In recent years, chemokines and their receptors have evolved into a major field in cancer research as these chemokine networks have been implicated in every aspect of cancer biology. CXCR4 plays a major role in ErbB-receptor mediated metastasis in several cancers, including ErbB2-mediated breast cancer (11
). Here, we evaluated the relationship between CXCR4 and the naturally occurring EGFR variant, EGFRvIII, a potent oncogene that has been shown to be associated with breast cancer progression and metastasis (4
). We have provided evidence that both an activated EGFR and ErbB2 have the ability to increase CXCR4 expression. We also demonstrated that up-regulation of CXCR4 is mediated by the activity of EGFRvIII, but not ErbB2 in all of the breast cancer model systems that we have examined. In addition to the role of CXCR4 in ErbB2-mediated breast cancer metastasis (11
), our study has demonstrated that CXCR4 is likely to play an essential role in EGFRvIII-mediated invasion in breast cancer cells.
CXCR4 expression is increased under hypoxic conditions by the transcriptional activity of the oxygen-dependent HIF-1α (17
). However, non-small cell lung cancer cells were shown to up-regulate the expression of CXCR4 through EGFR-dependent HIF-1α protein stability and increased DNA binding and transcriptional activity at the CXCR4 promoter irrespective of oxygen levels (17
). In this study, we report, for the first time, that EGFRvIII up-regulates HIF-1α protein levels and CXCR4 expression under both normoxic and hypoxic conditions in breast cancer cells. Increased CXCR4 transcripts were found to correlate with increased HIF-1α levels under normal oxygen tension in estrogen-dependent EGFRvIII-expressing breast cancer cells. Increased protein expression of other HIF-1α regulated genes, such as survivin (see Supplemental Fig. 7
), under normoxic conditions further supports the notion that increased HIF-1α activity is a novel signaling cascade in estrogen-dependent EGFRvIII-mediated oncogenesis. It is important to note that while EGF stimulation of parental breast cancer cells did increase HIF-1α levels at a short exposure time, EGF stimulation did not consistently increase CXCR4 levels in these cells at short or long exposure times (data not shown). In contrast, constitutively activated EGFRvIII mediates sustained downstream signaling (4
), and therefore, may allow for HIF-1α protein accumulation under normal oxygen levels and consequently promote the transcription of CXCR4. Alternatively, increased HIF-1α protein and/or CXCR4 transcripts may be a distinct and unique hallmark of EGFRvIII signaling which is not obtained through the activation of full length EGFR in breast cancer. This is possible as it has been shown that full length EGFR and EGFRvIII do not share common signaling pathways in gliomas (30
AIP4 overexpression suppresses CXCR4 expression in ErbB2-overexpressing breast cancer cells by decreasing the stability of CXCR4 protein levels (11
). We have discovered that EGFRvIII decreases endogenous levels of AIP4 in BT474/EGFRvIII cells, but AIP4 was undetectable in other model systems. However, β-arrestin 1/2 expression level was reduced in all the EGFRvIII-expressing model systems. Since, β-arrestin 1/2 plays an essential role in the trafficking of several GPCRs, these results provide a possible mechanism in which the reduction of AIP4 and β-arrestin 1/2 expression promote an aberrant degradation of CXCR4, thereby leading to its up-regulation in EGFRvIII-expressing breast cancer cells. It should be mentioned that although we did not detect increases in CXCR7 or CXCR3 (data not shown) in EGFRvIII cells, other GPCRs involved in breast cancer metastasis may be up-regulated in EGFRvIII expressing breast cancer cells. The use of the GPCR inhibitor, Pertussis Toxin, was able to abolish the increased invasive potential of EGFRvIII-expressing breast cancers, suggesting that EGFRvIII-enhanced invasion may be the result of increased GPCR expression and/or signaling. However, knocking-down CXCR4 also decreases the invasive potential of EGFRvIII-expressing breast cancer cells (data not shown), implicating a significant role for CXCR4 in EGFRvIII-mediated invasion. These studies are currently underway.
Taken together, our results also show that induction of CXCR4 protein expression is regulated both transcriptionally and post-translationally. In ER negative and estrogen-independent breast cancer cells, EGFRvIII-induced CXCR4 expression is likely to be regulated at the post-translational level by increasing the synthesis rate of CXCR4 protein and enhancing the CXCR4 protein stability through decreasing the expression levels of those molecules involved in the cellular trafficking and degradation of CXCR4. In contrast, in estrogen-dependent ER+/PgR+ breast cancer cells, EGFRvIII-mediated up-regulation of CXCR4 expression may be regulated at the transcriptional level through up-regulation of HIF-1α, as well as at the post-translational level. Therefore, both transcriptional and post-translational mechanisms are likely responsible for EGFRvIII-mediated up-regulation of CXCR4 protein levels in EGFRvIII-expressing breast cancer cells.
Our results have also demonstrated that p38 MAPK is one of the major down-stream signaling molecules involved in EGFRvIII/CXCR4-mediated invasion. p38 MAPK activity was markedly induced by CXCL12 stimulation under both normoxic and hypoxic conditions. Inhibition of p38 MAPK activity significantly reduced CXCR4 levels and attenuated the invasive potential of EGFRvIII-expressing breast cancer cells, whereas MAPK inhibitors do not have this effect on CXCR4 expression. These results suggest that p38 MAPK plays a role in EGFRvIII/CXCR4 induced invasion.
Finally, glioma cell lines ectopically expressing EGFRvIII did not exhibit enhanced CXCR4 expression in comparison to parental cells (data not shown). This observation appears to be unique to our EGFRvIII breast cancer model system. It is likely that the glioma cells express higher basal levels of CXCR4 in comparison to breast cancer cells, which is also reflected in the highly invasive phenotype of glioma cells. EGFRvIII may only enhance CXCR4 expression in cancer cells that have low invasive potential, as is the case for the cancer cell lines used in our studies. These results suggest that CXCR4 has an essential role in enhanced invasion of EGFRvIII-expressing breast cancer cells. Hence, further investigations are required in order to fully elucidate the relationship between CXCR4 and EGFRvIII in breast cancer and whether targeting these two molecules with pre-existing or novel therapies will prevent breast cancer metastasis and potentially other oncogenic functions involving both CXCR4 and EGFRvIII.