IL-24 inhibits lung cancer cell migration and invasion by disrupting the SDF-1/CXCR4 signaling axis

Abstract

Background: The stromal cell derived factor (SDF)-1/chemokine receptor (CXCR)-4 signaling pathway plays a key role in lung cancer metastasis and is molecular target for therapy. In the present study we investigated whether interleukin (IL)-24 can inhibit the SDF-1/CXCR4 axis and suppress lung cancer cell migration and invasion in vitro. Further, the efficacy of IL-24 in combination with CXCR4 antagonists was investigated.

Methods: Human H1299, A549, H460 and HCC827 lung cancer cell lines were used in the present study. The H1299 lung cancer cell line was stably transfected with doxycycline-inducible plasmid expression vector carrying the human IL-24 cDNA and used in the present study to determine the inhibitory effects of IL-24 on SDF-1/CXCR4 axis. H1299 and A549 cell lines were used in transient transfection studies. The inhibitory effects of IL-24 on SDF1/CXCR4 and its downstream targets were analyzed by quantitative RT-PCR, western blot, luciferase reporter assay, flow cytometry and immunocytochemistry. Functional studies included cell migration and invasion assays.

Principal findings: Endogenous CXCR4 protein expression levels varied among the four human lung cancer cell lines. Doxycycline-induced IL-24 expression in the H1299-IL24 cell line resulted in reduced CXCR4 mRNA and protein expression. IL-24 post-transcriptionally regulated CXCR4 mRNA expression by decreasing the half-life of CXCR4 mRNA (>40%). Functional studies showed IL-24 inhibited tumor cell migration and invasion concomitant with reduction in CXCR4 and its downstream targets (pAKTS473, pmTORS2448, pPRAS40T246 and HIF-1α). Additionally, IL-24 inhibited tumor cell migration both in the presence and absence of the CXCR4 agonist, SDF-1. Finally, IL-24 when combined with CXCR4 inhibitors (AMD3100, SJA5) or with CXCR4 siRNA demonstrated enhanced inhibitory activity on tumor cell migration.

Conclusions: IL-24 disrupts the SDF-1/CXCR4 signaling pathway and inhibits lung tumor cell migration and invasion. Additionally, IL-24, when combined with CXCR4 inhibitors exhibited enhanced anti-metastatic activity and is an attractive therapeutic strategy for lung metastasis.

Fig 1. CXCR4 expression in human lung cancer cells and its inhibition by IL-24.

A, Endogenous CXCR4 and AKT protein expression in human lung cancer cell lines. B, IL-24 reduced CXCR4 expression at 24 h and 48 h in doxycycline-treated H1299-IL24 cells but not in doxycycline untreated control cells. C, IL-24 reduced GRK6, phosphorylated (p) CXCR4 and total CXCR4 expression in doxycycline-treated H1299-IL24 cells compared to expression of these proteins in doxycycline untreated H1299-IL24 cells. D, Immunocytochemistry showing doxycycline-induced IL-24 expression in H1299-IL24 cell line reduced CXCR4 expression. Cells that were not treated with doxycycline served as control. Magnification IL-24- X 30; CXCR4- X 40. E, Time-course study showed CXCR4 expression was reduced as early as 4 h after IL-24 expression and the inhibitory activity was sustained up to 24 h in doxycycline-treated H1299-IL24 cells. Beta actin was used as protein loading control in Western blotting assays.

Fig 2. IL-24 suppresses lung cancer cell migration and invasion.

H1299-IL24 cells were either not treated or treated with doxycycline and observed for cell migration and invasion. IL-24 inhibited tumor cell migration (A) and invasion (B) starting from 6h and sustained its inhibitory activity till 48 h at which time point the experiment was terminated (P<0.05). Bars denote standard deviation (SD).

Fig 3. IL-24 inhibited the signaling proteins downstream of CXCR4.

Western blotting showed induction of IL-24 protein expression in H1299-IL24 cells resulted in marked reduction in the expression of phosphorylated (p) AKT^S473, pmTOR^S2448 and pPRAS40^T246 and HIF-1α at 24 h and 48 h after doxycycline treatment. Beta actin was used as protein loading control. Differences in the expression of the proteins was determined by semi-quantitative analysis and represented in graphical format (P<0.05). Bars denote standard deviation (SD).

Fig 4. IL-24 regulated CXCR4 at post-transcriptional level.

A, RT-PCR analysis showed IL-24 reduced CXCR4 mRNA levels at 6 h and 24 h (P<0.05). B, CXCR4 promoter activity was determined using a luciferase reporter vector. Induction of IL-24 showed no significant reduction in luciferase activity indicating IL-24 did not affect the CXCR4 promoter. C, mRNA stability studies showed IL-24 reduced the half-life of CXCR4 mRNA at approximately 4 h. Bars denote standard deviation (SD).

Fig 5. SDF-1 mediated CXCR4 activation in H1299 cells.

A, RT-PCR studies and B, Flow cytometry analysis showed SDF-1 activated CXCR4 mRNA and cell surface expression in H1299-IL24 cell line indicating CXCR4 is functionally active and intact (P<0.05). Bars denote standard deviation (SD).

Fig 6. IL-24 suppresses SDF-1/CXCR4 signaling and tumor cell migration.

A, IL-24 significantly inhibited tumor cell migration in the presence and absence of SDF-1. The inhibitory activity in the presence of SDF-1 however was less than that observed in the absence of SDF-1 (P<0.05). Error bars denote standard deviation. B, Induction of IL-24 protein expression in H1299-IL24 cells resulted in marked reduction in pAKT^S473 and pPRAS40^T246 protein expression at 24 h after doxycycline treatment. Beta actin was used as protein loading control. Differences in the expression of the proteins was determined by semi-quantitative analysis and represented in graphical format (P<0.05). Bars denote standard deviation (SD).

Fig 7. IL-24 combined with AMD3100 exhibited greater inhibitory activity on SDF-1 induced cell migration and SDF-1/CXCR4 signaling.

A, Combination of IL-24 and AMD3100 significantly suppressed SDF-1 induced cell migration compared to number of cells that migrated in the control group (P<0.05). Inhibitory effects by IL-24 treatment alone and AMD3100 treatment alone were also significant when compared to the control group. B, CXCR4 expression was markedly reduced in IL-24 expressing cells, cells treated with AMD3100, and in combination treatment of IL-24 and AMD3100 when compared to control cells. Greater reduction in the expression of pAKT^S473 and pPRAS40^T246 proteins were observed in combination treatment groups when compared to all other groups. Beta actin was used as protein loading control. Differences in the expression of the proteins was determined by semi-quantitative analysis and represented in graphical format (P< 0.05). Bars denote standard deviation (SD).

Fig 8. siRNA mediated CXCR4 inhibition in combination with IL-24 produced greater inhibitory activity on tumor cell migration.

A, siRNA-mediated CXCR4 knockdown when combined with IL-24 resulted in a significant suppression of SDF-1 induced tumor cell migration compared to cell migration in the control group (P<0.05). Inhibitory effects on tumor cell migration mediated by IL-24 treatment alone and CXCR4 siRNA treatment alone were also significant when compared to control group. B, Western blotting showed combination of CXCR4 siRNA and IL-24 produced greater reduction in the expression of CXCR4 and pAKT^S473 proteins when compared to all other groups. pPRAS40^T246 protein expression was reduced in all treatment groups when compared to control. However, its expression was slightly higher in the combination treatment group when compared to individual treatments. Beta actin was used as protein loading control. Differences in the expression of the proteins was determined by semi-quantitative analysis and represented in graphical format (P<0.05). Bars denote standard deviation (SD).