Epigenetic and genomic biomarker discovery in breast cancer

Breast cancer is the most common malignancy among females and the fifth most common cause of cancer death worldwide. The research for biomarker discovery for the diagnosis and prognosis of patients with breast cancer has been going on for decades, yielding only a few major breakthroughs. Our increasing understanding of cancer biology, including genetic, molecular and cellular mechanisms, and epigenetic background is now providing objectives for the early detection of some malignancies including breast cancer. Such a progress has a direct impact on current activities dedicated to the search for sensitive and specific biomarkers for the early detection and diagnosis of cancers.
In the present study, to find an efficient method for analyzing the methylation profile in breast cancer, we developed a method that allows for the simultaneous detection of multiple targets CpG residue by employing thymidine-specific cleavage on MALDI-TOF MS. Using this method we determined quantitative methylation changes of 22 candidate genes in breast cancer tissues. Firstly, we analyzed the methylation status of a total of 42,528 CpG dinucleotides on 22 genes in 96 different paraffin-embedded tissues (48 breast cancerous tissues and 48 paired normal tissues). In this study, 10 hypermethylated genes (APC, BIN1, BMP6, BRCA1, CST6, ESRb, GSTP1, P16, P21 and TIMP3) were identified to distinguish between cancerous and normal tissues according to the extent of methylation. These hypermethylated genes may serve as biomarkers for clinical molecular diagnosis and targeted treatments of patients with breast cancer.
To achieve a gene panel for developing a breast cancer blood-based test according to the pathologic methylation changes, we quantitatively assessed the DNA methylation proportion of 31,248 CpG sites on 10 candidate genes (APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P16, P21 and TIMP3). The number of 126 samples consisting of two different cohorts was used (first cohort: plasma samples from breast cancer patients and normal controls; second cohort: triple matched samples including cancerous tissue, matched normal tissue and serum samples). Circulating cell free methylated DNA of the 8 tumor suppressor genes (TSGs) was significantly higher in patients with breast cancer compared to normal controls. Using eight genes as a panel to develop a blood-based test for breast cancer, a sensitivity and specificity of more than 90% could be achieved in distinguishing between tumor and normal samples. Presented data is promising to design a gene panel and develop a blood-based screening method for breast cancer which relies on pathologic methylation changes.
Analyzing the relative telomere length in paired breast cancer and matched normal tissue demonstrated a significant shortening of telomere regions in the tumor compared to paired adjacent normal tissues. Moreover, promoter hypermethylation of the p16/Rb and p53/p21 pathways showed significant correlation with telomere shortening. The results suggested that inactivation of p16/Rb and/or p53/p21 pathways by hypermethylation may be linked to critical telomere shortening, leading to genome instability and ultimately to malignant transformation. Thus, shortened telomere length and hypermethylation of p53, p21 and p16 promoters might serve as biomarkers in breast cancer.
Epigenetic changes can be pharmacologically reversible and could be a useful target to develop new therapeutic strategies for cancer therapy. Reversal hypermethylation of silenced genes/miRNAs inhibiting malignant phenotypes is increasingly being targeted for cancer therapy and prevention strategies. In our study, the therapeutic value of detected hypermethylated tumor suppressor genes in different subtypes of breast cancer was assessed after treatment with a demethylating agent (5-aza-2-deoxycytidine; DAC). Additionally, we investigated multidimensional pan-omics models to predict effects of DAC treatment at the level of the genome, epigenome and proteome-wide alterations in highly aggressive and non-aggressive subtypes of breast cancer. The results provided early and late effects of this treatment in 3-dimentional follow-up omics model as cancer and non-cancer specific changes that may correlate with particular steps in breast neoplasm including cell proliferations, cell/tissue invasion, oncogenesis, angiogenesis, apoptosis, neoplasm metastasis and senescence. Additionally, beside the activation of epigenetically suppressed TSGs, we also showed significant down-regulation of some miRNAs with oncogenic functions in breast cancer cell lines as well as up-regulation of some other miRNAs with tumor suppressor functions that highlights the usefulness potential of a miRNA-based therapy in breast cancer. The obtained results provided a rationale for developing therapeutic strategies based on reactivation of epigenetically silenced genes/miRNAs in breast cancer.