Introduction: Wnt signaling pathway is often dysregulated in the pathogenesis of various malignancies, including breast cancer. This might be related to methylation of the genes encoding antagonists of this signaling pathway. Aim: The aim of the study was to analyze the methylation status of the promoter regions of Wnt antagonists-secreted frizzled-related protein 1 (sFRP1) and Dickkopf 3 (DKK3) and to determine their correlation with clinicopathological parameters and survival outcome in patients with primary invasive ductal breast cancer. Materials and Methods: The methylation status of sFRP1 and DKK3 was analyzed in 160 breast tumor samples using methylation-specific polymerase chain reaction. Statistical analysis was performed using SPSS software. P ≤ 0.05 was considered as statistically significant. Results: The promoter region of sFRP1 and DKK3 genes was found to be methylated in 76% and 64% of total invasive ductal breast cancer patients, respectively. The promoter methylation in sFRP1 and DKK3 genes was significantly associated with larger tumor size, positive lymph nodes, advanced stage, and perinodal extension of breast tumors. Further, sFRP1 methylation was associated with human epidermal growth factor receptor 2-positive tumors while DKK3 methylation was associated with Grade 3 tumors. Survival analysis demonstrated that sFRP1 methylation was correlated with reduced overall survival in breast cancer patients. Conclusion: Promoter methylation of Wnt pathway antagonists is frequent in breast cancer ultimately leading to probable upregulation of the pathway in these tumors. Hence, sFRP1 and DKK3 methylation may be used as a valuable biomarker in clinical breast cancer management.

Introduction

Breast cancer is a frequently diagnosed cancer contributing to 24.2% of total cancer cases and 13% of cancer deaths among females, worldwide. It is also the most common cancer among women in India with an estimated 162,468 (27.7%) new cases diagnosed and 87,090 (12.19%) deaths, according to GLOBOCAN 2018. The increasing trend of breast cancer owes to its clinically, molecularly, and biologically heterogeneous nature. This leads to disparate clinical behaviors and outcomes in breast cancer patients despite common histopathological features at diagnosis.[1] Hence, the aberrations at the genomic and molecular level results in dysregulated signaling pathways and thereby breast cancer initiation and progression are required to be explored.

In addition, breast carcinogenesis being a multistep process involves a combination of both genetic and epigenetic alterations.[2] The most common and well-defined epigenetic alteration is 5'-cytosine methylation that occurs within CpG islands in gene promoter regions and affects gene expression.[3] The genes affected by such alteration are mostly the tumor suppressor genes involved in regulation of several cellular pathways such as cell cycle, DNA repair, and growth factor signaling or cell adhesion involved in several cancer development including breast cancer.[4] Wnt signaling is one such developmental pathway that is predominantly disrupted, most importantly in breast cancer.[5],[6] The dysregulation of Wnt signaling pathway due to epigenetic aberrations owes to the promoter methylation of genes encoding pathway antagonists, such as secreted frizzled-related protein (sFRP), Dickkopf (DKK), and Wnt-inhibitory-factor which are reported to play a significant role in malignant behavior of breast cancer.

Methylation of sFRP1 gene leads to loss of its antagonistic effect on Wnt ligand, eventually resulting in elevated levels of β-catenin.[7] In breast cancer patients, it is shown to be associated with reduced overall survival (OS).[8],[9] Moreover, methylation of DKK3 gene results in downregulation of its protein expression which thereby causes anomalous Wnt signaling and poor survival in several human malignancies, prominently breast cancer.[10] Thus, the functional loss of sFRP1 and DKK3 genes contributing to Wnt pathway activation leads to dysregulation of cell proliferation and differentiation.[11] Based on these facts, in the present study, we aimed to evaluate the frequency of the promoter methylation of sFRP1 and DKK3 genes in patients with primary breast cancer. Further, the relation of promoter methylation status of these genes with various clinicopathological parameters is analyzed as well as their prognostic and predictive value is determined.

