Critical role of STAT3 in IL-6-mediated drug resistance in human neuroblastoma

Abstract

Drug resistance is a major cause of treatment failure in cancer. Here, we have evaluated the role of STAT3 in environment-mediated drug resistance (EMDR) in human neuroblastoma. We determined that STAT3 was not constitutively active in most neuroblastoma cell lines but was rapidly activated upon treatment with interleukin (IL)-6 alone and in combination with the soluble IL-6 receptor (sIL-6R). Treatment of neuroblastoma cells with IL-6 protected them from drug-induced apoptosis in a STAT3-dependent manner because the protective effect of IL-6 was abrogated in the presence of a STAT3 inhibitor and upon STAT3 knockdown. STAT3 was necessary for the upregulation of several survival factors such as survivin (BIRC5) and Bcl-xL (BCL2L1) when cells were exposed to IL-6. Importantly, IL-6-mediated STAT3 activation was enhanced by sIL-6R produced by human monocytes, pointing to an important function of monocytes in promoting IL-6-mediated EMDR. Our data also point to the presence of reciprocal activation of STAT3 between tumor cells and bone marrow stromal cells including not only monocytes but also regulatory T cells (Treg) and nonmyeloid stromal cells. Thus, the data identify an IL-6/sIL-6R/STAT3 interactive pathway between neuroblastoma cells and their microenvironment that contributes to drug resistance.

Figure 1

IL-6 and sIL-6R activate STAT3 in human neuroblastoma cells. A., The presence of phosphorylated pY⁷⁰⁵ STAT3 and STAT3 was detected by Western blot analysis in total cell lysates from 8 neuroblastoma cell lines. When indicated, cells were treated with IL-6 (10 ng/ml), sIL-6R (25 ng/ml). Lysates were obtained 30 minutes after treatment. The data are representative of 2 to 3 separate experiments showing similar results. B, The presence of STAT3 and pY⁷⁰⁵ STAT3 in cultured CHLA-255 (left panel) and CHLA-90 (right panel) cells untreated or treated for 30 minutes with IL-6 alone or with sIL-6R as described above was examined by immunocytofluorescence. Arrow: cytoplasmic STAT3. Arrowhead: nuclear pSTAT3. (Scale bar = 20 µm).

Figure 2

IL-6 and sIL-6R protect neuroblastoma cells from drug toxicity. CHLA-255 (A and B) and SK-N-SH cells (C and D) were treated with IL-6 (10 ng/mL) alone or with sIL-6R (25 ng/mL) for 24 hours before being exposed to etoposide (A and C) or melphalan (B and D) at indicated concentrations. Cell viability was determined after 24 hours of drug exposure by DIMSCAN analysis. The data represent the mean (±SD) survival fraction with a minimum of 10 replicates for each experimental condition.

Figure 3

IL-6 and sIL-6R protect neuroblastoma cells from drug-induced apoptosis. A and B, CHLA-255 cells were treated as in Figure 2. After 24 hours mitochondrial membrane depolarization (MΨP) was examined by JC-1 staining by flow cytometry. The data represent the mean (±SE) percent change in depolarization from control cells (untreated with cytotoxic drugs). C, CHLA-255 cells were treated as above indicated. After 24 hours cytosolic and mitochondrial extracts were examined for the presence of cytochrome C by Western blot. The data are representative of two separate experiments showing similar results. Cox-IV and β-actin were used as loading control for mitochondrial and cytosolic fractions respectively. D and E, CHLA-255 cells were treated with IL-6 and etoposide or melphalan. After 24 hours of drug exposure, cell lysates were examined for the expression of full length and cleaved caspases 3 and 9. The data are representative of two separate experiments showing similar results. In all gels (C to E), separation lines indicate different gels loaded with the same amount of cell lysate.

Figure 4

STAT3 is necessary for IL-6-mediated drug resistance. A, CHLA-255 cells were treated with IL-6 (10 ng/mL) and sIL-6R (25 ng/mL) for 30 minutes in the presence of stattic at indicated concentrations. Cell lysates were examined for pSTAT3 and STAT3 expression by Western blot. B, CHLA-255 cells were treated with stattic (2.5 µM) and IL-6 and sIL-6R for 24 hours before being exposed to etoposide (0.25 µg/mL). After 24 hours cells were examined for Annexin V expression by flow cytometry. The data represent the mean percentage (±SD) of Annexin V positive cells from 4 samples obtained in 3 independent experiments. C, CHLA-255 cells were transfected with STAT3 siRNA or with a scramble sequence. After 72 hours, cells were treated with IL-6 and sIL-6R for 30 minutes and cell lysates were examined for pSTAT3 and STAT3 expression by Western blot. The data are representative of three separate experiments showing similar results. D, siRNA transfected CHLA-255 cells were treated with IL-6 and sIL-6R for 24 hours before being exposed to etoposide (0.25 µg/mL) and examined for the presence of Annexin V at the cell surface. The data represent the mean percentage (±SD) of Annexin V positive cells compared with control (cells treated with etoposide alone) from 4 samples obtained from 3 independent experiments.

