Wnt信号通路抑制因子SFRP1在肿瘤中的研究进展

定位于染色体8p11.2上的分泌型卷曲相关蛋白1(secreted frizzled related protein 1,SFRP1)基因,是近来发现的新的抑癌基因。因其编码Wnt信号通路抑制因子SFRP1,SFRP1基因失活可导致Wnt信号转导途径的紊乱,影响肿瘤的发生发展。近年来国内外对SFRP1在恶性肿瘤的失活机制进行了一系列的研究,现对这方面的工作进展进行综述。     

Secreted frizzled-related protein 4 regulates two Wnt7a signaling pathways and inhibits proliferation in endometrial cancer cells

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication. Wnt7a is expressed in the luminal epithelium, whereas the extracellular modulator of Wnt signaling, secreted frizzled-related protein 4 (SFRP4), is localized to the stroma. Studies have reported that SFRP4 expression is significantly decreased in endometrial carcinoma and that both SFRP4 and Wnt7a genes are differentially regulated in response to estrogenic stimuli. Aberrant Wnt7a signaling irrevocably causes organ defects and infertility and contributes to the onset of disease. However, specific frizzled receptors (Fzd) that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of SFRP4 in the endometrium has not been addressed. We show here that Wnt7a coimmunoprecipitates with Fzd5, Fzd10, and SFRP4 in Ishikawa cells. Wnt7a binding to Fzd5 was shown to activate beta-catenin/canonical Wnt signaling and increase cellular proliferation. Conversely, Wnt7a signaling mediated by Fzd10 induced a noncanonical c-Jun NH2-terminal kinase-responsive pathway. SFRP4 suppresses activation of Wnt7a signaling in both an autocrine and paracrine manner. Stable overexpression of SFRP4 and treatment with recombinant SFRP4 protein inhibited endometrial cancer cell growth in vitro. These findings support a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent on the Fzd repertoire of the cell and can be regulated by SFRP4.     

Down-regulation of sfrp1 in a mammary epithelial cell line promotes the development of a cd44high/cd24low population which is invasive and resistant to anoikis

Background  

The Wnt family of secreted proteins is implicated in the regulation of cell fate during development, as well as in cell proliferation, morphology, and migration. Aberrant activation of the Wnt/β-catenin signaling pathway leads to the development of several human cancers, including breast cancer. Secreted frizzled-related protein 1 (SFRP1) antagonizes this pathway by competing with the Frizzled receptor for Wnt ligands resulting in an attenuation of the signal transduction cascade. Loss of SFRP1 expression is observed in breast cancer, along with several other cancers, and is associated with poor patient prognosis. However, it is not clear whether the loss of SFRP1 expression predisposes the mammary gland to tumorigenesis.     

CircSEMA4B targets miR-431 modulating IL-1β-induced degradative changes in nucleus pulposus cells in intervertebral disc degeneration via Wnt pathway

Intervertebral disc (IVD) degeneration (IDD), characterized by elevated levels of proinflammatory mediators, increased Aggrecan and collagen degradation, and increased degradation of extracellular matrix (ECM), has been widely regarded as a significant contributor to low back pain. Genetics are significant factors contribute to IDD. Based on previous data, circular RNA SEMA4B (circSEMA4B) is down-regulated in IDD specimens; herein, we demonstrated circSEMA4B overexpression could attenuate the effect of IL-1β on nucleus pulposus cell (NPC) proliferation, senescence, and ECM and Aggrecan degradation in IDD via Wnt signaling. Moreover, miR-431, a direct target of circSEMA4B, could bind to the 3′UTR of SFRP1 or GSK-3β, two inhibitory regulators of Wnt signaling, to inhibit their expression thus playing a role similar to the activator of Wnt signaling in NPCs. The effect of circSEMA4B knockdown on NPCs was partially reversed by miR-431 inhibition; circSEMA4B serves as a miR-431 sponge to compete with SFRP1 or GSK-3β for miR-431 binding, thus inhibiting IL-1β-induced degenerative process in NPCs through Wnt signaling. Rescuing circSEMA4B expression in NPCs in IDD might present a potential strategy for IDD improvement.     

Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells

Multipotential adult mesenchymal stem cells (MSCs) are able to differentiate along several known lineages, and lineage commitment is tightly regulated through specific cellular mediators and interactions. Recent observations of a low/high bone-mass phenotype in patients expressing a loss-/gain-of-function mutation in LRP5, a coreceptor of the Wnt family of signaling molecules, suggest the importance of Wnt signaling in bone formation, possibly involving MSCs. To analyze the role of Wnt signaling in mesenchymal osteogenesis, we have profiled the expression of WNTs and their receptors, FRIZZLEDs (FZDs), and several secreted Wnt inhibitors, such as SFRPs, and examined the effect of Wnt 3a, as a representative canonical Wnt member, during MSC osteogenesis in vitro. WNT11, FZD6, SFRP2, and SFRP3 are upregulated during MSC osteogenesis, while WNT9A and FZD7 are downregulated. MSCs also respond to exogenous Wnt 3a, based on increased beta-catenin nuclearization and activation of a Wnt-responsive promoter, and the magnitude of this response depends on the MSC differentiation state. Wnt 3a exposure inhibits MSC osteogenic differentiation, with decreased matrix mineralization and reduced alkaline phosphatase mRNA and activity. Wnt 3a treatment of fully osteogenically differentiated MSCs also suppresses osteoblastic marker gene expression. The Wnt 3a effect is accompanied by increased cell number, resulting from both increased proliferation and decreased apoptosis, particularly during expansion of undifferentiated MSCs. The osteo-suppressive effects of Wnt 3a are fully reversible, i.e., treatment prior to osteogenic induction does not compromise subsequent MSC osteogenesis. The results also showed that sFRP3 treatment attenuates some of the observed Wnt 3a effects on MSCs, and that inhibition of canonical Wnt signaling using a dominant negative TCF1 enhances MSC osteogenesis. Interestingly, expression of Wnt 5a, a non-canonical Wnt member, appeared to promote osteogenesis. Taken together, these findings suggest that canonical Wnt signaling functions in maintaining an undifferentiated, proliferating progenitor MSC population, whereas non-canonical Wnts facilitate osteogenic differentiation. Release from canonical Wnt regulation is a prerequisite for MSC differentiation. Thus, loss-/gain-of-function mutations of LRP5 would perturb Wnt signaling and depress/promote bone formation by affecting the progenitor cell pool. Elucidating Wnt regulation of MSC differentiation is important for their potential application in tissue regeneration.     

Methylation and loss of Secreted Frizzled-Related Protein 3 enhances melanoma cell migration and invasion

Background

Wnt signaling is important in development and can also contribute to the initiation and progression of cancer. The Secreted Frizzled Related Proteins (SFRPs) constitute a family of Wnt modulators, crucial for controlling Wnt signaling. Here we investigate the expression and role of SFRP3 in melanoma.

Methodology/Principal Findings

We show that SFRP3 mRNA is down-regulated in malignant melanoma tumors as compared to normal/benign tissue. Furthermore, we found that SFRP3 expression was lost in the malignant melanoma cell lines, A2058, HTB63 and A375, but not in the non-transformed melanocyte cell line, Hermes 3A. Methylated CpG rich areas were detected in the SFRP3 gene in melanoma cell lines and their SFRP3 expression could be restored using the demethylating agent, 5′aza-deoxycytidine. Addition of recombinant SFRP3 to melanoma cells had no effect on viable cell numbers, but decreased cell migration and invasion. Wnt5a signaling has been shown to increase the migration and invasion of malignant melanoma cells, and high expression of Wnt5a in melanoma tumors has been connected to a poor prognosis. We found that recombinant SFRP3 could inhibit Wnt5a signaling, and that it inhibited melanoma cell migration and invasion in a Wnt5a-dependent manner.

Conclusion/Significance

We conclude that SFRP3 functions as a melanoma migration and invasion suppressor by interfering with Wnt5a signaling.     

