The Roles Of FOXC2Expression In Breast Cancer Invasion And Migration And BCSCs Stemness Maintenance And Its Mechanism

PartⅠ The expressionof FOXC2in different molecular subtypesof breast cancer tissue and different invasive potential breastcancer cell linesFOXC2is a member of the fork (Forkhead Box, FOX) protein familymembers, which functions involves a variety of biological processes suchas embryo development, cell cycle regulation, sugar and lipid metabolism.Recently research showed that FOXC2expression was closely correlatedwith the invasion and metastasis in many malignant tumors. Mani andother research found that FOXC2over-expression promoted the invasionand metastasis of breast cancer, it might be used as an important molecularmarkers of basal-like breast cancer. Mego research groups memberscaptured a small number of Circulating tumor cells (CTCs) fromperipheral blood of27breast cancer patients by magnetic cell sortingmethod, found and identified many EMT related transcription factor(Twist,Snail1, Slug, ZEB, FOXC2) were highly expressed, the over-expressionsituation exists in at least21%patients. Yet, EMT related transcription factor was the more general existed in triple negative breast cancerpatients, neoadjuvant chemotherapy did not eliminate EMT induced CTCs.FOXC2expression is regulated by many EMT related factors(Snail1,Twist, TGF-beta), FOXC2over-expression in canine kidneycells(MDCK) significantly increased many mesenchymal markers(Vimentin, Fibronectin, N-cadherin)expression and reduced manyepidermal markers (E-cadherin, ZO-1)expression or translocation.Therefore, the expression of transcription factors FOXC2whether havesimilar functions to identified EMT transcription factors, whether as a newEMT transcription regulatory factors involved in invasion and metastasisof breast cancer, it needs further research.Objective：To explore the expression difference of FOXC2mRNA andprotein in different molecular subtypes of breast cancer tissue anddifferent invasive potential breast cancer cell lines, and stressed theassociations between FOXC2expression and stem cell related markersCD44and CD133protein expression in basal-like breast cancer, so as toidentified the relationship between FOXC2expression and aggressivephenotype of breast cancer and stemness characteristics, also to providenew clues to clarify the mechanism of invasion and metastasis of breastcancer.Methods: Taking87human primary breast cancer tissue samples asthe research object. Immunohistochemical single dye was used to detected the FOXC2protein expression in different molecular subtypes of breasttissue and analyze the correlation between FOXC2expression differenceand clinical prognosis factors; Immunohistochemical double dye was usedto examined the co-expression situation of FOXC2and CD44or CD133protein in basal-like breast cancer. Taking high invasive potential of breastcancer cell line MDA-MB-231and MDA-MB-435as the research object.Immunofluorescence, RT-PCR and Western blot was used to respectivelydetected the expression of FOXC2mRNA and protein in four differentaggressive potential of mammary gland cell lines.Results:(1) FOXC2protein was highly expressed in breast cancertissue and mainly expressed in cell nucleus or cytoplasm of basal-likebreast cancer. The significantly positively relationship between FOXC2protein expression and hormone receptor negative, high histologic gradehigh TNM staging, lymph node metastasis positive and tumor size inbreast cancer, but there was no correlation between menstrual status andpathological types;(2) The results from immunohistochemical double dyeand correlational analysis suggested that a significant positivelycorrelation between FOXC2and CD44orCD133protein expression wasrevealed in breast cancer;(3) FOXC2protein was highly expressed inbreast cancer MDA-MB-231cell and MDA-MB-435cell and mainlylocated in cell nucleus or cytoplasm;(4) whether in the gene or proteinlevel, FOXC2expression in breast cancer MDA-MB-231and MDA-MB-435cells expression was significantly higher than in MCF-7and MCF-10A cell.Conclusion: FOXC2was highly expressed in basal-like breast cancertissue and high aggressive potential breast cancer cell lines, prompted therelativity between FOXC2expression and breast cancer aggressivephenotypes. Breast malignant development mediated