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IL-8 confers resistance to EGFR inhibitors by inducing stem cell properties in lung cancer.

Liu YN, Chang TH, Tsai MF, Wu SG, Tsai TH, Chen HY, Yu SL, Yang JC, Shih JY - Oncotarget (2015)

Bottom Line: The effects of IL-8 on gefitinib-induced apoptosis, stemness, and in vivo tumorigenicity were investigated using established cell lines.IL-8 also increased stem-like characteristics including aldehyde dehydrogenase activity, expression of stemness-related genes, clonogenic activity, side-population, and in vivo tumorigenicity.Consistently, knockdown of IL-8 leads to loss of stem cell-like characteristics in gefitinib-resistant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Epidermal growth factor receptor (EGFR)-targeted strategy is limited by resistance. We identify the potential genes involved in EGFR TKI (tyrosine kinase inhibitor) resistance and study the therapeutic mechanism in the non-small cell lung cancers. Potential genes involved in resistance were examined by analyzing datasets from a pair of EGFR TKI-sensitive (PC9) and TKI-resistant cells (PC9/gef). Blood specimens from patients taking EGFR TKI as first-line treatment were used to examine the correlation between drug's efficacy and IL-8 level. The effects of IL-8 on gefitinib-induced apoptosis, stemness, and in vivo tumorigenicity were investigated using established cell lines. We identified IL-8 was up-regulated in gefitinib-resistant cells, and high plasma IL-8 level was correlated with shorter progression-free-survival time. IL-8 overexpression suppressed gefitinib-induced apoptosis in gefitinib-sensitive cells. By contrast, suppression of IL-8 enhanced gefitinib-induced cell death in gefitinib-resistant cells. IL-8 also increased stem-like characteristics including aldehyde dehydrogenase activity, expression of stemness-related genes, clonogenic activity, side-population, and in vivo tumorigenicity. Consistently, knockdown of IL-8 leads to loss of stem cell-like characteristics in gefitinib-resistant cells. Our study demonstrates an important role for IL-8, and suggests IL-8 is a potential therapeutic target for overcoming EGFR TKI resistance.

No MeSH data available.


Related in: MedlinePlus

IL-8 conferred stem cell-like characteristicsA. ALDH activity was examined by incubating PC9/mock and PC9/IL-8 cells with ALDH substrate in the presence and absence of DEAB. Plots show the results of three independent experiments (***p < 0.001). B. The levels of stem cell-related genes were analyzed in PC9/mock and PC9/IL-8 by RT-qPCR and normalized against that of TBP. The results shown represent the results of three independent experiments (***p < 0.001). C. Hoechst 33342 staining of PC9/mock and PC9/IL-8 cells. Left: Location of the side population in a representative experiment is indicated by gate and dot plots. Right: Quantification of results from four independent experiments (*p < 0.05). Verapamil (vera) was used to distinguish side population and non-side population as indicated. D. PC9/mock and PC9/IL-8 cells (2.5 × 103 cells/well) were analyzed for colony formation in soft agar. Left: Photographs of colonies were taken on days 14 and 28. Right: Quantification of total colonies per well from three independent experiments (***p < 0.001). E. Tumor formation incidence. NOD.SCID mice were injected subcutaneously with the indicated numbers (2.5 × 103 to 5 × 104 cells) of PC9/mock and PC9/IL-8 cells (n = 7). Tumors were examined 10 weeks after injection. F. Hematoxylin-Eosin (H&E) staining of tumor from mice implanted with 2.5 × 103 cells of PC9/mock or PC9/IL-8. Magnification is “400x”.
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Figure 4: IL-8 conferred stem cell-like characteristicsA. ALDH activity was examined by incubating PC9/mock and PC9/IL-8 cells with ALDH substrate in the presence and absence of DEAB. Plots show the results of three independent experiments (***p < 0.001). B. The levels of stem cell-related genes were analyzed in PC9/mock and PC9/IL-8 by RT-qPCR and normalized against that of TBP. The results shown represent the results of three independent experiments (***p < 0.001). C. Hoechst 33342 staining of PC9/mock and PC9/IL-8 cells. Left: Location of the side population in a representative experiment is indicated by gate and dot plots. Right: Quantification of results from four independent experiments (*p < 0.05). Verapamil (vera) was used to distinguish side population and non-side population as indicated. D. PC9/mock and PC9/IL-8 cells (2.5 × 103 cells/well) were analyzed for colony formation in soft agar. Left: Photographs of colonies were taken on days 14 and 28. Right: Quantification of total colonies per well from three independent experiments (***p < 0.001). E. Tumor formation incidence. NOD.SCID mice were injected subcutaneously with the indicated numbers (2.5 × 103 to 5 × 104 cells) of PC9/mock and PC9/IL-8 cells (n = 7). Tumors were examined 10 weeks after injection. F. Hematoxylin-Eosin (H&E) staining of tumor from mice implanted with 2.5 × 103 cells of PC9/mock or PC9/IL-8. Magnification is “400x”.