Figure 5

IL-6 upregulates the expression of anti-apoptotic proteins. A, CHLA-255 cells were treated with IL-6 (10ng/mL) and cells were lysed at indicated time points (hours). Total cell lysates (20µg/lane) were then examined by Western blot for the expression of indicated anti-apoptotic proteins. Actin was used as loading control. The data are representative of 3 separate experiments showing similar results. The bar graph represents the fold increase compared to control after the ratios of anti-apoptotic protein:actin were normalized for the ratio at 0 hour. B, CHLA-255 cells were treated with IL-6 and slL-6R and examined for survivin expression by immunofluorescence. Stattic (2.5 mM) was added 4 hours before IL-6 and sIL-6R when indicated (scale bar = 20µm). C, siRNA transfected CHLA-255 cells treated as above described were examined for pSTAT3, survivin and Bcl-xL expression by Western blot after 24 hours of stimulation with IL-6 and sIL-6R. The data are representative of 3 experiments showing similar results.

Figure 6

sIL-6R produced by monocytes sensitizes neuroblastoma cells to IL-6-mediated STAT3 activation. A, CHLA-255 cells were treated with IL-6 at indicated concentrations in the absence (top panel) or presence (lower panel) of sIL-6R (25ng/mL) for 30 minutes. The cell lysates were examined for expression of pSTAT3 and STAT3 by Western blot. B, CHLA-255 cells were treated with IL-6 alone (10 ng/ml) and in the presence of increased concentrations of sIL-6R (0.1 to 25 ng/mL) for 30 minutes. The cell lysates were then examined for expression of pSTAT3 and STAT3 by Western blot. The bar diagram represents the ratio pSTAT3/STAT3 obtained by scanning of the blot. C, CHLA-255 (4.5×10⁵) and human BMMSC or monocytes (4.5×10⁵) were cultured either alone or together in Transwell plates for 48 hours. The culture medium was collected and the concentrations of IL-6 and sIL-6R were determined by ELISA. The data represent the mean concentration (±SD) of duplicate (upper) and triplicate (lower) samples. D, CHLA-255 cells were co-cultured with monocytes in Transwell plates in the absence or presence of an anti-IL-6R antibody (tocilizumab). After 48 hours cell lysates were examined for pSTAT3 and STAT3 by Western blot. E. Monocytes isolated from the peripheral blood of patients with neuroblastoma and CHLA-255 cells were cultured alone or in contact (ratio tumor cells:monocytes 4:1) for 24 hours. Cells were then harvested and examined by flow cytometry for the presence of nuclear pSTAT3 and expression of cell surface CD56 and CD14 to separate tumor cells from monocytes. The data represent the average percent of pSTAT3 positive cells (±SD) from 3 separate samples.

Figure 7

STAT3 is activated in the bone marrow microenvironment in patients with metastatic neuroblastoma. A, Left panel: Paraffin-embedded sections of bone marrow biopsies from patients (n=10) with neuroblastoma were examined by immunofluorescence for the presence of nuclear pSTAT3 (white arrow) and by immunohistochemistry for the presence of TH positive tumor cells (black arrow) (bar = 50 µm). Right panel: The data represent the mean percentage (±SD) of positive nuclei determined in 5 high magnification (40×) fields per sample (bar = 20µm). B, Bone marrow biopsy infiltrated with > 90% of neuroblastoma cells was examined by dual immunohistochemistry as described in material and methods for the presence of pSTAT3 in PGP9.5⁺ tumor cells (Top) or CD45⁺ myeloid cells (bottom). The histogram on the right side represents the mean (±SD) percentage of PGP9.5 and CD45 positive cells that were also positive for pSTAT3 from 5 high power field areas (bar = 50 µm). C. The presence of pSTAT3 and CD68 positive cells in bone marrow samples was examined by dual immunofluorescence (white arrows indicate nuclear localization of pSTAT3 in tumor cells and yellow arrows in CD68 positive cells. Bar = 50 µm at 20× and 20 µm at 40×).