Herein, we demonstrate that androgens induce CXCR4 gene expression in TMPRSS2-ERG-positive VCaP cells. To our knowledge, this is the first report identifying the CXCR4
gene as a target for TMPRSS2-ERG activation in PC cells. In this study, we show that androgen-responsive VCaP cell lines coexpress higher levels of CXCR4 and ERG compared with androgen-unresponsive PC-3 cells. ERG protein expression is absent in PC-3 cells, whereas it is expressed in two forms by VCaP cells () as was previously shown by Tomlins et al. [1
]. The two ERG species expressed in VCaP cells are most likely due to the alternative splicing of the fusion transcript. There is significant heterogeneity in the expression of fusion transcripts in tumor cells with TMPRSS2-ERG alterations [1,7,27,28
], and the translation of these transcripts could give rise to several ERG species. For example, these ERG forms can lack 39 amino acids at the N-terminus [1
], fusion with the first five amino acids of TMPRSS2 protein and lack the N-terminus of ERG [7
], have an insertion of 24 amino acids in the central domain of ERG[29
], or have a deletion of the C-terminus Ets binding domain [27
]. A recent study by King et al. [13
] has shown that fast migrating ERG species in the doublet has been the translation product from the first AUG codon in the fourth exon. This translation product lacks 39 N-terminal amino acids. Overexpression studies in cell culture and animal models with several of these ERG forms, with the exception of the C-terminus Ets domain deletion, demonstrate that they play a key role in PC cell proliferation [29
], invasion [9,10
], and progression [12,13
]. Overall, the data strongly indicate that activation and alterations of TMPRSS2-ERG contribute to the lethal characteristics of PC development in patients.
Previous studies show that similar TMPRSS2-ERG deletions are present in both primary tumor cells and disseminated metastatic cells to several secondary sites [30
], which suggest that downstream target genes of TMPRSS2-ERG fusions facilitate the tumor cell invasion and dissemination process. Recent studies suggest that the chemokine receptor CXCR4 in tumor cells and its ligand CXCL12 expressed in secondary metastatic sites play a key role in the metastasis of primary tumor cells [16–18,25
]. Our data support the notion that TMPRSS2-ERG activation in PC cells regulates CXCR4 expression and subsequent metastasis to secondary sites.
Our study shows that the synthetic androgen upregulates the ERG expression in fusion-positive VCaP cells, which is in line with findings previously reported by Tomlins et al. [1
]. Interestingly, we found that CXCR4 is regulated in parallel with ERG in a panel of PC cells. In LNCaP cells, the synthetic androgen induced a very modest degree of CXCR4
gene expression in (). Because LNCaP cells have very low levels of ERG in the absence of TMPRSS2-ERG translocations, this regulation could be mediated indirectly by additional AR-dependent processes. Alternatively, other Ets factors could contribute to CXCR4 expression in these cells. In support of the potential regulation by other Ets factors, ETV1 has been shown to be expressed in LNCaP cells [1
] and could mediate CXCR4 expression through Ets binding sites in the CXCR4 promoter. Studies are in progress to identify the specific ERG and Ets binding sites in the CXCR4 promoter. The moderate levels of CXCR4 expression induced by R1881, coupled with the presence of ETV1 in these cells, support the notion that ERG and ETV1 translocations could be mutually exclusive in prostate tumor samples and could mediate prometastatic CXCR4
gene expression, as well as the subsequent invasion and metastasis of tumor cells. In VCaP cells, androgen-induced CXCR4 expression is mediated by the overexpression of ERG rather than by the general growth effects of androgens because we did not observe changes in the CXCR4 expression in VCaP cells in the presence of serum (data not shown).
