BPIFB1 (LPLUNC1) inhibits migration and invasion of nasopharyngeal carcinoma by interacting with VTN and VIM

Abstract

Background: Bactericidal/Permeability-increasing-fold-containing family B member 1 (BPIFB1, previously termed LPLUNC1) is highly expressed in the nasopharynx, significantly downregulated in nasopharyngeal carcinoma (NPC), and associated with prognosis in NPC patients. Because metastasis represents the primary cause of NPC-related death, we explored the role of BPIFB1 in NPC migration and invasion.

Methods: The role of BPIFB1 in NPC metastasis was investigated in vitro and in vivo. A co-immunoprecipitation assay coupled with mass spectrometry was used to identify BPIFB1-binding proteins. Additionally, western blotting, immunofluorescence, and immunohistochemistry allowed assessment of the molecular mechanisms associated with BPIFB1-specific metastatic inhibition via vitronectin (VTN) and vimentin (VIM) interactions.

Results: Our results showed that BPIFB1 expression markedly inhibited NPC cell migration, invasion, and lung-metastatic abilities. Additionally, identification of two BPIFB1-interacting proteins, VTN and VIM, showed that BPIFB1 reduced VTN expression and the formation of a VTN-integrin αV complex in NPC cells, leading to inhibition of the FAK/Src/ERK signalling pathway. Moreover, BPIFB1 attenuated NPC cell migration and invasion by inhibiting VTN- or VIM-induced epithelial-mesenchymal transition.

Conclusions: This study represents the first demonstration of BPIFB1 function in NPC migration, invasion, and lung metastasis. Our findings indicate that re-expression of BPIFB1 might represent a useful strategy for preventing and treating NPC.

Figure 1

BPIFB1 inhibits NPC cell migration and invasion in vitro. (A) BPIFB1 expression in 5-8F, HNE2, and HONE1 cells transfected with the BPIFB1-Flag vector was measured by qPCR and western blot using the anti-Flag antibody. (B) Wound healing assays were performed to measure the cell migration ability of 5-8F, HNE2, and HONE1 cells transfected with the BPIFB1-Flag vector or empty vector. The picture shows the scratch width at 0, 24, and 48 h. (C) A transwell assay was performed to detect the BPIFB1-specific invasion ability of 5-8F, HNE2, and HONE1 cells. Cells were transfected with BPIFB1-Flag or empty vector. The graph summarises data from three independent experiments. *P<0.05; **P<0.01; ***P<0.001. Scale bars=200 μm. (D) BPIFB1 re-expression inhibited the invasive progression of spheroids in a 3D cell culture model. Images of spheroids formed by 5-8F cells in Matrigel (left). The black arrows indicate the scattered protrusions formed on the spheroid surface. Scale bars=50 μm. Quantification of two spheroid types in NC and BPIFB1-overexpressing cells (right).

Figure 2

BPIFB1 inhibits NPC-cell lung metastasis in vivo. (A) Bright-field images of mouse lungs taken at 8-weeks post-injection of 1 × 10⁶ 5-8F cells transfected with BPIFB1-Flag or empty vector into the tail vein. (B) Representative images of visible nodules on the lung surface. Arrows indicate clusters of tumour cells that have colonised in the lung. (C) The number of lung-metastasis nodules on each lung surface was counted, and the data represent the mean±s.d. (each data point represents a different mouse; n=8 mice per group). *P<0.05. (D) Representative images of lung metastasis according to H&E staining. Rectangular boxes indicate clusters of micro-metastatic cells in the lung. Scale bars=200 and 50 μm.

Figure 3

Identification of BPIFB1-interacting proteins via IP-MS. (A) Co-IP was performed in 5-8F cells transfected with the BPIFB1-Flag plasmid using an anti-Flag antibody. The precipitated complex was subjected to SDS–PAGE and Coomassie blue staining. IgG heavy chain (IgG-H) and light chain (IgG-L) are 55 and 25 kDa, respectively. The molecular weight (MW) marker is shown on the left. (B) Representative image and charge states are shown according to the corresponding MS/MS spectrum. LC-MS/MS spectra of precursor ions at m/z 824.42 correspond to VTN residues 198 through 212 (DVWGIEGPIDAAFTR), and m/z 585.36 corresponds to VIM residues 130 through 139 (ILLAELEQLK). (C) BPIFB1 and VTN (or VIM) expression was examined at the protein level by western blot using 5-8F cells co-transfected with BPIFB1-Flag and His-VTN (or -VIM) vectors. (D and E) Co-IP was performed to detect interactions between BPIFB1 and exogenous VTN (or VIM) in 5-8F cells co-transfected with BPIFB1-Flag and His-VTN (or -VIM) using anti-Flag (BPIFB1) and anti-His (VTN or VIM) antibodies. (F and G) Co-IP was performed to detect interactions between BPIFB1 and endogenous VTN (or VIM) in 5-8F cells transfected with only BPIFB1-Flag vector using anti-Flag (BPIFB1) and anti-VTN or VIM antibodies.

