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Cell-line-specific stimulation of tumor cell aggressiveness by wound healing factors - a central role for STAT3.

Ekblad L, Lindgren G, Persson E, Kjellén E, Wennerberg J - BMC Cancer (2013)

Bottom Line: These effects were accompanied by a sharp increase in activation of signal transducer and activator of transcription 3 (STAT3).The involvement of c-Met or epidermal growth factor receptor family members was excluded, while the interleukin-6 receptor was found to be partly responsible for the activation of STAT3.In conclusion, we found cell-line-specific effects of wound healing factors on HNSCC, setting the stage for therapy development and predictive opportunities.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Lund University, Lund, Sweden. Lars.Ekblad@med.lu.se

ABSTRACT

Background: Local recurrence is a major factor affecting survival after treatment for head and neck squamous cell carcinoma (HNSCC). It is possible that the normal processes involved in wound healing after surgical removal of a primary tumor can boost the regrowth of residual cancer cells, thereby contributing to the recurrent growth. In this work, we collected human wound fluids and used them to investigate the effect of wound healing factors on HNSCC cell lines in vitro.

Methods: Wound fluids were collected from thyroidectomized patients diagnosed with benign disease and were included in assays of cell proliferation, migration, cell scattering, and invasion. The involvement of intracellular signaling pathways and membrane receptors were investigated by western blotting and the inclusion of specific inhibitors.

Results: One out of four cell lines was greatly stimulated in proliferation, migration, cell scattering, and invasion by the addition of wound fluid as compared with addition of fetal bovine or human serum. These effects were accompanied by a sharp increase in activation of signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 activation abolished the wound fluid response, showing that STAT3 plays an important role in the wound healing response. Several of the observed phenotypic changes were epithelial-to-mesenchymal transition (EMT)-like, but the appropriate changes were not seen in any of the EMT markers investigated. The involvement of c-Met or epidermal growth factor receptor family members was excluded, while the interleukin-6 receptor was found to be partly responsible for the activation of STAT3.

Conclusions: In conclusion, we found cell-line-specific effects of wound healing factors on HNSCC, setting the stage for therapy development and predictive opportunities.

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The role of IL-6 as an initiator of the HWF effect on HN-7 cells. (a) Inhibition of STAT3 phosphorylation by tocilizumab. HN-7 cells were grown with 10% HWF, HS, or FBS and the indicated concentrations of tocilizumab. Phospho-STAT3 was analyzed by western blotting (upper panel) and quantified in relation to the loading control (lower panel). (b) The effect of tocilizumab on migration analyzed by the scratch assay (N=16). (c) Stimulation of migration by IL-6. HN-7 cells were analyzed in the presence of 10% HS or FBS and the indicated concentrations of IL-6 and tocilizumab (N=16). (d) The effect of tocilizumab on HWF-supported cell proliferation analyzed by SRB (N=10). Error bars represent SEM. (e) The expression of IL6Rα in HNSCC cell lines. Cell lysates (10 μg protein per lane) from cells grown with 10% FBS were analyzed by western blotting with an anti-IL6Rα antibody. Specific binding was determined using a 100-fold molar excess of an antibody blocking peptide. Relative expression of IL6Rα normalized to protein loading control is shown in the lower panel.
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Figure 6: The role of IL-6 as an initiator of the HWF effect on HN-7 cells. (a) Inhibition of STAT3 phosphorylation by tocilizumab. HN-7 cells were grown with 10% HWF, HS, or FBS and the indicated concentrations of tocilizumab. Phospho-STAT3 was analyzed by western blotting (upper panel) and quantified in relation to the loading control (lower panel). (b) The effect of tocilizumab on migration analyzed by the scratch assay (N=16). (c) Stimulation of migration by IL-6. HN-7 cells were analyzed in the presence of 10% HS or FBS and the indicated concentrations of IL-6 and tocilizumab (N=16). (d) The effect of tocilizumab on HWF-supported cell proliferation analyzed by SRB (N=10). Error bars represent SEM. (e) The expression of IL6Rα in HNSCC cell lines. Cell lysates (10 μg protein per lane) from cells grown with 10% FBS were analyzed by western blotting with an anti-IL6Rα antibody. Specific binding was determined using a 100-fold molar excess of an antibody blocking peptide. Relative expression of IL6Rα normalized to protein loading control is shown in the lower panel.

Mentions: As a third alternative inducer of STAT3 activation, we investigated the role of IL-6 using the IL-6 receptor antagonist tocilizumab. Incubation of HN-7 cells with tocilizumab decreased the HWF-stimulated STAT3 phosphorylation by approximately 70% (Figure 6a). In line with this, HWF-stimulated migration was also reduced by the addition of tocilizumab (Figure 6b). IL-6 measurement showed a concentration near the detection limit in HS (approx. 8 pg/mL), while the early wound fluids used in the study contained an average of 73 ng/mL. Addition of recombinant IL-6 increased the HS-driven migration by 26% (p<0.0001), and this effect was completely blocked by 1 μmol/L tocilizumab (Figure 6c). Interestingly, IL-6 did not affect migration in combination with FBS, indicating that other factors present in HS and HWF are necessary to facilitate the IL-6 effect.


