Genome-wide methylation analyses identifies Non-coding RNA genes dysregulated in breast tumours that metastasise to the brain

Abstract

Brain metastases comprise 40% of all metastatic tumours and breast tumours are among the tumours that most commonly metastasise to the brain, the role that epigenetic gene dysregulation plays in this process is not well understood. We carried out 450 K methylation array analysis to investigate epigenetically dysregulated genes in breast to brain metastases (BBM) compared to normal breast tissues (BN) and primary breast tumours (BP). For this, we referenced 450 K methylation data for BBM tumours prepared in our laboratory with BN and BP from The Cancer Genome Atlas. Experimental validation on our initially identified genes, in an independent cohort of BP and in BBM and their originating primary breast tumours using Combined Bisulphite and Restriction Analysis (CoBRA) and Methylation Specific PCR identified three genes (RP11-713P17.4, MIR124-2, NUS1P3) that are hypermethylated and three genes (MIR3193, CTD-2023M8.1 and MTND6P4) that are hypomethylated in breast to brain metastases. In addition, methylation differences in candidate genes between BBM tumours and originating primary tumours shows dysregulation of DNA methylation occurs either at an early stage of tumour evolution (in the primary tumour) or at a later evolutionary stage (where the epigenetic change is only observed in the brain metastasis). Epigentic changes identified could also be found when analysing tumour free circulating DNA (tfcDNA) in patient's serum taken during BBM biopsies. Epigenetic dysregulation of RP11-713P17.4, MIR3193, MTND6P4 are early events suggesting a potential use for these genes as prognostic markers.

Figure 1

Genome-wide DNA methylation analyses by 450 K array for BBM, primary breast tumours (BP) and normal breast tissues (BN) identified CpG loci which have either gained methylation (hypermethylated) or have lost methylatioin (hypomethylated) in BBM compared to BP and BN tumours. Representative examples of gene-associated CpG loci that have gained methylation (hypermethylated) in BBM (A,B) or have lost methylation (hypomethylated) in BBM (C,D) compared to BP and BN (as measured by β-value). (E) Regional methylation pattern of MIR124-2 showing the the location of all CpG loci present on the 450 K array relative to the transcription start of the gene and the differing methylation levels for each loci in BBM, BP and BN. * denotes the location of the differentially methylated CpG that was identified by the bioinformatic analysis of 450 K-array data from BN, BP and BBM.

Figure 2

Representative examples of experimental validation of methylation status of candidate genes hypermethylated in breast to brain metastases (BBM) in a cohort of non-metastatic primary breast tumours (BP) (n = 20) and BBM (n = 30) using Combined Bisulphite and Restriction Analyses (CoBRA). (A) The two genes RP11-713P17.4 and NUS1P3 are infrequently methylated in BP and frequently methylated in BBM (In addition, MIR124-2 methylation is enriched in BBM compared to primary breast tumours). In contrast, (B) MIR3193, CTD-2023M8, and MTND6P are frequently methylated in BP and infrequently methylated (hypomethylated) in BBM. (C) An overview of the methylation status of genes that are hypermethylated or hypomethylated in BBM in all BBM and BP samples analysed by CoBRA (each box represents a tumour). (D) Summary of methylation status of genes in primary and metastatic brain tumours, *: Indicates statistical significant difference between primary and BBM tumours. BBM: Breast to Brain Metastases, BP: Primary breast tumours, U: Uncut/control sample, C: cut by restriction enzyme, , HyperM: hypermethylated in BBM, HypoM: Hypomethylated in BBM.

Figure 3

Methylation status of BBM-Hypermethylated and BBM-Hypomethylated genes in BBM and their originating BP tumours from individual patients. (A) Early events; Methylation of MTND6P4 (left panel), RP11-713P14.4 (middle panel) and MIR3193 (right panel) in originating BP is the same as found in their corresponding BBM in individual patients (B) Late events; Methylation status of CTD-2023M8.1 in orginating BP is different to their corresponding BBM tumours (loss of methylation at this region occurs in the metastasis). (C) Summary of methylation status of genes denoting early events and late events in individual patients/matched pairs (MP) and an independent cohort (IC) of primary breast tumours. (D) Expression analyses of genes by quantative reverse transcriptase PCR (qRT-PCR) in representative BBM tumours showing methylation-expression correlation, tumours with genes that are methylated (M) have a low level of expression. BP: Breast Primary tumour, BBM: Brain Metastases, U: Uncut/Control sample, C: cut by methylation specific restriction enzyme, *: Methylated samples, IC: independent cohort, MP: matched pairs from individual patients.

Figure 4

Methylation status of candidate genes in BBM, and tumour free circulationg DNA (tfcDNA) in patients’ serum determined by Methylation Specific PCR (MSP). (A,B) Methylation status of candidate genes denoting early events (CCDC8 and MIR124-2) and (C,D) late events (CTD-2023M8 and MIR3193) in BBM and their corresponding tumour free circulating DNA in individual patients (presence of a PCR product in the MSP lane is indicative of methylation in the region analysed). (E) Summary of methylation status of BBM and serum DNA in individual patients; Red suqares indicate methylation identified, Green squares indicate no methylation identified. BBM: Brain Metastases, SD: Serum DNA, U: Unmethylation Specific PCR; USP, M: Methylation Specific PCR; MSP, *: Methylated samples.

Figure 5

Loss of expression of BBM-hypermethylated genes (candidate metastasis suppressor genes) correlates with survival in breast cancer patients. (A) Kaplan–Meier analysis shows that low expression of MIR124-2 correlates to poor relapse free survival (RFS) of breast cancer patients (p = 0.00004). (B) Kaplan–Meier analysis using a combined four-metastasis suppressor gene signature (BNC1, CCDC8, GALNT9 and MIR124-2) shows that loss of expression of these genes in combination correlates to poor RFS of breast cancer patients (p = 0.0046). (C) Low expression of MIR124-2 is associated with poor prognosis of ER + and ER- breast cancer patients (p = 0.007 and 0.017 respectively).