Function Of δEFL In Breast Cancer Proliferation And Bone Metastasis

PartⅠ：δEF1promotes breast cancer cell proliferation throughdown-regulating p21expressionBreast cancer is the most common cancer in women. Abnormalproliferation of breast cancer cells and subsequent transfer process is amulti-gene participation through the completion of complex multi-stepprocess, its principle is not very clear. δEF1(δ-crystallin enhancer factor1) is a member of the zinc finger homeodomain transcription factorfamily. Studies revealed that δEF1is a widely expressed transcriptionalrepressor, working through its zinc finger clusters binding to consensusE2-box-like sequences. Although δEF1is implied as a regulatory factorat the crossroad between proliferation and differentiation incarcinogenesis, its potential effect in the regulation of cell cycleprogression has not been well elucidated. First, we assessed the effect of δEF1in regulating cell cycleprogression of breast cancer using cell proliferation assay. CCK-8asssyindicated that in δEF1-overexpressing cells, cell number increasedsignificantly, in comparison to the empty vector transfected cells. Incontrast, depletion of δEF1in MDA-MB-231cells exhibited a decreasedproliferative rate, compared to the control cells, indicating a promotingeffect of δEF1on proliferation of MDA-MB-231cells. Furthermore, flowcytometric measurements after BrdU incorporation confirmed an increasein the number of cells in the S phase after δEF1overexpression inMDA-MB-231cells. That knockdown of δEF1using RNA interferencewould result in a decrease of cell number in the S phase. Those resultshighlighting a potent role of δEF1to promote G1-S transition of breastcancer cells.Second,After known that δEF1can promote G1-S transition ofbreast cancer cells, we further studied the molecular mechanisms: Overexpressing δEF1in MDA-MB-231cells, we found thatCDK2,CDK4expression levels increased and the p21expressionreduced both at the transcriptional level and protein level; whereas δEF1knockdown exhibited an opposite effect.Further, having found that δEF1inhibited p21expression inMDA-MB-231cells, we were curious about whether δEF1is a bona fiderepressor of p21transcription. In this present report, a search using thetranscription factor database TRANSFAC and TESS identified an E2-box(CAGGTG) that is located at position545/540of human p21promoter.Luciferase and CHIP assays confirmed that δEF1inhibited thetranscription of p21through engaging the E2box on the p21promoter.To test whether δEF1mediates the transcriptional repression of p21in human tumor cells, we found that the expressions of δEF1and p21isinverse correlation in MDA-MB-231,MCF-7, T47D and ZR-75-1fourhuman breast cancer cell lines. We are further found a strong inverse relationship between the expressions of δEF1and p21in human breastcancer tissue samples. p21is one of critical regulatory molecules in cellcycle regulation. δEF1may play an irreplaceable role in breast cancercell cycle regulation trough regulate p21expression.Moreover, we demonstrated that δEF1down-regulated p21andconcurrently up-regulated the expressions of CDK2and CDK4duringthis process. δEF1inhibited p21transcription by recruiting to the E2-boxelement on the p21promoter. Depletion of endogenous δEF1inMDA-MB-231cells was sufficient to allow an inherent release of p21expression, thus resulting in the cell cycle arrest. In addition, thestimulatory effect of δEF1on cell proliferation through p21regulationwas supported by an inverse correlation of δEF1and p21expressionsobserved in both breast cancer cell lines and clinical tumor specimens.Taken together, these observations suggest a dual effect of δEF1inpromoting breast cancer cell proliferation, by differentially regulating the cell cycle regulatory proteins. This lay a good foundation for us to furtherresearch and development a novel target molecule for repression ofsystemic breast cancer progression.Part Ⅱ：δEF1promotes osteolytic metastasis of MDA-MB-231breast cancer cells by regulating MMP-1expressionBreast cancer metastasis is supposed to involve several stages inwhich epithelial-mesenchymal transition (EMT) is regarded as themechanistic basis for the behavior of cancer cells. Our recent studieshave implicated that δEF1is required for governing both breast cancerEMT and bone remodeling.We found that δEF1was highly expressed in MDA-MB-231, anosteolytic breast cancer cell line, but it was weakly expressed in theosteoblastic cell line, ZR-75-1. Its expression was also hardly detected inMCF-7and T47-D, two cell lines with low-metastatic potential. The results of cell migration and Matrigel invasion assays indicated thatMDA-MB-231cells overexpressing δEF1presented a distinct increase ofmigrating and invasive ability in MDA-MB-23cells, whereas depletionof δEF1revealed a decreased mobility and invasive ability inMDA-MB-231cells. the results confirmed that high expression of δEF1in MDA-MB-231cells might promote invasion and migration in vitro,thus triggering osteolytic bone metastasis of breast cancer.Bone metastase of breast cancer cells is closely related to theactivity of osteoblasts and osteoclasts. Therefore, we went on to studyhow changes in δEF1expression in breast cancer cells could affect theextent of breast cancer cell-induced osteoclast maturation, osteoblastmaturation and calcium. RAW264.7cells cultured in conditioned mediafrom δEF1overexpressing MDA-MB-231cells had significantly higherTRACP activities; and C2C12cells had a lower activity of ALP whentreated with conditioned medium from δEF1overexpressing MDA-MB-231cells;In addition, quantitative RT-PCR results showedthat the conditioned media from δEF1overexpressing MDA-MB-231cells exhibited an inhibition on BMP-2-induced expressions of osterixand osteocalcin, which are two othermarker genes of osteoblastdifferentiation; Alizarin red S staining assay show that retained dye ofMC3T3-E1cells treated with the conditioned medium from δEF1overexpressing MDA-MB-231was much less. In the present study, wefurther demonstrated that conditioned medium derived fromδEF1-overexpressing MDA-MB-231cells significantly inducesosteoclast maturation and concurrently represses osteoblastdifferentiation and mineralization. On the contrary, conditioned mediumderived from δEF1-interfered MDA-MB-231cells exhibits an oppositeeffect, thus confirming the effect of δEF to mediate osteolytic metastasisof breast cancer.To understand the molecular changes, we examined the MDA-MB-231-secreted factors in medium, we found that, δEF1remarkably up-regulates matrix metalloproteinase-1(MMP-1) expressionin MDA-MB-231cells. A search using the transcription factor databaseTESS identified an AP-1site (CATGAGTCAG) that is located atposition70/60of the MMP-1promoter. Luciferase assay resultsindicated that TPA up-regulated the promoter activity of MMP-1,whereas Curcumin repressed its activity in MDA-MB-231cells. Theresults of the luciferase assay also demonstrated that the mutation of theAP-1element on human MMP-1promoter totally depleted its response toTPA, Curcumin, and δEF1. CHIP assays show that overexpression ofδEF1in MDA-MB-231cells significantly increases the recruitment ofthe AP-1components, c-Jun/c-Fos, to the endogenous MMP-1promoter.Importantly, the results of luciferase and CHIP assays indicated thatδEF1activates MMP-1promoter activity through the AP-1responseelement. The AP-1components have been demonstrated to functiondownstream of the MAPK pathway.Western blot result show thatoverexpression of δEF1in MDA-MB-231cells significantly activatedphosphorylation of JNK signaling. The results of quantitative RT-PCRand luciferase assay showed that the blockade of the JNK signaling byspecific JNK inhibitor sp600125dramatically weakened δEF1-inducedexpression of MMP-1. δEF1up-regulates MMP-1expression could bemediated via the MAPK signaling pathway. In conclusion, theseobservations suggest a potent role of δEF1to promote breast cancermetastasis to bone by regulating secretion of growth factors in the tumormicroenvironment.