Secreted frizzled-related protein 2: a key player in noncanonical Wnt signaling and tumor angiogenesis.

Overactivation of the Wnt signaling pathway is linked to cancer initiation, progression, and metastasis formation. For example, active Wnt signaling can provide cells with a growth advantage and suppress their differentiation process [ 39 ]. In colorectal cancer (CRC), the majority of cases present with overactivation of the canonical Wnt signaling pathway caused by mutations of, e.g., adenomatous polyposis coli (APC) or β-catenin [ 39 ]. Mutations in β-catenin are also often observed in many other cancer types, such as hepatocellular carcinoma (HCC), gastric carcinoma, ovarian carcinoma, and melanoma [ 39 , 40 ]. These gain-of-function mutations disrupt phosphorylation sites and make β-catenin often refractory to proteasomal degradation. On the other hand, mutations of β-catenin or APC are uncommon in lung cancer [ 41 ]. Hyperactivation of the Wnt signaling pathway in this type of cancer is a result of overexpression of DvL proteins or downregulation of Wnt antagonists, eventually leading to an increase of β-catenin. Noncanonical pathways are also likely to be involved in tumorigenesis. The noncanonical PCP and Wnt/Ca 2+ signaling pathways are involved in cell motility and cell proliferation, respectively. However, there is a high need for further research in this field to fill in the large information gaps that currently exist [ 41 ]. As suggested earlier, the effect of SFRP2 on Wnt signaling seems to be context dependent. In cervical cancer cell lines, overexpression of SFRP2 was found to decrease nuclear β-catenin levels, and consequently downregulated gene expression of the cell cycle regulators C-myc and Cyclin D1 [ 42 ]. Similarly, overexpression of SFRP2 in oral squamous cell carcinoma cells leads to a downregulation of C yclin D1 expression [ 43 ]. Using a different approach, the treatment of melanoma cells with recombinant SFRP2 also inhibited the expression of β-catenin [ 44 ]. Further investigations are needed to see whether a difference in Wnt receptor expression can be observed. Nevertheless, being a key player in the Wnt signaling pathway, SFRP2 is able to influence several branches of tumorigenesis. However, evidence is quite contradictory, describing both tumor promoting and suppressive roles. Many studies have investigated SFRP2 downregulation by promotor hypermethylation in several types of cancer. The SFRP2 promotor has been described to be (hyper)methylated in bladder cancer [ 45 ], breast cancer [ 46 ], cervical cancer [ 47 ], CRC [ 48 ], esophageal cancer [ 49 ], gallbladder cancer [ 50 ], gastric cancer [ 51 ], HCC [ 52 ], lung cancer [ 53 ], ovarian cancer [ 54 ], pancreatic cancer [ 55 ], prostate cancer [ 56 ], endometrial cancer [ 57 ], osteosarcoma [ 58 ], oral carcinoma [ 43 ], skin cancer [ 59 ], and brain tumors [ 60 ]. Similarly, SFRP2 mRNA was decreased in osteosarcoma cell lines compared to primary osteoblast cells [ 58 ]. A reduced expression has also been observed in pituitary adenoma [ 61 ], choriocarcinoma [ 62 ], non-small-cell lung carcinoma [ 63 ], and glioblastoma [ 64 ] compared to their healthy counterparts. When comparing subgroups within the same cancer type, expression of SFRP2 was found to be lower in high grade-, as compared to low grade glioma [ 64 , 65 ]. SFRP2 expression also seems to be involved in tumor aggressiveness and invasiveness, indicated by the largest SFRP2 downregulation in aggressive [ 66 ] and invasive [ 61 ] pituitary adenoma compared to their less aggressive or invasive tumor types, respectively. The fact that SFRP2 is found to be downregulated in a large number of tumor types suggests a tumor suppressor role of the glycoprotein [ 67 ]. Indeed, low expression of SFRP2 was associated with a poor clinical outcome in glioblastoma patients [ 64 ]. The relationship between SFRP2 expression and tumor growth was further explored in murine tumor models. When nude mice were subcutaneously inoculated with oral squamous cell carcinoma cells or gastric cancer cells overexpressing SFRP2, tumor size was greatly reduced compared to control