FOXC2might beassociated with cancer cell invasive and stemness enhancement; FOXC2could be as a new EMT transcription factor involved in the program ofinvasion and metastasis of breast cancer.PartⅡ The effects of FOXC2over-expression on the invasion andmigration ability of less aggressive potential breast cancer MCF-7cell and its mechanismBy detecting the expression difference of FOXC2mRNA and proteinin different molecular subtypes of breast cancer tissue and differentinvasive potential of breast cancer cell lines. We found that FOXC2washighly expressed in basal-like breast cancer, suggests its expression wasassociated with breast aggressive phenotype; Other Studies have shownthat breast cancer epithelial cells could obtained molecular phenotypicheterogeneity by EMT. Therefore, to investigate whether FOXC2wasimplicated in the regulation of the malignant phenotype of breast cancer by EMT. It has important significance to elucidate the mechanism ofinvasion and metastasis in breast cancer.Objective: To explore the effects of FOXC2over-expression on theinvasion and migration ability of less aggressive potential of breast cancerMCF-7cell and its mechanism, so as to determine whether FOXC2wasinvolved in invasion and metastasis of breast cancer mediated by EMT.Methods: To construct over-expression lentivirus vector for FOXC2gene and obtain stable transfection of MCF-7cell line; The MCF-7cellmorphological changes before and after transfection was observed underinverted phase contrast microscope; Immunofluorescence and RT-PCRand Western blot method was used to respectively detected the expressionof EMT related markers mRNA and protein in MCF-7cell before and aftertransfection. Transwell Chambers model was used to detected the invasionand migration ability in MCF-7cells before and after transfection; Scratchexperiment was used to detected the movement ability in MCF-7cellsbefore and after transfection.Results: The FOXC2gene recombinant lentivirus expression vectorwas constructed successfully and obtained stable transfection of MCF-7cell line; Phase contrast microscope observation results showed thatFOXC2over-expression could triggered epithelial phenotype tomesenchymal phenotype transform in MCF-7cell; Immunofluorescence results showed that FOXC2over-expression increased mesenchymalmarker Vimentin protein expression and reduced epithelial markersE-cadherin and Claudin-1protein expression; Transwell Chambers modeltest results showed that FOXC2over-expression significantly enhancedthe invasion and migration ability of MCF-7cell; Scratch experiments alsoshowed that FOXC2over-expression significantly enhanced themovement ability of MCF-7cell; RT-PCR and Western blot resultsindicated that FOXC2over-expressed increased the mesenchymal markerN-cadherin Vimentin and Fibronectin-1protein expression and reducedepithelial markers E-cadherin and Claudin-1protein expression at geneand protein level.Conclusion: The FOXC2gene recombinant lentivirus expressionvector was constructed successfully and obtained stable transfectionMCF-7cell line, to provide reliable guarantee for FOXC2functionresearch; FOXC2over-expression enhanced the invasion and migrationability of breast cancer MCF-7cell, its mechanism may be mediated byEMT pathway and participated in malignant progression of breast cancer.PartⅢ The effects of of FOXC2over-expression on BCSCsmicrospheres enrichment and its mechanismMore and more studies have shown that EMT was not only promote the invasion and metastasis of malignant tumor and radiation andchemotherapy resistance, but also conducive to the enrichment of CSCs;Previous studies have confirmed that FOXC2enhanced the invasion andmigration ability mediated by EMT pathway in MCF-7cell, Which can bespeculated that FOXC2whether participated in the enrichment of BCSCsand stemness maintenance regulation mediated by EMT. Therefore, toinvestigate the effects of FOXC2over-expression on BCSCs microspheresenrichment and stemness maintenance and possible mechanism. To clarifythe significance of FOXC2in invasion and metastasis and recurrence ofbreast cancer.Objective: To explore the effects of FOXC2over-expression onenrichment of BCSCs microspheres derived from MCF-7cells and itsself-renewal ability and multi-differentiation potential, so as to determinethe roles of FOXC2expression in BCSCs