CXCR4 has been shown to be regulated at the transcriptional level by the growth factor through hypoxic [31
] and nuclear factor κB transcription factor activities [32
]. In contrast with those models, our data with cycloheximide () suggest that androgen-mediated protein synthesis is required forCXCR4 expression inPC cells. Furthermore, such a requirement is only present in PC cells exhibiting the TMPRSS2-ERG translocations. Our analysis (data not shown) suggests that CXCR4 promoter does not contain consensus AR binding sites [33
], and thus, this regulation is most likely to be mediated indirectly. Conversely, Akashi et al. [34
] have shown that overexpression of AR in DU145 cells downregulated CXCR4 expression, although it is not clear whether the overexpressed AR is active in these cells. Although the lack of an AR binding site in the CXCR4 promoter region suggests that such down-regulation could be indirectly mediated by AR activation, which would be independent of ERG function in these cells, overexpression of ERG in TMPRSS2-ERG fusion-positive cells could override these inhibitory effects on CXCR4 expression.
In agreement with our conclusions, Carver et al. [12
] recently reported, while this article was in preparation, that ERG-transfected PC-3 cells have higher functional CXCR4 expression. Our data also demonstrate that CXCR4
is a target gene for the ERG transcription factor, and our results with the synthetic androgen regulation of CXCR4 () further suggest that CXCR4 is a physiological target of androgens in prostate tumor cells and that this process could facilitate the pathological progression of tumor cell metastasis through the CXCL12/CXCR4 axis. Carver et al. reported that ERG binds to Ets binding sites in the -2683 to -2531 promoter of CXCR4, whereas the data from chromatin immunoprecipitation analysis in this study identified ERG binding sites in the CXCR4 promoter between the transcription start site and the -513 of the CXCR4 promoter. We identified eight potential ERG binding sites in the 1-kb CXCR4 promoter, and three of these putative sites were present in the transcription start site to the -513 of the CXCR4 promoter. Maroni et al. [35
] reported that Ets1 factor binds to -397 to -412 and -478 to -481 sites in the CXCR4 promoter, suggesting that Erg could also bind to these sequences. Because ERGis under androgen control in TMPRSS2-ERG-positive cells, it is highly likely that ERG could be the relevant factor interacting with the CXCR4 promoter in PC cells. The sequences between -513 and -996 have four potential ERG binding sites, but our attempts to design primers to amplify this region have been unsuccessful so far because of the high percentage of GC content at this region. Studies are in progress to determine the relative contribution of these putative ERG binding sites in the regulation of the CXCR4 promoter.
ERG knock down by siRNA has been shown to decrease invasive and proliferative functions in VCaP cells. Our previous data demonstrated that the CXCL12/CXCR4 axis promotes PC cell invasion through activation of signaling pathways leading to protease expression [17
]. CXCR4 expression also has been shown to contribute to the growth of tumor cells in bone metastatic sites [18
]. Our present data demonstrate that ERG knockdown attenuates androgen-dependent CXCR4 expression without significantly changing PSA
expression. These results suggest that the TMPRSS2-ERG fusion facilitates tumor cell invasion and metastasis through the regulation of CXCR4 expression and function in PC cells. To test this concept, our data with an in vitro
invasion assay () demonstrate that R1881 alone can induce VCaP cell invasion. This supports previously published reports that R1881-induced ERG expression contributes to in vitro
invasion of VCaP cells and is mediated by the expression of proteases [9,10
]. Interestingly, the R1881-treated cells invaded more efficiently in the presence of CXCL12. CXCR4 inhibition suppressed the CXCL12 effect, suggesting that androgen-induced CXCR4 expression is functional in VCaP cells and contributes to PC cell invasion. These data are in line with previous reports, demonstrating the role of ERG in CXCR4 function in PC cells [12
In summary, we show that TMPRSS2-ERG activation in fusion-positive cancer cells induces the expression of the prometastatic gene CXCR4, which is functionally active in the chemoinvasion process. Targeting CXCR4, a relevant target for androgen activation of TMPRSS2-ERG, could be an advantageous strategy for lethal phenotypes associated with these chromosomal translocations in PC patients.