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 is a paracrine modulator of epithelial branching morphogenesis, proliferation, and secretory gene expression in the prostate

Previous in vitro studies identified secreted frizzled related protein 1 (SFRP1) as a candidate pro-proliferative signal during prostatic development and cancer progression. This study determined the in vivo roles of SFRP1 in the prostate using expression studies in mice and by creating loss- and gain-of-function mouse genetic models. Expression studies using an Sfrp1^lacZ knock-in allele showed that Sfrp1 is expressed in the developing mesenchyme/stroma of the prostate. Nevertheless, Sfrp1 null prostates exhibited multiple prostatic developmental defects in the epithelium including reduced branching morphogenesis, delayed proliferation, and increased expression of genes encoding prostate-specific secretory proteins. Interestingly, over-expression of SFRP1 in the adult prostates of transgenic mice yielded opposite effects including prolonged epithelial proliferation and decreased expression of genes encoding secretory proteins. These data demonstrated a previously unrecognized role for Sfrp1 as a stromal-to-epithelial paracrine modulator of epithelial growth, branching morphogenesis, and epithelial gene expression. To clarify the mechanism of SFRP1 action in the prostate, the response of WNT signaling pathways to SFRP1 was examined. Forced expression of SFRP1 in prostatic epithelial cells did not alter canonical WNT/β-catenin signaling or the activation of CamKII. However, forced expression of SFRP1 led to sustained activation of JNK, and inhibition of JNK activity blocked the SFRP1-induced proliferation of prostatic epithelial cells, suggesting that SFRP1 acts through the non-canonical WNT/JNK pathway in the prostate.     

Secreted frizzled related protein 1 (Sfrp1) and Wnt signaling in innervated and denervated skeletal muscle

Wnts are secreted proteins with functions in differentiation, development and cell proliferation. Wnt signaling has also been implicated in neuromuscular junction formation and may function in synaptic plasticity in the adult as well. Secreted frizzled-related proteins (Sfrps) such as Sfrp1 can function as inhibitors of Wnt signaling. In the present study a potential role of Wnt signaling in denervation was examined by comparing the expression levels of Sfrp1 and key proteins in the canonical Wnt pathway, Dishevelled, glycogen synthase kinase 3β and β-catenin, in innervated and denervated rodent skeletal muscle. Sfrp1 mRNA and immunoreactivity were found to be up-regulated in mouse hemidiaphragm muscle following denervation. Immunoreactivity, detected by Western blots, and mRNA, detected by Northern blots, were both expressed in extrasynaptic as well as perisynaptic parts of the denervated muscle. Immunoreactivity on tissue sections was, however, found to be concentrated postsynaptically at neuromuscular junctions. Using β-catenin levels as a readout for canonical Wnt signaling no evidence for decreased canonical Wnt signaling was obtained in denervated muscle. A role for Sfrp1 in denervated muscle, other than interfering with canonical Wnt signaling, is discussed.     

Secreted frizzled related protein 4 (sFRP4) update: A brief review

Secreted frizzled-related proteins control a multitude of biological phenomena throughout development and adult life in humans. In parallel, aberrant gene expression and abnormal secreted protein levels accompany a wide range of pathologies in humans. In this review, we provide a brief introduction to sFRP4, an update of the pathways it's involved, its various physiological actions that are reported to contribute to diseases, outlining the importance of its wider research and specific modulation by pharmacologic interventions. First recognized as a novel molecule that co-purified with a disparate protein, its identity was based on its sequence homology to the frizzled receptors. Once multiple members of the family were cloned, their genetic loci, tissue and subcellular distributions were located. Nucleotide and amino acid sequences were characterized and homology to different organisms was found to be present that helped elucidate their actions. Following subsequent experimental studies, they were found to be secreted proteins with an affinity to bind to the Wnt ligands, participating in different developmental and adult homeostatic pathways by the virtue of their regulatory function to the Wnt signal transduction system. Secreted frizzled related protein 4 has garnered considerable attention in the recent years following breakthrough discoveries implicating them in the pathogenesis of various diseases. Studies investigating them can provide information not only regarding their association with a disease but can also help use them as potential biomarkers and therapeutic targets.     

