Therefore, we validated the methylation and resul tant decreased expression of BMX and SOX1 in the non invasive cells. Functional role of Bmx and Sox1 during invasion To further determine the role of Bmx and Sox1 during the process of invasion we performed the invasion assay with DU145 cells stably infected with shRNAs directed against Sox1or different Bmx. A significant decrease in expression of SOX1 and BMX following induction with 1 ug/mL of doxycycline for 24 hours was first verified using western blotting. Upon induction with Dox, the shRNA is turned on and a downstream red fluorescent protein demonstrates efficiency of this induction. Densitometry analysis was per formed to compare expression of individual clones with the NS cells, and no significant differences in protein expression were seen using the non silencing con trols.

In addition, SOX1 shRNA cells demonstrated a significant decrease in proliferation compared to either the parental Inhibitors,Modulators,Libraries cell line or the NS infected line, as well as a significant decrease in invasion toward SCM. However, there was not a significant difference using the shBMX lines, except for a slight reduction in invasion using clone 3. Interestingly, a small increase in proliferation Inhibitors,Modulators,Libraries was seen with the shBMX clones. Further promoter tiling array analysis using two short term cultures primary prostate tumor cell lines, PCSC1 and PCSC2, determined that Sox1, and not Bmx, was methylated in the invasive population of cells. Overall, we demonstrate that Sox1is differentially methylated Inhibitors,Modulators,Libraries within the invasive CSC population and the shRNA studies indicate it could be selectively targeted to block invasion.

Role of SOX1 during differentiation In addition to the method presented here, prostate TICs can also be isolated by culturing total cells Inhibitors,Modulators,Libraries in SCM where structures called prostato spheres are generated. The prostatospheres are multicellular globes that develop from cells that sur vive anchorage independent conditions in vitro, and are frequently used when analyzing the ability of TICs to self renew or differentiate upon the addition of serum. Using this assay as a model, a greater number of prosta tospheres were isolated from DU145 NS cells compared to shSOX1 cells. When invasive DU145 cells were isolated and cultured in SCM, prostatospheres were maintained for up to 3 passages and if these cells were further cultured in the presence of 1% human serum, the vector control cells rapidly differentiated and proliferated, while the shSOX1 cells did not.

These observations suggest that not only does Sox1 play a role in regulating invasion, but it can also regulate the maintenance of stem ness in culture. Ingenuity pathway analysis defines pathways of differentially Inhibitors,Modulators,Libraries methylated genes within invasive sub populations of cells Each data set of differentially methylated genes was then extracted leave a message and uploaded to the Ingenuity server to identify common gene pathways that are regulated during the process of invasion.