The Wnt signaling pathway antagonists have been studied extensively in development and their involvement in oncogenesis has been demonstrated. There are four families of Wnt antagonists that can be divided into two sub-groups according to their mode of action. The first group includes the secreted-Frizzled related protein (sFRP) family, Wnt-inhibitory factor (WIF-1), and Cerberus. They inhibit Wnt signaling by direct binding to Wnt molecules. The second group, consisting of the Dickkopf (DKK) family, inhibits Wnt signaling by binding to the LRP5/LRP6 component of the Wnt receptor complex [
83‐
85]. In mammalian cells, sFRP1 has been found to specifically bind to Wnt-1 protein, but not Wnt-5a protein, and it modulated the Wnt-1 signaling. sFRP-1 efficiently inhibited the Wnt-1 mediated increase in cytoplasmic β-catenin levels as well as the Wnt-1 induction of transcription from a Lef/Tcf reporter gene [
86]. However binding specificity may not relate to functional specificity, as SFRP-3 associated with Wnt-5a but was unable to inhibit its activity [
87]. Even the significance of specific functional interactions might be suspect based on titration experiments with purified soluble sFRP-1. At low concentrations sFRP-1 enhanced signaling activity by soluble wingless protein, whereas at higher concentrations it was inhibitory [
88]. The authors proposed high and low states of binding affinity that involved the carboxy-terminal heparin binding domain and the amino-terminal cysteine-rich domain of sFRP-1, respectively. Binding to the cysteine-rich domain might confer inhibition while binding to the carboxy-terminal region could facilitate presentation of active ligand to receptor. Thus sFRP-1 exerts a biphasic effect on Wingless (Wg) activity [
88]. Reports from other investigators and recent publications from our laboratory show the biphasic role of sFRPs in renal cancer. Gumz et al. showed that stable re-expression of sFRP-1 in clear cell RCC cells resulted in decreased expression of Wnt target genes, decreased growth in cell culture, inhibition of anchorage-independent growth, and decreased tumor growth in athymic nude mice. Thus sFRP-1 acts as a tumor suppressor and its restoration attenuated the clear cell renal cancer tumor phenotype [
89]. Other studies reported that sFRP-1 expression loss is a common event in renal cancer [
90,
91].
Abnormal promoter methylation of tumor suppressor genes contributes to tightly heritable gene silencing and can cause loss of gene function, which thereby contributes to tumorigenesis. Various Wnt pathway components that regulate proper WNT/β-catenin signaling are frequently disrupted in human cancers through either genetic or epigenetic alterations [
92,
93]. Constitutive activation of WNT/β-catenin signaling as a result of mutations in APC and β-catenin was first documented in both inherited familial adenomatous polyposis (FAP) [
37,
94] and sporadic colon cancers [
38,
95]. Mutations of pathway components including
APC, AXIN1/2 and β-catenin are well established in colorectal [
35,
38,
96,
97], gastric [
98], hepatocellular [
39] as well as other tumors [
99]. Most of the human cancers show elevated levels of nuclear β-catenin, a hallmark of active WNT/β-catenin signaling, although mutations of
APC, AXIN or β-catenin are substantially less frequent. In renal cell carcinoma APC and β-catenin mutations are uncommon events [
100,
101]. Whereas, CpG island hypermethylation at the promoter of a gene is a common and early event in kidney tumorigenesis [
90,
102‐
105]. Independent studies have reported hypermethylation of atleast one of a set of different genes (VHL, p16/INK4a, p14ARF, APC, RASSF-1A, TIMp-3, MGMT, GSTP1, CDH1, and ARF RARbeta2) in over 95% of tumor samples representing all major biological and histological types, grades and stages compared to no methylation in corresponding normal renal or urethral tissues. Results have been found to correlate DNA sediment from pre-operative urine samples, serum and tissue [
102‐
104] highlighting their biomarker potential. Functional loss of negative WNT regulators by epigenetic gene silencing [
92] has been frequently reported to contribute to the activation or amplification of aberrant WNT/β-catenin signaling in tumors. CpG promoter hypermethylation has been often found in antagonists of the Wnt pathway, the SFRP family, WNT inhibitory factor-1 and DICKKOPF family members after comparing primary renal cancer samples to the corresponding normal renal tissues. The methylation levels of six Wnt antagonist genes (sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wif-1, and Dkk-3) were significantly higher in renal cancer compared to normal renal tissues. sFRP-1 methylation was found to be a significant independent predictor of RCC. In RCC patients, the methylation results were identical in samples of tumor and serum DNA. In addition, the methylation status of Wnt antagonist genes in serum DNA was significantly correlated with tumor grade and stage showing their potential as useful epigenetic biomarkers [
106]. Loss of sFRP-1 due to hypermethylation is common in renal carcinoma [
103] than other cancers [
90]. The expression of sFRP-1 was decreased 89% at the mRNA level and 75% at the protein level while the promoter was found to be methylated in 68% of RCC samples [
90]. sFRP-5 was epigenetically suppressed in RCC and its overexpression induced apoptosis in renal cancer cell lines [
107]. The Wnt inhibitory factor-1 (WIF-1) promoter was found to be hypermethylated in RCC and its over-expression inhibited Wnt activity and induces apoptosis in renal cancer cells [
108]. The Dickkopf class of Wnt antagonists including DKK1, DKK2 and DKK3 are also epigenetically silenced in renal cancer and their over-expression induced apoptosis and inhibited renal cell growth
in-vitro and
in-vivo [
22,
23,
109], whereas DKK4 was found higher in renal cancer compared to normal tissue samples and it activated the non-canonical Wnt pathway in renal cancer thereby promoting the invasive and migratory capability of renal cancer cells [
24]. The biphasic effects of some Wnt antagonists and their potential to activate Wnt signaling have been demonstrated in some recent reports from our lab. Saini et al. reported that sFRP1 is related to invasiveness and metastatic behavior in RCC [
110]. The authors showed that the invasive capability of a metastatic renal cancer cell line was decreased by attenuating sFRP1 with a concomitant decrease in the levels of metastasis related gene
MMP10 [
110]. sFRP-2 activated the Wnt pathway and promoted renal cancer growth [
111] whereas sFRP-3 expression induced the
MMP-3 and
ANGPT1 genes in renal cancer and thus contributed to the invasive capability of RCC [
112]. Uren et al. observed that sFRP-1 exerted a biphasic effect on Wnt activity increasing armadillo level at low concentrations but reducing it at higher concentrations. Depending on the expression levels and molecular, cellular and tissue context, the SFRPs may promote Wnt signaling by protecting Wnts from degradation or by facilitating Wnt secretion or transport [
88]. Rubin et al. have reviewed some possible mechanisms to the contradictory behavior of Wnt antagonists [
113]. The functional consequences of over-expression patterns of Wnt antagonists with regard to tomorigenesis are largely unknown and much work will be required to define the specific relationships that govern the interactions of the Wnts, sFRPs, Dickkopfs and Fdzs.