ETV4 protein was also present at high levels in CWR22Rv1. Of the four lines with high onco genic ETS Seliciclib CDK2 protein expression, all had high levels of pAKT, but only one had high levels of pERK, con Inhibitors,Modulators,Libraries sistent with the analysis of prostate tumors in Table 1. Surprisingly, despite an ETV1 gene rearrangement, and high ETV1 mRNA levels, ETV1 protein was not observed in LNCaP cells. Inhibitors,Modulators,Libraries However, this is consistent with results from Vitari et al. who showed low ETV1 protein levels in LNCaP cells due to proteasomal targeting by the COP1 E3 ubiquitin ligase. Long exposures could identify pERK, pAKT, and some ETS proteins at low levels in immunoblots from most cell lines. To more quantitatively establish the high level threshold shown in Figure 1B, ETS proteins in cell ex tracts were compared with purified standards.

All high level expression for ETS pro teins exceeded 50,000 proteins per cell, and Inhibitors,Modulators,Libraries was highest at 330,000 proteins per cell for ERG in VCaP. Low level ETS expression was 10,000 proteins per cell or less. It is possible that oncogenic ETS expression and sig naling pathway activation could influence each other. To test this, RWPE 1 cells derived from normal prostate or variations of this line that express either Ki RAS or ERG were compared. ERG levels in RWPE ERG cells were similar to VCaP cells. None of the oncogenic ETS were expressed at high levels in RWPE or RWPE KRAS cells, and only ERG was expressed in RWPE ERG cells. As expected, KRAS increased both pERK and pAKT levels. Interestingly, over expression of ERG also resulted in activation of AKT and a small increase in pERK.

In other cell types, the RAS ERK Inhibitors,Modulators,Libraries pathway activates ETV1, ETV4, and ETV5 expression. Therefore, high ETV4 expression in CWR22Rv1 cells could be the result of ERK activation. To test this, CWR22Rv1 and DU145 cells were treated Inhibitors,Modulators,Libraries with the MEK inhibitor U0126 for 24 hours. In both cell lines, U0126 decreased pERK levels, but did not alter levels of ETV4. Therefore, RAS ERK activation does not drive oncogenic ETS read this expression in prostate cancer cell lines, however in at least one context an oncogenic ETS could induce the phosphorylation of both AKT and, to a lesser degree, ERK. Oncogenic ETS proteins and KRAS drive prostate cell migration, but not synergistically We next tested the role of signaling pathways in the ability of oncogenic ETS proteins to drive cell migration. Because cancer derived cell lines have many mutations and copy number alterations that affect cellular pheno types, we used the RWPE ERG and RWPE KRAS cell lines to compare the ability of oncogenic ETS and RAS signaling to promote cell migration in the same cellular background. RWPE ERG and RWPE KRAS cells mi grated 5 and 10 fold more than RWPE cells, indicating that both ERG and KRAS induce cell migration.