The CBP/β-Catenin Antagonist, ICG-001, Inhibits Tumor Metastasis via Blocking of the miR-134/ITGB1 Axis-Mediated Cell Adhesion in Nasopharyngeal Carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy ranking as the 23rd most common cancer globally, while its incidence rate ranked the 9th in southeast Asia. Tumor metastasis is the dominant cause for treatment failure in NPC and metastatic NPC is yet incurable. The Wnt/β-catenin signaling pathway plays an important role in many processes such as cell proliferation, differentiation, epithelial-mesenchymal transition (EMT), and self-renewal of stem cells and cancer stem cells (CSCs). Both the EMT process and CSCs are believed to play a critical role in cancer metastasis. We here investigated whether the specific CBP/β-catenin Wnt antagonist, IGC-001, affects the metastasis of NPC cells. We found that ICG-001 treatment could reduce the adhesion capability of NPC cells to extracellular matrix and to capillary endothelial cells and reduce the tumor cell migration and invasion, events which are closely associated with distant metastasis. Through a screening of EMT and CSC-related microRNAs, it was found that miR-134 was consistently upregulated by ICG-001 treatment in NPC cells. Very few reports have mentioned the functional role of miR-134 in NPC, except that the expression was found to be downregulated in NPC. Transient transfection of miR-134 into NPC cells reduced their cell adhesion, migration, and invasion capability, but did not affect the growth of CSC-enriched tumor spheres. Subsequently, we found that the ICG-001-induced miR-134 expression resulting in downregulation of integrin β1 (ITGB1). Such downregulation reduced cell adhesion and migration capability, as demonstrated by siRNA-mediated knockdown of ITGB1. Direct targeting of ITGB1 by miR-134 was confirmed by the 3'-UTR luciferase assay. Lastly, using an in vivo lung metastasis assay, we showed that ICG-001 transient overexpression of miR-134 or stable overexpression of miR-134 could significantly reduce the lung metastasis of NPC cells. Taken together, we present here evidence that modulation of Wnt/β-catenin signaling pathway could inhibit the metastasis of NPC through the miR-134/ITGB1 axis.

Keywords: ICG-001; ITGB1; NPC; Wnt; miR-134.

Figure 1

The Wnt/CBP/β-catenin antagonist ICG-001 suppresses the cell–matrix and tumor cell–endothelial cell adhesion of NPC cells. (A) ICG-001 (10 μM, 3-day treatment) inhibited the adhesion of C666-1, HK-1, HONE-1 cells to fibronectin-coated 96-well plate. Scale bar, 40 μm. (B) Statistical summary of inhibitory effects of ICG-001 on adhesion of NPC cells. (C) ICG-001 (3-day treatment) inhibited the adhesion of HONE-1 cells to HPMEC. Scale bar, 20 μm. (D) Statistical summary of inhibitory effects of ICG-001 on adhesion of NPC HONE-1 cell to HPMEC. Results were obtained from three independent experiments. * p < 0.05, ** p < 0.01.

Figure 2

ICG-001 inhibits NPC adhesion and migration via upregulating the expression of miR-134. (A) ICG-001 treatment increased expression of miR-134 in three NPC cell lines. qPCR was used to study the miRNA expression. Control, solvent control. (B) miR-134 overexpression inhibited adhesion of NPC cells. Scale bar, 40 μm. (C) Statistical summaries of the inhibitory effects on NPC adhesion from three independent experiments. (D) miR-134 overexpression inhibited migration of NPC cells. Scale bar, 100 μm. Extracellular matrix adhesion assay was used to study the tumor cell–fibronectin adhesion as described in Figure 1A. A transwell migration assay was used to analyze the tumor cell migration. (E) Statistical summaries of the inhibitory effects on NPC migration from three independent experiments. * p < 0.05, ** p < 0.01.

Figure 3

ICG-001 and miR-134 inhibit NPC invasion in a 3D Matrigel culture model. (A) A schematic illustration of the 3D Matrigel culture. NPC cells were seeded into the bottom layer Matrigel. This first layer of Matrigel was allowed to solidify before layering a second layer of Matrigel on top of it. Cells were allowed to grow for one week for spheroid formation before imposing a chemo-gradient for spheroid invasion towards the upper layer Matrigel. Both (B) ICG-001 and (C) transient overexpression of miR-134 could inhibit the invasion of NPC C666-1 spheroids. Results were shown from three independent experiments. Scale bar in (B) and (C), 500 μm. * p < 0.05; ** p < 0.01.

