P53 pathway alterations in breast cancer for biomarker discovery

Breast cancer has the highest incidence rate amongst female cancers worldwide. Following the advent of mammography and the utilization of advanced treatment strategies breast cancer mortality rates have markedly declined. However, in order to treat the refractory and more aggressive forms of the disease, development of detection methods using novel biomarkers and more efficacious therapeutic protocols are still needed. P53 dysfunction and impaired p53 signalling has been reported in over 50% of human cancers. Impairment of p53 activity in various cancers has primarily been attributed to P53 gene mutations. In addition to gene mutations, however, alterations in the activity of upstream and/or downstream TP53 regulators (e.g. autoregulatory loops, proteins and miRNAs) also modulate the activity of the p53 pathway. The frequency of p53 mutations in breast cancer is lower than other cancers (approximately 20%). The impact of these additional p53 regulatory mechanisms on breast cancer pathology is poorly understood. To address this issue, the present study examined the following:
I. Identify novel mechanisms of p53 pathway inactivation in breast cancer, independent of P53 gene mutations.
II. Establish an association between aberrant DNA methylation of p53 regulatory pathways and downstream p53 activity.
III. Evaluate differences in aberrant DNA methylation signatures between primary tumors and metastatic lesions and its contribution to pathology.
In the first part of the study, we demonstrated that both P14ARF and PTEN (P <0.05 and P <0.01, respectively) promoters were hypermethylated in breast cancer patients. Hypermethylation of both promoters could potentially mediate p53 inactivation via the dysregulation of p53 auto-regulatory feedback loops. Indeed, proteomic characterization of breast cancer cell lines upon demethylation therapy revealed the overexpression of p53 regulatory proteins - Atic, Calr and Pcna. This indicates a putative role of DNA methylation on the regulation of p53 pathway activity. Furthermore, we identified a subset of differentially expressed miRNAs that targeted p53 pathway components. In this context, over-expression of miR-21 (P<0.05) is potentially important for the pathogenesis of wt-p53 breast cancer patients.
Second, we were able to demonstrate a significant correlation between the hypermethylation of P16/Rb and P53/P21 promoters and the shortening of telomere length in breast cancer patients (r=-0.33, P=0.001; r=-0.70, P<0.0001 and r=-0.71, P<0.0001; respectively). Moreover, hypermethylation of P14ARF and PTEN was associated with increased mitochondrial DNA (mt-DNA) damages including: depletion of mt-DNA content (11.88-fold changes; P< 0.01) and enhancement of mutations in the D-loop region (36.36%).
Lastly, we explored the methylation signature of 12 breast cancer candidate genes (APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P14, P16, P21, PTEN and TIMP3). There was considerable heterogeneity in the methylation status of primary tumors and metastatic lesions. Higher methylation levels at promoter regions of APC, BIN1, BMP6, BRCA1, CST6, ESR-b, P16, PTEN and TIMP3 were identified in primary tumor tissues (P<0.05, P<0.05, P<0.01, P<0.0001, P<0.01, P<0.01, P<0.05, P<0.05, P<0.01; respectively), whereas in metastatic lymph node lesions only BMP6, BRCA1 and P16 were hypermethylated (P<0.05 and P<0.01, P<0.0001, P<0.05; respectively).
In conclusion, the present study identified novel biomarkers for tumor specific and metastatic breast cancers. Aberrant methylation signature of APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P14ARF, P16, P21, PTEN and TIMP3, as well as overexpression of miR-21, increased mt-DNA damages and shortened telomere length specifically identified tumor specific lesions. Moreover, hypermethylation of BMP6, BRCA1 and P16 gene promoters indicate their potential use as metastatic biomarkers. These novel biomarkers will be further examined for the effect of specific hypermethylated CpG sites on the transcriptional activity of each gene. Furthermore, establishing absolute percent methylation cutoffs for each breast tumor sub-type would enable their utilization in clinical laboratory settings.