Loss of sfrp1 promotes ductal branching in the murine mammary gland

ABSTRACT: BACKGROUND: Secreted frizzled-related proteins (SFRPs) are a family of proteins that block the Wnt signaling pathway and loss of SFRP1 expression is found in breast cancer along with a multitude of other human cancers. Activated Wnt signaling leads to inappropriate mammary gland development and mammary tumorigenesis in mice. When SFRP1 is knocked down in immortalized non-malignant mammary epithelial cells, the cells exhibit a malignant phenotype which resembles the characteristics observed in metastatic breast cancer stem-like cells. However, the effects of SFRP1 loss on mammary gland development in vivo are yet to be elucidated. The work described here was initiated to investigate the role of SFRP1 in mammary gland development and whether SFRP1/ mice exhibit changes in mammary gland morphology and cell signaling pathways shown to be associated with SFRP1 loss in vitro. RESULTS: 10 week old nulliparous SFRP1/ mammary glands exhibited branching with clear lobulo-alveolar development, which normally only occurs in hormonally stimulated mid-pregnant wt mammary glands. Explant cultures of SFRP1/ mammary glands display increased levels of a well known Wnt signaling target gene, Axin2. Histomorphologic evaluation of virgin glands revealed that by 10 weeks of age, the duct profile is markedly altered in SFRP1/ mice showing a significantly higher density of ducts with distinct alveoli present throughout the mammary gland, and with focal ductal epithelial hyperplasia. These findings persist as the mice age and are evident at 23 weeks of age. Changes in gene expression, including c-Myc, TGFbeta-2, Wnt4, RANKL, and Rspo2 early in mammary gland development are consistent with the excessive hyper branching phenotype. Finally, we found that loss of SFRP1 significantly increases the number of mammary epithelial cells capable of mammosphere formation. CONCLUSIONS: Our study indicates that SFRP1 gene is critical for maintaining proper mammary gland development, and that reduced levels of SFRP1 results in hyperplastic lesions and its loss may be a critical event in cancer initiation.     

Methylation of secreted frizzled-related protein 1 (SFRP1) promoter downregulates Wnt/β-catenin activity in keloids

Keloid, a benign skin disorder, forms during wound healing in genetically susceptible individuals. To better control keloid and understand the molecular mechanisms, this study screened gene hypermethylations of GEO database microarray data on keloids and identified the hypermethylation of the secreted frizzled related protein-1 (SFRP1) promoter. Subsequently, hypermethylation and mRNA and protein levels were assessed in 57 cases of keloid vs. normal skin tissues. Fibroblasts from tissues were isolated for the assessment of gene regulation in vitro. The methods used were bioinformatic analysis, lentiviral infection carrying SFRP1 cDNA, qRT-PCR, western blot, immunohistochemistry, luciferase reporter assay, methylation-specific PCR and methylated DNA immunoprecipitation-qPCR, ELISA, and/or 5-Aza-2′-deoxycytidine treatment. The data revealed that the SFRP1 promoter was hypermethylated in keloid tissues, compared with that in normal skin tissues. The SFRP1 promoter methylation contributed to the downregulation of SFRP1 mRNA and protein in keloid tissues and keloid fibroblasts. The 5-Aza treatment significantly upregulated SFRP1 mRNA and protein level in keloid fibroblasts. Furthermore, the knockdown of DNMT1 expression, and not the expression of DNMT3a or DMNT3b, was responsible for the hypermethylation of the SFRP1 promoter and upregulation of SFRP1 mRNA and protein in keloid fibroblasts. In addition, the infection of lentivirus carrying SFRP1 cDNA significantly inhibited the signaling activity of Wnt/β-catenin and the mRNA and protein expression of β-catenin and α-SMA in keloid fibroblasts. In summary, the lost SFRP1 expression-induced Wnt/β-catenin signaling due to the hypermethylation of the SFRP1 promoter could associate with keloid development, suggesting that SFRP1 might be a therapeutic target for keloid treatment.     

SFRP分子对心脏发育的影响

Wnt信号通路对心脏发育起着重要的作用.分泌型卷曲相关蛋白（SFRP）家族作为调控 Wnt信号的重要分子,对心脏发育和心肌分化的作用也越来越被人们所重视.最近,研究人员们对SFRP家族蛋白有了新的认识,认为它们不仅具有拮抗Wnt的作用,还对Wnt信号的转导有着复杂的调节作用.本文就SFRP分子与Wnt信号转导对心脏发育的影响进行综述.     

