Cloning And Expression Of MMP-26 And Its Effect On Biological Behavior Of Breast Carcinoma

The invasion and metastasis of malignant tumor is a multi-stage andcomplicated process with the enhancement of proteolytic enzymes' activityand the degradation of extracellular matrix (ECM). There are four maingroups of proteolytic enzymes that take part in the degradation of ECM.Among these, matrix metalloproteinases (MMPs) are the hugest family withthe most complicated functions and high proteolytic activities. The functionsof MMPs in tumor metastasis and invasion include degradation of ECM,regulating cell adherence, promoting angiogenesis and so on. MMP-26 is anovel member of MMPs, which was cloned in 2001 by Dr. Strongin who is aninternational famous specialist in MMPs. In 2003, Professor Yulin Li beganthe cooperation with Dr. Strongin on the research of the function of MMP-26in tumor progression. We have got significant improvement in the prokaryoticexpression of MMP-26, in vitro activation and the substrate cleavagespecificity. However, the research on MMP-26 has just begun so far. Thefunctions of MMP-26 in tumor progression and its potential clinicalsignificance are still unknown.We introduced the recombinant plasmid, pcDNA3.1(+)-neo expressionplasmid carrying the proMMP-26 coding sequence, into breast cancer MCF-7cells, successfully. Multiple transfected clones resistant to G418 were selectedfor the subsequent analysis and the expression of MMP-26 was identified by aseries of experiments. After that we performed systemic studies in vivo and invitro to further elucidate the role of MMP-26 on tumor progression. All ourstudies aimed at determining the effect of MMP-26 on breast carcinoma andproviding a basis for further studies.1. Transfection, selection and identification of MMP-26 gene inMCF-7 cells.Cell line choosing for transfection The expression levels ofMMP-26 were detected in a series of human breast carcinoma cell lines,including MCF-7, MDA-MB231, by RT-PCR and immunofluorescence. Theresults showed that MMP-26 expressed weakly in MCF-7 cells comparing toother cell lines and MCF-7 cells were selected as the target cells fortransfection accordingly.Transfection of MMP-26 gene The pcDNA3.1(+)-neo expressionvector carrying the proMMP-26 coding sequence was constructed in ourprevious research. The vector was transfected by liposome transfection agent.After selection by G418, several pcDNA-3.1-MMP-26 transfected clones werechosen to identify the expression of MMP-26 by following methods.RT-PCR MMP-26 mRNA was detected in the 9 neomycin-resistantclones. The results showed that MMP-26 mRNA was significantly increased inclone ③,⑦and⑨ .Immunofluorescence The expression of MMP-26 protein wasdetected and localized in the cell using anti-MMP-26 polyclonal antibody andFITC labeled secondary antibody. The results showed that the fluorescenceintensity in the MMP-26 transfected cells was much higher than in the controlgroups (MCF-7 group and pcDNA-3.1 group). The positive fluorescence wasdistributed in the whole cytoplasm.Flow cytometry The expression of MMP-26 protein was furtherdetected by flow cytometry. The cellular fluorescence peak moved to the rightside and the average intensity was one time increased comparing to controlgroups.Gelatin zymography The cultured cells were collected and lysed toextract cytoplasm protein for gelatin zymography analysis. The results showedthat there were two bright bands in the transfected cell lane, 29.6KD and18KD respectively, corresponding to the molecular weight of MMP-26enzymogen and its active form. It suggested that the MMP-26 transfected genehas been expressed at a high level as proenzyme and could be activated incytoplasm.2. Effect of MMP-26 protein on biological characters of MCF-7 cellsLight microscope changes The MMP-26 transfected cells showedcellular pleomorphism, including larger in size and more irregular in shape. Alarge number of mitoses and tumor giant cells appeared in the transfected cells.The tranfected cells showed more atypia than the control groups.Electron microscope changes The MMP-26 transfected cells showedultrastructure changes as follows. (1) Large numbers of mitoses appeared inthe MMP-26 transfected cells including many bizarre mitotic figures. (2)There are more dead cells in the MMP-26 transfected cells than in the controls.