The Overexpression Of TRPV6 In MCF-7 Promoted Its Motility

Background Breast cancer has climbed to be the leading cause of cancer mortality for females in many countries,and the disease with metastasis is the most fatal for patients with breast cancer.More than 60%patients with advanced breast cancer suffer from bone metastasis.And these patients subsequently undergo pain,fractures,and even cord compression,which can be deadly for them.Metastatic lesions in the brain and the lung are also common in advanced breast cancer.As blood-brain barrier is sturdy for drugs to cross,and brain edema due to brain lesion is an emergency for oncologist,the metastasis of tumor has become a thorny problem in the treatment of breast cancer.Transient receptor potential(TRP)channels are mammalian homologues of the Drosophila transient receptor.The TRP family consists of three main subfamilies:classic(TRPC),vanilloid(TRPV),and melastatin(TRPM).TRP channels has been indicated to be widely involved in malignant tumor progression,and distinct roles are played by them.TRPM7 was found to be essential for metastasis of breast cancer cells,via regulating cell cytoskeleton and cell-cell adhesion.This channel was also reported to be key in epidermal growth factor(EGF)and hypoxia-induced epithelial mesenchymal transition(EMT)of breast cancer cells.It has been shown that TRPV6 was a predictor of poor outcome for patients with prostate cancer,and knockdown of TRPV6 has been reported to inhibit proliferation of prostate cancer cell lines.However,TRPV2 seemed to be a favorable marker compared to many other members of TRP family.It was indicated to function as an inhibitor of glioma cell proliferation and resistance to Fas-induced apoptosis.TRPV6,which has been found to regulate Ca2+ homeostasis in cells,belongs to TRPV family.Abundantly expressed in the kidney and the intestine,TRPV6 was supposed to play a role in Ca2+ absortion,and it is distinguished from other TRP members for its consecutive activated Ca2+permeability,and extreme Ca2+ selectivity.Mostly,in patients with malignant tumors,metastatic lesions were the main cause of death.Cancer cells struggle to escape,to invade,to metastasize,and to disseminate.Numerous studies have shown that Ca2+ is central to cell motility,which is key in cancer cell migration.Cell migration is an integrated process of cytoskeleton and cell adhesions.When cells migrate,they initially polarize and extend their protrusions forwards,which is an result of actin polymerizing into f-actin,and cytoskeleton attaching to the extracellular matrix.And this is followed by the disassembly of adhesions at rear of the cell,leading to the rear retraction.Both extracellular and intracellular Ca2+ are indispensable in cell movement.In polarized cells,the concentration of intracellular Ca2+ displays a gradient,with highest at rear and lowest at the front of the cell.Increased concentration of Ca2+ at rear of the cell,is responsible for contraction of myosin II,followed by the rear-retraction.This process is regulated by myosin light-chain(MLC)phosphorylation,which is Ca2+dependent.Local intracellular Ca2+ concentration does not sustain stable,as it fluctuates in a way of calcium flickers.Following the signals of Ca2+ wave,are the adhesions turnover,actin assembly,concomitantly extending the protrusion of cells.Objectives We aimed to investigate the role of TRPV6 in breast cancer metastasis and the mechanism involved,in the level of breast cancer tissue and cell lines.Since the metastasis of breast cancer has become a thorny problem in the treatment of breast cancer,to figure out the mechanism involves and to find effective drugs targeting the metastatic mechanism has become an urgent task for oncologists.TRPV6 enjoys abundant expression in human kidney and intestine,and contributes to calcium absortion and intracellular calcium homeostasis.Dhennin-Duthille reported that there was a higher expression of TRPV6 in breast duct carcinoma than in normal mammary gland,especially in invasive area.And knockdown of TRPV6 in MDA-MB-231 cells and MCF-7 cells inhibit their motility.We focused in our experiment on studying the expression of TRPV6 in breast cancer tissue and its correlation with clinical pathologic indexes,and investigating the influence of the overexpression of TRPV6 in MCF-7 on calcium homeostasis and motility of MCF-7,as well as the underlying mechanism.Methods(1)A total of 46 paraffin-embedded breast cancer and 30 adjacent normal tissue samples were obtained from the Nanfang Hospital Affiliated to Southern Medical University,Guangzhou,China.Each specimen was assigned a score according to the percentage of positive cells(none,0;1-25,1;26%-50%,2;51%-75%,3;>75%,4),and staining intensity(none,0;weak,1;moderate,2;strong,3).Points for percentage of positive cells and staining intensity were added,and these samples were then attributed to two groups according to their overall score:low expression,0-3 points;and high expression,4-7 points.(2)Western blot was performed to assess the expression of TRPV6 in three cell lines with different invasion ability.Besides,we construct a stable cell line V6,expressing TRPV6 after transfection with lentivirus,and set up siV6 group with siRNA transfection into V6 cells for the following experiments.