Mentions: EMT is a biological process by which epithelial cells lose cell-cell adhesion, and have less E-cadherin expression. Both the EMT regulator (Slug) and IL-8 were up-regulated in PC9/gef cells and conferred resistance to gefitinib [4]. To investigate whether IL-8 initiated EMT to result in gefitinib resistance, we examined the expression of EMT-related genes. However, overexpression of IL-8 didn't induce EMT or up-regulation of EMT-related genes in PC9/IL-8 cells compared with PC9/mock cells (Supplementary Fig. S4). The EMT regulator (Slug) and IL-8 are involved in cell motility, and invasion in previous reports and this study, respectively [4]. Slug initiates cell invasion through repression of E-cadherin and activation of EMT, but IL-8 promotes cell motility by directly activating Rac GTPase (one member of Rho family) instead of regulating EMT mediators in previous studies [24, 25]. We previously showed that PC9/gef presented a higher proportion of ALDH-positive compared with PC9 cells and ALDH-positive cells in PC9 cells were resistant to gefitinib [18]. Here, we isolated ALDH-positive and ALDH-negative sub-populations from PC9/gef to determine the correlation between IL-8 expression and ALDH activity. Intriguingly, the ALDH-positive sub-population from PC9/gef showed simultaneously increased IL-8 (Supplementary Fig. S5), supporting a positive correlation between IL-8 and stem-like characteristic. To investigate whether IL-8 contributes to stem-cell like activity, we found that the ALDH-positive cell population was increased in PC9/IL-8 cells compared with PC9/mock cells (Fig. 4a). A number of stem cell-associated genes, including Nanog, Oct4, and Sox2, were also significantly up-regulated in PC9/IL-8 cells (Fig. 4b); others, including Bmi-1, c-Myc, Klf4 and Nestin, were not different.


IL-8 confers resistance to EGFR inhibitors by inducing stem cell properties in lung cancer.

Liu YN, Chang TH, Tsai MF, Wu SG, Tsai TH, Chen HY, Yu SL, Yang JC, Shih JY - Oncotarget (2015)