(3) Glucogen pools in the cytoplasm occurred in some MMP-26 transfectedcells. (4)Special lysosomes appeared in the cytoplasm of MMP-26 transfectedcells.Growth curve The growth curve of MMP-26 transfected cells wasdecreased comparing to the control groups, which suggest that transfectMMP-26 cells grew slower in total number than the control cells.Cell cycle analysis by Flow cytometry The results of cell cycleanalysis by Flow cytometry showed that the MMP-26 transfected cells peakbecame wide and the number of cells in S phase and G2-M phase increasedsignificantly. It implied that the MMP-26 transfected cells remained in highproliferous state. The increase of dead cells number was also detected by Flowcytometry.3. Effect of MMP-26 protein on biological behavior of tumor cells invitroAdherence and spreading of tumor cells The adherent ability ofMMP-26 transfected cells were extremely increased comparing to controlgroups on Matrigel precoated wells. The cells attached more quickly andshowed a large size, a polygonal shape with more projections. There was nodifference between the two control groups.Tumor cell migration and invasion assay by Boyden chamber Ourresults showed that the MMP-26 transfected cells had an increased migrationand invasion ability through Matrigel. However, the migration speed ofMMP-26 transfected cells slowed down extremely in the Matrigel containinganti-MMP-26 antibody and it was even slower than control groups. At the endof the four hours, the number of invasive cells under the chamber showed thatthe MMP-26 transfected cells had the most powerful migration ability. Thecontrol pcDNA-3.1 transfected cells took second place and the MMP-26transfected cells in Matrigel containing antibodies possessed the weakestmigration ability even lower than control groups. This indicated that MMP-26could promote tumor cell migration and invasion in Matrigel by its directdegradation of Matrigel .4. Effect of MMP-26 protein on biological behavior of breastcarcinoma in vivoHuman breast carcinoma nude mice heterogeneous transplantationmodel We injected the MMP-26 transfected cells and the other two groupsinto nude mice groins to observe the tumor growth. The data showed that therewas no difference in tumor size among them (P>0.05).The experimental invasive model under murine nephric theca.Transplant a clot (1 mm3)of tumor cells under the Balb/c murine nephrictheca. Kill the mice on the sixth day and measure the invasive depth ofinvasion by the tumor cells. The results showed that the invasion depth ofMMP-26 transfected cells was much deeper than the controls. It indicated thatMMP-26 could promote tumor's local invasion.Auto metastasis model Inject tumor cells of three experimentalgroups respectively into the tail vein of nude mice. Kill the mice on the 21stday and remove the organs such as lung, liver, heart, etc. HE staining showedthat there were tumor emboli in the pulmonary vessels. Some of thempenetrated the vessels and hematogenous spread occurred. There was nodifference between the MMP-26 cells and the controls.Angiogenesis induced by tumor cells in vivo Inject tumor cells ofdifferent groups respectively under the back skin of nude mice. Four days later,remove the skin and measure the vessels. The result showed that MMP-26protein could induce angiogenesis efficiently. The new vessels were wide indiameter and had abundant branches which connected into net. The statisticsdata showed the number of vascular branches and the total length of vessels byMMP-26 transfected cells were significantly different from the controls(P<0.01).5. The expression of MMP-26 in human breast tissues and itscorrelations with some clinical features. By immunohistochemistry weshowed that the rate of expression of MMP-26 protein was low in normalmammary tissue and hyperplasia. But it was much higher in carcinoma in situ,as well as in IDC (61.2%). The expression of the MMP-26 had no correlationwith the age of patient, the size of tumor, the pathological grading. But therewas a significant correlation between the expression of MMP-26 and thelymph node metastasis,as well as a reverse correlation between MMP-26 andestrogen receptor.Taken together, the original results were concluded as followed.1. The significance of MMP-26 cloning and expression in eukaryoticcells.In our previous work, we cloned MMP-26 gene into prokaryoticexpression vector and expressed it in E.coli as inclusion bodies. The activeform was produced by in vitro activation. Since the mechanism of expressionand activation in vivo is not clear, the cloning of MMP-26 into eukaryotes,achievement of proenzymes and active enzymes are not successful so far..Based on our previous experience in prokaryotic study, we transfected thepcDNA-3.1 vector carrying the full-length gene of MMP-26 into MCF-7successfully, and identified the expression of mRNA and protein by RT-PCR,immunofluorescence, flow cytometry and zymography. We are the first whoclone the proMMP-26 into eukaryotes and detect the proenzymes and activeenzymes in the world. This provided not only a model and tool for the studyof the effect of MMP-26 on tumor progression, but also a basis for furtherstudies on mechanism of MMP-26 activation in vivo.2. The effect of MMP-26 on the biological characters and behaviorof breast carcinoma.MMP-26 can promote