Methylation Analysis In Breast Cancer Based On High-density Genomic DNA Methyaltion Chips And Clinical Verification Of Important Functional Genes

Introduction:Introduction:According to the data from International Union of Counter Cancer, breast cancer now strikes more women in the world than any other type of cancer. There are currently about1.2million women worldwide fighting breast cancer per year, accounting for the highest incidence (23%) of the global burden of cancers among women. Breast cancer is the leading cause of cancer deaths among women worldwide, with0.3%-8%increasing rate every year. Breast cancer is becoming more and more common among Chinese women and the main killer for their health, making up32%of all new cases. According to the data reported to the National Cancer Registry Center received from a total of49tumor registries in2006, breast cancer contributed to an estimated23.3/100,000new cases and4.71/100,000deaths, resulting in the highest proportion of female cancer in China. The Gail model (GM) for predicting the absolute risk of invasive breast cancer was the most commonly used method in worldwide. Although the GM has been validated in western populations, its performance in other populations is unclear because of the wide variation in international breast cancer rates. However, receiver operating characteristic analysis estimated a prediction accuracy of about58-59%for the Gail model. Mutations in high penetrance cancer susceptibility genes, such as the BRCA1and BRCA2genes, confer a substantially elevated risk to familial breast cancer, accounting for5-10%of breast cancer cases. Genomewide association studies (GWASs) have identified multiple genetic variants associated with breast cancer. The area under the curve (AUC) estimated for10single nucleotide polymorphism (SNPs) from genome-wide association studies of breast was59.7%. Therefore, the known genetic risk and environmental factors in prediction of breast cancer is limited. Early diagnosis and treatment is the key to improve the survival rate and quality of life in patients with breast cancer. Currently the main means of early diagnosis for breast cancer is self examination and imaging examination. However, there are30%misdiagnosis cases. And even15-25%Stage I and II breast cancer will eventually develop distant metastases. Therefore, to further understand the molecular mechanism of breast carcinomas, search the useful targets for the development of novel therapies and improve systemic adjuvant therapy scientifically, is an important content in breast cancer research. Detection of circulating tumor biomarkers is one of the current hot spots in tumor research. Detection of biomarkers in peripheral blood might be used as potential clinical application for cancer prediction, early diagnosis, disease monitoring and decide prognosis. However, there is no ideal result so far.Malignant tumor is the accumulation of environmental and genetic factors with multi stage, multi steps and abnormal changes in genes. One of the most important mechanisms is the activation of oncogenes and inactivation of tumor suppressor genes. According to Knudson’s hypothesis, a tumor suppressor gene generally requires "two hits" to lose its function, which means both alleles must be inactivated. Two pathways by which suppressors become disabled have been widely studied: intragenic mutations and loss of chromosomal material. With more research appeared, people found that DNA sequence is complete and without any mutation or deletion in some cancer. The "two hit theory" cannot explain the reason for the inactivation of tumor suppressor gene. The fact that the traditional genetics cannot answer this phenomenon results in a new discipline-epigenetics, which corresponds with the genetics. DNA methylation is an important epigenetic modification and regulates the function of genome. DNA methylation occurs mainly in the CpG dinucleotide sites with rich G/C content. CpG islands play a role in regulation of gene expression and have special significance. In vertebrate genomes, DNA methylation occurs as a result of the post-replicative addition of a methyl group to selected cytosines to form an altered nucleotide-5-methyl cytosine residue. Approximately70-80%of CpG sites in the human genome are methylated. High concentrations of unmethylated CpG dinucleotides exist in CpG islands. DNA methylation is a regulatory mechanism used to control gene expression and plays a role in such diverse functions as gene imprinting, X-chromosome inactivation, normal development and repression of gene transcription. Much experimental evidence has documented promoter methylation of CpG islands as the third mechanism of inactivation of tumor suppressor, combined with LOH and mutation, and the only mechanism in some cases. It contributes to the carcinogenesis in many tumors. Yan et al. reported the identification of somatic mutations of DNMT3A by exome sequencing and methyaltion microarray analysis in acute monocytic leukemia. They discovered high mutations in DNMT3A together with gene abnormal methylation, which further prove the importance of promoter methylation in cancer research.Therefore, more and more studies on the correlation between promoter methylation and breast cancer are carrying out. Evidences indicate that DNA hypermethylation is a common phenomenon in the development of breast cancer. It always results in the unexpression of key genes; including cell cycle regulation genes, tumor susceptibility genes, tumor metabolic enzyme genes and cell adhesion molecule gene etc. van Hoesel et al. demonstrated that promoter methylation is an early event in breast cancer with cell and tissue heterogeneity. It might be earlier than the malignant transformation. MINT17, MINT31, RARbeta2and RASSF1A methylation could be used as biomarkers for early detection and present a predictor of malignant potential. Xu et al. found that triple-negative breast cancer patients with BRCA1-methylated tumors are sensitive to adjuvant chemotherapy and have a favorable survival compared with patients with