The secreted frizzled related protein 2 (SFRP2) gene is a target of the Pax2 transcription factor

Despite their essential role in vertebrate development, the function of Pax proteins in gene regulation is not well understood. To identify potential genes regulated by the Pax2 protein, we screened embryonic kidney cells transformed with Pax2-expressing retroviruses for genes activated in response to Pax2 expression. In this system, the gene encoding the secreted frizzled related protein, Sfrp2, was strongly activated in all Pax2b-expressing cells. This activation of Sfrp2 expression correlated with changes in chromatin structure at the Sfrp2 locus, particularly in and around regions of Pax2 binding. Although the amount of Pax2-dependent transactivation was low in transient assays, the data suggests that local alterations of chromatin structure by Pax proteins can greatly enhance expression when presented in the right cellular context.     

Obscurin-like 1, OBSL1, is a novel cytoskeletal protein related to obscurin

Cytoskeletal adaptor proteins serve vital functions in linking the internal cytoskeleton of cells to the cell membrane, particularly at sites of cell-cell and cell-matrix interactions. The importance of these adaptors to the structural integrity of the cell is evident from the number of clinical disease states attributable to defects in these networks. In the heart, defects in the cytoskeletal support system that surrounds and supports the myofibril result in dilated cardiomyopathy and congestive heart failure. In this study, we report the cloning and characterization of a novel cytoskeletal adaptor, obscurin-like 1 (OBSL1), which is closely related to obscurin, a giant structural protein required for sarcomere assembly. Multiple isoforms arise from alternative splicing, ranging in predicted molecular mass from 130 to 230 kDa. OBSL1 is located on human chromosome 2q35 within 100 kb of SPEG, another gene related to obscurin. It is expressed in a broad range of tissues and localizes to the intercalated discs, to the perinuclear region, and overlying the Z lines and M bands of adult rat cardiac myocytes. Further characterization of this novel cytoskeletal linker will have important implications for understanding the physical interactions that stabilize and support cell-matrix, cell-cell, and intracellular cytoskeletal connections.     

The GATA-binding protein CGF-1 is closely related to GT-1

Many light-regulated genes contain a conserved GATA motif in their 5-upstream region. We have characterized in detail the GATA-binding factor, CGF-1, which binds within a 73 bp TATA-proximal light/circadian regulatory element in the Arabidopsis cab2 promoter and to two more sites farther upstream. CGF-1 was found to be distinct from other metal-dependent GATA-binding factors, but to have the same sequence requirements for binding and similar physical and chemical properties as GT-1, a factor required for light regulation of the tobacco rbcS-3A gene. CGF-1 was found to be constitutively present in extracts and was shown to be immunologically related to GT-1. The close similarity between CGF-1 and GT-1 suggests that a GT-1-like factor is involved in the phytochrome/circadian regulation of the cab2 gene. CGF-1 and GT-1 were also found to have similar sequence specificities to another constitutively-regulated GATA factor, IBF-2b, which binds the I box region of the tomato nitrate reductase gene. Of three complexes detected using an IBF-2b-specific probe, only one was identical to CGF-1/GT-1. The other two were similar to IBF-2b, demonstrating that CGF-1/GT-1, although very similar, are actually distinct from IBF-2b. These data indicate that more than one factor can bind to the same short sequence and may indicate how constitutively present factors like GT-1 can play a role in light regulation.     