Cell-line-specific stimulation of tumor cell aggressiveness by wound healing factors - a central role for STAT3.

Ekblad L, Lindgren G, Persson E, Kjellén E, Wennerberg J - BMC Cancer (2013)

The role of IL-6 as an initiator of the HWF effect on HN-7 cells. (a) Inhibition of STAT3 phosphorylation by tocilizumab. HN-7 cells were grown with 10% HWF, HS, or FBS and the indicated concentrations of tocilizumab. Phospho-STAT3 was analyzed by western blotting (upper panel) and quantified in relation to the loading control (lower panel). (b) The effect of tocilizumab on migration analyzed by the scratch assay (N=16). (c) Stimulation of migration by IL-6. HN-7 cells were analyzed in the presence of 10% HS or FBS and the indicated concentrations of IL-6 and tocilizumab (N=16). (d) The effect of tocilizumab on HWF-supported cell proliferation analyzed by SRB (N=10). Error bars represent SEM. (e) The expression of IL6Rα in HNSCC cell lines. Cell lysates (10 μg protein per lane) from cells grown with 10% FBS were analyzed by western blotting with an anti-IL6Rα antibody. Specific binding was determined using a 100-fold molar excess of an antibody blocking peptide. Relative expression of IL6Rα normalized to protein loading control is shown in the lower panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585883&req=5

Figure 6: The role of IL-6 as an initiator of the HWF effect on HN-7 cells. (a) Inhibition of STAT3 phosphorylation by tocilizumab. HN-7 cells were grown with 10% HWF, HS, or FBS and the indicated concentrations of tocilizumab. Phospho-STAT3 was analyzed by western blotting (upper panel) and quantified in relation to the loading control (lower panel). (b) The effect of tocilizumab on migration analyzed by the scratch assay (N=16). (c) Stimulation of migration by IL-6. HN-7 cells were analyzed in the presence of 10% HS or FBS and the indicated concentrations of IL-6 and tocilizumab (N=16). (d) The effect of tocilizumab on HWF-supported cell proliferation analyzed by SRB (N=10). Error bars represent SEM. (e) The expression of IL6Rα in HNSCC cell lines. Cell lysates (10 μg protein per lane) from cells grown with 10% FBS were analyzed by western blotting with an anti-IL6Rα antibody. Specific binding was determined using a 100-fold molar excess of an antibody blocking peptide. Relative expression of IL6Rα normalized to protein loading control is shown in the lower panel.
Mentions: As a third alternative inducer of STAT3 activation, we investigated the role of IL-6 using the IL-6 receptor antagonist tocilizumab. Incubation of HN-7 cells with tocilizumab decreased the HWF-stimulated STAT3 phosphorylation by approximately 70% (Figure 6a). In line with this, HWF-stimulated migration was also reduced by the addition of tocilizumab (Figure 6b). IL-6 measurement showed a concentration near the detection limit in HS (approx. 8 pg/mL), while the early wound fluids used in the study contained an average of 73 ng/mL. Addition of recombinant IL-6 increased the HS-driven migration by 26% (p<0.0001), and this effect was completely blocked by 1 μmol/L tocilizumab (Figure 6c). Interestingly, IL-6 did not affect migration in combination with FBS, indicating that other factors present in HS and HWF are necessary to facilitate the IL-6 effect.

Bottom Line: These effects were accompanied by a sharp increase in activation of signal transducer and activator of transcription 3 (STAT3).The involvement of c-Met or epidermal growth factor receptor family members was excluded, while the interleukin-6 receptor was found to be partly responsible for the activation of STAT3.In conclusion, we found cell-line-specific effects of wound healing factors on HNSCC, setting the stage for therapy development and predictive opportunities.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Lund University, Lund, Sweden. Lars.Ekblad@med.lu.se

ABSTRACT

Background: Local recurrence is a major factor affecting survival after treatment for head and neck squamous cell carcinoma (HNSCC). It is possible that the normal processes involved in wound healing after surgical removal of a primary tumor can boost the regrowth of residual cancer cells, thereby contributing to the recurrent growth. In this work, we collected human wound fluids and used them to investigate the effect of wound healing factors on HNSCC cell lines in vitro.

Methods: Wound fluids were collected from thyroidectomized patients diagnosed with benign disease and were included in assays of cell proliferation, migration, cell scattering, and invasion. The involvement of intracellular signaling pathways and membrane receptors were investigated by western blotting and the inclusion of specific inhibitors.

Results: One out of four cell lines was greatly stimulated in proliferation, migration, cell scattering, and invasion by the addition of wound fluid as compared with addition of fetal bovine or human serum. These effects were accompanied by a sharp increase in activation of signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 activation abolished the wound fluid response, showing that STAT3 plays an important role in the wound healing response. Several of the observed phenotypic changes were epithelial-to-mesenchymal transition (EMT)-like, but the appropriate changes were not seen in any of the EMT markers investigated. The involvement of c-Met or epidermal growth factor receptor family members was excluded, while the interleukin-6 receptor was found to be partly responsible for the activation of STAT3.

Conclusions: In conclusion, we found cell-line-specific effects of wound healing factors on HNSCC, setting the stage for therapy development and predictive opportunities.

Show MeSH
Related in: MedlinePlus