A galectin-3-dependent pathway upregulates interleukin-6 in the microenvironment of human neuroblastoma

Abstract

Interleukin-6 (IL-6) is a pleiotropic cytokine with a broad range of physiologic and pathologic functions. Because in cancer, IL-6 contributes to a microenvironment that promotes tumor cell survival, angiogenesis, and inflammation, understanding the mechanism responsible for its production is important. In neuroblastoma, the second most common solid tumor in children, IL-6 is produced not by tumor cells but by stromal cells such as monocytes and bone marrow mesenchymal stem cells (BMMSC). Here we show that the production of IL-6 in BMMSCs is in part stimulated by galectin-3 binding protein (Gal-3BP) secreted by neuroblastoma cells. We identified a distal region of the IL-6 promoter that contains 3 CCATT/enhancer binding protein (C/EBP) binding domains involved in the transcriptional upregulation of IL-6 by Gal-3BP. Gal-3BP interacted with Galectin-3 (Gal-3) present in BMMSCs, and a Gal-3BP/Gal-3/Ras/MEK/ERK signaling pathway was responsible for the transcriptional upregulation of IL-6 in BMMSCs in which Gal-3 has a necessary function. In support of the role of this pathway in human neuroblastoma tumors, Gal-3BP was found to be present in tumor cells and in the adjacent extracellular matrix of 96% of 78 primary neuroblastoma tumor samples examined by immunohistochemistry. Considering the protumorigenic function of IL-6 in cancer, this tumor cell-stromal cell interactive pathway could be a target for anticancer therapy.

Figure 1. Gal-3BP is secreted by several neuroblastoma cell lines that upregulate IL-6 in mesenchymal stem cells

A. immunofluorescence analysis of Gal-3BP in 9 neuroblastoma cell lines permeabilized with Triton-X100. Bar=50µm. B. The data represent the mean (±SD) amount of Gal-3BP produced by 10⁴ cells over 24 hours. Aliquots of the medium containing 10 µg of proteins were also examined for the presence of a 93 kDa Gal-3BP protein by Western blot (bottom of graph). C. Serum-free CM of the 9 neuroblastoma cell lines shown in panel A and B was incubated with BMMSC for 24 hours. The graph is a plot of the mean concentrations of Gal-3BP in the neuroblastoma serum-free CM (in ng/ml; x axis) and the mean concentration of IL-6 present after 24 hours of incubation with BMMSC (in pg/ml; y axis) from triplicate samples.

Figure 2. Gal-3BP is one regulator of IL-6 expression and increases its transcriptional regulation in BMMSC

A. Western blot analysis of 10× serum-free CM from CHLA-255 immunodepleted of Gal-3BP as described in Materials and Methods. B. Concentration of IL-6 in the medium of BMMSC exposed for 24 hours to the CM of CHLA-255 cells treated as described in A. The data represent the mean concentration (± SD) of IL-6 in triplicate samples and are representative of one among two independent experiments showing similar results. C. Increase in IL-6 mRNA expression in BMMSC 4 hours after treatment with CM or rGal-3BP (2.5 µg/ml) determined by qRT-PCR. The data represent the mean fold increase in the ratio IL-6: GAPDH (± SD) of duplicate samples from 2 independent experiments. D. IL-6 promoter Luciferase activity in BMMSC and PC3 cells transfected with pGL2 or pGL2-IL-6-Luc and pRV and incubated in the absence or presence of 5× serum-free CM from CHLA-255 cells or rGal-3BP (2.5 µg/ml) for 24 hours. The data represent the mean fold increase in the FLuc/RLuc ratio (± SD) over control (no treatment) from 5 separate experiments each performed in triplicate (*p<0.002, **p<0.03, ***p<0.004 and ****p<0.025).

Figure 3. The distal region of the IL-6 promoter contains functional C/EBP motifs

A. IL-6 promoter Luciferase activity in PC3 cells transfected with the pGL2-IL-6 deletion mutants shown on the left and pRV with and without treatment with rGal-3BP (2.5 µg/ml) for 24 hours. The data represent the mean (±SD) ratio Firefly Luciferase/Renilla Luciferase and are representative of one among three separate experiments each performed in triplicate samples. B. Sequencing analysis of the region of the IL-6 promoter extending from position −1586 to −1255 showing the presence of 3 C/EBP domains. C. EMSA of nuclear extracts from PC3 cells treated with rGAL-3BP (5 µg/ml) using biotinylated oligonucleotides in the presence of 10× fold excess of non-biotinylated oligonucleotides when indicated.