Secreted frizzled related proteins: Implications in cancers

The Wnt (wingless-type) signaling pathway plays an important role in embryonic development, tissue homeostasis, and tumor progression becaluse of its effect on cell proliferation, migration, and differentiation. Secreted frizzled-related proteins (SFRPs) are extracellular inhibitors of Wnt signaling that act by binding directly to Wnt ligands or to Frizzled receptors. In recent years, aberrant expression of SFRPs has been reported to be associated with numerous cancers. As gene expression of SFRP members is often lost through promoter hypermethylation, inhibition of methylation through the use of epigenetic modifying agents could renew the expression of SFRP members and further antagonize deleterious Wnt signaling. Several reports have described epigenetic silencing of these Wnt signaling antagonists in various human cancers, suggesting their possible role as tumor suppressors. SFRP family members thus come across as potential tools in combating Wnt-driven tumorigenesis. However, little is known about SFRP family members and their role in different cancers. This review comprehensively covers all the available information on the role of SFRP molecules in various human cancers.     

Secreted Frizzled Related Protein 1 Modulates Taxane Resistance of Human Lung Adenocarcinoma

Taxanes, such as docetaxel and taxol, have been used as firstline chemotherapies in advanced lung adenocarcinoma (LAD), but limited responses to chemotherapy remain a major impediment in the clinic. Treatment with 5-azacytidine increases the sensitivity of SPC-A1/DTX cell line to taxanes. The results of DNA methylation microarray and cDNA array analysis indicate that DNA methylation contributes to the downregulation of secreted frizzled related protein 1 (SFRP1) in SPC-A1/DTX cells. Overexpression of SFRP1 reverses the chemoresistance of taxane-resistant LAD cell lines and enhances the in vivo sensitivity of taxane-resistant LAD cells to taxanes. Meanwhile, short hairpin RNA (shRNA)-mediated SFRP1 knockdown decreases the sensitivity of parental LAD cell lines to taxanes. Furthermore, FH535, a reversible Wnt signaling inhibitor, enhances the sensitivity of taxane-resistant LAD cells to taxanes. The level of SFRP1 in tumors of nonresponding patients is significantly lower than that in tumors of responders. Taken together, our results provide the direct evidence that SFRP1 is a clinically important determinant of taxanes resistance in human LAD cells, suggesting that SFRP1 might be a novel therapeutic target for the treatment of taxane-resistant LAD patients.     

Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6.

BACKGROUND: Dickkopf-1 (Dkk-1) is a head inducer secreted from the vertebrate head organizer and induces anterior development by antagonizing Wnt signaling. Although several families of secreted antagonists have been shown to inhibit Wnt signal transduction by binding to Wnt, the molecular mechanism of Dkk-1 action is unknown. The Wnt family of secreted growth factors initiates signaling via the Frizzled (Fz) receptor and its candidate coreceptor, LDL receptor-related protein 6 (LRP6), presumably through Fz-LRP6 complex formation induced by Wnt. The significance of the Fz-LRP6 complex in signal transduction remains to be established. RESULTS: We report that Dkk-1 is a high-affinity ligand for LRP6 and inhibits Wnt signaling by preventing Fz-LRP6 complex formation induced by Wnt. Dkk-1 binds neither Wnt nor Fz, nor does it affect Wnt-Fz interaction. Dkk-1 function in head induction and Wnt signaling inhibition strictly correlates with its ability to bind LRP6 and to disrupt the Fz-LRP6 association. LRP6 function and Dkk-1 inhibition appear to be specific for the Wnt/Fz beta-catenin pathway. CONCLUSIONS: Our results demonstrate that Dkk-1 is an LRP6 ligand and inhibits Wnt signaling by blocking Wnt-induced Fz-LRP6 complex formation. Our findings thus reveal a novel mechanism for Wnt signal modulation. LRP6 is a Wnt coreceptor that appears to specify Wnt/Fz signaling to the beta-catenin pathway, and Dkk-1, distinct from Wnt binding antagonists, may be a specific inhibitor for Wnt/beta-catenin signaling. Our findings suggest that Wnt-Fz-LRP6 complex formation, but not Wnt-Fz interaction, triggers Wnt/beta-catenin signaling.     