IL-8 conferred stem cell-like characteristicsA. ALDH activity was examined by incubating PC9/mock and PC9/IL-8 cells with ALDH substrate in the presence and absence of DEAB. Plots show the results of three independent experiments (***p < 0.001). B. The levels of stem cell-related genes were analyzed in PC9/mock and PC9/IL-8 by RT-qPCR and normalized against that of TBP. The results shown represent the results of three independent experiments (***p < 0.001). C. Hoechst 33342 staining of PC9/mock and PC9/IL-8 cells. Left: Location of the side population in a representative experiment is indicated by gate and dot plots. Right: Quantification of results from four independent experiments (*p < 0.05). Verapamil (vera) was used to distinguish side population and non-side population as indicated. D. PC9/mock and PC9/IL-8 cells (2.5 × 103 cells/well) were analyzed for colony formation in soft agar. Left: Photographs of colonies were taken on days 14 and 28. Right: Quantification of total colonies per well from three independent experiments (***p < 0.001). E. Tumor formation incidence. NOD.SCID mice were injected subcutaneously with the indicated numbers (2.5 × 103 to 5 × 104 cells) of PC9/mock and PC9/IL-8 cells (n = 7). Tumors were examined 10 weeks after injection. F. Hematoxylin-Eosin (H&E) staining of tumor from mice implanted with 2.5 × 103 cells of PC9/mock or PC9/IL-8. Magnification is “400x”.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: IL-8 conferred stem cell-like characteristicsA. ALDH activity was examined by incubating PC9/mock and PC9/IL-8 cells with ALDH substrate in the presence and absence of DEAB. Plots show the results of three independent experiments (***p < 0.001). B. The levels of stem cell-related genes were analyzed in PC9/mock and PC9/IL-8 by RT-qPCR and normalized against that of TBP. The results shown represent the results of three independent experiments (***p < 0.001). C. Hoechst 33342 staining of PC9/mock and PC9/IL-8 cells. Left: Location of the side population in a representative experiment is indicated by gate and dot plots. Right: Quantification of results from four independent experiments (*p < 0.05). Verapamil (vera) was used to distinguish side population and non-side population as indicated. D. PC9/mock and PC9/IL-8 cells (2.5 × 103 cells/well) were analyzed for colony formation in soft agar. Left: Photographs of colonies were taken on days 14 and 28. Right: Quantification of total colonies per well from three independent experiments (***p < 0.001). E. Tumor formation incidence. NOD.SCID mice were injected subcutaneously with the indicated numbers (2.5 × 103 to 5 × 104 cells) of PC9/mock and PC9/IL-8 cells (n = 7). Tumors were examined 10 weeks after injection. F. Hematoxylin-Eosin (H&E) staining of tumor from mice implanted with 2.5 × 103 cells of PC9/mock or PC9/IL-8. Magnification is “400x”.
Mentions: EMT is a biological process by which epithelial cells lose cell-cell adhesion, and have less E-cadherin expression. Both the EMT regulator (Slug) and IL-8 were up-regulated in PC9/gef cells and conferred resistance to gefitinib [4]. To investigate whether IL-8 initiated EMT to result in gefitinib resistance, we examined the expression of EMT-related genes. However, overexpression of IL-8 didn't induce EMT or up-regulation of EMT-related genes in PC9/IL-8 cells compared with PC9/mock cells (Supplementary Fig. S4). The EMT regulator (Slug) and IL-8 are involved in cell motility, and invasion in previous reports and this study, respectively [4]. Slug initiates cell invasion through repression of E-cadherin and activation of EMT, but IL-8 promotes cell motility by directly activating Rac GTPase (one member of Rho family) instead of regulating EMT mediators in previous studies [24, 25]. We previously showed that PC9/gef presented a higher proportion of ALDH-positive compared with PC9 cells and ALDH-positive cells in PC9 cells were resistant to gefitinib [18]. Here, we isolated ALDH-positive and ALDH-negative sub-populations from PC9/gef to determine the correlation between IL-8 expression and ALDH activity. Intriguingly, the ALDH-positive sub-population from PC9/gef showed simultaneously increased IL-8 (Supplementary Fig. S5), supporting a positive correlation between IL-8 and stem-like characteristic. To investigate whether IL-8 contributes to stem-cell like activity, we found that the ALDH-positive cell population was increased in PC9/IL-8 cells compared with PC9/mock cells (Fig. 4a). A number of stem cell-associated genes, including Nanog, Oct4, and Sox2, were also significantly up-regulated in PC9/IL-8 cells (Fig. 4b); others, including Bmi-1, c-Myc, Klf4 and Nestin, were not different.

Bottom Line: The effects of IL-8 on gefitinib-induced apoptosis, stemness, and in vivo tumorigenicity were investigated using established cell lines.IL-8 also increased stem-like characteristics including aldehyde dehydrogenase activity, expression of stemness-related genes, clonogenic activity, side-population, and in vivo tumorigenicity.Consistently, knockdown of IL-8 leads to loss of stem cell-like characteristics in gefitinib-resistant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Epidermal growth factor receptor (EGFR)-targeted strategy is limited by resistance. We identify the potential genes involved in EGFR TKI (tyrosine kinase inhibitor) resistance and study the therapeutic mechanism in the non-small cell lung cancers. Potential genes involved in resistance were examined by analyzing datasets from a pair of EGFR TKI-sensitive (PC9) and TKI-resistant cells (PC9/gef). Blood specimens from patients taking EGFR TKI as first-line treatment were used to examine the correlation between drug's efficacy and IL-8 level. The effects of IL-8 on gefitinib-induced apoptosis, stemness, and in vivo tumorigenicity were investigated using established cell lines. We identified IL-8 was up-regulated in gefitinib-resistant cells, and high plasma IL-8 level was correlated with shorter progression-free-survival time. IL-8 overexpression suppressed gefitinib-induced apoptosis in gefitinib-sensitive cells. By contrast, suppression of IL-8 enhanced gefitinib-induced cell death in gefitinib-resistant cells. IL-8 also increased stem-like characteristics including aldehyde dehydrogenase activity, expression of stemness-related genes, clonogenic activity, side-population, and in vivo tumorigenicity. Consistently, knockdown of IL-8 leads to loss of stem cell-like characteristics in gefitinib-resistant cells. Our study demonstrates an important role for IL-8, and suggests IL-8 is a potential therapeutic target for overcoming EGFR TKI resistance.

No MeSH data available.


Related in: MedlinePlus