Therapeutic targeting of CBP/β-catenin signaling reduces cancer stem-like population and synergistically suppresses growth of EBV-positive nasopharyngeal carcinoma cells with cisplatin

Abstract

Nasopharyngeal carcinoma (NPC) is an EBV-associated epithelial malignancy prevalent in southern China. Presence of treatment-resistant cancer stem cells (CSC) may associate with tumor relapse and metastasis in NPC. ICG-001 is a specific CBP/β-catenin antagonist that can block CBP/β-catenin-mediated transcription of stem cell associated genes and enhance p300/β-catenin-mediated transcription, thereby reducing the CSC-like population via forced differentiation. In this study, we aimed to evaluate the effect of ICG-001 on the CSC-like population, and the combination effect of ICG-001 with cisplatin in the C666-1 EBV-positive NPC cells. Results showed that ICG-001 inhibited C666-1 cell growth and reduced expression of CSC-associated proteins with altered expression of epithelial-mesenchymal transition (EMT) markers. ICG-001 also inhibited C666-1 tumor sphere formation, accompanied with reduced SOX2(hi)/CD44(hi) CSC-like population. ICG-001 was also found to restore the expression of a tumor suppressive microRNA-145 (miR-145). Ectopic expression of miR-145 effectively repressed SOX2 protein expression and inhibited tumor sphere formation. Combination of ICG-001 with cisplatin synergistically suppressed in vitro growth of C666-1 cells and significantly suppressed growth of NPC xenografts. These results suggested that therapeutically targeting of the CBP/β-catenin signaling pathway with ICG-001 can effectively reduce the CSC-like population and combination with cisplatin can effectively suppress the growth of NPC.

Figure 1. Effect of ICG-001 on the growth of EBV-positive C666-1 cells.

(a) Cell growth assay. Total number of viable cells was counted on days 3, 5, and 7 after ICG-001 treatment. Results were expressed as the mean ± S.D. of three experiments; *p < 0.05. (b) Western blotting analysis of SOX2, CD44, Survivin, EGFR, FOXM1, and EZH2 expression on day-7. (c) Western blotting analysis of epithelial-mesenchymal transition (EMT) markers, E-cadherin and vimentin on day-7. The gels have been run under the same experimental conditions with different percentage of gels. The full-length blots were presented in the supplementary Figure S1.

Figure 2. Effect of ICG-001 on the growth of C666-1 in the 3-D tumor sphere formation and tumor sphere growth assay.

(a) Effect on the tumor sphere formation. ICG-001 was added on day 0, when plating the cell into ultra-low attachment plate. The number and size of tumor sphere formed from C666-1 with and without ICG-001 treatment were compared. (b) Effect on the growth of established spheroids. Tumor spheres were allowed to form for 7 days, then the established tumor spheres were incubated with ICG-001 for another 7 days. The number and diameter of tumor spheres measured were presented as tumor sphere size profiles. Results were expressed as the mean ± S.D. of three experiments; **p < 0.02.

Figure 3. ICG-001 reduced SOX2 and CD44 expression in C666-1 tumor sphere.

(a) Confocal staining showing pseudocolor red for SOX2 and green for CD44. Loss of both SOX2 and CD44 expression was observed in ICG-001-treated C666-1 tumor sphere. (b) FACS analysis of spheroid cells stained with Alex Fluor® 647 conjugated SOX2 and Alexa Fluor® 488 conjugated CD44 and gated against the non-stained tumor sphere cells. The SOX2^hi/CD44^hi population was significantly decreased after ICG-001 treatment. Results were expressed as the mean ± S.D. of three experiments; *p < 0.05.

Figure 4. The role of SOX2 in tumor sphere formation.

Transfection of C666-1 with SOX2 siRNA led to (a) decreased SOX2 expression and (b) decreased number and size of tumor sphere formation. Results were expressed as the mean ± S.D. of three experiments; *p< 0.05; **p < 0.02. The gels have been run under the same experimental conditions. The full-length blots are presented in the supplementary Figure S2.

Figure 5. Restoration of miR-145 by ICG-001 and the role of miR-145 in suppression of SOX2 and tumor sphere formation.

(a) Expression of miR-145 in ICG-001-treated EBV-positive and -negative NPC cell lines. (b) SOX2 3'UTR Luciferase reporter assay. (c) and (d) Transfection of miR-145 precursor suppressed SOX2 protein expression and reduced number and size of tumor sphere formed in ultralow attachment plate. Results were expressed as the mean ± S.D. of three experiments; *p < 0.05; **p < 0.02. The gels have been run under the same experimental conditions. The full-length blots are presented in the supplementary Figure S3.

Figure 6. Combination of ICG-001 and cisplatin showed a synergistic growth inhibitory effect on C666-1 cells.

(a) MTT assay showing the percentage of cell proliferation after 7 days treatment with various concentrations of ICG-001 and cisplatin. When comparing the IC₆₀, the calculated CI value is 0.7, indicating a synergistic growth inhibitory effect of ICG-001 and cisplatin. (b) Cell growth assay showing the total number of proliferating cells counted on day-7 after ICG-001 and cisplatin drug treatment. Combination of ICG-001 and cisplatin showed a greater significant growth inhibitory effect in C666-1 than either drug treatment alone. Results are expressed as the mean ± S.D. of three experiments; *p < 0.05.

Figure 7. Combination of ICG-001 and cisplatin significantly suppressed tumor growth in EBV-positive NPC xenografts.

(a) C666-1 cells and xeno-2117 were subcutaneously injected into the right flanks of nude mice, respectively (n = 4 for C666-1 xenograft and n = 5 for xeno-2117). Drug treatments were started when the tumors became palpable, and the size of tumors was measured twice weekly and plotted as the average tumor volumes versus number of days post-treatment. (b) Percentage change of mouse body weight measured throughout the experiment. (c) Photos of tumor-bearing mice and tumors dissected from mice at the end of experiment.