BRCA1-unmethylated triple-negative tumors. Aran and Hellman analyzed the relationship between DNA methylation of transcriptional enhancers and breast cancer predisposition. Gene promoter methylation is not only an important epigenetic change in the development of many cancers, but a suitable sensitively biomarker for early diagnosis, even with small sample volume compared with the detection of protein and RNA. Since hypermethylated DNA may serve as a potential molecular tumor marker due to its high specificity in differentiating cancer from normal tissues, detection of aberrant methylation of tumor suppressor genes in the bodily fluids of cancer patients is attracting increasing attention. Xu et al. collected peripheral blood samples from298breast cancer patients and612healthy women and examined the methylation status. Receiver operating characteristic analysis estimated a prediction accuracy of65.8%for methylation, compared with56.0%for the Gail model and58.8%for genome-wide association study polymorphisms (GWASs). Ther concluded methylation profiling of blood holds promise for breast cancer detection and risk prediction. Although some progress has been made from studies on aberrant methylation of related genes in breast cancer, most of the research is aimed at several or a group of candidate genes. So far, there is no direct or comprehensively systematic analysis on aberrant methylation at the whole genome level. The construction of methylation profile of breast cancer not only helps to reveal the molecular mechanism, but more importantly to provide a valuable clue and reliable scientific basis for diagnosis, treatment, disease monitoring and prognosis of breast cancer. This study screened the genes with aberrant methylation of breast cancer and normal breast tissue preliminarily using genome wide high-density methylation microaaray technique. Specific genes with aberrant methylation for breast cancer were selected, which initially laid the foundation for the establishment of methylation profiles in breast cancer and provide possible clues for the diagnosis, treatment and prognosis of breast cancer. The focus of our attention is to further understand the molecular mechanism of breast cancer and verify the functions of specific genes. The following will be divided into three aspects in detail.Part1Methylation analysis in breast cancer based on50K Infinium Methylation BeadChipObjective:To analyze the methylation level of human breast cancer tissue and normal breast tissue using high-density DNA methylation chip (450K Infinium Methylation BeadChip). Methods:6breast cancer samples and6normal breast tissues were selected for DNA extraction and bisulfite conversion. Illumina HD450K Infinium Methylation BeadChip was applied to detect450,000methylation sites of the whole human genome, covering96%CpG islands. The cluster analysis and significance test were carried out to analyze the different methylation level between human breast cancer tissue and normal breast tissue. And then, the Delta Data was screened for statistics. Results:3268genes showed significant differences in the degree of methylation between breast cancer tissue and normal breast tissue (Diff Score=50, P<0.00001), which included1926hypermethylation genes and1342hypomethylation genes. The average methylation level of significant different genes is higher in the experimental group compared to the control. The chromosome distribution of aberrant methylation genes is quite even,14%at chromosome1and8%at chromosome3. Cluster analysis of the candidate genes showed that:aberrant methylation genes in breast cancer mainly participated in cell adhesion molecules, signal transduction, cell cycle, apoptosis, cell cycle proteins. These genes may be one of the reasons that leaded to breast cancer. Conclusion:Based on the systemic analyses by high-density genome-wide methylation chips between breast cancer tissue and normal breast tissue, we screened out a batch of abnormal methylation genes related to the process of breast caner. This study lays the foundation for further exploration of the molecular mechanism into breast cancer. According the current results, these aberrant methylation genes need to be further studied so that the roles of them in the process of breast carcinogenesis might be understood in the future. Part2RARβ Gene Methylation can Predict the Prognosis of Breast Cancer Patients with Nodal InvolvementObjective:Retinoic acid receptor (RAR) gene plays a role in the process of reverse transcription and inhibits the cell proliferation in malignant tumor. RARβ gene was considered as a tumor suppressor gene and to be significantly associated with breast cancer, which causes the great interests in recent research. Carcinomas will develop if without the regulation of RARβ gene. RARβ gene locates at3p24. There is high loss of heterozygosity (LOH) rate (45%) in this area. However, LOH couldn’t be the independent factor to suppress the expression of RARβ. Therefore, further researches proved that RARP methylation was importantly correlated with breast cancer. The purpose of this study is to determine the roles of RARβ gene in the occurrence and development of breast cancer and provide the foundation for further exploration of the molecular mechanism between breast cancer and RARβ methylation. Methods:192primary invasive breast tumor and the corresponding normal tissues were colleted for DNA extraction and bisulfite conversion. Two sets of specific primers for methylation and unmethylation were decided for PCR. We screened the primary human breast tumors and normal breast tissues for RARβ gene promoter mehtylation using methylation specify PCR (MSP), and the results were analyzed with corresponding clinical pathological data. Results:The frequency of RARβ gene mehtylation among192cases was26%(50/192), however no RARβ gene mehtylation was found in normal breast tissues. The difference was significant (P<0.05). RARβ methylation were associated with tumor histological type, differentiation and tumor size (P<0.05). Patients with lymph node metastasis, ER(-), PR (-) and erbB2amplified had more RARβ methylation, but not significant. No significant association was found between aberrant methylation of RARβ gene and patient age, ploidy and TP53mutation (P>0.05). Average ER value was significantly higher in patients with RARβ methylation (Mean±D=119±151fmol/μg), compared with those with RARβ unmethylation (Mean±SD=137±169fmol/μg). Average PR value was significantly higher in patients with RARP methylation (Mean±SD=91±154fmol/μg), compared with those with RARβ unmethylation (Mean±SD=132±205fmol/μg), but not significant. Kaplan-Meier survival analysis revealed that breast cancer patients with RARβ unmethylation had longer survival of69.06(65.84～72.28) months, compared with65.53(59.21～71.85) months for those with RARβ methylation. However, it was not significant after Long-rank test (χ2=1.23, P=0.268). Then with the subgroup analysis, we found that patients with RARβ methylation [52.43(40.56～64.30)] showed significantly worse survival compared with those with RARβ unmethylation [73.13(65.12～81.14)] in nodal involvement groups (χ2=4.230, P=0.040). Conclusion:RARβ gene promoter mehtylation may play an important role in the carcinogenesis and development of breast cancer. The association with poor differentiation, large tumor size and poor survival indicates that RARβ methylation could predict the prognosis of breast cancer patients with nodal involvement. Part3Aberrant PTPRO gene methylation predicts clinical outcome in breast cancer patients and its detection in peripheral bloodObjective:Protein tyrosine phosphatase receptor-type O (PTPRO) gene is a member of the family of receptor-type protein tyrosine phosphatases (PTPs). It is an important signaling medium that can affect various cellular processes, including proliferation, differentiation, metabolism, communication, transcription, survival, contact inhibition, cell cycle and oncogene transformation. PTPRO has been described as a new potential tumor suppressor gene in many recent hot researches on carcinogenesis. Since free tumor DNA may exist in the peripheral blood of malignant tumor patients, detection of potential tumor makers in peripheral blood is one of the hot spots in tumor research currently. Recent studies reported that aberrant DNA changes in peripheral blood were associated with those in primary tumors The purpose of this study is to determine the roles of PTPRO gene methylation as a potential biomarker in the detection of breast cancer. And its association with clincopathologic features including prognosis. Methods:We screened the primary human breast tumors, normal breast tissues and peripheral blood for PTPRO gene promoter mehtylation and its expression using methylation specify PCR (MSP) and reverse transcription PCR (RT-PCR). And the results were analyzed with corresponding clinical pathological data. Cell culture and treatment with5-azacytidine was used to analyze the relationship between PTPRO gene mehtylation and its expression. Results:The frequency of PTPRO gene mehtylation among98breast cancer tissues was55%(54/98), and34%(33/98) in peripheral blood, however no PTPRO gene mehtylation was found in normal breast tissues. PTPRO gene methylation in peripheral blood was significantly correlated to that in tumor tissue (c=0.435, P=0.000). PTPRO methylation were associated with patient age (χ2=4.178, P=0.041), tumor stage (χ2=8.616, P=0.003), histological grade (χ2=5.139, P=0.023), lymph node metastasis (χ2=6.273, P=0.012) and erbB2amplified(χ2=12.124, P=0.0005). Patients with ER(-)and PR(-)had more PTPRO methylation, but not significant. No aberrant methylation of PTPRO gene was found in the plasma samples from healthy control and the patients without gene methylation in tumor tissues. In univariate analysis, PTPRO methylation was associated with significantly worse cancer-specific survival in the overall tumor group（χ2=17.240, p=0.000) Subgroup analysis revealed that PTPRO methylation also showed significant prognostic value within the ER+(χ2=12.844, p=0.000), PR+(χ2=7.195, p=0.007) and HER2amplified (χ2=8.603, p=0.003) patient groups. Multivariate analysis showed that PTPRO methylation was an independent factor for worse survival in the overall patient group (χ2=5.506; p=0.019). In addition, demethylation by5-azacytidine treatment led to gene reactivation in PTPRO-methylated and-silenced breast cancer cell lines. Our data suggests that PTPRO methylation is responsible for its inactivation. Conclusion:PTPRO gene promoter mehtylation may play an important role in the carcinogenesis and development of breast cancer. It could be used to predict the prognosis of breast cancer patients. Further, this is the first report of methylated PTPRO as a noninvasive tumor biomarker in peripheral blood of breast tumor patients for detection and disease monitoring. Conclusion:The new high-density DNA methylation chip was applied to detect450,000methylation sites of the whole human genome, covering96%CpG islands. The cluster analysis and significance test were carried out to analyze the different methylation level between human breast cancer tissue and normal breast tissue. And then, the Delta Data was screened for statistics. Specific genes with aberrant methylation for breast cancer were selected. Combined with study on the molecular functions during the development of breast cancer, the two significant genes were sletected for further clinical verification. And the conclusion as following:1. RARβ methylation could predict the prognosis of breast cancer patients with nodal involvement.2. PTPRO gene promoter mehtylation could be used to predict the prognosis of breast cancer patients. This is the first report of methylated PTPRO as a noninvasive tumor biomarker in peripheral blood of breast tumor patients for detection and disease monitoring.Therefore, this study initially laid the foundation for the establishment of methylation profiles in breast cancer and provide possible clues for the diagnosis, treatment and prognosis of breast cancer, with the hope of development of screening serum kits for breast cancer.