Materials and Methods

Patients

In the present study, a total of 160 untreated histologically confirmed Invasive Ductal Carcinoma patients of breast registered at The Gujarat Cancer and Research Institute from March 2014 to December 2015 were enrolled. The study was approved by the Institute's Ethics Committee Board, and written consent forms were obtained from all the patients before treatment administration. Detailed clinicopathological history of the patients was obtained from the case files maintained at the medical record department of the institute. Histopathological details such as tumor size, lymph node status, disease stage, Bloom–Richardson score (BR score), histological grade and status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2) were evaluated and reported on routine basis by the pathologists of our institute. All patients underwent surgery, and adjuvant treatment decision was based on molecular subtypes of breast cancer patients by clinicians of the institute. The clinicopathological characteristics of the enrolled patients are enlisted in [Table 1]. Complete follow-up details were obtained for 69% (111/160) patients and were included in OS analysis. Among these, 3% (2/111) patients had persistent disease and so only 68% (109/160) patients were analyzed for relapse-free survival (RFS).{Table 1}

Bisulfite-modification and methylation-specific polymerase chain reaction

For methylation-specific polymerase chain reaction (MS-PCR) study, breast tumor tissues were collected immediately after surgery and tumor portion selected by the pathologist was snap frozen in the liquid nitrogen and stored at −80°C till further procedure. DNA extraction was performed using phenol: chloroform extraction method and quantified by agarose gel electrophoresis using Lambda DNA-HindIII digest ladder. The isolated genomic DNA was modified by bisulfite treatment using EpiJET Bisulfite Conversion Kit (ThermoScientific, Lithuania, Europe). Manufacturer's protocol was strictly followed. Thereafter, bisulfite modified DNA was amplified by MS-PCR as described by Herman et al.[12] The primers used [Table 2] were specific for either the unmethylated or the methylated promoter regions of sFRP1 and DKK3 and the PCR reaction was carried out in a 25 μl system using Platinum® PCR SuperMix (Invitrogen, CA, USA) according to the manufacturer's protocol. The reaction conditions for each PCR are described in [Table 3].{Table 2}{Table 3}

Statistical analysis

The statistical evaluation of the data was carried out using Statistical Package for Social Sciences (SPSS) software version 16 (SPSS Inc, USA). Two-tailed Chi-square test and Spearman's correlation method were used to correlate the promoter methylation status of the molecules with various clinicopathological characteristics of breast cancer patients. Survival analysis was performed using Kaplan–Meier survival function, and the differences in survival were tested for statistical significance using log-rank statistic. P ≤ 0.05 was considered to be statistically significant.

Results

Secreted frizzled-related protein 1 and Dickkopf 3 promoter methylation in breast cancer

In total breast cancer patients, sFRP1 promoter region gene was found to be methylated in 76% (122/160) patients and unmethylated in 24% (38/160) patients. Further, DKK3 promoter region gene was observed to be methylated in 64% (103/160) of breast cancer patients compared to 36% (57/160) of patients with unmethylated DKK3 promoter. Representative gel images for sFRP1 and DKK3 are shown in [Figure 1].{Figure 1}

Association of secreted frizzled-related protein 1 and Dickkopf3 promoter methylation with clinicopathological parameters

As depicted in [Table 4], correlation with clinical parameters revealed that the incidence of methylated DKK3 promoter was significantly higher in premenopausal women as compared to postmenopausal women (P = 0.016) while it showed a trend of association with the younger age group as compared to older age group of breast cancer patients (P = 0.077). On the other hand, incidence of methylated sFRP1 promoter was not associated with the clinical parameters of breast cancer patients. Further, on correlating with pathological parameters, methylated sFRP1 showed significant association (P = 0.039) and methylated DKK3 showed a trend of association (P = 0.070) with larger tumor size as compared to smaller size of breast tumors, respectively. The methylation frequency of sFRP1 and DKK3 gene promoter was also significantly higher in breast cancer patients with the presence of metastatic nodes (P = 0.036 and P = 0.05, respectively), advanced disease stage (P = 0.036 and P = 0.008 respectively), and perinodal extension (P = 0.032 and P = 0.029 respectively) as compared to their respective counterparts. Furthermore, methylated DKK3 promoter was significantly higher in breast cancer patients with Grade 3 tumors (P = 0.004) and methylated sFRP1 promoter showed a trend of association with the presence of lymphatic permeation (P = 0.061) as compared to their respective counterparts [Table 4].{Table 4}

Next, correlation with ER, PR and Her2 expression revealed that incidence of methylated sFRP1 was significantly associated with Her2-positive tumors as compared to Her2-negative tumors (P = 0.016) while no significant association of either methylated sFRP1 or DKK3 was observed with ER or PR expression. In addition, on the basis of ER, PR, and Her2 expression, methylated sFRP1 (P = 0.074) and DKK3 (P = 0.058) genes showed a trend of association with different molecular subtypes of breast cancer patients. Further analysis of the molecular subtypes revealed that Luminal B and Her2-positive breast cancers exhibited increased sFRP1 promoter methylation as compared to Luminal A (P = 0.043 and P = 0.009 respectively). However, increased DKK3 promoter methylation was observed in triple-negative breast cancer (TNBC) patients when compared to Luminal A (P = 0.050) [Table 5].{Table 5}

Intercorrelation of secreted frizzled-related protein 1 and Dickkopf3 promoter methylation in breast cancer

The intercorrelation of sFRP1 and DKK3 promoter region did not show significant correlation between their methylation statuses.