(3)After the verification of transfection efficiency,we performed Transwell experiments to assess the motility of V6 cells,NC cells,siV6 cells and V6+EGTA cells.Cells were counted in 5 random fields and expressed as the average number of cells per field under a light microscope.Calcium imaging was performed for V6 cells,NC cells and V6+EGTA cells with Fluo-3 AM,and we used SPSS 13.0 software to made curve graphs demonstrating the changing tendency of intracellular calcium concentration.(4)The expression of f-actin in the lamellipodia of V6 cells,NC cells and V6+EGTA cells was observed by laser confocal scanning microscope with FITC-phalloidine,and evaluated with the use of Image J software.We performed immunofluorescence assay to evaluate the expression of p-MYL9 in V6 cells,NC cells and V6+EGTA cells.As well,the expression of p-MYL9 was detected by Western blot,and quantified by Image J software.SPSS 13.0 software was used for statistical analysis.All data are expressed as mean ± SD.Significance was established by independent t-test,one-way anova or Chi square test as appropriate.Results(1)TRPV6 was expressed both in breast cancer tissue and mammary gland,mainly on cell membrane,and little intracellularly.Specimens incubated with PBS in place of the primary antibody displayed no staining.The ratio of high expression of TRPV6 in breast cancer was 52.2%(24/46),and 26.7%(8/30)in mammary gland tissue.The expression of TRPV6 in breast cancer tissue is significantly higher than in mammary gland tissue(p=0.028,χ2 test).TRPV6 demonstrated no significant correlation with the clinical pathologic indexes ER,Her-2,Ki67 and pathologic grades(p>0.05,χ2 test).But high expression of TRPV6 in breast cancer was found to be implicated with N3-N4 lymph node metastasis(p=0.012,χ2 test).(2)In Western blot analysis,invasive cell lines MDA-MB-468(2.47±0.08),MDA-MB-231(2.45±0.18)were revealed to express high level of TRPV6,and conversely the least invasive cell line MCF-7 showed little expression(1.14±0.11).(3)The overexpression of TRPV6 in MCF-7(2.85±0.35)and the silencing of TRPV6 in MCF-7 cells expressing TRPV6(0.74±0.17)was validated by Western blot.(4)MCF-7 cells overexpressing TRPV6(121.60±25.54 cells per field)had stronger motility compared to NC(77.80±12.07 cells per field,p<0.001)and siV6(67.40±19.53 cells per field)groups,revealed by Transwell experiment(p<0.001).But extracellular calcium depletion by EGTA completely compensated this effect(14.00±5.48 cells per field,p<0.001).(5)Calcium imaging demonstrated that,increased expression of TRPV6 brought a boost to calcium flickers in MCF-7,making a steeper concentration peak,and this could be offset by extracellular calcium depletion.(6)The fluorescence density of f-actin in V6 is 0.066 ± 0.010,indicated by FITC-labeled phalloidin,compared to 0.061± 0.036 in NC cells,and 0.030 ± 0.011 in V6+EGTA(V6 vs NC,p=0.001;V6 vs V6+EGTA,p<0.001).(7)Overexpression of TRPV6 was associated with significantly increased expression of p-MYL9,showed by immunofluorescence(0.10±0.027),in comparison with NC(0.061 ±0.036)and V6+EGTA(0.036±0.010),V6 vs NC,p=0.030;V6 vs V6+EGTA,p=0.008.Then We performed Western blot analysis to confirm the results of immunofluorescence and found that the expression of p-MYL9 in V6 cells protein(5.43±0.15)was significantly higher than that in NC(4.37±0.04)and V6+EGTA(3.82±0.82),V6 vs NC,p<0.01,V6 vs V6+EGTA,p<0.01.Conclusion We discovered in our experiments that,the expression level of TRPV6 in breast cancer tissue is significantly higher than in normal mammary gland tissue,and its expression in breast cancer is associated with more lymph node metastasis.Western blot analysis also verified the expression of TRPV6 in invasive breast cancer cell lines.With lentivirus-mediated transfection,the MCF-7 cell line expressing TRPV6 was constructed,and with siRNA-mediated gene silencing,the expression of TRPV6 in MCF-7 transfected with TRPV6 DNA was inhibited.MCF-7 cells transfected with empty vector was set as negative control group(NC group),and MCF-7 cells expressing TRPV6 with 5mmol/L EGTA added into medium,was set as V6+EGTA group.Calcium imaging showed that,overexpression of TRPV6 improved calcium flickers in MCF-7,and silencing of TRPV6 gene and extracellular calcium chelation by EGTA offset this change.It was shown in Transwell experiment that the overexpression of TRPV6 in MCF-7 improved its motility,compared to NC group,siV6 group and V6+EGTA group.When exploring the underlying mechanism of the overexpression of TRPV6 improved MCF-7’s motility,we found that MCF-7 cells expressing TRPV6 had higher f-actin and p-MYL9 expression,than NC group and V6+EGTA group.We also found that,after calcium chelation by EGTA,MCF-7 cells expressing TRPV6 no longer had advance in expressing f-actin and p-MYL9.So we assumed that,TRPV6 promoted MCF-7 cells’ motility,by increasing calcium influx.Chelation of extracellular calcium more greatly diminished MCF-7 cells’ motility,than siRNA interference.This suggested that calcium is key to cell motility and it contributes to cell motility through various ion channels.Furthermore,we put forward that,the increase of calcium influx in MCF-7 cells due to TRPV6 overexpression,might promote the polymerization of actin into f-actin,accordingly forming the cell lamellipodia to extend the protrusion.Besides,the increase of calcium influx possibly promoted cell contraction by activating myosins,and subsequently the rear part of the cell detached from extracellular matrix,which is dispensable for cell movement.