Promoter Methylation and Phosphorylation Status of PTEN in Taiwanese Breast Cancer.

Abstract Background: The PTEN (phosphatase and tensin homolog deleted on chromosome Ten) gene is a novel candidate of tumor suppressor that plays an important role in cell cycle regulation and apoptosis by modulating protein kinase-B/AKT activity. In western breast cancer population, PTEN downregulation is frequently associated with high-grade tumor, distant metastases and poorer disease-free survival. It is also noted that the methylation of the PTEN promoter results in PTEN inactivation in a subset of breast cancers. Taiwan is the low-incidence area of breast cancer. However, breast cancer has been jumped to the first place in the incidence of female malignancies in the past 3 years. Hence, we aim to explore the potential role of PTEN in Taiwanese breast cancer.Material and Method: Firstly, we used tissue microarrays containing 192 specimens (PTEN) and 193 samples (p-PTEN) from the Department of Pathology, Kaohsiung Medical University to analyze the expression patterns of PTEN and p-PTEN (Ser380/Thr382/Thr383) protein by immunohistochemistry (anti-PTEN antibody, A2B1, and anti-p-PTEN R, Santa Cruz). Clinical data, such as stage, grade, recurrence, lymph-node metastasis, and patient's age, tumor-specific survival (in months) and others were included in our statistic analysis. The genomic DNA (1 μg) obtained from 318 fresh specimens containing 159 matched cancer and non-cancer tissues were treated with sodium bisulfite for 16 hours and purified using methylation-specific PCR (MSP) kit (Millipore). Separate primers were used for amplification of methylated and unmethylated promoter regions. Primers that amplified a region of the promoter specific to PTEN gene but not the PTEN pseudogene were used. Primers used to amplify the methylated PTEN promoter were 5'-GTATTTCGAGTAAAGGAAGAAGACG-3' and 5'-GATAAAAAACTACAACC CAACGAA-3'; those used to amplify the unmethylated promoter were 5'- TATTTTGAGTA AAGGAAGAAGATGA-3' and 5'-CAATAAAAAACTACAACCCAACAAA-3'.Results: Our results showed that an increased nuclear expression of PTEN was positively correlated with an increased tumor staging (p=0.010). Furthermore, an increased p-PTEN expression in the cytoplasm was inversely correlated with tumor staging (p=0.003), while the decreased expression of p-PTEN in the nucleus was significantly correlated with positive recurrence rate (p=0.009) and metastasis to lymph node (p=0.033). Notably, the expression level of p-PTEN was positively correlated with that of PTEN. We also observed that the methylation of PTEN promoter was increased in 70% of breast cancer tissues as compared with the matched non-cancer breast tissues. Moreover, the increased methylation of PTEN promoter was positively correlated with tumor grade (p=0.037).Discussion: Taken together, our results suggest that an altered PTEN expression through methylation or phosphorylation may have potential roles in Taiwanese breast cancer. The underlying mechanism requires further investigation. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4060.