Survival analysis

Univariate survival analysis revealed that breast cancer patients with methylated sFRP1 promoter had poor OS as compared to patients with unmethylated sFRP1 promoter (P = 0.082) while no such association was observed for methylated DKK3 promoter [Figure 2] and [Table 6]. Moreover, sFRP1 and DKK3 promoter methylation failed to predict the RFS in breast cancer patients.{Figure 2}{Table 6}

Discussion

Wnt/β-catenin signaling is reportedly a crucial pathway in tumorigenesis and embryogenesis which is found to be inhibited by the antagonists, namely sFRP1 and DKK3.[7],[8],[13],[14],[15],[16] Experimentally, it has been postulated that sFRP1 and DKK3 are putative tumor suppressor genes and frequent targets of epigenetic inactivation through promoter methylation in a variety of solid tumors such as colorectal cancer (CRC),[17] ovarian cancer,[18] mesotheliomas,[19] lung cancer,[20] and prostate cancer[21] including breast cancer.[8] Thus, identification of sFRP1 and DKK3 methylated genes could provide vital information specifically for breast cancer detection and targeted therapy. Hence, the present study examined the methylation status of sFRP1 and DKK3 in breast cancer patients. Dahl et al.[22] and Jeong et al.[23] reported a frequency of 75% and 83.3% for sFRP1 promoter methylation in breast cancer cases which is in accordance to that observed in the current study (76%) of sFRP1 in breast cancer patients. However, Lo et al.[24] and Veeck et al.[8] noted a slightly lower incidence (68% and 61%) of methylated sFRP1 gene in patients with breast cancers. On the other hand, 64% of breast cancer patients exhibited methylated DKK3 gene in the present study, which is similar to that observed by Veeck et al. in breast cancer patients.[25] In addition, methylated DKK3 gene has been reported in other malignancies in prostate cancer (68%)[21] and in gastric cancer (67.6%).[13]

The correlation with clinicopathological parameters revealed that incidence of methylated DKK3 was significantly higher in premenopausal breast cancer patients than postmenopausal patients. Similarly, Kloten et al. reported that sensitivity of DKK3 methylation was more frequent in premenopausal women with breast cancer, indicating a pronounced benefit of DKK3 for the early detection of breast cancer in premenopausal women.[26] Further, the present study observed a trend of higher incidence of methylated DKK3 in patients of younger age group as compared to older age group. Contradictorily, Veeck et al. showed the association of methylated DKK3 with advanced age of breast cancer patients.[25] On the other hand, a study by Yin et al. reported the association of methylated DKK3 promoter with younger age group of papillary thyroid carcinoma patients, which is consistent with the current study.[27] Moreover, the current study observed a significant predominance of methylated sFRP1 and DKK3 in high-risk prognostic variables such as larger tumor size, positive lymph node status, advanced stage, high BR score, and presence of perinodal extension as compared to their respective counterparts. This signifies that aberrant sFRP1 and DKK3 promoter methylation plays a contributing role toward tumor aggressiveness leading to progressive breast cancer. Similar findings are being reported in literature by Xiang et al.[28] and Saied et al.,[29] demonstrating the association of methylated DKK3 promoter with aggressive characteristics of breast cancer. Besides breast cancer, methylated DKK3 promoter was significantly associated with advanced tumor stages in gastric cancer patients, larger tumor size in cervical cancer patients and with advanced stage, high-tumor grade and lymph node metastasis in papillary thyroid carcinoma patients; confirming the relation of methylated DKK3 promoter with poor prognosticators.[13],[27],[30] Likewise, sFRP1 methylation was reported to be more frequent in glioma patients with higher grade tumors, suggesting a direct correlation of sFRP1 methylation with tumor aggressiveness.[31] However, Veeck et al.[8] and Kloten et al.[26] showed significant association of hypermethylated sFRP1 and DKK3 promoter, respectively, with smaller tumor size in breast cancer patients. Nevertheless, several other authors did not find any significant relationships between sFRP1 and DKK3 methylation and clinicopathological characteristics in breast cancer patients[25],[32],[33] and hepatocellular carcinoma (HCC) patients.[34],[35]

