Background

Breast cancer (BC) is considered the most common malignancy in females, and it accounts for approximately 32.04% of the reported cancers in Egyptian women [1]. Cancer biomarkers’ research has shown that not only genetic mutations, but also epigenetic alterations such as promoter methylation patterns have potential value in early recognition of malignant growth. Silencing of tumor suppressor genes through hypermethylation and activation of oncogenes by hypomethylation are hallmarks of early molecular epimarkers in BC development [2].

Mounting evidence emphasizes the significance of DNA methylation, not only for being potentially modifiable but also because it is related to age, the strongest BC risk predictor [3]. The Cancer Genome Atlas studied the whole genome methylation of more than 10,000 human samples from more than 30 distinct tumors and paired normal controls [4]. With the accessibility of such immense amount of genetic data, the challenge is to capture the most significant genes with relevance to diagnosis and personalized medicine.

One of the most frequent epigenetic alterations described is increased methylation of breast cancer gene (BRCA1). By using peripheral blood, BRAC1 promoter methylation could serve as a screening tool as well as predictive biomarker for poly ADP ribose polymerase (PARP1) inhibitor therapy, which has been used for management of BRCA1 and BRCA2 mutations to reduce tumor growth and vascularization [5, 6].

Yes-associated protein (YAP) gene is mapped to chromosome region 11q22 amplicon [7]. YAP1 is considered a specific transcriptional activator and a leading effector of the Hippo tumor suppressor pathway. The Hippo pathway is an evolutionarily conserved kinase cascade which is implicated in cell proliferation, organ development, regeneration, and stem cell biology. YAP1 regulates nuclear cellular responses by interacting with transcription factors. It also modulates actin filament architecture and cell migration [8, 9].

Hippo pathway is inhibited by distinct signaling pathways, and this causes hyperactivation of YAP1 with its subsequent shift to the nucleus. The hyperactivated YAP1 interacts with transcription regulators such as TEA domain/Transcription Enhancer Factor (TEAD) family. The complex between YAP1 and TEAD molecules is crucial for growth-enhancing function of YAP1 [10]. The mechanism by which YAP1 performs its tumor suppressor or oncogenic functions is not yet completely understood.

Aim

Regarding the aforementioned points, study of DNA methylation seems to be important in developing new diagnostic biomarkers; therefore, this work aimed at analyzing the methylation profile of YAP1 in BC patients using MSP analysis. Furthermore, the correlation of clinicopathological parameters and aberrant methylation was examined.

Methods

Fifty females, genetically unrelated, with primary BC undergoing breast surgery at the Medical Research Institute Teaching Hospital, Alexandria, Egypt, during the period 2016–2017, were included in our study. BC was diagnosed according to standard clinical, radiological, and histological parameters. Patients were subjected to thorough physical examinations, chest radiography, bilateral mammography, and ultrasonography of the breasts, axilla, cervical region, and abdomen before surgical resection. Breast tumor tissue and normal adjacent breast tissue not infiltrated with tumor, serving as control, were obtained from subjects during surgery. Exclusion criteria were previous exposure to neoadjuvant chemotherapy, radiotherapy, or hormonal therapy. Patients with multiple cancers and patients with acute myocardial or surgical complications were excluded. The tumor histological types, grading, and TNM staging were assessed [11]. Late stage was defined as stage III and stage IV. The study was approved by the Ethical Committee of Medical Research Institute, Alexandria University (IORG#: IORG008812). All subjects have signed the informed consent and agreed the research purposes of clinical data and samples of our study. Postoperative pathology examination, as well as hormonal receptors (ER and PR) and epidermal growth factor receptor 2(HER-2), was done by immunohistochemistry [12].

DNA extraction and sodium bisulfite modification

Briefly, extraction of genomic deoxyribonucleic acid (DNA) from breast tumor and adjacent normal breast tissue was done using the Gene JET™ Genomic DNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The isolated DNA was evaluated by Nanodrop ND 1000 spectrophotometer (Thermo Scientific, USA) and confirmed by gel electrophoresis running. The ratio of absorbance at 260 nm and 280 nm (A260/A280) was taken to assess the purity of the DNA.

Bisulfite conversion for the extracted DNA was done using the EZ DNA Methylation™ Kit (Zymo Research Corp) according to the manufacturer’s instructions. Treatment of DNA with sodium bisulfite converted unmethylated cytosines into uracil, while methylated cytosines remained unchanged. Bisulfite modification step creates sequence differences in the DNA. The converted DNA was then stored at − 20 °C till use. MSP was carried out according to the method described earlier by Herman et al. [13] and performed previously by the author Ramadan et al. [14]. Separate MSP reactions were carried out for both methylated and unmethylated DNA sequences utilizing primer sets specific for each reaction.

