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Methylation in promoter regions of PITX2 and RASSF1A genes in association with clinicopathological features in breast cancer patients

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Tumor Biology

Abstract

Breast cancer is a heterogeneous disease with very different responses to therapy and different length of survival. In many cases, however, the determination of the stage and histopathological characteristics of breast cancer is insufficient to predict prognosis and response to treatment for the vast heterogeneity of the disease. To understand the molecular signature of subtypes of breast cancer, we attempted to identify the methylation status of key tumour suppressor gene Ras association (RalGDS/AF-6) domain family member 1 isoform a (RASSF1A) and a member of the paired-like homeodomain transcription factor family which functions in left–right asymmetry development (PITX2) and to correlate results with known clinicopathological features of breast cancer. Formalin-fixed, paraffin-embedded (FFPE) tissues of breast carcinomas (n = 149) were used for DNA extraction. DNA was modified by bisulphite conversion. Detection of the methylation level of the genes mentioned above was performed by methylation-sensitive high-resolution melting assay (MS-HRM). Based on MS-HRM results for RASSF1A and PITX2, we subdivided the samples into four groups according to methylation level (≤50 % methylated, >50 % methylated, 100 % methylated and completely unmethylated alleles). All degrees of methylation status for both genes underwent analysis of dependence with known clinicopathological features, and we found significant associations. In 134 of 149 (89.9 %) primary breast carcinomas, the RASSF1A promoter was methylated. Total hypermethylation of PITX2 was observed in 60 of 135 (44.4 %) breast cancer cases. RASSF1A hypermethylation had significant association with increased age (p < 0.05), tumour grade (p < 0.0001) and stage (p < 0.0001) in the 100 % methylated group. There was significant association of PITX2 hypermethylation with tumour grade (p < 0.0001) and stage (p < 0.0001). Association between the methylation level of both investigated genes and tumour type was significant for ductal invasive carcinoma cases only (p < 0.0001). This study shows different levels of heterogeneous methylation acquired by MS-HRM assay of the promoter region of RASSF1A and PITX2 and its relationship with clinicopathological features of 149 breast cancer patients. We noticed that immunohistopathological subtypes of breast cancer contain distinct promoter methylation patterns. All these data suggest that hypermethylation of the CpG island promoters of RASSF1A and PITX2 might play an essential role in the very early stages of breast cancer pathogenesis.

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Acknowledgments

The present study was supported by the Slovak Research and Development Agency (contract no. APVV-14-0815), the Scientific Grant Agency (VEGA 1/0243/12) and Comenius University (grant UK/156/2010).

Authors’ contributions

EJ wrote this paper. KK and LP were involved in histopathological analysis and data collection. MG statistically evaluated data. ZL and BM participated in laboratory procedures. JD and KD critically revised the manuscript. PZ revised the final manuscript. All authors have read and approved the final manuscript.

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Authors and Affiliations

  1. Department of Oncology JFM CU, Biomedical Center Martin JFM CU, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Mala Hora 4C, 036 01, Martin, Slovakia

    Eva Jezkova, Pavol Zubor, Marian Grendar, Bibiana Malicherova & Zora Lasabova

  2. Clinic of Gynaecology and Obstetrics, Jessenius Faculty of Medicine, Martin University Hospital, Kollarova 2, 036 01, Martin, Slovakia

    Eva Jezkova, Pavol Zubor, Karol Dokus & Jan Danko

  3. St. Elizabeth Cancer Institute Hospital, Heydukova 10, 812 50, Bratislava, Slovakia

    Karol Kajo

  4. Department of Molecular Medicine JFM CU, Biomedical Center Martin JFM CU, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Mala Hora 4C, 036 01, Martin, Slovakia

    Andrea Mendelova

  5. Department of Pathological Anatomy, Jessenius Faculty of Medicine, Martin University Hospital, Kollarova 2, 036 01, Martin, Slovakia

    Lukas Plank

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  1. Eva Jezkova
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Jezkova, E., Kajo, K., Zubor, P. et al. Methylation in promoter regions of PITX2 and RASSF1A genes in association with clinicopathological features in breast cancer patients. Tumor Biol. 37, 15707–15718 (2016). https://doi.org/10.1007/s13277-016-5324-3

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