Figure 4

miR-134 targets ITGB1 in NPC. (A)Transient overexpression of miR-134 in NPC cells reduced the expression of ITGB1 mRNA. (B) Overexpression of miR-134 in NPC cells reduced the expression of ITGB1 protein. The protein expression was detected by Western blot analysis, and actin serves as a loading control. Statistical summary from three independent experiments was also shown. (C) miR-134 specifically targeted ITGB1 in NPC. The sequences of wild-type and mutant ITGB1 3′-UTR and the base-paring between miR-134 and ITGB1 3′-UTR were indicated in the top panel; the specific inhibition of miR-134 on the wild type ITGB1 3′-UTR containing luciferase activity was displayed in the bottom panel. MUT, mutant; WT, wild type. * p < 0.05, ** p < 0.01.

Figure 5

ICG-001 downregulates the expression of ITGB1 in NPC cells. (A) ICG-001 treatment (10 μM, 3-day) inhibited the expression of ITGB1 in NPC cells. The ITGB1 protein expression was detected by Western blot analysis, and actin serves as a loading control. A statistical summary is displayed in (B). (C) ICG-001 treatment (10 μM, 7-day) reduced the expression of integrin α5β1 and phalloidin in NPC cells. The expression of integrin α5β1 and phalloidin was detected by immunofluorescent imaging. * p < 0.05.

Figure 6

ITGB1 is involved in the suppression of tumor cell adhesion and migration of NPC cells by ICG-001. (A) Effects of ITGB1 siRNA on the expression of ITGB1 in NPC cells. The protein expression was detected by Western blot analysis, and actin serves as a loading control. si-Ctrl, scramble siRNA control. (B) Statistical summary of knockdown efficiency from three independent experiments. (C) ITGB1 siRNA inhibited adhesion of NPC cells. Extracellular matrix adhesion assay was used to study the tumor cell-matrix adhesion as described in Figure 1A. Scale bar, 40 μm. (D) Statistical summaries of the effects of ITGB1 knockdown on cell adhesion from three independent experiments. * p < 0.05, ** p < 0.01. (E) ITGB1 siRNA inhibited migration of NPC cells; a transwell migration assay was used. Scale bar, 50 μm. (F) Statistical summaries of the effects of ITGB1 knockdown on tumor cell migration from three independent experiments. * p < 0.05, ** p < 0.01.

Figure 7

Inhibitory effects of ICG-001 treatment and the upregulated miR-134 expression on lung metastasis of NPC cells. (A) The ICG-001 pre-treatment (10 μM, 7 days) inhibited the metastasis of HONE-1-luc cells in nude mice (n = 8). (B) Transient overexpression of miR-134 inhibited the metastasis of HONE-1-luc cells in nude mice (n = 12). (C) Stable overexpression of miR-134 inhibited the metastasis of HONE-1-luc cells in nude mice (n = 10). (D) Statistical summary of the suppressive effects of ICG-001, transient/stable miR-134 transfection on the metastasis of NPC cells to the lung of mice. (E) Representative H&E staining of the mice lung. (Scale bar, 2 mm).

Figure 8

ICG-001 inhibits NPC tumor metastasis via the miR-134/ITGB1 axis. A β-catenin/TCF complex in the nucleus could either bind with CBP or p300. The CBP-bound form favors cell proliferation while the p300-bound form favors cell differentiation. ICG-001, via blocking the binding between β-catenin and CBP, favors the formation of the p300/β-catenin/TCF complex. As a result, the miR-134 expression is upregulated by ICG-001 and that subsequently mediates downregulation of ITGB1. Reduced expression of ITGB1, including the functional heterodimer of ITGB1 with an alpha subunit (e.g., Integrin α5β1), can result in reduced cell–matrix adhesion, cell–cell adhesion, cell migration and invasion, and lung metastasis of NPC cells.