The primers used for amplification were:

  • Forward: 5′-AGTTCGTATAGGCGTTTCGTTC-3′ (methylated), F 5′-AAGTTTGTATAGGTGTTTTGTTTGG-3 (unmethylated).

  • Backward: F 5′-CTTAACTACAAAAAATTCTTCCGCT-3′ (methylated), F 5′-CTTAACTACAAAAAATTCTTCCACT-3′(unmethylated) [15]

PCR was done by using a total volume of 25 μl (12.5 μl of ZymoTaq™ PreMix, 5 μl of modified bisulfate DNA, 1 μl of each primer, 5.5 μl of nuclease-free H2O). The PCR reaction was performed using thermocycler (Quanta Biotech, UK) under the following conditions: initial denaturation at 95 °C for 10 min followed by 35 cycles with denaturation at 95 °C for 45 s, annealing at 57 °C for 30 s, and extension at 72 °C for 45 s, and finally extension at 72 °C for 7 min. The resultant PCR products were identified by electrophoresis on 2% agarose gel and stained with ethidium bromide for visualization under UV light.

With each run, positive and negative controls were incorporated. Completely unmethylated and methylated human genomic DNA (EpiTect Control DNA (human); Qiagen) were applied respectively for unmethylation- and methylation-positive control, while nuclease-free sterile water and non-bisulfite-modified DNA were employed as negative control. The PCR generated a 188-bp products for both methylated and unmethylated DNA.

Amplification was successful if a band appeared with the same size as that of the control (100 bp). If amplification was successful in the unmethylated run and failed in the methylated run, this was considered as the unmethylated status (UU genotype) of the YAP1 promoter. If the reverse occurred (successful amplification in the methylated run and failed in the unmethylated run), that was hypermethylation (MM genotype). A successful amplification in both runs denoted hemimethylation (MU genotype), and this could suggest methylation heterogeneity where methylation is not inclusive of all CpG sites between the primers, hereby allowing amplification with both primer sets. An aberrant methylation was considered in hypermethylation and hemimethylation situations. All tissue samples were processed and analyzed in a blinded manner. Methylation status was further correlated with clinicopathological criteria.

Statistics

We compared YAP1 methylation frequency in tumor and non-tumor breast tissue. MSP results were analyzed as a dichotomous variable based on the occurrence of methylation. The methylation status of YAP1 and clinicopathological data were analyzed with the Pearson chi-square test, Fisher’s exact test, and Monte Carlo test. The diagnostic performance of YAP1 methylation was evaluated. SPSS version 20 was applied, and a p value < 0.05 was considered statistically significant. Each p value was statistically adjusted with the Bonferroni correction.

Results

The study included 50 paired tumor and adjacent normal breast tissues. The participants had a mean age of 46.6 ± 8.45 years. The demographic, biochemical, and clinical parameters of the participants are indicated in Table 1.

Table 1 Correlation analysis of YAP1 promoter methylation with different variables
Full size table

Promoter aberrant methylation of the YAP1 gene in BC tumor tissue was found in 34 of 50 (68%), while adjacent normal tissue showed methylation in 4 of 50 (8%). The frequency of methylation of the YAP1 gene was significantly higher in BC tissue compared to normal breast tissue with (p < 0.001).

The analytic results of the correlation between clinicopathological characteristics and methylation status of YAP1 gene showed that the aberrant methylation pattern was independent of the age, body mass index, menopausal status, histopathological type, tumor size, axillary lymph node involvement, and serum CA15–3 (p > 0.05). However, methylation status was significantly correlated with tumor stage (p < 0.001) (Table 1). YAP1 methylation showed high accuracy (92%) in discriminating between early and late BC (stage II vs stages II and IV) (Table 2). Although methylation status was significantly correlated with metastasis (p = 0.02), it showed low accuracy (52%) in distinguishing those with and without metastasis (Table 3).

Table 2 Diagnostic performance of methylation to differentiate early and late tumor stages
Full size table
Table 3 Diagnostic performance of methylation in relation to metastasis
Full size table

Discussion

It is known that YAP1 regulates the downstream effect of Hippo signaling. YAP1 regulates organ size by controlling cellular proliferation and survival. Kinase-mediated phosphorylation of YAP1 promotes its sequestration from the nucleus to the cytoplasm, with subsequent downregulation of its target genes [8, 9].

