Panoply™ Human CXCR4 Knockdown Stable Cell Line

Chemokine signaling regulates cell migration and tumor metastasis. The chemokine family member CXCL12 and its receptor CXCR4, a G protein-coupled receptor (GPCR), are key mediators of prostate cancer (PC) bone metastasis. To identify novel lipid raft-associated CXCR4 regulators that support invasion/metastasis, researchers performed SILAC-based quantitative proteomic analysis of lipid rafts derived from stable CXCR4 overexpressing or knockdown PC3 cell lines. This analysis identified the evolutionarily conserved phosphatidylinositol 4-kinase IIIα (PI4KIIIα) and the SAC1 phosphatase, which dephosphorylates phosphatidylinositol-4-phosphate, as potential CXCR4 regulators. CXCR4 interacts with the PI4KIIIα membrane targeting machinery, recruiting it to the plasma membrane for PI4P production. Consistent with this interaction, PI4KIIIα was found to be critically involved in CXCR4-induced PC cell invasion. Consequently, depletion of PI4KIIIα in CXCR4-expressing PC3 cells reduced the invasive response to multiple chemokines. Immunofluorescence microscopy in CXCR4-expressing cells revealed localized production of PI4P at invasive protrusions. Studies in human tumors have shown that PI4KIIIα expression is higher in metastatic tumors than in primary tumors, further supporting a role for PI4KIIIα in tumor metastasis.

To identify proteins interacting with CXCR4 in lipid rafts, researchers generated CXCR4-overexpressing (CXCR4) and CXCR4-knockdown (shCXCR4) PC3 stable cell lines. FACS analysis of the CXCR4 and shCXCR4 cell lines revealed a significant positive shift in the median fluorescence intensity (MFI) of CXCR4-1 and CXCR4-2, indicating a high level of CXCR4 overexpression. In contrast, the shCXCR4 cell line exhibited a significant negative shift in the MFI, consistent with CXCR4 knockdown (Figure 1a, b). Quantitative RT-PCR (Figure 1c) and Western blot analysis confirmed CXCR4 overexpression and knockdown in these cell lines. SILAC-based proteomic analysis of lipid raft microdomains purified from the CXCR4-2 and shCXCR4-1 cell lines was performed using sucrose gradient buoyant density ultracentrifugation. Of the 277 proteins identified, 126 showed changes greater than 1.5-fold, with 79 proteins showing changes greater than 1.5-fold and 47 proteins showing changes less than 1.5-fold, respectively, with increased or decreased expression in CXCR4-overexpressing and shCXCR4-knockdown cells. Unexpectedly, SILAC analysis revealed that the phosphatidylinositol 4-kinase type IIIα (PI4KA) isoform was overexpressed in CXCR4 cells (Figure 1d). Its counterpart, Sac1 phosphatase, was 11.0% more abundant in CXCR4 cells. Similarly, Western blot analysis of lipid rafts revealed elevated expression of PI4KIIIα and Sac1 in CXCR4-overexpressing cells (Figure 1e). Proteomic data from CXCR4 cells suggest a potential functional link between CXCR4 and PI4KIIIα/Sac1 in prostate cancer cells.

Figure 1. Characterization of PC3 low and high CXCR4 cells and proteomics analysis. (Sbrissa D, et al., 2019)