cells [ 43 , 51 ]. In an orthotopic model of glioblastoma, overexpression of SFRP2 was also associated with reduced tumor growth and prolonged survival of mice [ 64 ]. In a reversed approach, when SFRP2 expression was silenced in choriocarcinoma cells, subcutaneous xenografts grew significantly larger compared to those from cells expressing SFRP2 [ 62 ]. Together, these data provide evidence that SFRP2 can function as a tumor suppressor. On the contrary, (over)expression of SFRP2 in cancer cell lines and tumor tissues has also been described. Canine mammary tumor cell lines have an abundant SFRP2 expression, while this was not observed in normal mammary gland cells [ 68 ]. Similarly, SFRP2 expression was significantly higher in osteosarcoma tumors compared to mesenchymal stem cells [ 69 ]. In bone marrow samples from multiple myeloma patients, SFRP2 could be detected in 10/14 specimens, while only 1/5 bone marrow samples from patients without bone lesions scored positive for SFRP2 expression [ 70 ]. When investigating the levels of SFRP2 in serum of breast cancer patients [ 71 ], levels were found to be elevated in patients, as compared to controls. High levels of SFRP2 in serum were associated with a poor prognosis. So, in the context of breast cancer, SFRP2 levels in serum may be a promising biomarker and prognostic prediction tool. The relationship between SFRP2 expression and poor survival in breast cancer was further confirmed by Hill et al. [ 72 ] and Mohammed et al. [ 73 ] . A similar correlation between protein expression and prognosis was observed in osteosarcoma [ 74 ] and CRC patients [ 75 ]. This further confirms that SFRP2 can play a role as tumor promotor. Direct proof for the tumor promoting effect of SFRP2 can be obtained from in vivo tumor mouse models. Yamamura et al. transfected renal carcinoma cells with SFRP2 and monitored their tumor growth potential in nude mice [ 76 ]. SFRP2-overexpressing cells generated significantly larger tumors compared to regular renal carcinoma cells, consistent with activated Wnt signaling. Similarly, glioma cells that were experimentally designed to overexpress SFRP2 did generate larger xenografts in athymic mice compared to their non-mutated counterparts [ 77 ]. Switching to another approach, treatment of angiosarcoma or breast cancer, using an anti-SFRP2 antibody, results in a significant tumor growth inhibition [ 78 , 79 ]. Treated tumors displayed no differences in proliferation, but apoptosis was greatly enhanced, as compared to control tumors [ 79 ]. Even though this study did not investigate the effect on the Wnt signaling pathway in vivo , their in vitro data suggest that SFRP2 antagonism results in a reduced level of nuclear β-catenin, unmasking SFRP2 as a Wnt agonist in this setting [ 78 ]. Xiao et al. did confirm this agonistic effect on Wnt signaling in lung cancer cell lines [ 80 ]. As mentioned earlier, Wnt signaling can also play a major role in the formation of metastasis. Indeed, in late stage cancers, Wnt5a is often upregulated and is known to promote invasion and metastasis formation in breast cancer, melanoma, and gastric cancer [ 81 ]. This highlights the role of the noncanonical PCP signaling pathway in this process. Similar effects have been described for the noncanonical Wnt/Ca 2+ signaling pathway in the context of melanoma [ 82 ]. Specifically looking at SFRP2, Techavichit et al. compared the expression levels in both cell lines and tissues samples of metastatic and non-metastatic osteosarcoma, and hit upon a significantly higher SFRP2 expression in metastatic tumors [ 69 ]. While overexpression of SFRP2 in osteosarcoma cells did not significantly affect primary tumor growth, a larger number of lung metastases occurred [ 69 ]. Similar pro-metastatic effects of SFRP2 were also observed for breast cancer cells [ 83 ] and melanoma cells [ 44 ]. These data indicate that SFRP2 is a potent stimulator of cell migration and invasion. Montagner et al. investigated which mechanism was responsible for the pro-metastatic effect in melanoma and did not see any effect on canonical Wnt signaling when cells were depleted of SFRP2 [ 83 ].

