| Breast cancer remains the most common female malignant tumor in the worldwide and it still one of the leading causes of cancer-related death. It is a complex disease, involving a variety of changes in gene expression and structure. The primary tumor and the metastasis tumor at distant sites are the main cause of death. Breast cancer proliferation and metastasis are multi-steps, complex process, and the invasive breast carcinoma cells need an independent migratory phenotype associated with increased expression of certain genes involved in breast cancer cell progression. The grade, stage, rate of growth, and other characteristics of the tumor determined the kinds of treatment. Targeted cancer therapies may be more effective than current treatments and less harmful to normal cells. The next stage of targeted therapies will focus on finding which gene silencing will respond to therapies. In this study, we focused on the mechanisms how HBXIP and DEK genes promote breast cancer.Hepatitis B x-interacting protein (HBXIP), a cellular18KD protein, was originally identified by its interaction with the C-terminus of hepatitis B virus (HBV) X protein (HBx). HBXIP also forms a complex with survivin, an anti-apoptotic protein, resulting in the suppression of cell apoptosis through the mitochondrial/cytochrome pathway. And S100A4is associated with breast carcinoma progression by contributing proliferation, invasion and metastasis. In this study, we showed that HBXIP increased the S100A4promoter activity in a dose-dependent manner by luciferase reporter gene assay. Then the data indicated that S100A4expression correlated well with HBXIP in breast cancer cell lines by RT-PCR, qRT-PCR and Western blot. Chromatin immunoprecipitation (ChIP), luciferase reporter assay and electrophoresis mobility shift assay (EMSA) identified that the promoter core region of S100A4 was located at nt+210/+334and HBXIP up-regulated S100A4through STAT-4. Moreover, HBXIP over-expression cells showed activation of the PI3K/Akt pathway. PTEN is the key gene of this signaling pathway, and Western blot assay about the PTEN over-expression in MCF-7cell lines demonstrated that S100A4was a target gene of PTEN. Using Methylation-specific PCR and bisulfate sequencing, we found abnormal DNA methylation in upstream region of PTEN resulted in the upregulation of HBXIP in MCF-7cells. Moreover, HBXIP enhanced growth of breast cells through S100A4in vivo and in vitro. The enhanced proliferation of cells mediated by HBXIP could be blocked by siRNA targeting S100A4. Tissue microarray analysis showed that the positive rates of S100A4in HBXIP over-expressed cases is73.7%(28/38) in primary tumor tissues and83.3%(30/36) in metastatic lymph tissues. In conclusion, these data suggest that the proliferation of breast cancer cells promoted by HBXIP is associated with activation of S100A4.The DEK oncoprotein was identified as a fusion with the CAN/NUP214nucleoporin in a subset of acute myeloid leukemia patients originally. The DEK gene on chromosome6encodes a375amino acid protein with an estimated molecular weight of43kD that has not been classified into any known protein family. Recently, DEK was reported as a gene that is frequently upregulated in aggressive human tumors. To investigate the role and mechanism of DEK oncogene and the correlation to the clinicopathological status in breast cancers, total262cases, including25of normal tissues,16of intraductal hyperplasia,40of ductal carcinoma in situ (DCIS) and181of invasive ductal carcinoma of breast, were selected from Department of Pathology, Yanbian Tumor Hospital for immunohistochemical detection of DEK, ER, PR, and Ki-67proteins. Additionally, the breast cancer cell lines of MCF-7, MDA-MB-231, SKBR3, BT549and T47D were also included in this study. In results, DEK protein had higher positivity in DCIS, compared with the adjacent normal breast tissues. Also, DEK protein was strongly positive in invasive ductal carcinoma of breast on immunohistochemistry, which was significantly higher than normal breast tissues. However, DEK protein was only weak positive in the intraductal hyperplasia of breast. DEK protein over-expression was significantly correlated with the increased proliferating index of Ki-67. For the histological grade, DEK positive rate was only39.6%in G1breast cancers, however significantly higher in G2(90.4%) and G3(96.6%) cases (P<0.05). Additionally, strong DEK positive rate was lower in Stage-0(20.8%) and Stage-I (36.7%) compared with Stage-IIa (88.1%), Stage-Ⅱb (93.9%), and Stage-IIIa (97.2%)(P<0.01). Meanwhile, DEK protein showed higher expression level in<3years disease free survival breast cancers than it in≥3years disease free survival cases. However, no statistically difference was found between DEK expression and lymph node metastasis, ER and PR expressions. For the further study, we also investigated the mechanism of DEK on the progression of breast cancer in vitro. The results showed that DEK protein was co-localized with Ki-67in the nucleus of MCF-7cells by Immunofluoresence staining and confocal observation, and DEK mRNA was expressed in many breast cancer cell lines, including MCF-7, MDA-MB-231, SKBR3and MDA-MB-453by RT-PCR and western blotting. Meanwhile, silencing of DEK by siRNA transfection could reduce the proliferation and induce the early-stage apoptosis of MCF-7cells by MTT and FCM assay, respectively, and interestingly, DEK knockdown could also down-regulate the expression of Ki-67; however the detailed mechanism needs the further study to verify. In conclusion, DEK over-expression appears to associate with breast cancer progression, and DEK may potentially be used as a breast cancer biomarker for the early diagnosis, prognostic evaluation and therapeutic target for breast cancer. |
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