Figure 2

Epigenetic silencing of the PRKD1 gene promoter correlates with breast tumor invasiveness. (A) Patient samples (invasive ductal carcinoma (IDC) and normal tissue) were analyzed for PRKD1 gene promoter methylation. Genomic DNA was extracted from 25 mg of tissue and modified by bisulfite treatment. Methylation-specific PCR (MSP-PCR) was performed using methylation-specific primers for PRKD1 promoter. Universal methylated DNA (Univ. Met. DNA) served as a positive control. All samples were prepared at the same time and were analyzed on the same agarose gel. (B) PRKD1 gene promoter methylation was determined in confirmed human breast cancer and normal human breast tissue by tissue microarray. DNA was bisulfite-modified in situ. In situ MSP-PCR and hybridization were performed using methylation-specific primers and probes. (C) Statistical analysis of PRKD1 promoter methylation was conducted using the Aperio Positive Pixel Count algorithm in ImageScope viewing software. P values were acquired using Student’s t-test using GraphPad Prism version 5 software. *P ≤ 0.005, indicating extreme statistical significance. DCIS = ductal carcinoma in situ, IDC = invasive ductal carcinoma, ILC = invasive lobular carcinoma. (D) PRKD1 gene promoter methylation was determined in human tissue from IDC with positive or negative lymph nodes. (E) PRKD1 gene promoter methylation was determined in normal human breast tissue (adjacent to tumor), IDC and lymph nodes metastases. For (D) and (E), statistical analysis was performed using the Aperio Positive Pixel Count algorithm in the ImageScope viewing software. P values were calculated using Student’s t-test in GraphPad Prism version 5 software. *P ≤ 0.0001, indicating extreme statistical significance compared to “normal.” **P ≤ 0.005, indicating statistical significance compared to IDC.