Figure 4. Gal-3 is necessary for Gal-3BP mediated IL-6 expression

A. Immunocytofluorescence analysis of Gal-3 (red) and Gal-3 BP (green) in BMMSC treated with rGal-3BP (5 µg/ml), fixed without permeabilization as described in Materials and Methods. a to c. Untreated cells. d to f. Cells treated with rGal-3BP. B. Western blot analysis of cell – associated Gal-3 in BMMSC examined 72 hours after transfection with indicated siRNA as described in Materials and Methods. C. Amount of IL-6 produced over 24 hours by BMMSC Parallel transfected with siRNA as shown in B and incubated in the absence or presence of 5× CM from CHLA-255 cells or rGal-3BP (2.5 µg/ml). The data represent the mean IL-6 concentration (±SD) in the supernatant of triplicate samples and are representative of three independent experiments showing similar results. D. IL-6 Luciferase promoter activity in PC3 cells transfected with a Gal-3 siRNA and treated with rGal-3BP (5 µg/ml). The data represent the mean fold increase in the FLuc/RLuc from triplicate samples from 3 separate experiments.

Figure 5. Gal-3BP signals through the Ras/MEK/ERK pathway

A. Western blot analysis of lysates of BMMSC treated with rGal-3BP (10 to 25 µg/ml) subjected to pull down assay and examined for Ras-GTP, N, K and H-Ras as described in Materials and Methods. The data are representative of 2 experiments showing similar results (left panel). The bar diagram on the right represents the fold increase in Ras-GTP/total Ras and N-Ras/total Ras from analysis of data by scanning densitometry. B. Western blot analysis of BMMSC treated as in A and examined for pMEK1/2, MEK1/2, pERK1/2 and ERK1/2 expression (left panel). The bar diagrams on the right represents the mean fold increase (±SD) in the ratio phosphorylated protein : total protein over time for pMEK1/2 (10 µg/ml, n=6; 25 µg/ml, n=3) and pERK1/2 (10 µg/ml, n=7; 25 µg/ml, n=5). The bar diagram on the right lower panel represents the ratio pERK1/ERK1 and pERK2/ERK2 obtained by scanning densitometry of the data shown on the Western blot.

Figure 6. Inhibition of Gal-3BP-mediated IL-6 stimulation by Ras and MEK inhibition

A. Amount of IL-6 produced by BMMSC cultured with 12.5 µM FTS or DMSO for 12 hours before being exposed to 5× CM from CHLA-255 cells or rGal-3BP (5 µg/ml) for 24 hours. The data represent the mean (±SD) concentration from triplicate samples and are representative of one from three independent experiments showing similar results. B. IL-6 Luciferase promoter activity in PC3 cells treated with FTS for 4 hours before exposure to CHLA-255 CM or rGal-3BP. The data represent the mean (±SD) increase from control (no treatment) in the FLuc/RLuc ratio of triplicate samples and is representative of one among 3 independent experiments showing similar results. C. Same as B except that PC3 cells were treated with PD98058 (50 µM). The data represent the mean (±SD) increase from control in triplicate samples and is representative of 2 independent experiments showing similar results.

Figure 7. Gal-3BP is present in neuroblastoma tumors

A. Digital photomicrographs of representative neuroblastoma tumor sections stained for Gal-3BP. Panel a: absence of Gal-3BP; panel b: score 1; panel c: score 2; panel d: score 3. Bar=50 µm. B. Sections were stained for Gal-3BP (red) and IL-6 (green). Bar=10 µm, inset: 30 µm. C. Bar diagram showing the mean score (±SD) in 78 neuroblastoma samples according to categories: age at diagnosis (<18 months vs. ≥18 months); DNA index (1 or >1); Diff = grade of neuroblastic differentiation: D (differentiated), P (poorly differentiated), and U (undifferentiated); Histo: F (Favorable Histology) and U (Unfavorable Histology); MKI = mitosis-karyorrhexis index; H (high, ≥200/5,000 cells), I (intermediate, 100–200/5,000 cells), and L (low, < 100/5,000 cells); MYCN: A (amplified) and NA (non-amplified); and Stage: 1 to 3: non-metastatic; 4: metastatic; 4S: metastatic with good prognosis. D. Kaplan-Meier graph of survival probability by time in years from diagnosis within each of the three Gal-3BP expression groups.