The secreted glycoprotein SFRP2 is known to regulate Wnt signaling, both via the canonical and noncanonical pathways. It is therefore not unexpected that this protein plays important roles in embryonic development and cancer initiation and metastasis formation. A major recent finding is the upregulation of SFRP2 in the tumor vasculature, suggesting it being a specific marker of tumor endothelial cells [ 5 ]. The upregulation of SFRP2 can enhance noncanonical Wnt/Ca 2+ signaling, resulting in enhanced tumor angiogenesis, a crucial step in tumorigenesis [ 87 ]. Therefore, targeting of SFRP2 with anti-SFRP2 antibodies or small molecules can disrupt this process, making it a promising approach in anti-cancer therapy. However, besides upregulation on the tumor vessels, it is important that target candidates are absent or only limitedly expressed on regular blood vessels or other tissues to prevent toxicity. The studies of Fontenot [ 78 ] and Garcia [ 79 ] provide valuable information on the potential use of anti-SFRP2 antibodies to reduce tumor growth and tumor angiogenesis. Importantly, they did not observe any weight loss or pathological abnormalities in mice treated with these antibodies, suggesting that targeting SFRP2 is a safe anti-cancer strategy. Likewise, molecular imaging in mice using a SFRP2-targeted contrast agent showed specific imaging of the tumor vessels which enhanced by increasing tumor size [ 6 ]. This provides evidence that SFRP2 expression is specific for blood vessels in the tumor.

The glycoprotein SFRP2 is shown to be a key player in the process of tumor angiogensis, an important process in tumor formation and progression. This tumor promoting effect can likely be contributed to the upregulation SFRP2 on the tumor vasculature and, consequently, activation of the noncanonical Wnt/Ca 2+ pathway. We believe that vaccination against specific tumor endothelial markers is a promising approach to treat or even prevent cancer [ 101 ]. Due to its specific expression on the tumor vasculature and the absence of toxicity when treating mice with anti-SFPR2 antibodies, we propose SFRP2 might be a valuable target for vaccination. We previously showed that vaccination against tumor endothelial cell markers leads to the production of target specific antibodies, which are efficient in reducing tumor growth and tumor vessel density in mouse models [ 9 , 101 , 102 ]. Future studies are needed to confirm the potential anti-angiogenic effect of vaccination against SFRP2 in solid tumors.

The secreted frizzled-related protein (SFRP) family consists of five secreted glycoproteins: SFRP1, SFRP2, SFRP3, SFRP4, and SFRP5 (Fig.  1 ). From a phylogenetic perspective, SFRP1, SFPR2, and SFRP5 form an SFRP subfamily based on their sequence similarities [ 1 ]. All five family members contain a signal peptide, a netrin domain (NTR), and a frizzled-like cysteine-rich domain (Fz/CRD) (Fig. 1 ). The signal peptide is important in the secretion process of SFRP2 and is likely to be absent in the mature secreted protein. The C-terminal NTR domain contains six conserved cysteine residues, able to form a total of three disulfide bridges. This domain shows homology to the netrin domain found in complement proteins C3, C4, C5, type I procollagen C-proteinase enhancer proteins, and tissue inhibitors of metalloproteinases [ 2 ]. The Fz/CRD domain present in SFRP proteins is highly similar to the extracellular Wnt binding domain of FzD receptors [ 3 ], enabling binding between SFRP proteins to Wnt ligands. The SFRP family is known to be involved in the regulation of Wingless-related integration site (Wnt) signaling, an important pathway not only in embryonic development, tissue regeneration, and cell proliferation, but also in carcinogenesis [ 4 ]. This pathway is activated by binding of soluble Wnt ligands to frizzled (FzD) receptors on the cell surface, and eventually leads to the transcription of Wnt target genes. Fig. 1 The SFRP family members contain a signal peptide (green), frizzled-like cysteine-rich domain (Fz/CRD; blue), and netrin domain (NTR; purple). Synonyms for each protein are indicated between brackets The SFRP family members contain a signal peptide (green), frizzled-like cysteine-rich domain (Fz/CRD; blue), and netrin domain (NTR; purple). Synonyms for each protein are indicated between brackets Although overactivation of the Wnt signaling pathway is inextricably linked to cancer initiation and progression, it also plays an important role in the tumor vasculature. Interestingly, SFRP2 is described to be overexpressed in the tumor vasculature of breast cancer tissues [ 5 ]. In addition, SFRP2-directed ultrasound imaging clearly shows specific signal in the tumor vasculature, while normal vessels are not visualized [ 6 ]. Proteins such as SFRP2, that seem to be specifically (over)expressed in the tumor vasculature, show great promise as therapeutic targets in the fight against cancer [ 7 – 9 ]. To enhance insight in oncogenic role of SFRP2, we performed a literature study about SFRP2 in Wnt signaling and tumor angiogenesis.