Membrane Associated Collagen ?? Promotes Breast Cancer Metastasis By Enhancing Cancer Cell Invasion And Stemness

BACKGROUND: Increased collagen expression and deposition are associated with cancer progression and poor prognosis in breast cancer patients.Dense breast is a risk factor for breast cancer,and the increased breast density is due in part to increased deposition of collagen proteins.Collagen XIII m RNA has been shown to be expressed in higher levels in the colon,cervix,bladder,endometrium and ovary.However,the function and regulation of collagen XIII in breast cancer have not been determined.Although the function of collagen XIII is thought to have a role in cell-cell and cell-matrix interactions,whether collagen XIII plays a role in breast cancer development and progression still need to be studied.OBJECTIVE: In this study,we investigated the function of collagen XIII in malignant phenotype and stemness of breast cancer cell,using collagen XIII silencing TNBC cell and collagen XIII-expressing normal breast epithelial cell.We explored collagen XIII activates TGF-? pathway through ?1 integrin,using activated integrin ?1functionalblocking antibody.We inspected the role of collagen XIII in primary tumor growth and metastasis,throuth xenograft experiment and lung metastasis experimentof mice.To determine the molecular mechanism of breast cancer metastasis promoted by collagen XIII.METHODS:(1)Western blot was used to investigatetheexpression of collagen XIII inmultiplenormal mammary epithelial cell lines and different types of breast cancer cell lines.The difference of collagen XIII expression between normal breast tissue and breast cancer tissue,malignant breast cancer tissue and breast cancer patients with different overall survival werestatistically analyzed using bioinformatics method.To determine the relationship of collagen XIII and breast cancer progression.(2)Establishment MDA-MB-231-Col13-/-cell line,using Crispr/cas9 gene knockout technology,and two monoclonal MDA-MB-231-Col13-/-(25)and(28)were chosen.Genomic DNA sequencing and western blot were used to confirm COL13A1 has been knocked out successfully.MCF-10 A cellswere infected with collagen XIIIexpressing lentivirus,and we got MCF-10A-Col13 cell lines.Western blot and immunofluorescence staining were used to confirm expression of collagen XIII was successfuly.Using Crispr/cas9 gene knockout andlentivirustechnology,establishment T4-2-Ctrl,T4-2-Col13-/-and T4-2-Col13 cell lines.Western blot was used to confirm collagen XIII knockout and expression.(3)In vitro3 D cell culture of MDA-MB-231-Col13-/-(25/28)and MCF-10A-Col13 cell,as well as Transwell invasion assay,single cell migration assay were used to investigate the invasive phenotypes which were induced by collagen XIII.Ed U and caspase3 immunofluorescence staining were used to statisticate the positive cell numbers of Ed U at different time points and the positive cell numbers of caspase3,we detected collagen XIII influences cell proliferation and apoptosis.Transwell invasion assay was used to detect the invasive ability of T4-2-Ctrl,T4-2-Col13-/-and T4-2-Col13 cells.(4)Through mammosphere/tumorsphere assay under the condition of suspension,we study collagen XIII influenced cancer cell colony formation activity.Flow cytometry was used to determine the changes of cell surface-expressed phosphatidylserine under detachment environment,and western blot was used to investigate the expression of PARP-cleaved.Tumorsphere assay was used to detect the colony formation activity of T4-2-Ctrl,T4-2-Col13-/-and T4-2-Col13 cells.To make a decision of collagen XIII enhances cancer cell stemness.(5)Immunofluorescence staining was used to detect collagen XIII activates integrin ?1.Through active-integrin ?1 functional blocking antibody,we determine that collagen XIII enhances cell invasion,migration,mammosphere formation and anoikis resistance at least partially through the ?1 integrin.(6)Western blot was used to observe the ratio of phosphorylated SMAD2/3 compared to total SMAD at different time points,combined with TGF-?-induced double luciferase reporter assay,we studied collagen XIII activates TGF-? signaling pathway.How can this kind of activationbe inhibited when active-integrin ?1 functional blocking antibody was present,we determinedwhether collagen XIII may enhance the TGF-? signaling pathway through ?1 integrin.(7)Setted up MDA-MB-231-luc-D3H2LN-Ctrl/Col13-/-and MDA-MB-231-GFPCtrl/Col13-/-cell lines,which can expression bioluciferase and green fluorescence protein respectively.TNBC cell xenograft and lung metastasis model were setted up through injected those cells into SCID mice from their tail vain and fat pad.Cardiac injection was used to set up TNBC bone metastasis model.In vivo imaging system(IVIS)and haemotoxylin-eosin(H&E)staining were used to test primary tumor growth,lung colonization and bone metastasis.To determine the role of collagen XIII promotes primary tumor growth and metastasis.RESULTS:(1)Collagen XIII expression is induced during breast cancer development:Western blot result showed triple-negative breast cancer cell lines expressed higher levels of collagen XIII protein than luminal type breast cancer cell lines and nonmalignant mammary epithelial cell lines.By analyzing microarray datasets,we found that collagen XIII m RNA levels were significantly increased in human breast cancer tissue compared to normal mammary gland tissue(p<0.01).Collagen XIII m RNA level in ER negative breast cancer was higher than the expression in ER positive breast cancer(p<0.01).Collagen XIII m RNA level in triple-negative breast cancer tissue were higher when compared with other subtypes(p<0.01).Kaplan-Meier log rank analysis showed that breastcancerpatients whose tumors had higher collagen XIII expression levels should have a significantly shorter overall survival period than those collagen XIII lower(p<0.001).For the breast cancer patients with negative estrogen receptor,higher collagen XIII expression levels lead to a shorter overall survival period(p<0.001).