In the present study, we investigated the usefulness of YAP1 as a biomarker in BC. Our results demonstrated that YAP1 methylation was significantly higher in BC tissue compared to normal breast tissue and showed a significant correlation with tumor stage. Moreover, the methylation status had good diagnostic performance (accuracy of 92%) in differentiating between early and late stages. Although methylation profile was significantly associated with metastasis and showed 100% sensitivity and negative predictive value, the specificity and positive predictive value were low. This may be attributed to the relatively small percent (10 cases) of participants with metastasis.

The relation of YAP1 with BC was previously examined by immunohistochemical assay, Western blotting, and by real-time quantitative PCR. Different expression levels as well as different subcellular localization were observed [16,17,18,19]. The general conclusion of several reports was the association of dysregulation of YAP1 with tumor aggressiveness in BC [20, 21].

In accordance with our findings, Real et al., by using MSP, reported methylation in 70.8% of the studied BC tissue samples. They found a significantly high correlation between YAP hypermethylation and YAP immunohistochemical expression. Although 84.13% cases of advanced BC stages III and IV had YAP promoter methylation and subsequent protein loss, they did not prove any significant association between YAP methylation and clinicopathological criteria [15].

The function of YAP1 on being a tumor suppressor or oncogene is quite controversial. Overexpression of YAP1 was observed in lung [22], esophageal [23], and ovarian tumors [24]. The assumption that YAP1 may act as a tumor suppressor was based on the finding that human BC tissue exhibits decreased YAP1 expression compared with normal breast tissue [17]. Moreover, knockdown of YAP1 in BC cell line and in mouse model demonstrated increased cellular migration and tumor invasiveness [25]. On the contrary, other studies advocated the oncogenic potentials of YAP1, since its overexpression in BC cell line [26] and in mice xenografts [18] increased proliferation and tumorigenesis.

Numerous mechanisms were postulated to explain the relation with BC tumorigenesis. One of which was reported by Shen et al. [27]; their findings showed that YAP1 enhanced tumor invasiveness by modulating thrombospondin 1 transcription, thus leading to focal adhesion kinase phosphorylation in a TEAD-dependent manner. Wang et al. showed that YAP1 promotes metastasis of BC by inhibiting the transcription of growth differentiation factor-15 which is a divergent element in the transforming growth factor superfamily [21].

We studied the relation of YAP1 methylation with the expression of hormone receptors, and we found a mild significant association with HER2 expression (p = 0.043), but no association was observed with ER or PR status. On the other hand, Kim et al. [28] reported that hyper-activation of YAP1 in BC tissues was associated directly to the PR status and inversely to HER2 and Ki67 levels. Others described the relation between reduced YAP1 expression and decreased recurrence-free survival in luminal cancers [20, 29]. This emphasizes the assumption that, at least in triple-negative BC, YAP1 may possess an oncogenic character. It is plausible that YAP1 acts differently in distinct BC subtypes.

Many laboratory methodologies exist for DNA methylation determination. However, the majority of the methods depend on bisulfite conversion of DNA. Then, the modified DNA is subsequently incorporated to additional molecular techniques; PCR, microarray technology, mass spectrometry or next-generation sequencing. The choice of the appropriate analytical methodology depends on the nature and number of the samples, information required, and cost. MSP is considered a quite sensitive and cost-effective tool without the need for expensive instruments, thus making it applicable to most clinical laboratories [2].

Based on the association of YAP1 with cancer proliferation and drug resistance, there were several trials to identify therapeutic small molecule inhibitors of YAP1 [30, 31]. Verteporfin (VP), a FDA-approved photosensitizing agent used in treatment of neo-vascular macular degeneration, blocks YAP-TEAD binding. Liu-Chittenden et al reported thatVP inhibited liver overgrowth resulting from the overexpression of YAP1 by silencing Hippo kinase components [32]. Surprisingly, statins were reported to have inhibitory effects on YAP1 by promoting its cytoplasmic sequestration and degradation [33]. Studies in animal models of cancers indicate that statins have tumor suppressor effects. This was ascertained in clinical studies showing the negative association of statins with cancer occurrence or survival [34].

Certain points are worth mentioning such as the selection of CpG containing regions with respect to differences observed in methylation levels of different locations. Further large-scale studies on different population in addition to validation with different methods, and functional analyses of methylation profile are required in order to determine if the specific methylation patterns are exclusively associated with BC.

Conclusion

YAP1 methylation was significantly higher in BC tissue compared to normal breast tissue and showed a significant correlation with tumor stage. Moreover, the methylation status had good diagnostic performance in discriminating early and late stages. Our study pinpoints the value of promoter methylation of YAP1 in BC particularly in view of its future therapeutic potentials.