Expression of secreted frizzled related protein 1, a Wnt antagonist, in brain, kidney, and skeleton is dispensable for normal embryonic development

Secreted frizzled related protein-1 (sFRP1), an antagonist of Wnt signaling, regulates cell proliferation, differentiation and apoptosis and negatively regulates bone formation. The spatial and temporal pattern of endogenous sFRP1 expression and loss-of-function were examined in the sFRP1-LacZ knock-in mouse (sFRP1-/-) during embryonic development and post-natal growth. beta-gal activity representing sFRP1 expression is robust in brain, skeleton, kidney, eye, spleen, abdomen, heart and somites in early embryos, but sFRP1 gene inactivation in these tissues did not compromise normal embryonic and post-natal development. Kidney histology revealed increased numbers of glomeruli in KO mice, observed after 5 years of breeding. In the skeleton, we show sFRP1 expression is found in relation to the mineralizing front of bone tissue during skeletal development from E15.5 to birth. Trabecular bone volume and bone mineral density in the sFRP1-/- mouse compared to WT was slightly increased during post-natal growth. Calvarial osteoblasts from newborn sFRP1-/- mice exhibited a 20% increase in cell proliferation and differentiation at the early stages of osteoblast maturation. sFRP1 expression was observed in osteoclasts, but this did not affect osteoclast number or activity. These findings have identified functions for sFRP1 in kidney and bone that are not redundant with other sFRPs. In summary, the absence of major organ abnormalities, the enhanced bone formation and a normal life span with no detection of spontaneous tumors suggests that targeting sFRP1 can be used as a therapeutic strategy for increasing bone mass in metabolic bone disorders or promoting fracture healing by modulating Wnt signaling.     

Identification of iminooxothiazolidines as secreted frizzled related protein-1 inhibitors

Secreted frizzled related protein-1 (sFRP-1) inhibitors have the potential to be used for the treatment of osteoporosis or other bone related disorders, since the level of sFRP-1 affects osteoblast apoptosis and proliferation. From high throughput screening, we have identified a class of iminooxothiazolidines as sFRP-1 inhibitors. Structure–activity relationships were established for various regions of the scaffold along with the biochemical characterization of this class to probe selectivity, binding and ex vivo activity.     

Schizosaccharomyces pombe Spk1 is a tyrosine-phosphorylated protein functionally related to Xenopus mitogen-activated protein kinase.

Mitogen-activated protein kinase (MAPK) and its direct activator, MAPK kinase (MAPKK), have been suggested to play a pivotal role in a variety of signal transduction pathways in higher eukaryotes. The fission yeast Schizosaccharomyces pombe carries a gene, named spk1, whose product is structurally related to vertebrate MAPK. Here we show that Spk1 is functionally related to Xenopus MAPK. (i) Xenopus MAPK partially complemented a defect in the spk1- mutant. An spk1- diploid strain could not sporulate, but one carrying Xenopus MAPK could. (ii) Both Spk1 and Xenopus MAPK interfered with sporulation if overexpressed in S. pombe cells. (iii) Spk1 underwent tyrosine phosphorylation as does Xenopus MAPK. Tyrosine phosphorylation of Spk1 appeared to be dependent upon mating signals because it occurred in homothallic cells but not in heterothallic cells. Furthermore, this phosphorylation was diminished in a byr1 disruptant strain, suggesting that spk1 lies downstream of byr1, which encodes a MAPKK homolog in S. pombe. Taken together, the MAPKK-MAPK cascade may be evolutionarily conserved in signaling pathways in yeasts and vertebrates.     

Alterations of frizzled (FzE3) and secreted frizzled related protein (hsFRP) expression in gastric cancer.

Wnt signaling pathway is important for development and carcinogenesis. Alterations of this pathway, such as mutations in adenomatous polyposis coli (APC) gene and activation mutations of beta-catenin, would result in stabilization of beta-catenin and subsequent translocation to nucleus where genes are transcribed. Recently, a receptor of Wnt, FzE3 was found to be up-regulated in esophageal carcinoma while a non-receptor antagonist of Wnt, secreted frizzled related protein (hsFRP) was found to be down-regulated in some cancer. These findings suggested that FzE3 is a potential oncogene while hsFRP is a potential tumor suppressor gene. We aimed to investigate whether FzE3 and hsFRP were altered in gastric cancer. Twelve cases of gastric cancer, including 7 cases of intestinal type, 4 cases of diffuse type and I case of mixed type, were studied. FzE3 and hsFRP mRNAs were expressed in most of the paired normal gastric tissues. FzE3 was over-expressed in 9 cases (75%) of gastric carcinoma tissues while hsFRP was down-regulated in 2 cases (16%). Beta-catenin nuclear staining was identified in 3 cases (27%) and cyclin D1 was expressed in 5 cases (41%) of cancer samples. All these cases were associated with either up-regulation of FzE3 or down-regulation of hsFRP. Our results suggested that alterations of FzE3 or hsFRP were frequent in gastric cancer. These provide alternative mechanisms leading to activation of Wnt signaling pathway in gastric carcinogenesis.     