microspheres enrichment andstemness maintenance and its mechanism.Methods: Serum-free suspension culture methods was used tocultivate BCSCs microspheres, inverted phase contrast microscope toobserved the morphology before and after transfection BCSCsmicrospheres; Immunofluorescence to test stem cell related markersexpression before and after transfection BCSCs microsphere cell; Limiteddilution and serum induction differentiation method were detectedrespectively self-renewal and multi-differentiation potential before and after transfection BCSCs microsphere cell; Flow cytometry were detectedBCSCs microsphere CD44+/CD24-/lowin the cell phenotype of BCSCssubgroup ratio and cell cycle distribution before and after transfection;RT-PCR and Western blot method were detected respectively stem cellrelated markers mRNA and protein expression before and aftertransfection.Results: BCSCs microsphere were cultured successfully in serum-freesuspension. FOXC2over-expression promoted the enrichment of BCSCsmicrospheres. Immunofluorescence showed that BCSCs microspherescells were highly expressed stem cell related markers CD44andCD133protein; Serum induced differentiation results showed that FOXC2over-expression enhanced the multi-differentiation potential of BCSCsmicrosphere cells; FACS results showed that FOXC2over-expressionincreased the proportion of CD44+/CD24-/lowphenotype BCSCs andG0/G1phase cells; RT-PCR and Western blot results showed that FOXC2over-expression promoted the expression of stem cell related markerCD44, CD133, OCT-4in BCSCs microspheres cells.Conclusion: Serum-free suspension culture can be used as a reliablemethod for sorting BCSCs; FOXC2over-expression promoted theenrichment of BCSCs microsphere and enhanced the self-renewal abilityand multi-differentiation potential of BCSCs microsphere cells; FOXC2might participated in the enrichment of BCSCs and its stemness maintenance by up-regulating stem cell related markers expression.PartⅣ The effects of FOXC2over-expression on BCSCsmicrospheres cells tumorigenicity and invasiveness in nude miceand its mechanismThe first three part research have clarified FOXC2over-expression notonly enhanced the invasion and migration ability in breast cancer MCF-7cell, but also promoted the enrichment of BCSCs microspheres andstemness characteristic maintenance. Therefore, this part intended toidentified the effects of FOXC2over-expression on tumorigenicity andinvasivenss of BCSCs microspheres cells tumor growth in nude mice andpossible mechanism.Objective: To explore the effects of FOXC2over-expression on BCSCsmicrospheres cells tumor growth in nude mice and EMT and stem cellrelated markers expression, so as to clarify the effects of FOXC2over-expression on tumorigenicity and invasivenss of BCSCsmicrospheres cells tumor growth in nude mice and its mechanism.Methods: Taking BCSCs microsphere cells deriving from MCF-7cellstransfected empty vector or FOXC2gene as the research object.Toestablish transplantation model of human BCSCs microsphere cells innude mice. To observe tumor growth and draw the tumor growth curve;HE staining and fluorescence microscopy were used to identified the pathologic condition of tumor tissue; Immunofluorescence and Westernblot methods r to respectively detected EMT related markers and stem cellrelated markers expression in transplanted tumor tissue.Results: FOXC2over-expression promoted the growth oftransplantation tumor derived from BCSCs microspheres cells. HEstaining result showed that obvious ischemia necrosis existed inover-expression group; Immunofluorescence results showed that FOXC2over-expression enhanced the mesenchymal markers N-cadherin proteinexpression and stem cell related markers CD44and CD133proteinexpression, reduced epithelial markers E-cadherin protein expression;RT-PCR and Western blot method to respectively indicated that FOXC2over-expression increased mesenchymal markers N-cadherin, Vimentinand Fibronectin-1protein expression and stem cell related markers CD44,CD13, OCT-4及KLF-4protein expression and reduced epithelial markersE-cadherin and Claudin-1protein expression at gene and protein level.Conclusion: FOXC2up-regulated the expression of stem cell relatedmarkers mediated by EMT, and enhanced the tumorigenicity andinvasiveness of BCSCs transplantation tumor in nude mice.