As predicted for invasive cancer, we detected a high percentage of positive tumor cells for methylated PRKD1 promoter in both sam ples. However, the percentage of promoter methylation was significantly higher in IDC with Nutlin-3a (-)-Nutlin-3 positive lymph nodes as compared to IDC with negative lymph nodes. Next, we compared PRKD1 promoter methylation in normal tissue adjacent Inhibitors,Modulators,Libraries to tumor, primary tumor and lymph node metastases from patients with IDC. In these samples, we ob served a significant increase in the percentage of positive cells for PRKD1 promoter methylation in primary tumors and a further Inhibitors,Modulators,Libraries increase in lymph node me tastasis compared to adjacent normal tissue. Hypermethylation of the PRKD1 promoter correlated with loss of PKD1 expression in the same tissue.

In summary, our analysis of patient data indicates that decrease or loss of PKD1 expression in human breast cancer is due to hypermethylation of the PRKD1 promoter. Such silencing correlates with the invasive po tential of tumors. This suggests Inhibitors,Modulators,Libraries that both PKD1 expres sion and methylation of its promoter could serve to determine the invasive potential of breast tumors. Pharmacologic inhibition of PRKD1 methylation leads to PKD1 dependent reversion of the invasive phenotype On the basis of the preceding experiments, we hypothe sized that inhibition of methylation of the PRKD1 pro moter with DNA methyltransferase inhibitors can lead to reexpression of PKD1 and reversion of the invasive phenotype. To test this hypothesis, we treated MDA MB 231 cells with decitabine and tested its effect on PRKD1 promoter methylation using MSP PCR.

Decitabine induced the demeth ylation of the PRKD1 promoter, and this correlated with the reexpression of PKD1 at the transcriptional level and at the protein level Inhibitors,Modulators,Libraries without affecting the levels of expression of PKD2 and PKD3. Similar results were obtained with two additional inva sive breast cancer cell lines. Inhibition of methyltransferases can lead to induction of multiple genes in cancer, including the estrogen re ceptor. To distinguish between decitabine Inhibitors,Modulators,Libraries induced PKD1 17-AAG Tanespimycin dependent and PKD1 independent effects, we next compared control MDA MB 231 cells to cells previously infected with shRNA targeting PKD1. Expression of shRNA specific for PKD1 in these cells blocks decitabine induced reexpression of PKD1 as com pared to parental or control cells. Treatment with decitabine slightly decreased MDA MB 231 cell viability, and this effect was independent of the PKD1 expression status. However, the inhibitory effects of decitabine on tumor cell invasion were partially restored in PKD1 knockdown cells. This suggests that the inhibitory effects of decitabine on cell invasion are due in part to PRKD1 promoter demethylation and reexpression of PKD1.