Plasmodium DDI1 is a potential therapeutic target and important chromatin-associated protein

DNA damage inducible 1 protein (DDI1) is involved in a variety of cellular processes including proteasomal degradation of specific proteins. All DDI1 proteins contain a ubiquitin-like (UBL) domain and a retroviral protease (RVP) domain. Some DDI1 proteins also contain a ubiquitin-associated (UBA) domain. The three domains confer distinct activities to DDI1 proteins. The presence of a RVP domain makes DDI1 a potential target of HIV protease inhibitors, which also block the development of malaria parasites. Hence, we investigated the DDI1 of malaria parasites to identify its roles during parasite development and potential as a therapeutic target. DDI1 proteins of Plasmodium and other apicomplexan parasites share the UBL-RVP domain architecture, and some also contain the UBA domain. Plasmodium DDI1 is expressed across all the major life cycle stages and is important for parasite survival, as conditional depletion of DDI1 protein in the mouse malaria parasite Plasmodium berghei and the human malaria parasite Plasmodium falciparum compromised parasite development. Infection of mice with DDI1 knock-down P. berghei was self-limiting and protected the recovered mice from subsequent infection with homologous as well as heterologous parasites, indicating the potential of DDI1 knock-down parasites as a whole organism vaccine. Plasmodium falciparum DDI1 (PfDDI1) is associated with chromatin and DNA-protein crosslinks. PfDDI1-depleted parasites accumulated DNA-protein crosslinks and showed enhanced susceptibility to DNA-damaging chemicals, indicating a role of PfDDI1 in removal of DNA-protein crosslinks. Knock-down of PfDDI1 increased susceptibility to the retroviral protease inhibitor lopinavir and antimalarial artemisinin, which suggests that simultaneous inhibition of DDI1 could potentiate antimalarial activity of these drugs. As DDI1 knock-down parasites confer protective immunity and it could be a target of HIV protease inhibitors, Plasmodium DDI1 is a potential therapeutic target for malaria control.     

Kermit, a frizzled interacting protein, regulates frizzled 3 signaling in neural crest development

Wnts are a family of secreted glycoproteins that are important for multiple steps in early development. Accumulating evidence suggests that frizzled genes encode receptors for Wnts. However, the mechanism through which frizzleds transduce a signal and the immediate downstream components that convey that signal are unclear. We have identified a new protein, Kermit, that interacts specifically with the C-terminus of Xenopus frizzled-3 (Xfz3). Kermit is a 331 amino acid protein with a central PDZ domain. Kermit mRNA is expressed throughout Xenopus development and is localized to neural tissue in a pattern that overlaps Xfz3 expression temporally and spatially. Co-expression of Xfz3 and Kermit results in a dramatic translocation of Kermit to the plasma membrane. Inhibition of Kermit function with morpholino antisense oligonucleotides directed against the 5' untranslated region of Kermit mRNA blocks neural crest induction by Xfz3, and this is rescued by co-injection of mRNA encoding the Kermit open reading frame. These observations suggest that Kermit is required for Wnt/frizzled signaling in neural crest development. To the best of our knowledge, Kermit is the first protein identified that interacts directly with the cytoplasmic portion of frizzleds to modulate their signaling activity.     

Secreted protein acidic and rich in cysteine is a matrix scavenger chaperone

Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca(2+) concentrations are low, high extracellular concentrations of Ca(2+) activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca(2+) concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimensional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the pathogenesis of matrix-associated disorders and lead to the novel treatment strategies.