To identify the expression level of the four oncogenic ETS protei

To identify the expression level of the four oncogenic ETS proteins, we first tested available antibodies using puri fied recombinant proteins. We identified antibodies to ERG, ETV1, ETV4, and ETV5 that could detect each protein at femtomolar levels. Because ETV1, ETV4, and ETV5 are homologous proteins, sellectchem the sensitiv ity and specificity of these antibodies were compared. ETV1 and ETV4 antibodies were specific, but the ETV5 antibody recognized ETV4 and ETV5 equally. We then examined oncogenic ETS protein levels, along with phosphorylated ERK and phosphorylated AKT levels in six prostate cancer cell lines. DU145 cells, which have a KRAS gene rearrangement, did not have high levels of any onco genic ETS protein, or pAKT, but did have pERK, consist ent with the small fraction of prostate cancers with RAS ERK pathway mutations.

Of the remaining five prostate cancer cell lines, four had high expression of a single oncogenic protein. These included ERG in VCaP, consistent with a TMRPSS2ERG rearrangement, ETV1 in MDA PCa Inhibitors,Modulators,Libraries 2B, consistent with an ETV1 gene re arrangement, and ETV4 in PC3, consistent with high ETV4 mRNA. ETV4 protein was also present at high levels in CWR22Rv1. Of the four lines with high onco genic ETS protein expression, all had high levels of pAKT, but only one had high levels of pERK, con 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. However, this is consistent with results from Vitari et al.

who showed low ETV1 protein levels in LNCaP cells due to proteasomal Inhibitors,Modulators,Libraries targeting by the COP1 E3 ubiquitin ligase. Long exposures could identify pERK, pAKT, Inhibitors,Modulators,Libraries 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 Inhibitors,Modulators,Libraries expression for ETS pro teins exceeded 50,000 proteins per cell, and 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 Inhibitors,Modulators,Libraries 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 BAY 87-2243? 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 RASERK pathway activates ETV1, ETV4, and ETV5 expression.

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