| Wingless-type mouse mammary tumor virus integration site family (Wnt) proteins are a large family of cysteine-rich, secreted signaling glycoproteins which control essential developmental and normal physiologic processes. Abnormalities in the Wnt signaling are frequently observed in human cancers. Vertebrate Wnts are divided into canonical signaling and non-canonical members. The canonical Wnt signaling pathway with transforming activities has been extensively studied, which is thought to activate a signal-transduction pathway that induces the nuclear accumulation and transcriptional activation of β-Catenin and T cell factor (TCF). Activation of canonical Wnts, including wnt1, wnt2, wnt3, wnt3a, wnt8, leads to the tumorigenesis of multiple carcinomas including breast cancer. The non-canonical wnts, including wnt4, wnt5a, wnt5b, wnt7b, wnt11, signal through Planar Cell Polarity and Wnt/Ca2+ pathways. The switch of wnt signaling controls the expression of many genes involved in growth and metabolism. Meanwhile, it could affect the expression of series genes indirectly through interaction with various signaling pathway, including TGF-beta/BMP, Hedgehog, PI3K, RTK, etc. Thus, wnts are involved in a variety of processes in embryonic growth and morphological development, the maintenance of normal tissue homeostasis, energy metabolism, and stem/ progenitor cell capability, while the misfuntion of wnt signaling could result in serious diseases.In the absence of a Wnt ligand, p-Catenin is bound in a cytosolic protein complex containing glycogen synthase kinase-3b (GSK-3b), the adenomatous polyposis coli gene product (APC), and Axin. β-Catenin is phosphorylated by GSK-3b and then targeted for ubiquitination degradation in the proteasome, thus the cytosolic β-Catenin protein levels are kept low. Wnt proteins, such as wntl, wnt3a, bind to Frizzled (Frz, seven-transmembrane protein) and low-density lipoprotein receptor-related protein 5/6 (LRP5/6, single-transmembrane protein) co-receptors, leading to the activation of the Dishevelled (Dvl) protein, which then inhibits GSK-3p mediated phosphorylation of β-Catenin. Cytosolic P-Catenin protein becomes stabilized and accumulated, then translocate to the nucleus leading to the activation of series target gene expression by binding to T cell factor (TCF).β-Catenin/TCF could activate target genes including c-myc, cyclin D1, and MMP7, accordingly mediate cell differentiation and proliferation. The abnormal activation of wnt/β-Catenin pathway has been regarded as hallmark of cancer development, and is involved in the proliferation and migration of tumor cells. Besides the presence of wnt canonical proteins, mutation in APC, Axin and CTNNB1 (β-Catenin coding gene) could also impede p-Catenin/APC/Axin formation, resulting in switch-on of wnt/β-Catenin pathway. However, these mutations are rarely found in breast cancer. In the mean time, multiple studies verified the abnormal activation of wnt/β-Catenin pathway in breast cancer, leaving the underground mechanism unclear.Wnt5a is an important non-transforming member of the Wnt non-canonical signaling. It exerts effect through a special Fzr receptor, and an intracellular signaling pathway that roles in cell growth and differentiation. Wnt-5a-/- mice embryos could not survive, and showed developmental defects such as dwarfism, facial abnormalities, short limbs and tails, indicating its vital role in development. However, on account of the inability of Wnt-5a to transform cells, less has been focused on this gene than the canonical wnt members, and little is known about its signaling mechanism. In addition to wnt/polar and wnt/Ca2+ pathway, wnt5a could also roles by mediating the canonical pathway in some occasions. It could promote or inhibit wnt/β-Catenin canonical pathway through combination with different receptors.The role of wnt5a in tumorigenesis is not come to light until recently. Nevertheless, recent work has pointed to a critical role of Wnt-5a in malignant progression but whether it is afforded by a tumour-suppressing effect or an oncogenic effect is questionable. Wnt5a has been shown as a potent oncogene as detected up-regulated in cancers of melanoma, pancreatic cancer, non-small cell lung cancer and prostate cancer. On the other hand, In cancers depending on wnt/β-Catenin canonical pathway to maintain cell malignant proliferation, wnt5a could inhibit cancer development through antagonizing wnt/β-Catenin signaling. In colorectal cancer, neuroblastoma and acute myeloid leukemia, wnt5a has been shown to decrease or postpone tumor cell proliferation and metastases, acting as a tumor suppressor, while majorly silenced by promoter methylation.The expression and exact role of Wnt5a in human breast cancer has been subject to much debate. Some study finds that wnt5a is up-regulated in non-immortalized breast cancer cells, indicating it as a member of oncogenes. Meanwhile, others detected down-regulated or silenced wnt5a expression in invasive ductal carcinoma. Down-regulation of wnt5a has been associated early relapse and poor prognosis, supporting the notion that the Wnt-5a gene is related to tumor suppression.Aberrant epigenetic alterations could result in a change in expression patterns of genes implicated in cellular proliferation, survival and differentiation, which takes place frequently in tumor development. Among those alterations, silencing of tumor suppressor genes by promoter methylation represents an important mechanism of tumor suppressor gene inactivation during tumorigenesis. Multiple tumor suppressor genes acting in various biological processes and pathways have been shown to be silenced by aberrant promoter CpG methylation in virtually all tumor types. Promoter methylation has been verified as vital factors in breast tumorigenesis, as it could silence the expression of varies tumor suppressor genes, including cell cycle regulators, steroids hormone receptors, and genes involved in cell invasion.The present study aims to figure out the expression and regulate mechanism