Reduction of RKIP expression promotes nasopharyngeal carcinoma invasion and metastasis by activating Stat3 signaling

Abstract

The role and underlying mechanism of Raf kinase inhibitory protein (RKIP) in nasopharyngeal carcinoma (NPC) metastasis remain unclear. Here, we showed that RKIP was downregulated in the NPC with high metastatic potentials, and its decrement correlated with NPC metastasis and poor patient survival, and was an independent predictor for reduced overall survival. With a combination of loss-of-function and gain-of-function approaches, we observed that high expression of RKIP reduced invasion, metastasis and epithelial to mesenchymal transition (EMT) marker alternations of NPC cells. We further showed that RKIP overexpression attenuated while RKIP knockdown enhanced Stat3 phosphorylation and activation in NPC cells; RKIP reduced Stat3 phosphorylation through interacting with Stat3; Stattic attenuated NPC cell migration, invasion and EMT marker alternations induced by RKIP knockdown, whereas Stat3 overexpression restored NPC cell migration, invasion and EMT marker alternations reduced by RKIP overexpression. In addition, there was an inverse correlation between RKIP and phospho-Stat3 expression in the NPC tissues and xenograft metastases. Our data demonstrate that RKIP is a metastatic suppressor and predictor for metastasis and prognosis in NPC, and RKIP downregulation promotes NPC invasion, metastasis and EMT by activating Stat3 signaling, suggesting that RKIP/Stat3 signaling could be used as a therapeutic target for NPC metastasis.

Keywords: RKIP; Stat3; metastasis; metastatic suppressor; nasopharyngeal carcinoma.

Figure 1. Association of RKIP expression levels with NPC metastasis and overall survival of the patients

A. a representative result of RKIP and phospho-Stat3 immunohistochemical staining in normal nasopharyngeal mucosa (a), NPC without metastasis (b), NPC with metastasis (c), and lymphonode metastasis (d). Original magnification, ×200. B. Kaplan-Meier survival analysis for NPC patients according to the expression levels of RKIP. NPC patients with low RKIP expression have a significantly worse overall survival than those with high RKIP expression. The log-rank test was used to calculate p value.

Figure 2. The effects of RKIP expression changes on in vitro NPC cells migration and invasion and in vivo metastasis

A. (left) a representative result of Western blotting shows the levels of RKIP expression in 5-8F and 6-10B NPC cells and their transfectants; (right) histogram of relative RKIP expression levels in 5-8F and 6-10B NPC cells and their transfectants as determined by densitometric analysis. B. (left top) a representative result of scratch wound-healing assay shows migration of 6-10B and 5-8F cells and their transfectants. Images were taken at 0, 24 and 48h after wounding under the inverted microscope; (left bottom) a representative result of Matrigel invasion assay shows invasion of 6-10B and 5-8F cells and their transfectants. Invasive cancer cells were photographed at 48h after incubation; (right) histogram of average numbers of invasive cancer cells per microscopic field. C. in vivo metastasis assays of NPC cells with RKIP expression changes. (left) RKIP overexpression 5-8F cells, RKIP knockdown 6-10B cells, and their corresponding empty vector-transfected cells were injected into the tail vein of nude mice, and the representative photography of lung is shown; (middle) histogram of average numbers of lung metastases per mouse; (right) the representative H&E staining of lung tissues shows metastatic tumors. Metastases generated by RKIP overexpression 5-8F cells are significantly less than those generated by empty vector-transfected 5-8F cells. The empty vector-transfected 6-10B cells do not generate metastases, but RKIP knockdown 6-10B cells can generate metastases. Columns, mean values; bars, S.D. *, p < 0.01. Vector, transfcted with an empty vector; OE, overexpression; KD, knockdown.

Figure 3. The regulation of RKIP on the expression of EMT-like cellular markers in NPC cells and their xenograft metastases

A. mRNA expression levels of E-cadherin, N-cadherin and Vimentin in 5-8F and 6-10B NPC cells and their transfectants detected by qRT-PCR. Columns, mean values from triplicate experiments; bars, S.D. *, p < 0.01. B. a representative result of Western blotting shows the levels of E-cadherin, N-cadherin and Vimentin in 5-8F and 6-10B NPC cells and their transfectants. C. a representative result of immunofluorescent staining shows the levels of E-cadherin, N-cadherin and Vimentin in RKIP overexpression 5-8F cells, RKIP knockdown 6-10B cells and their corresponding empty vector-transfected cells. D. (left) a representative result of immunohistochemical staining of RKIP, E-cadherin, N-cadherin, Vimentin and phospho-Stat3 in the lung metastases of RKIP overexpression 5-8F cells, RKIP knockdown 6-10B cells and empty vector-transfected 5-8F cells; (right) histogram of expression levels of RKIP, E-cadherin, N-cadherin, Vimentin and phospho-Stat3 in the lung metastases. Original magnification, ×200. Columns, mean values from 10 mice; bars, S.D. *, p < 0.01. E-cad, E-cadherin; N-cad, N-cadherin; Vim, Vimentin. Vector, transfcted with an empty vector; OE, overexpression; KD, knockdown.

Figure 4. The regulation of RKIP on the activity of NPC cellular signaling pathways

A. a representative result of Western blotting shows the phosphorylated and total levels of Stat3, ERK-1/2, IKK-α/β, IκB-α, and GSK-3β in RKIP overexpression 5-8F cells, RKIP knockdown 6-10B cells and their corresponding empty vector-transfected cells. B. a representative result of immunofluorescent staining shows the nuclear translocation of phospho-Stat3 in RKIP overexpression 5-8F cells, RKIP knockdown 6-10B cells and their corresponding empty vector-transfected cells. C. Stat3 luciferase reporter activity in RKIP overexpression 5-8F cells, RKIP knockdown 6-10B cells and their corresponding empty vector-transfected cells. Columns, mean values from triplicate experiments; bars, S.D. *, p < 0.01. D. a representative result of Co-IP shows RKIP interacting with and inhibiting Stat3 phosphorylation in RKIP overexpression 5-8F cells. Cells were lysed in RIPA buffer, a portion of the sample was removed as the IP input and the remaining supernatant was immunoprecipitated with RKIP antibody and protein A agarose. Immunocomplexes were separated by SDS-PAGE, transferred onto PVDF membrane, and detected with Stat3, phospho-stat3 or RKIP antibody. The input samples were examined for the expression of the indicated proteins. Vector, transfcted with an empty vector; OE, overexpression; KD, knockdown.

Figure 5. The regulation of RKIP on migration, invasion, and EMT marker expressions mediated by Stat3 signaling in NPC Cells

A. a representative result of Western blotting shows the levels of E-cadherin, N-cadherin and Vimentin in RKIP overexpression 5-8F cells transfected with pReceiver-M13-Stat3 or empty vector pReceiver-M13, and RKIP knockdown 6-10B cells treated with a range 0-7.5 μM Stat3 inhibitor Stattic. B. a representative result of scratch wound-healing assay (top) and Matrigel invasion assay (middle) of RKIP overexpression 5-8F cells transfected with pReceiver-M13-Stat3 or pReceiver-M13, and RKIP knockdown 6-10B cells treated with Stat3 inhibitor Stattic; (bottom) histogram of average numbers of invasive cancer cells per microscopic field in these cells. Columns, mean values; bars, S.D. *, p < 0.01. Vector, transfcted with an empty vector; OE, overexpression; KD, knockdown.