Figure 4

BPIFB1 inhibits NPC-cell migration and invasion by downregulating VTN expression. (A) BPIFB1 and VTN colocalisation in 5-8F cells. DAPI-stained nuclei: blue; anti-Flag-BPIFB1: red; anti-His-VTN: green; merged image represents the overlay of DAPI, Flag, and His signals; BPIFB1 and VTN colocalisation: yellow. Scatter analysis shows the signals of Channel 561 (BPIFB1) and 488 (VTN). Pearson’s correlation coefficient of colocalisation is indicated in the top right corner of the plot. Scale bar=29 μm. (B) BPIFB1 downregulates VTN expression in 5-8F, HNE2, and HONE1 cells transfected with the BPIFB1-Flag overexpression vector as compared with the control group. (C) BPIFB1 and VTN protein expression was confirmed by western blot in 5-8F cells transfected or co-transfected with BPIFB1-Flag and VTN-His vectors using anti-Flag and anti-His primary antibodies. (D and E) Representative images of migration and transwell Matrigel invasion assays on 5-8F cells transfected or co-transfected with BPIFB1-Flag and VTN-His vectors. The graph summarises the data from three independent experiments. **P<0.01; ***P<0.001; n.s., no significance. Scale bars=200 μm.

Figure 5

BPIFB1 inhibits VTN-ITGAV-complex formation and downstream activation of the FAK signalling pathway. (A) The ITGAV protein was immunoprecipitated by the VTN antibody in 5-8F cells. (B) Expression of ITGAV and some proteins associated with the FAK-signalling pathway, including FAK, p-FAK, Src, p-Src, MEK, and ERK, was detected by western blot in 5-8F cells transfected or co-transfected with BPIFB1-Flag and VTN-His vectors. (C) BPIFB1, VTN, ITGAV, and Src expression was detected by immunofluorescence in 5-8F cells transfected or co-transfected with BPIFB1-Flag and VTN-His vectors. Scale bar=29 μm. Five randomly selected areas were scanned, and the data are shown as the mean±s.d. **P<0.01; ***P<0.001; n.s., no significance. (D) BPIFB1, VTN, ITGAV, FAK, and Src expression as determined in 20 NPC and 11 NPE tissues by immunohistochemistry. H&E staining was also performed on these tissues. Data shown are representative images of the respective molecular expression analyses. Normal rabbit immunoglobulin G was used as the isotype control. Magnification= × 200, scale bars=50 μm; Magnification= × 400, scale bars=20 μm. (E) Administration of the FAK inhibitor PND1186 decreased p-FAK expression. (F and G) PND1186 suppresses NPC cell migration and invasion in 5-8F cells induced by BPIFB1 loss or VTN overexpression. The graph summarises data from three independent experiments. Scale bars=200 μm.

Figure 5

BPIFB1 inhibits VTN-ITGAV-complex formation and downstream activation of the FAK signalling pathway. (A) The ITGAV protein was immunoprecipitated by the VTN antibody in 5-8F cells. (B) Expression of ITGAV and some proteins associated with the FAK-signalling pathway, including FAK, p-FAK, Src, p-Src, MEK, and ERK, was detected by western blot in 5-8F cells transfected or co-transfected with BPIFB1-Flag and VTN-His vectors. (C) BPIFB1, VTN, ITGAV, and Src expression was detected by immunofluorescence in 5-8F cells transfected or co-transfected with BPIFB1-Flag and VTN-His vectors. Scale bar=29 μm. Five randomly selected areas were scanned, and the data are shown as the mean±s.d. **P<0.01; ***P<0.001; n.s., no significance. (D) BPIFB1, VTN, ITGAV, FAK, and Src expression as determined in 20 NPC and 11 NPE tissues by immunohistochemistry. H&E staining was also performed on these tissues. Data shown are representative images of the respective molecular expression analyses. Normal rabbit immunoglobulin G was used as the isotype control. Magnification= × 200, scale bars=50 μm; Magnification= × 400, scale bars=20 μm. (E) Administration of the FAK inhibitor PND1186 decreased p-FAK expression. (F and G) PND1186 suppresses NPC cell migration and invasion in 5-8F cells induced by BPIFB1 loss or VTN overexpression. The graph summarises data from three independent experiments. Scale bars=200 μm.

Figure 6

BPIFB1 inhibits VIM-induced migration and invasion of NPC cells. (A) BPIFB1 and VIM colocalisation in 5-8F cells. DAPI-stained nuclei: blue; anti-Flag-BPIFB1: red; anti-His-VIM: green; merged image represents the overlay of DAPI, Flag, and His signals; BPIFB1 and VIM colocalisation: yellow. Scatter analysis shows the signals of channel 561 (BPIFB1) and 488 (VIM). Pearson’s correlation coefficient of colocalisation is indicated in the top right corner of the plot. Scale bar=29 μm. (B) BPIFB1 and VIM expression was confirmed by western blot in 5-8F cells transfected or co-transfected with BPIFB1-Flag and VIM-His vectors using anti-Flag and anti-His primary antibodies. (C and D) Representative images of migration and transwell Matrigel invasion assays of 5-8F cells transfected or co-transfected with BPIFB1-Flag and VIM-His vectors. Data represent the mean±s.d. and are representative of three independent experiments. *P<0.05; **P<0.01; ***P<0.001; n.s., no significance.

Figure 7

BPIFB1 inhibits the EMT process through VTN and VIM in NPC cells. Expression of typical EMT markers, including E-cadherin, N-cadherin, BPIFB1, VIM, β-catenin, Slug, and Snail, according to western blot proteins from of 5-8F, HNE2, and HONE1 cells transfected with the (A) BPIFB1-Flag overexpression vector or (B) co-transfected with the BPIFB1-Flag and VTN-His vectors. GAPDH was used as an internal control. (C) Schematic model illustrating the role of BPIFB1 in regulating NPC migration and invasion by binding and interacting with VTN and VIM.