Heterogeneity is an important predictor of tumor treatment failure and drug resistance, and genomic mutations (such as copy number variation) are important causal factors of heterogeneity among tumors. Previous studies have shown that CNV can affect the expression level of proteins through epigenetic regulation, and the key mechanism is to affect epigenetic modifications (such as DNA methylation). The overall hypomethylation of oncogenes and hypermethylation of tumor suppressor genes are characteristic of most cancer types. Molecular understanding of BRCA heterogeneity is the key to effective treatment and personalized medicine. In this study, BRCA-related methylation data downloaded from the TCGA database included 883 samples, comprising 96 normal tissue samples and 787 BRCA tissue samples, the difference analysis results obtained a total of 122 protein-coding genes. The CNV data included 2201 samples, 1103 normal tissue samples, and 1098 BRCA tissue samples, a total of 19178 genes with CNV were found. The difference analysis of gene expression data between 112 normal tissue samples and 1096 cancer tissue samples showed that 2138 dysregulated genes, including 1375 upregulated genes and 763 downregulated genes. We used TCGA high-throughput molecular profiling data to characterize inter-tumor heterogeneity and analyzed the effects of CNV on DNA methylation and gene expression.
In our analysis, CNV of HPDL and SOX17 affected methylation and gene expression levels in BRCA, and CNV and methylation of HPDL and SOX17 can lead to poor prognosis in patients with BRCA. The CNV of SOX17 and HPDL can influence the expression of genes through genetic regulation. The CNV of SOX17 showed copy number amplification on chromosome 8, and further analysis showed that the prognosis of BRCA patients was poor when SOX17 copy number was increased. The CNV of HPDL showed a decrease in copy number on chromosome 1, and further analysis indicated that the survival rate of BRCA patients was lower when HPDL copy number was reduced. The CNV of SOX17 and HPDL can affect the expression of genes through epigenetic modification, and DNA methylation is an important pathway for epigenetic modification. The methylation sites of SOX17 that we characterized with BRCA OS included cg00123055, cg02222728, cg03329976, cg08044907, cg15377283, cg24150172, cg24891539, and cg24928317. The methylation sites of HPDL included cg12178578 and cg15071854. Survival analysis showed that the OS of BRCA patients hypermethylated in SOX17 and HPDL was poorer. Therefore, CNV and methylation of SOX17 and HPDL are early events of BRCA and can predictrecurrence, metastasis, and prognosis of BRCA patients.
SOX17, a transcriptional regulator, binds to target promoter DNA and inhibits Wnt signaling. SOX17 gene promoter methylation can be used as a tumor suppressor and dysregulated oncogene (via aberrant DNA methylation) in many tumors, such as lung adenocarcinoma , cholangiocarcinoma , esophageal squamous cell carcinoma , colorectal cancer , non-small cell lung cancer , endometrial cancer , and so on. In BRCA, Fu Deyuan et al. used methylation-specific polymerase chain reaction to assess the relationship between the methylation of the SOX17gene promoter and the onset and prognosis of BRCA. Abnormal SOX17 methylation in cancer tissues and plasma DNA was found to be significantly associated with tumor lymph node metastasis and lymph node metastasis, associated with poor disease-free survival (P < 0.005) and overall survival (P < 0.005). In addition, SOX17 methylation in plasma DNA is an independent prognostic factor for DFS in BRCA . Chimonidou Maria et al. found that the SOX17 promoter is highly methylated in primary breast tumors, in CTCs isolated from patients with BRCA, and in corresponding cfDNA samples, which provides new predictive ideas for recurrence and prognosis in patients with operable BRCA and metastatic patients [33, 34]. HPDL may have dioxygenase activity. Previous studies have found that HPDL exhibits differential expression in CNS lymphoma compared with non-primary central nervous system (CNS) lymphoma . However, understanding the role of HPDL in BRCA needs further research and interpretation, which provides an idea for the in-depth study of the molecular mechanism of BRCA.
Intracellular signaling pathways regulate various cellular activities. We performed GSEA identification on SOX17 and HPDL to further explore the small molecule regulation mechanism of BRCA and found that signaling pathways with significant changes in enrichment exist between patients with low expression and high expression. It is well known that the JAK-STAT signaling pathway, a signal transduction pathway stimulated by cytokines, is involved in biological processes, such as cell proliferation, differentiation, apoptosis, and immune regulation, and is associated with pathogenesis of many tumors, such as liver cancer, ovarian cancer, and BRCA [36–38].The major cellular processes during BRCA development rely on JAK/STAT signaling to coordinate growth factor function. Previous studies have found that activation of the JAK/STAT pathway is common in triple-negative BRCA, which can affect the expression of genes controlling immune signals. Dysregulated JAK/STAT signaling has been implicated in BRCA metastasis, associated with high risk of recurrence [39–41]. The Wnt signaling pathway plays a crucial role in early embryonic development, organ formation, tissue regeneration, and other physiological processes, often involving stem cell control, which may induce cancer if a key protein is mutated . Wnt signaling pathway involves the onset and treatment of colorectal cancer, pancreatic cancer, gastric cancer, and other tumors [43–45]. Yang Feibiao et al. confirmed that SOX17 is a target gene of miR-194-5p. In mouse studies, knockdown of miR-194-5p in BRCA cells may increase SOX17 expression and regulate the signaling pathway of Wnt/β-catenin . Therefore, increased expression of SOX17 is associated with activation of the Wnt signaling pathway and is thus involved in the pathogenesis of BRCA. In addition, the enrichment results of SOX17 include pathways related to cell growth, division, and proliferation of oocyte meiosis, ABC transporters, and neuroactive ligand receptor interaction. HPDL upregulation is related to cell cycle and P53 signaling pathway. HPDL down-regulation is related to MAPK signaling pathway and TGF-β signaling pathway. Both cell cycle and p53 signaling pathways are involved in cell division and proliferation. The p53 gene is called the “guardian of the genome”, but when p53 is deregulated, it participates in the development and proliferation of various tumor cells .Both MAPK and TGF-βsignaling pathway are involved in cell growth, differentiation, and apoptosis. In recent studies, abnormal activation of the MAPK signaling pathway signal has been found to favor the abnormal proliferation of malignant cells . TGF-βsignaling acts as suppressor and inducer of tumor progression during the early and late stages of cancer, and can trigger a cascade of reactions that mobilize cancer cells [49, 50].
Recent studies have demonstrated the consequences of genetic variation in regulating overall risk associated with BRCA patients. In the study so far, we explored the effects of CNV and DNA methylation on gene expression levels and OS of BRCA patients and found that CNV can affect DNA methylation levels. CNV and methylation of SOX17 and HPDL are related to expression and regulation. In addition, the CNV of SOX17 and HPDL were also correlated with methylation levels. In addition, we found methylation sites for SOX17 and HPDL associated with BRCA prognosis. DNA methylation is an effective regulator of gene expression, If the CpG island is located in the promoter region of a gene, the methylation of the CpG island will significantly reduce or even completely silence the transcription of the gene, and then affect the protein expression. In this study, due to data and conditional restrictions, we did not distinguish whether it was on the promoter or DNA when screening prognostic related methylation sites, which is what we will explore in the next study. Finally, by enriching the low and high expression pathways of SOX17 and HPDL, pathways related to BRCA progression have been discovered, including the JAK-STAT / Wnt / P53 / MAPK signaling pathway.