the proliferation of tumor cells and increasetheir malignant phenotypes. Morphologic changes occurred aftertransfection with MMP-26 gene in vivo and in vitro, including cellularpleomorphism, distinct atypia, increased mitoses tumor giant cells andappearance of bizarre mitotic figures. Electron microscope showed the samechanges and the finding of glucogen pools may be another hint for malignanttransformation. Flow cytometry further demonstrated that MMP-26 promotedthe proliferation of tumor cells.MMP-26 may play a key role in the process of early invasion ofbreast carcinoma by promoting the infiltration of basement membrane.MMP-26 tranfected cells showed more attached, mobile and invasive than thecontrol cells in Matrigel in vitro. But the ability of migration and invasion canbe effectively inhibited in the Matrigel by anti-MMP-26 antibody. Thissuggested that MMP-26 could promote the adherence, migration and invasionof breast carcinoma cells in Matrigel. Therefore, we presumed that MMP-26might play a key role in the process of early invasion of breast carcinoma cellsby promoting the infiltration of basement membrane. We also confirmed thepromotion of MMP-26 to the local invasion of breast carcinoma by theexperimental invasive model under murine nephric theca. Furthermore, wedetected the expression of MMP-26 protein in breast carcinoma byimmunohistochemistry and the result showed that MMP-26 was highexpressed in carcinoma in situ, which agreed with Zhao YG. All our datasuggested that MMP-26 may play a key role in the progress of breastcarcinoma in situ to infiltrating ductal carcinoma.Tumor angiogenesis induced by MMP-26. Our angiogenesis modelshowed that MMP-26 transfected cells could induce the angiogenesis of tumor.The vessels induced by MMP-26 transfected cells are abundant in number,wider in diameter, longer in total length than by MCF-7 cells and pcDNA-3.1cells and they connected into vascular net. This demonstrates that MMP-26can induce tumor angiogenesis efficiently.3. MMP-26 could be a significant predictor in judging malignantdegree and invasion and metastasis capability, further to evaluate theprognosis of breast carcinoma.MMP-26 protein is correlated with the malignant grade of breastcarcinoma. Our studies showed that MMP-26 could influence the biologiccharacters and behaviors of tumor cells and promote their malignant grade,proliferation, adherence and invasion. The expression of MMP-26 proteinturned the MCF-7, which showed low malignant and weakly invasive, intocells with high malignant degree and strong invasive ability. Therefore, weconcluded that MMP-26 protein could predict the malignant characters andbehaviors of breast carcinoma. It could be an indicator for malignanttransformation and prognosis evaluation.The effect of MMP-26 on metastasis of breast carcinoma. Byimmunohistochemistry staining of MMP-26 and its correlation analysis withsome clinical features, we confirmed that MMP-26 protein was significantlycorrelated with lymph node metastasis. MMP-26 expressed higher in positivecases of lymph metastasis than in negative cases, which suggested thatMMP-26 may play an important role in promoting the lymphatic spread ofbreast carcinoma. The effect of MMP-26 on the hematogenous spread ofbreast carcinoma need to be further demonstrated.The expression of MMP-26 protein is reverse correlated with ER inbreast carcinoma. Our results showed that there was a significant reversecorrelation between the expression of MMP-26 protein and estrogen receptor(ER). ER was negative in most positive cases of MMP-26 protein, whereas theexpression of MMP-26 protein was negative or weak in the cases of ERpositive(P<0.01).It suggested that estrogen and ER may down-regulate theexpression of MMP-26 protein. These results clarified the correlation betweenthe MMP-26 protein expression and ER and provided a morphologic basis forthe transcriptional regulation of MMP-26. Nowadays ER is one of the clinicalsignals for the prediction of patient's prognosis, which also suggested thatMMP-26 may be a new signal for the patient's prognosis.Overall, we cloned and expressed proMMP-26 gene in breast carcinomacells successfully, and confirmed that MMP-26 could transform the malignantphenotype of breast carcinoma. We also demonstrated that MMP-26 played animportant role in promoting the infiltration through basement membrane, localinvasion and angiogenesis of breast carcinoma. We illuminated that theexpression of MMP-26 protein was related to the lymphatic spread of breastcarcinoma and there was a reverse correlation between MMP-26 and estrogenreceptor by detecting the benign and malignant mammary tissues byimmnohistochemistry. MMP-26 could be a significant predictor in judgingmalignant degree and invasion and metastasis capability, further to evaluatethe prognosis of breast carcinoma.