IL-1β promotes stemness and invasiveness of colon cancer cells through Zeb1 activation

Abstract

Background: IL-1β is a pleiotropic pro-inflammatory cytokine and its up-regulation is closely associated with various cancers including gastrointestinal tumors. However, it remains unclear how IL-1β may contribute to the initiation and development of these inflammation-associated cancers. Here we investigated the role of IL-1β in colon cancer stem cell (CSC) development.

Methods: Using self-renewal assay, soft-agar assay, invasion assay, real-time PCR analysis, immunoblot assay and shRNA knockdown, we determined the effects of IL-1β on cancer stem cell development and epithelial-mesenchymal transition (EMT) in human primary colon cancer cells and colon cancer cell line HCT-116.

Results: We found that IL-1β can increase sphere-forming capability of colon cancer cells in serum-free medium. IL-1β-induced spheres displayed an up-regulation of stemness factor genes (Bmi1 and Nestin) and increased drug resistance, hallmarks of CSCs. Importantly, expression of EMT activator Zeb1 was increased in IL-1β-induced spheres, indicating that there might be a close association between EMT and IL-1β-induced CSC self-renewal. Indeed, IL-1β treatment led to EMT of colon cancer cells with loss of E-cadherin, up-regulation of Zeb1, and gain of the mesenchymal phenotype. Furthermore, shRNA-mediated knockdown of Zeb1 in HCT-116 cells reversed IL-1β-induced EMT and stem cell formation.

Conclusion: Our findings indicate that IL-1β may promote colon tumor growth and invasion through activation of CSC self-renewal and EMT, and Zeb1 plays a critical role in these two processes. Thus, IL-1β and Zeb1 might be new therapeutic targets against colon cancer stem cells.

Figure 1

Characterization of human primary colon cancer cells (HPCC). (A) HPCC cells exhibit epithelial-like morphology as human colon cancer cell line HCT-116. During initial growth in culture (Upper panel), HPCC cells formed small islets; while in late stage of growth (lower panel) they formed multilayer culture, similar to HCT-116 cells. Scale bar = 200 μm. (B) HPCC cells react only with cytokeratin but not vimentin as determined using an immunofluoresent staining assay. Nuclei (blue staining) were counterstained with DAPI. Scale bar = 20 μm. (C) HPCC display transformation capability as determined using a soft-agar assay. HPCC cells (1x10⁴/well) were plated in soft agar containing the medium with 10% FBS in 6-well plates for 14 days. Scale bar = 50 μm.

Figure 2

IL-1β induces sphere formation and proliferation of colon cancer cells in serum-free medium (SFM). (A) Representative images of HCT-116 and HPCC cells in the absence or presence of IL-1β. HCT-116 and HPCC cells (1 cell/μl) were cultured in 96-well plates containing 100 μl SFM in each well with or without IL-1β for seven days. The number of spheres was counted under a microscope and size was measured using ImageJ (B and C). Then sphere cells were dissociated and stained with Trypan blue and counted under a microscope. Error bars represent SEM. *p < 0.05.

Figure 3

IL-1β-induced sphere cells express stem cell markers and Zeb1. (A) Transcription levels of stem cell markers in control and IL-1β-treated colon cancer cells. HCT-116 and HPCC cells were cultured in SFM with or without IL-1β for seven days. mRNA levels of stem cell markers were determined by real-time PCR. β-actin was used as an internal normalization control. Error bars represent SEM. *p < 0.05. (B) Immunoblot analysis for Bmi1 and Nestin from control and IL-1β-treated cells. β-actin was used as an sample loading control. (C) IL-1β-induced sphere cells develop drug resistance. The control and IL-1β-induced sphere cells were treated with various concentrations of carboplatin for two days. Then, cells were dissociated and stained with Trypan blue, and counted under a microscope. The viability was determined by the percentage of live cells over the sum of live and dead cells. *p < 0.05, **p < 0.01. (D). Transcription levels of Zeb1 in control and IL-1β-treated colon cancer cells. HCT-116 and HPCC cells were cultured in SFM with or without IL-1β for seven days. mRNA levels of Zeb1 were determined by real-time PCR. β-actin was used as an internal normalization control. Error bars represent SEM. *p < 0.05. (E). Immunoblot analysis for Zeb1 from control and IL-1β-treated cells. β-actin was used as an sample loading control. Normalized quantification of band intensities on immunoblots for Zeb1 is listed under each band.

Figure 4

IL-1β induces EMT in colon cancer cells. (A) IL-1β induces morphological changes from epithelial-like to fibroblast-like appearances in colon cancer cells. HCT-116 and HPCC cells were cultured in the medium with 1% FBS in the presence or absence of IL-β for seven days. Scale bar = 200 μm. (B) Transcription levels of Zeb1 and E-cadherin as determined by real-time PCR. β-actin was used as an internal normalization control. Error bars represent SEM. *p < 0.05. (C) Immunoblot analysis for E-cadherin from control and IL-1β-treated cells. β-actin was used as an sample loading control.

Figure 5

IL-1β enhances invasiveness and proliferation of colon cancer cells. (A) Representative images of wounds at 0 and 48 h in the presence or absence of IL-1β. Confluent monolayers of HCT-116 cells were scraped by a pipette tip to generate wounds and then were cultured in the presence or absence of IL-1β for 48 h. (B) Relative wound width represented as percentages compared to the wound width at 0 h. (C) Proliferation of HCT-116 cells in the presence or absence of IL-1β as measured by cell count. Error bars represent SEM. *p < 0.05.

Figure 6

Knockdown of Zeb1 inhibits IL-1β-induced EMT in HCT-116 cells. (A) Zeb1 knockdown cells still maintain epithelial appearance seven days after treatment with IL-1β. Scale bar = 200 μm. Scramble and shZeb1 HCT-116 cells were cultured in the medium with 1% FBS in the presence or absence of IL-1β for seven days. (B) Zeb1 knockdown inhibits IL-β-induced suppression of E-cadherin expression. Immunoblot analysis for Zeb1 and E-cadherin was performed on lysates from control and IL-1β-treated cells. β-actin was used as an sample loading control. Normalized quantification of band intensities on immunoblot for Zeb1 and E-cadherin is listed under each band.

Figure 7

Zeb1 knockdown decreases Bmi1 expression as well as sphere-forming capability in HCT-116 cells treated with or without IL-1β. (A) Zeb1 knockdown inhibits Bmi1 expression in either IL-1β-treated or untreated cells. Scramble and shZeb1 HCT-116 cells were cultured in SFM in the presence or absence of IL-1β for seven days. Expression of Zeb1 and Bmi1 was determined using immunoblot assay on lysates from scramble and shZeb1 cells treated with or without IL-1β. β-actin was used as an sample loading control. Normalized quantification of band intensities on immunoblot for Zeb1 and Bmi1 is listed under each band. (B) Zeb1 knockdown reduces sphere-forming capability in HCT-116 cells as well as inhibits IL-1β-induced sphere formation. Scramble or shZeb1 cells (1 cell/μl) were cultured in 96-well plates containing 100 μl SFM in each well with or without IL-1β for seven days. The number of spheres was counted under a microscope and size was measured using ImageJ. The data are presented as relative sphere number and size as compared to scramble cells cultured in the absence of IL-1β. Error bars represent SEM. *p < 0.05.