NF-κB p65 Subunit Is Modulated by Latent Transforming Growth Factor-β Binding Protein 2 (LTBP2) in Nasopharyngeal Carcinoma HONE1 and HK1 Cells

Abstract

NF-κB is a well-characterized transcription factor, widely known as a key player in tumor-derived inflammation and cancer development. Herein, we present the functional and molecular relevance of the canonical NF-κB p65 subunit in nasopharyngeal carcinoma (NPC). Loss- and gain-of-function approaches were utilized to reveal the functional characteristics of p65 in propagating tumor growth, tumor-associated angiogenesis, and epithelial-to-mesenchymal transition in NPC cells. Extracellular inflammatory stimuli are critical factors that trigger the NF-κB p65 signaling; hence, we investigated the components of the tumor microenvironment that might potentially influence the p65 signaling pathway. This led to the identification of an extracellular matrix (ECM) protein that was previously reported as a candidate tumor suppressor in NPC. Our studies on the Latent Transforming Growth Factor-β Binding Protein 2 (LTBP2) protein provides substantial evidence that it can modulate the p65 transcriptional activity. Re-expression of LTBP2 elicits tumor suppressive effects that parallel the inactivation of p65 in NPC cells. LTBP2 was able to reduce phosphorylation of p65 at Serine 536, inhibit nuclear localization of active phosphorylated p65, and impair the p65 DNA-binding ability. This results in a consequential down-regulation of p65-related gene expression. Therefore, the data suggest that the overall up-regulation of p65 expression and the loss of this candidate ECM tumor suppressor are milestone events contributing to NPC development.

Fig 1. Knockdown of p65 reduces the abilities of in vitro colony formation, in vivo tumor formation, and angiogenesis in NPC.

(A) Western blot analysis shows the transient shRNA-mediated knockdown of p65 in HONE1 cells using shRNA. The p84 serves as a loading control. (B) Two-dimensional (2D) colony formation assay shows that stable and transient knockdown of p65 reduces the number of colonies formed compared to scramble-transduced HONE1 cells. Data represented on the bar graph is the average of triplicate experiments ± S.E.M. (C) Nude mice were inoculated subcutaneously with p65 shRNA and scramble HONE1 cells. p65 shRNA-transduced cells showed delayed and reduced tumor growth compared to scramble control cells. Each data point represents an average tumor volume of six injection sites inoculated for each cell population ± S.E.M. (D) HUVEC tube formation is abrogated with p65 shRNA conditioned medium compared to the scramble control conditioned medium from HONE1 cells. The bar charts indicate data obtained from an average of triplicate experiments ± S.E.M. The (*) indicates P-value < 0.05 for all graphs.

Fig 2. Inactivation of p65 reduces the abilities of in vitro colony formation, in vivo tumor formation, migration, invasion, and angiogenesis in NPC.

(A) Western blot analysis shows the expression of exogenous IκBα-SR with triple HA-tag (^) in HONE1 cells. The p84 served as a loading control. (B) 2D colony formation assay shows that IκBα-SR suppressed the colony-forming ability of HONE1 cells compared to pWPI-vector alone (VA). Bar graphs indicate data obtained from an average of triplicate experiments ± S.E.M. (C) Nude mice were inoculated subcutaneously with IκBα-SR and pWPI-VA HONE1 cells. IκBα-SR-transduced cells showed delayed and reduced tumor growth kinetics compared to pWPI-VA. Each data point represents an average tumor volume of six injection sites inoculated for each cell population ± S.E.M. (D) Wound healing analysis for pWPI-VA and IκBα-SR showed delayed migration of IκBα-SR cells compared to VA. Bar graphs show the percentage difference between IκBα-SR and VA cells ± S.E.M. (E) Migration chamber assays showed that IκBα-SR-expressing HONE1 cells reduced migration compared to VA cells. Data represented on the bar graph are the average of triplicate experiments ± S.E.M. (F) HUVEC tube formation was suppressed with IκBα-SR conditioned medium compared to that of VA cells. Data represented on the bar graph are the average of triplicate experiments ± S.E.M. The (*) for all graphs indicate P-value < 0.05.

Fig 3. Enhanced p65 expression promotes in vitro tumorigenic responses.

(A) Confocal visualization of cytolocalization of RFP-fusion p65 WT (63x magnification). Scale bar represents 1μm. (B) 2D colony formation assay showed increased number of colonies in p65 WT-overexpressing cells compared to RFP-tagged pLVX-VA cells. Bar graphs show the percentage difference between the number of colonies in p65 WT and VA cells ± S.E.M. (C) Migration chamber assays showed that p65 WT-overexpressing HONE1 cells enhanced migration compared to VA cells. Bar graphs illustrate the percentage difference between the relative migration abilities of p65 WT and VA cells ± S.E.M. (D) Wound healing analysis for pLVX-VA and p65 WT showed an increase in migratory potential of p65 WT transduced cells compared to VA. Bar graphs show the percentage difference between p65 WT and VA cells ± S.E.M. (E) HUVEC tube formation was augmented with p65 WT conditioned media compared to that of VA cells. Bar graphs illustrate the percentage difference between the tube measurements of p65 WT and VA conditioned media-induced HUVEC cells ± S.E.M. The above data were obtained from three independent experiments and the (*) for all graphs indicate P-value < 0.05.

Fig 4. p65 signaling pathway regulates the protein expression of EGFR and EMT markers.

