Chemokines have been reported to be associated with breast cancer metastasis. The chemokine and chemokine receptor axis of CXCL12/CXCR4 is believed to home CXCR4-expressing cells to organ sites expressing CXCL12 [1
]. The expression of CXCR4 in human breast cancer specimens are also associated with metastatic spread and poor prognosis [2
]. CXCR4 neutralizing antibodies and other CXCR4 antagonists developed primarily to inhibit HIV-viral entry have shown to inhibit the invasive potential of breast cancer cells in vitro
as well as reduce breast cancer metastasis in nude mice [1
Silencing of CXCR4 expression with RNA-interference (RNAi) has shown to inhibit breast cancer cell migration, invasion, and metastasis in vitro
and in vivo
. Initially, an inducible knock-down of CXCR4 using RNAi technology resulted in a significant reduction of breast cancer cell migration [13
]. Down-regulation of CXCR4 with short hairpin RNA (shRNA) in breast cancer cells with high metastatic potential (MDA-MB-231) not only lowered lung metastasis, it also reduced breast cancer cell proliferation [14
]. Decreased proliferation of breast cancer cells by suppressed expression of CXCR4 also resulted in the failure of these cells to grow tumors in SCID mice [15
]. Targeting CXCR4 with the combination of two small interfering RNA (siRNA) duplexes also impaired breast cancer cell invasion using matrigel invasion assays and breast cancer metastasis in an animal model [16
]. ErbB2-mediated breast cancer metastasis is also dependent on CXCR4 up-regulation as it was shown that shRNA knock-down of CXCR4 in ErbB2-overexpressing breast cancer cells inhibited breast cancer metastasis to the lung [9
]. However, the signaling cascades or other molecular events by which this inhibition occurs are not well described.
Our previous studies have found increased expression of the Epidermal Growth Factor Receptor (EGFR) variant, EGFRvIII, in breast cancer metastasis [17
]. Expression of this variant has also been detected in circulating breast cancer cells and was found to be correlated with metastatic disease, however, the mechanism by which this tumor specific, constitutively active oncoprotein promotes breast cancer metastasis is not understood [18
]. Recently, we reported that EGFRvIII up-regulates CXCR4 in breast cancer cells regardless of estrogen and progesterone receptor (ER/PgR) status or levels of endogenous ErbB-receptors [20
]. Furthermore, CXCR4 was transcriptionally up-regulated in EGFRvIII-expressing breast cancer cells via up-regulation of HIF-1α and/or post-translationally through decreased protein degradation and presumably increased receptor recycling and protein trafficking involving p38 MAPK activity and down-regulation of the endosomal sorting molecules β-Arrestin 1/2 and the Nedd4-like E3 ubiquitin ligase AIP4 [20
]. More importantly, these cells also had enhanced SDF-1/CXCR4-mediated invasion [20
]. Since EGFRvIII-expressing breast cancer cells universally had an up-regulation of CXCR4 expression and metastatic breast cancer cells that require CXCR4 for breast cancer metastasis often express high levels of ErbB-receptors such as EGFR and ErbB2, we were interested in understanding potential changes in the activity or expression of these ErbB-receptors upon suppression of CXCR4 expression.
Here we report that suppression of CXCR4 with shRNA significantly reduces not only the invasive potential of EGFRvIII-expressing breast cancer cells, it also reduces the proliferation of the cells. Cross-talk between CXCR4 and EGFRvIII is bidirectional. Suppression of CXCR4 expression reduces the protein levels of EGFRvIII through enhanced EGFRvIII protein degradation. Inhibition of the proteasome and protein trafficking revealed that both of these pathways are involved in the turnover of EGFRvIII in CXCR4-shRNA cells. Furthermore, inhibition of p38 MAPK, a key molecule which has been shown to be involved in EGFR internalization and down-regulation, reverses EGFRvIII protein suppression by CXCR4 knockdown. However, activation of p38 MAPK under hypoxic conditions increases EGFRvIII protein levels suggesting an essential role of p38 MAPK in EGFRvIII protein trafficking.