Expression of secreted frizzled-related protein 2 in a primary canine mammary tumor cell line: a candidate tumor marker for mammary tumor cells

Canine mammary tumors (CMTs) have been proposed to be a good animal model for human breast cancer. To provide a basis for the tumorigenic study of CMTs, cell lines were established using a modified cell culture technique. The epithelial morphology and immunostaining with cytokeratin 18 confirmed the epithelial origin of the cells. In an investigation of possible mammary tumorigenesis-related factors, the expression of Wnt signaling-related proteins was detected in cell lines. Secreted frizzled-related protein 2 (SFRP2) was abundantly expressed in CMT cells but not in normal canine mammary gland (MG) cells. Secreted frizzled-related protein 2 was secreted into the culture medium and was associated with the extracellular matrix. In addition, increased expressions of beta-catenin and cyclin D1 were observed in cells overexpressing SFRP2. The marked differential expression of SFRP2 reveals that this protein may be a potential candidate marker for CMTs. The CMT cell line established in this study provides a useful tool and experimental model for understanding both the tumorigenesis of CMTs and the role of Wnt signaling in cancers.     

SFRP2 enhances the osteogenic differentiation of apical papilla stem cells by antagonizing the canonical WNT pathway

Background

Exploring the molecular mechanisms underlying directed differentiation is helpful in the development of clinical applications of mesenchymal stem cells (MSCs). Our previous study on dental tissue-derived MSCs demonstrated that secreted frizzled-related protein 2 (SFRP2), a Wnt inhibitor, could enhance osteogenic differentiation in stem cells from the apical papilla (SCAPs). However, how SFRP2 promotes osteogenic differentiation of dental tissue-derived MSCs remains unclear. In this study, we used SCAPs to investigate the underlying mechanisms.

Methods

SCAPs were isolated from the apical papilla of immature third molars. Western blot and real-time RT-PCR were applied to detect the expression of β-catenin and Wnt target genes. Alizarin Red staining, quantitative calcium analysis, transwell cultures and in vivo transplantation experiments were used to study the osteogenic differentiation potential of SCAPs.

Results

SFRP2 inhibited canonical Wnt signaling by enhancing phosphorylation and decreasing the expression of nuclear β-catenin in vitro and in vivo. In addition, the target genes of the Wnt signaling pathway, AXIN2 (axin-related protein 2) and MMP7 (matrix metalloproteinase-7), were downregulated by SFRP2. WNT1 inhibited the osteogenic differentiation potential of SCAPs. SFRP2 could rescue this WNT1-impaired osteogenic differentiation potential.

Conclusions

The results suggest that SFRP2 could bind to locally present Wnt ligands and alter the balance of intracellular Wnt signaling to antagonize the canonical Wnt pathway in SCAPs. This elucidates the molecular mechanism underlying the SFRP2-mediated directed differentiation of SCAPs and indicates potential target genes for improving dental tissue regeneration.

     

Downregulation of Wnt signaling by sonic hedgehog activation promotes repopulation of human tumor cell lines

Tumor repopulation after radiotherapy is a big obstacle for clinical cancer therapy. The molecular mechanisms of tumor cell repopulation after radiotherapy remain unclear. This study investigated the role of sonic hedgehog (SHH) and Wnt signaling pathways in tumor repopulation after radiotherapy in an in vitro repopulation model. In this model, irradiated dying tumor cells functioned as feeder cells, whereas luciferase-labeled living tumor cells acted as reporter cells. Proliferation of reporter cells was measured by bioluminescence imaging. Results showed that irradiated dying HT29 and Panc1 tumor cells significantly stimulated the repopulation of living cells in their respective cultures. In HT29 and Panc1 cells, radiation significantly inhibited Wnt activity. In the irradiated dying HT29 and Panc1 cells, the level of the activated nuclear β-catenin was significantly decreased. Treatment with the Wnt agonist 68166 significantly decreased, whereas treatment with Wnt antagonist significantly increased, repopulation in HT29 and Panc1 tumor cells in a dose-dependent manner. β-catenin short-hairpin RNA (shRNA) also significantly promoted tumor cell repopulation. The level of secreted frizzled related protein-1 (SFRP1), hedgehog and Gli1 were increased in irradiated cells. Our results highlight the interaction between Wnt and SHH signaling pathways in dying tumor cells and suggest that downregulation of Wnt signaling after SHH activation is negatively associated with tumor repopulation.KEY WORDS: Colon cancer, Pancreatic cancer, Radiotherapy, Repopulation, Wnt signaling