(A) Western blot analysis of p65shRNA- HONE1 cells showed reduced levels of phosphorylated p65 S536, total p65, and total IκBα compared to scramble control. IκBα-SR- HONE1 cells only showed a decreased amount of phosphorylated p65 S536, but did not affect the overall amount of total p65 when compared to pWPI-VA. Endogenous and exogenous IκBα are labeled as shown. The p84 was used as a loading control for scramble and p65 shRNA, pWPI-VA and IκBα-SR, separately. Protein expression of N-cadherin and total EGFR were all reduced in p65 shRNA and IκBα-SR HONE1 cells compared to scramble and pWPI-VA control. The (^) indicates the exogenous 3xHA-IκBα-SR. (B) Western blot analysis of p65 WT-overexpressing HONE1 cells showed increased acetylation at K310 and enhanced phosphorylation at S536 compared to VA cells. The p65 WT overexpression increases the protein levels of IκBα, snail, slug, twist, N-cadherin, and sox9, compared to VA cells. The p84 was used as a loading control. The (✚) indicates the exogenous RFP-p65 WT. The (★) indicates the acetylation band in the exogenously expressed RFP-p65 WT.

Fig 5. LTBP2 regulates the p65 signaling pathway.

(A) Phosphorylated IKKα/β S176/180 and phosphorylated IκBα SS32/36 are also reduced in LTBP2-infected cells. Phosphorylated p65 serine 536 level is reduced in LTBP2-infected cells compared to VA in HONE1 and HK1. The p84 and α-tubulin were used as loading controls. (B) Matrigel plug tumors show a reduction in phosphorylated p65 S536 in LTBP2-transduced cells compared to VA. The p84 was used as loading control. (C) Subcellular fractionation results show that phosphorylated p65 S536 was reduced in the nuclear fraction of LTBP2-transduced cells compared to VA. The p84 was used as control to determine nuclear fraction, while α-tubulin was used as the control for the cytoplasmic fraction.

Fig 6. LTBP2 regulates the translocation and activities of p65.

(A) Immunofluorescence staining of phosphorylated p65 S536 (green fluorescence) revealed weaker staining intensity as well as decreased nuclear staining in LTBP2-infected cells compared to VA. Nuclei were stained with 4, 5-diamidino-2-phenylindole (DAPI, blue fluorescence). Scale bar represents 5μm. (B) NF-κB binding reporter assay showed reduced chemiluminescence intensity in the nuclear fraction of LTBP2-transduced cells compared to VA in both HONE1 and HK1. Data represented on the bar graph are the average of triplicate experiments ± S.E.M. (C) Real-time PCR of NF-κB target genes showed that the transcriptional activities of LTBP2-infected cells were reduced compared to VA in both HONE1 (top) and HK1 (bottom). The panel of genes include: RELA, IκBα, TWIST, MMP3, SOX9, ICAM, MCAM, EGFR, and FN1. GAPDH was used as an internal control. The above data were obtained from three independent experiments done in duplicate and the (*) for all graphs indicate P-value < 0.05.

Fig 7. LTBP2 suppresses tumorigenic properties in vitro and in vivo.

(A) Restoration of LTBP2 expression in HONE1 and HK1 cells using the lentiviral system, as detected by Western blot analysis. The p84 was used as a loading control. (B) 2D colony formation assay showed reduced number of colonies in LTBP2-expressing cells compared to pLVX-VA cells. Bar graphs show the percentage difference between number of colonies in LTBP2 and VA cells ± S.E.M. (C) 3D colony formation assay showed reduced number and size of LTBP2 cell colonies compared to VA. Percentage difference is represented by the bar graphs ± S.E.M. (D) In vivo tumor growth kinetics of LTBP2 and VA HONE1 and HK1 cells. Each data point represents an average tumor volume of six injection sites inoculated for each cell population ± S.E.M. (E) Wound healing analysis for HONE1 and HK1 cell lines showed delayed migration of LTBP2-transduced cells compared to VA. Bar graphs show the percentage difference between LTBP2 and VA cells ± S.E.M. (F) Real-time monitoring of cell migration using xCELLigence RTCA DP analyzer in LTBP2-transduced HONE1 and HK1 cells. The above data were obtained from three independent experiments and the (*) for all graphs indicate P-value < 0.05.

Fig 8. LTBP2 reduces angiogenesis in vitro and in vivo.

(A) Expression of secreted LTBP2 in conditioned media from HONE1 and HK1 LTBP2-transduced cells. Coomassie blue staining of total protein in conditioned media was used to indicate equal loading. (B) LTBP2-conditioned media from HONE1 and HK1 cells suppressed the tube formation ability of HUVEC cultures. Bar graph represents an average of triplicate experiments ± S.E.M. (C) Matrigel plug tumors were stained for CD34 to visualize the microvessels, as indicated by the black arrows. LTBP2-transduced cells displayed significantly fewer microvessels in matrigel plugs compared to VA cells, as indicated by the bar graphs. (D) Real-time PCR of HONE1 (top) and HK1 (bottom) LTBP2- and VA-transduced cells showed the down-regulation of angiogenesis-related genes with LTBP2 expression: IL6, IL8, VEGF 165, VEGF 189, total VEGF, ANG, TSP1, uPAR, PDGB, RANTES, and MCP1. The housekeeping gene, GAPDH, was used as an internal control. The above data were obtained from three independent experiments in duplicate and the (*) for all graphs indicate P-value < 0.05.