Panoply™ Human CXCR4 Over-expressing Stable Cell Line

Overexpression of the chemokine receptor CXCR4 in solid tumors is closely associated with poor prognosis and clinical outcomes. Here, researchers found that the presence of intracellular CXCR4 protein, but not CXCR4-CXCL12 signaling, enhances paclitaxel resistance and pro-tumorigenic function. In exploring the pro-apoptotic mechanism of CXCR4-mediated drug resistance, researchers identified DR5 as a novel, selective target of CXCR4 in breast and colon cancers. Furthermore, during transcriptional repression, CXCR4 directs the differential recruitment of the transcription factors p53 and YY1 to the DR5 promoter. Notably, inhibition of CXCR4 ligand-mediated signaling did not completely abrogate these phenotypes. Overexpression of different signaling-deficient mutants of CXCR4 also resulted in significant downregulation of DR5 expression in colon cancer, confirming the inverse relationship between DR5 and intracellular CXCR4 protein expression. Using stable gain- and loss-of-function approaches, researchers observed that intracellular CXCR4 selectively resistant and enhanced sensitivity to paclitaxel treatment in colon cancer cells in vitro and in vivo, regardless of CXCR4 surface expression. Together, these data suggest that targeting the intracellular CXCR4 protein may be crucial for inhibiting the pro-tumorigenic functions of CXCR4.

Here, the researchers selected MCF-7 cells for overexpression studies because they express very low levels of CXCR4 and CXCR7 on their cell surfaces. FACS analysis and Western blotting (Figure 1A) confirmed that overexpression of CXCR4 and CXCR7 in MCF-7 cells was stable compared to a vector control. Subsequently, these cells were treated with FDA-approved anticancer drugs, and their cytotoxic effects were assessed. As shown in Figure 1B, CXCR4-overexpressing MCF-7 cells were significantly resistant to paclitaxel-induced cell death compared to control cells. Furthermore, CXCR4-overexpressing cells exhibited a degree of resistance to doxorubicin and 5-fluorouracil, whereas CXCR7-overexpressing cells showed no similar responses. Next, to verify the impact of overexpression of CXCR4 or CXCR7 on drug resistance in cancer cells, control MCF7 cells were stained with DiO (green dye) and CXCR4- or CXCR7-overexpressing cells were stained with DiL (red dye), respectively. Equal numbers of DiO-stained control cells were mixed and plated with DiL-stained CXCR4- or CXCR7-overexpressing cells. Cells were treated with vehicle control (DMSO) or paclitaxel (10 nmol) for 3 or 5 days and then monitored by fluorescence microscopy (Figure 1C) or flow cytometry (Figure 1D), respectively. Fewer CXCR4-overexpressing cells died compared to other cells, indicating a significant contribution of CXCR4 to paclitaxel resistance (Figure 1C). Quantitative flow cytometric analysis revealed a higher number of CXCR4-overexpressing MCF7 cells than control cells, while the number of CXCR7-overexpressing cells remained unchanged compared to control cells (Figure 1D). Enhanced CXCR4 function significantly induced resistance to cell death at all three paclitaxel doses tested (Figure 1E). These experiments suggest that selective CXCR4 enhancement, rather than CXCR7 overexpression, contributes to chemoresistance, particularly paclitaxel resistance, in breast cancer.

Figure 1. CXCR4 regulates paclitaxel resistance in cancer. (Nengroo M A, et al., 2021)