Further, in relation to ER, PR, Her2 expression, the present study revealed that incidence of methylated sFRP1 was significantly higher in Her2-positive tumors as compared to Her2 negative and was not associated with ER or PR status while methylated DKK3 gene promoter had no influence on the expression of ER, PR, and Her2 status of breast cancer patients. Likewise, several other studies also failed to find any significant correlation of methylated sFRP1 and DKK3 promoter with ER, PR, and Her2 expression in breast cancer patients.[8],[25],[26],[29] However, Jeong et al.[23] has shown significant association of ER, PR, and Her2-negative tumors with low level of sFRP1 gene methylation. Furthermore, Holm et al. has described the association of various gene methylation with the molecular subtypes of breast cancer, with a significant high frequency in Luminal B tumors and a low frequency in basal-like tumors.[36] Hence, it was important to explore whether there exists any difference in sFRP1 and DKK3 methylation pattern between the molecular subtypes. Indeed, sFRP1 methylation displayed a trend toward higher frequency in Her2-positive subtype, followed by Luminal B, TNBC, and Luminal A subtypes. Jeong et al. indicated significantly low sFRP1 gene methylation in basal-like subtype compared to the Luminal A, Luminal B, and Her2-positive subtypes.[23] Wang et al. reported that patients with TNBC have decreased sFRP1 methylation as compared to other molecular subtypes.[37] The current study also observed decreased incidence of sFRP1 methylation in patients with TNBC subtype than those with Luminal B or Her2-positive subtype. However, a trend of higher incidence of methylated DKK3 gene in TNBC patients was noted as compared to those with Luminal A subtype. This could also be explained from a study by Lorsy et al. who observed loss of DKK3 expression in aggressive TNBC subtype which was suggested to be due to increased promoter methylation of DKK3 in this subtype.[38] Hence, the above findings suggest that biologically molecular subtypes not only display genetic aberrations but also harbor epigenetic aberrations.

In addition, the value of sFRP1 and DKK3 was elucidated as possible molecular markers of prognosis in breast cancer. Survival analysis revealed that methylated sFRP1 promoter was associated with decreased OS in breast cancer patients, but with borderline significance. Consistent with present results, Veeck et al. observed significant association of sFRP1 methylation with shorter OS in breast cancer and it emerged as an independent adverse prognostic factor.[8] Likewise, Majchrzak-Celińska et al. also observed a significant negative correlation of sFRP1 methylation with survival time in glioma patients.[31] However, few other studies did not observe any significant associations of sFRP1 methylation status with clinical outcome in CRC and acute myeloid leukemia patients.[7],[39] Moreover, Vincent and Postovit performed pancancer analysis of different types of cancer and showed that sFRP1 was associated with tumor suppressive functions but not with prognosis.[40] Furthermore, the current study did not observe significant difference in the survival of breast cancer patients with methylated DKK3 promoter. However, several studies in literature reported significant association of DKK3 methylation with poor survival in breast cancer, gastric cancer, and HCC patients.[13],[25],[41]

Hence, summarizing the present observations, high frequency of sFRP1 and DKK3 methylation was found in breast cancer patients, with significant associations with tumor aggressiveness contributing to the malignant behavior of the disease. Furthermore, methylation of both the genes was differentially present in various molecular subtypes of breast cancer patients that may suggest epigenetic aberrations and differences in the molecular pathogenesis in various subtypes. Moreover, not DKK3 but sFRP1 emerged as a prognosticator for breast cancer patients and thus might be useful as a potential prognostic marker in clinical oncology for breast cancer detection and therapy, thereby assisting to improve patient outcome. Moreover, as sFRP1 and DKK3 genes particularly regulate Wnt signaling pathway, it could be concluded that aberrant promoter methylation of these genes leads to dysregulation of the Wnt pathway in breast cancer

Acknowledgment

We would like to thank Gujarat Cancer Society and Gujarat Cancer and Research Institute.

Financial support and sponsorship

This study was financially supported by Gujarat Cancer Society, Gujarat Cancer and Research Institute.

Conflicts of interest

There are no conflicts of interest