CCR4 promotes metastasis via ERK/NF-κB/MMP13 pathway and acts downstream of TNF-α in colorectal cancer

Abstract

Chemokines and chemokine receptors are causally involved in the metastasis of human malignancies. As a crucial chemokine receptor for mediating immune homeostasis, however, the role of CCR4 in colorectal cancer (CRC) remains unknown. In this study, we found that high expression of CCR4 in CRC tissues was correlated with shorter overall survival and disease free survival. In vitro and in vivo experiments revealed that silencing CCR4 attenuated the invasion and metastasis of CRC cells, whereas ectopic overexpression of CCR4 contributed to the forced metastasis of these cells. We further demonstrated that matrix metalloproteinase 13 (MMP13) played an important role in CCR4-mediated cancer cell invasion, which is up-regulated by ERK/NF-κB signaling. Positive correlation between CCR4 and MMP13 expression was also observed in CRC tissues. Moreover, our investigations showed that the level of CCR4 could be induced by TNF-α dependent of NF-κB activation in CRC cells. CCR4 might be implicated in TNF-α-regulated cancer cells metastasis. Combination of CCR4 and TNF-α is a more powerful prognostic marker for CRC patients. These findings suggest that CCR4 facilitates metastasis through ERK/NF-κB/MMP13 signaling and acts as a downstream target of TNF-α. CCR4 inhibition may be a promising therapeutic option for suppressing CRC metastasis.

Keywords: CCR4; MMP13; TNF-α; colorectal cancer; metastasis.

Figure 1. Expression of CCR4 and its clinical significance in CRC patients

(A) qRT-PCR analysis showing CCR4 expression in 16 paired CRC samples that were randomly selected from the 116 CRC cases, by random numbers generated with SAS software. (B) Representative western blot images of CCR4 expression in 16 paired CRC samples. (C) Quantification of relative grey value of bands compared with GAPDH, as detected by western blot. (D) CCR4 expression level in tumor tissues and the paired normal tissues was evaluated by immunohistochemical staining with tissue microarray. (E) CRC patients with positive expression of CCR4 presented with worse overall survival, and disease free survival compared with that of negative expression of CCR4. Data represent the mean ± SD and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. CCR4 plays a crucial role in the metastasis of SW1116 and SW480 cells

(A) CCR4 expression in eight CRC cell lines detected by western blot. (B) SW1116 and SW480 cells transfected with pcDNA-CCR4 and sh-CCR4, respectively, were subject to western blot. (C) Invasive behavior was evaluated using matrigel invasion assays after overexpression or knockdown of CCR4 in SW1116 or SW480 (magnification, ×200). (D) The migratory capacity of SW1116/CCR4 and SW480/sh-CCR4 cells was analyzed by wound-healing assay.

Figure 3. CCR4 facilitates CRC cells metastasis in vitro and in vivo

(A) HT29 and SW620 cells transfected with pcDNA-CCR4 and sh-CCR4, respectively, were tested by western blot. (B) Matrigel invasion assays were performed to assess invasive ability of HT29/CCR4 and SW620/sh-CCR4 (magnification, ×200). (C) Wound-healing assay shows a significant increase or decrease in healing rate of the scramble wound in HT29/CCR4 and SW620/sh-CCR4, respectively. (D) Representative images of hematoxylin & eosin staining of liver tissue sections. Black arrows indicated liver metastasis. (E) Number of metastasis in the liver. (F) Number of tumors on the surface of liver given orthotopic implantation of xenograft. Data represent the mean ± SD and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. MMP13 plays a crucial role in CRC cells invasion mediated by CCR4

(A) Five metastasis-related genes (VEGF, MMP13, CDH1, IL1B, ITGA7) showed a more than 2-fold mRNA differential expression in PCR array. (B) The effect of MMP13 knockdown in SW1116/CCR4 and SW480 cells, detected by western blot. Densitometry represents the expression of the proteins relative to GAPDH. (C) Results of invasion assays showed the inhibitory roles of si-MMP13 or CL82198 on SW1116/CCR4 and SW480 cells (magnification, ×200). (D) Representative images of MMP13 staining in the cohort of 116 CRC tissues. (E) Expression correlation of CCR4 and MMP13 was analyzed in 116 CRC patients using IHC.

Figure 5. CCR4 up-regulates MMP13 expression through Erk1/2/ NF-κB activation

(A) p-ERK, p-p38, p-JNK, p-Akt, p-p65 and MMP13 expressions were determined by western blot analysis. Densitometry represents the expression of the proteins relative to GAPDH. (B) p-p65, β-catenin, Runx2, and MMP13 expressions were analyzed using western blot. Densitometry represents the expression of the proteins relative to GAPDH. (C) Results of invasion assays showed the inhibitory roles of U0126 or TCPA-1 on SW1116/CCR4 and SW480 cells (magnification, ×200). (D) Representative images of IHC staining of p-ERK, p-p65 and MMP13 in the CRC tissues of orthotopic implantation model in nude mice. Scare bars = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. CCR4 could be induced by the cytokine TNF-α

(A) Cells were incubated with different doses of TNF-α for 12 h, and p-p65, CCR4 or MMP13 expression is examined by western blot. (B) Representative images of IHC staining of TNF-α in tissue microarray. (C) Prognostic values of CCR4 combined with TNF-α. (D) DNA fragments pulled down with NF-κB antibody from SW1116 cells treated with TNF-α (50 ng/ml) or PBS were amplified by PCR. (E) The promoter activity was evaluated by transfection with different CCR4 luciferase expression vectors. Cells were treated with TNF-α (50 ng/ml) or PBS for 12 h.