of wnt5a in breast cancer, and explore the underground mechanism of aberrant activation of wnt/β-Catenin pathway. The study is comprised of two parts.Part Ⅰ:The expression and its regulation mechanism of wnt5a in breast cancer were explored, and whether it acts as a tumor-promoter or suppressor was determined. Breast cancer and para-cancer tissues were collected from 136 women who had undergone surgery at the Affiliated Hospital of Qingdao University (Qingdao, China), Feb.2008 to Oct.2009. Real-time RT-PCR was explored to detect the differentiated expression of wnt5a between cancer and para-cancer tissues, and the methylation status of wnt5a promoter was determined by both MSP (methylation-specific PCR) and BGS (bisulfite genomic sequencing). The association between wnt5a expression and the cancer biological characters was analyzed, and survival analysis was also performed. Data in this part will illuminate the expression regulation of wnt5a in breast cancer, and its participation in cancer development as well.Real-time RT-PCR of the 136 pairs of cancer and para-cancer tissues showed that the mean Wnt5a levels of breast cancer tissues was down-regulated to about 36.35% percent of those in para-cancer tissues. No association was found between down-regulation of Wnt-5a expression and tumor type or tumor size, while a significant association between Wnt-5a down-regulation and increasing histological grade was found. Besides, wnt5a tends to be poorly expressed in young patients or those with lymphnode metastasis. Furthermore, down-regulation of Wnt-5a mRNA was significantly associated with absence of ER and PR, while in companion with increasing ratio of Ki-67-positive cells. All these data support the notion that the Wnt-5a gene act as tumor suppresser in breast cancer.MSP analysis targeting two domains in wnt5a promoter both indicated a higher methylation status in breast cancer than matched para-cancer tissues. Further detailed methylation analyses of individual CpG sites in the Wnt5a promoter using BGS showed that CpGs were densely methylated in cancer tissues, while only rarely in paired normal tissues. Thus, promoter methylation of Wnt5a is frequent and tumor specific in breast cancer, resulting in its down-regulation.According to the mean relative expression of wnt5a in breast cancer as compared with para-cancer tissues, the cases are divided into 3 groups with low, medium or high wnt5a expression were indicated to censored cases. A survival analysis was performed using Kaplan-Meier method, and log-rank tests was taken to compare among the survival curves established based on wnt5a expression. Kaplan-Meier estimates of the 5-year survival rate were 67.74%(95% CI=48.34-81.36),82.43% (95% CI-68.93-90.45), and 94.08%(95% CI= 82.74-98.95) for the groups with low, medium, and high wnt5a expression, respectively. The log-rank test verifies a significant difference among the three curves (x2=9.934, P=0.007), and also a significant linear trend between wnt5a expression and survival time (x2=9.285, P=0.0023).Part Ⅱ:The molecular mechanism of aberrant activation of wnt/β-Catenin canonical pathway was investigated, together with the underground mechanism of the participation of wnt5a in breast cancer development. Wnt5a and its nonfunctional short isoform, Wnt5a-SI, were transfected into MCF-7 cell lines. MTT assay was explored to determine the affect of ectopic wnt5a expression on cell proliferation. β-Catenin and its downstream factors, including c-myc, cyclin D1, and MMP7, were detected by western blot. Data from this part would decide whether wnt5a could inhibit wnt/β-Catenin pathway, thus shed light on the underground mechanism of both the aberrant activation of wnt/β-Catenin signaling and tumor-inhibiting role of wnt5a.Ectopic expression of wnt5a substantially inhibited tumor cell proliferation, with statistically significant cell number decrease showed on day 3-5 compared with those in wnt5a-SI or not transfected cells, while the dysfunctional short isoform of wnt5a showed no cell growth suppression. Western blotting indicated that wnt5a could antagonize wnt/β-Catenin signaling pathway by decreased β-Catenin protein level and the target genes, including cyclinD1, c-myc, and MMP13 as well. These data suggest that wnt5a act as tumor suppressor through inhibiting wnt/β-Catenin signaling, while the aberrant activation of wnt/β-Catenin in breast cancer could be related to the epigenetic silence of wnt5a.Several conclusions were drawn in the present study:(1). Wnt5a is down-regulated in breast cancer. The down-regulation of wnt5a expression is significantly associated with features indicative of an aggressive tumor phenotype, thus supporting the notion that wnt-5a act as tumor suppresser in breast cancer.(2). Epigenetic mechanism plays a part in the expression regulation of wnt5a in breast cancer, as promoter methylation being involved in its down-regulation in breast cancer.(3). Wnt5a act as tumor suppressor through antagonizing wnt/β-Catenin signaling, and could inhibit cell proliferation and invasion in breast cancer.(4).The aberrant activation of wnt/β-Catenin pathway in breast cancer could be related to the epigenetic silence of wnt5a.(5). Epigenetic therapy holds a promising potential for the successful treatment of cancer since epigenetic changes are reversible as opposed to mutation. Wnt signaling could be supposed as target in breast cancer treatment as the aberrant activation of wnt/β-Catenin pathway could be related to the epigenetic silence of wnt5a.The abnormal activation of wnt/β-Catenin pathway is a significant event in breast tumorigenesis. The present study suggests the non-canonical wnt members, wnt5a, retains tumor suppressor function in breast cancer, in which the activation of wnt/β-Catenin pathway could be resulted in the epigenetic silence of wnt5a. These findings shed light on the underground mechanism of breast tumorigenesis, and provide a candidate target for epigenetic tumor therapy. |
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