In order to determine the epigenetic profile of these invasive pros tate cancer cells, we isolated DOT1L DNA and performed a very sensitive MeDIP assay coupled with Agilents 244 K Human Promo ter Tiling Arrays. This allowed for an in depth analysis of the methylation status within promoter elements, upstream as well as down, in these cells. Differences between the invaded and non invaded cells, as well as the bulk tumor cell line were compared. In our analysis, the LNCaP and DU145 cell lines were used, as well as confirmation analysis in two primary prostate cancer cell lines. A unique set of genes were found to be expressed in the invasive cells, yet methylated in the non invasive cells and parental cell lines.

This included genes involved in embryonic Inhibitors,Modulators,Libraries and tissue/organ development, and specifically in neurogenesis including bone marrow X kinase, Iroquois homeobox 3, Sine oculis homeobox homolog 1 and Sex determining region Y box 1. Using the available online expression Inhibitors,Modulators,Libraries databases in Oncomine, it was determined that Sox1 plays a significant role in prostate cancer pro gression and metastasis. Furthermore, Ingenuity pathway analysis determined that the set of differentially methy lated genes are involved in cellular functions such as cell to cell interaction and cell morphology, as well as development of the hematological system and cancer. The most intriguing data identified many of the methy lated targets as members of the IL 6/STAT3 signaling pathway. Further investigation demonstrated that Stat3 was increased in these invasive cells, and cells infected with an shRNA against either BMX or SOX1 resulted in decreased levels of activated STAT3.

However, only the differentially Inhibitors,Modulators,Libraries methylated Sox1 directly interacts with STAT3. Thus, in our model SOX1 plays a critical role in regulating invasive prostate cancer cells. These aggressive sub populations of cells may be linked to the cancer stem cell hypothesis, making their patterns of epigenetic regulation very attractive for biomarker analysis. Materials and methods Inhibitors,Modulators,Libraries Cell Lines and Reagents LNCaP and DU145 human prostate cancer cell lines were obtained from ATCC and cultured Inhibitors,Modulators,Libraries accordingly. Primary human prostate cancer cells were acquired from Celprogen and maintained as recommended using spe cific coated culture plates and defined media. Human bone marrow derived mesenchymal stem cells were obtained from Lonza and maintained using their recommended conditions. The cultures were maintained in 5% CO2 air at 37 C. Human serum was obtained from Gemini Bioproducts. The following inhibitors were also used Anti human IL 6 antibody, PI3K inhibitor LY294002, Tec Kinase inhibitor LFM A13, MEK inhibitor PD98059, JAK inhibitor AG490, and STAT3 inhibitor sellekchem Stattic.