CD44 and CD44 gene expression using TCGA RNA-seq datasets of 1,215 primary human breast tumors and Kaplan Meier Plotter of 249 triple negative breast tumors,to analyze the relationship between CD44、CD24 gene expression and patience prognosis、liver metastasis.We then performed a bioinformatics analysis of CD44 and CD44 gene expression using GOBO microarray datasets of 51 breast cancer cell lines.Finally,we performed a bioinformatics analysis of TGFβ1 and CDK4 gene expression using GOBO microarray datasets of 51 breast cancer cell lines.Results:We found that high expression of CD44 or low expression of CD24 suggested poor prognosis of triple negative breast cancer patients.Moreover,triple negative breast cancer patients with liver metastasis expressed high level of CD44 gene and low level of CD24.Furthermore,we found TGFβ1 and CDK4 gene were highly expressed in TNBC cell lines.Conclusions:Cancer stem cells may play an important role in liver metastasis and poor prognosis of TNBC patients.TGFβ1/CDK4 may play important role in TNBC stem cells regulation.