The Wnt signaling pathway is modulated by extracellular and intracellular control mechanisms. Extracellular antagonists are Dkk, sFRP, Cerebrus, and WIF-1. Inside the cell, Dvl, APC, Axin, CK-1 α, GSK-3 β Wnt play a role in the signal pathway. Especially sFRP and Dkk-1 genes are the most important extracellular antagonists. Dkk-1 and sFRP genes, which play a role in the Wnt pathway, have a tumor-suppressive role, and inhibit the Wnt signaling pathway . Dkk-1 inhibits the canonical Wnt signaling pathway by binding to the Frizzled receptor and LRP 5/6. On the other hand, sFRP genes, bind directly to the Wnt ligand and show an antagonistic effect. sFRP genes inhibit both canonical and non-canonical signaling pathways . The Wnt signaling pathway plays a key role in the development of lung adenocarcinoma [22,23]. In our study, expression levels of APC1, APC2, Dkk-1, Dkk-3, sFRP-2, sFRP-4, sFRP-5 genes involved in the Wnt signaling pathway were evaluated in paraffin-embedded lung adenocarcinoma and adjacent normal lung tissue. The expression levels of the Dkk-1 gene in tumor tissue, especially in stage I-II, were statistically significantly suppressed compared to normal tissue. Although Dkk-1 gene expression was suppressed in tumor tissue of patients with early-stage lung adenocarcinoma, the level of expression of sFRP -5 gene, another extracellular antagonist, was found to be statistically significantly higher. This reverse regulation between Dkk-1 and sFRP -5 gene expressions was striking in our study. In the literature, cross-regulation between the sFRP-1 and Dkk-1 gene has also been associated with multidrug resistance .
Another remarkable result is that while the expression level of the Dkk-1 gene in tumor tissues is generally reported to be high in the literature, the opposite of the literature Dkk-1 gene expression was suppressed in our study. Studies of the Wnt signaling pathway in lung cancers have generally been studied in NSCLC cell cultures or adenocancer cell cultures, and studies in human tissues with homogeneous adenocarcinoma tumors are limited in the literature [25–28]. Differences in Dkk-1 gene expression levels have also been reported in NSCLC cell cultures . Dkk-1 gene expression levels in NSCLC tissues of Xiao Yao et al. were in line with the results of our study and were suppressed . Xiao Yao et al. pointed out that this difference in other studies may be ascribed to different sources of tissue samples . The proportion of lung adenocarcinoma tissue is also higher in the authors' studies [29–31]. Our study was homogeneous and consisted of lung adenocarcinoma tissues and mostly stage I. As the stage progresses in lung cancers, the promoter methylation of the Dkk-1 gene increases advanced in other stages compared to the first stage. Overexpression of the Dkk-1 gene in advanced stages leads to increased cancer invasion and migration . It fulfills this role through the PI3K / Akt signaling pathway [14,37]. For this, BAX and Bcl-2 signal with the AKT-1 pathway by inducing the increase of mRNA in apoptotic gene levels . In our study, the protein-protein relationship of Dkk-1 and sFRP-5 was enriched with the String database  (Figure 5). These two proteins with dysregulation have agonistic and antagonistic roles in the Wnt signaling pathway in the GO process analyses  (Table 9). Dkk-1 protein is closely related to pro-oncogenes such as Wnt 3a, Wnt 8b, Wnt 8a, Wnt 1 as shown in figure 5.
The protein-protein relationship of the Dkk-1 and sFRP-5 genes and the enriched analysis of the KEGG signaling pathway database also revealed their relationship with the mTOR, Hippo signaling pathway  (Table 10).
The role of the Dkk-1 gene in human cancer cells is still controversial. In mouse tumor models with advanced NSCLC and in vitro studies, blockade of Dkk-1 gene activity (DKN-01) reduced tumor proliferation and invasion . Again, due to the role of the Dkk-1 gene in bone metabolism, it has been proposed as a biomarker in the early diagnosis of bone metastasis, where high expression levels play a role in bone metastasis in patients with lung cancer [14,30,32]. In our study, high expression levels of sFRP-5, one of the sFRP proteins, which is an extracellular antagonist of the Wnt ligand, were detected in early-stage adenocarcinoma tissues. The sFRP-5 gene is mostly involved in lipoprotein metabolism and plays a protective role in the development of cardiovascular disease [39,40]. There is no study on the relation with Wnt-mediated lung adenocarcinoma development and the sFRP -5 gene in the PubMed database ("SFRP5 protein, human" [Supplementary Concept])AND "Adenocarcinoma of Lung"[Mesh]. Especially in the sFRP-5 gene and NSCLC cancer development, sFRP genes are prone to methylation, and methylation of the gene has been reported to be correlated with EGFR mutations. Patients with the methylated sFRP-5 gene have lower disease-free survival rates and higher EGFR_TKI drug resistance than other non-methylated patients . Although sFRP genes are Wnt antagonists, biphasic effects on the Wnt signaling pathway have also been reported. Wnt agonistic effects of sFRP genes by increasing c-JUN-terminal kinase activity through the non-canonical Wnt signaling pathway and inducing an increase in indirect Dkk-1 gene expression levels, increased Dkk gene expression levels in advanced stages have been tried to be explained in the literature .
On the other hand, studies by Liang et al. found that sFRP genes play an active role not only outside the cell but also inside the cell, bind B-catenin in the nucleus, and increase the expression of Wnt target genes, showing agonistic effects . Contrary to what is known, the sFRP gene is not antagonistic, but an oncogenic role in the development of adenocarcinoma in the Wnt signaling pathway was also detected at an early stage in our study.
Also, in our study, as an explanation of difference or dysregulation between sFRP 5 and Dkk-1 gene expressions, it was reported that methylation and mRNA expression levels of sFRP 2 and Dkk-2 genes in gastric cancer tissues by Wang H et al. , methylation of these two genes were simultaneous, methylation rates were high, and the mRNA gene expression levels of the Dkk-2 gene were negatively correlated with the methylation rate. These authors argued that this signaling pathway and hypermethylation of genes were the underlying cause of gastric carcinogenesis .