(2)Collagen XIII promotes invasive 3D malignant phenotypes in TNBC cells: Compared with control group,the number of invasive branches of MDA-MB-231-Col13-/-(25/28)cells reduced significantly(clone 25:p<0.001;clone 28:p<0.001).Collagen XIII overexpression increased 3D colony size of MCF10 A cells(day4,p<0.05;day5~day11,p<0.001).The ratio of Ed U positive cells was significantly higher in MCF-10A-Col13 group compared with the control group(day6,p<0.001;day8,p<0.001;day10,p<0.001).A reduction trend of activated caspase 3 was detected in collagen XIII overexpression MCF-10 A cells(p=0.098).Silencing collagen XIII significantly reduced invasion of MDA-MB-231 cells(clone 25:p<0.01;clone 28:p<0.01).In contrast,MCF-10 A cells with collagen XIII overexpression were more invasive than control cells(p<0.05).Collagen XIII silencing reduced the velocity and the distance of cell migration in MDA-MB-231 cells(velocity: clone 25:p<0.05;clone 28:p<0.05;distance: clone 25:p<0.05;clone 28:p<0.05).While collagen XIII overexpression significantly increased the cell migration velocity and distance in MCF-10A(velocity: p<0.001;distance: p<0.001).Silencing collagen XIII reduced invasion of T4-2 cells(p<0.05).However,collagen XIII overexpression can not promote that kind of ability.(3)Collagen XIII expression enhances cancer cell stemness:Silencing collagen XIII significantly reduced tumorsphere formation in MDA-MB-231 cells compared with control group(clone 25,p<0.001;clone 28,p<0.001).In contrast,overexpression of collagen XIII enhanced mammosphere formation in MCF10 A cells(p<0.001).Collagen XIII expression reduced detachment-induced cell surface-expressed phosphatidylserine(p<0.01)and PARP cleaved in MCF-10 A cells.While silencing collagen XIII increased phosphatidylserine expression in MDA-MB-231 cells surface(p<0.001).Silencing collagen XIII reduced tumorsphere formation of T4-2 cells(p<0.05).However,collagen XIII overexpression can not promote that kind of ability.(4)Collagen XIII enhances tumorsphere formation and TGF-? signaling through ?1 integrin:Collagen XIII silencing reduced the activated ?1 integrin foci in MDA-MB-231 cells(clone 25,p<0.01;clone 28,p<0.001).In contrast,overexpression of collagen XIII induced ?1 integrin activation in MCF-10 A cells(p<0.01).Integrin ?1 functional blocking antibody reduced collagen XIII-induced cell migration(velocity,p<0.001;distance,p<0.001),invasion(p<0.01),and mammosphere formation(p<0.05)in MCF10 A cells.Inhibition of ?1 integrin activation enhanced PARP cleavage in collagen XIII-expressing MCF-10 A cells.The TGF-?-induced SMAD2/3 phosphorylation was decreased in the collagen XIII knockout clones compared to control MDA-MB-231 cells(clone 25: 0.5h,p<0.05;1.0h,p<0.05;2.0h,p<0.05;24h,p<0.05;clone 28:0.5h,p<0.05;1.0h,p<0.05;2.0h,p<0.05;24h,n.s.).Consistent with western blot data,luciferase activities were significantly reduced in collagen XIIIsilenced MDA-MB-231 cells(p<0.01).These experiments were repeated in the MCF-10 A cell lines.TGF-?-induced SMAD2/3 phosphorylation(0.5h,p<0.05;1.0h,p<0.05;2.0h,p<0.05)and reporteractivities(p<0.001)were enhanced by exogenous collagen XIII expression.Importantly,integrin ?1 functional blocking antibody significantly reduced the collagen XIII-enhanced SMAD2/3 phosphorylation.(5)Silencing collagen XIII inhibits breast cancer metastasis in mice:In lung colonization model,the mice who were injected with control cells through tail vain developed lung colonization within 5 weeks,while silencing collagen XIII group significantly reduced the lung coloization(p<0.05).Haemotoxylin and Eosin(H&E)staining further confirmed that silencing collagen XIII inhibited the lung colonization of cancer cells in SCID mice(p<0.05).In the xenograft mammary tumor model,silencing collagen XIII slightly reduced tumor growth(day23,p<0.05;day25,p<0.05),and also showeda reduction trend of lung metastases(p=0.164).In the model of breast cancer bone metastasis,the whole body metastasis of silencing collagen XIII group reduced than that of control group(p=0.09),but there was no significant difference in bone metastasis.CONCLUSIONS:(1)Breast cancer development and progression are accompanied by increased expression of collagen XIII;(2)Theincreased expression of collagen XIII in TNBC cells promotes malignant phenotypes in 3D culture by enhancing cancer cell migration and invasion;(3)Collagen XIII promotes breast cancer progression by enhancing cancer cell stemness and its associated anoikis resistance;(4)Collagen XIII expression may enhance the TGF-? signalingpathway through ?1 integrin which subsequently promotes cancer progression;(5)Increased collagen XIII expression in TNBC may promote cancer colonization and metastasis by enhancing cancer cell invasion and stemness.