Toll-like receptor 3 (TLR3) functions as a pivotal target in latent membrane protein 1 (LMP1)-mediated nasopharyngeal carcinoma cell proliferation

Abstract

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) activation of NF-κB is pivotal for EBV-infected B lymphocyte survival. Herein, we found that LMP1 markedly rescued the suppressed the proliferation of several nasopharyngeal carcinoma (NPC) cell lines caused by a Toll-like receptor 3 (TLR3) ligand poly (I:C). We profiled the expression alterations of TLR3 and LMP1 within these NPC cell lines in response to poly (I:C) treatment, and found a high correlation between them ws found, suggesting potential involvement of TLR3 in LMP1 signaling. Then, cells deficient in TLR3 were used to assess its role in poly (I:C)-induced inhibition of cell proliferation and LMP1-mediated NF-κB activation. NF-κB p65 activation and the consequent pro-inflammatory responses were unresponsive to poly (I:C) stimulation after TLR3 knockdown (KD), and NOS2 and MMP9 were substantially suppressed in CNE1-745, but nearly normal in LMP1-overexpressed CNE1-LMP1-745 cells. This suggests an alternative pathway that LMP1 may depend on, in terms of NOS2 and MMP9 regulation, whereas an unusual TLR3-dependent expression of c-Myc was identified. Consistently, poly (I:C)-induced retarded growth was reversed by TLR3 silencing, which was especially enhanced in LMP1-overexpressed cells. TLR3 is essential for poly (I:C)-incited NPC cell death, and occupies a critical role in LMP1-mediated NF-κB activation. Our findings provide new insight into the mechanism underlying LMP1-involved EBV-associated pathogenesis of refractory NPC, thereby potentially improving treatment outcome.

Keywords: EB virus; NF-κB; Nasopharyngeal carcinoma (NPC); TLR3; latent membrane protein 1 (LMP1).

Figure 1

LMP1 rescued the inhibited NPC cell proliferation caused by poly (I:C) treatment. The proliferation rates of CNE1 (A), CNE1-LMP1 (B), HNE2 (C), and HNE2-LMP1 (D) in the presence and absence of 50 μg/mL poly (I:C) at varying time intervals were assessed by CCK-8 assay. Data are presented as the mean ± SD of triplicate determinations.

Figure 2

LMP1 promoted TLR3 expression. A. LMP1 overexpression in CNE1 and HNE2 substantially increased TLR3 production. For sake of comparison, proteins blotted to the same piece of X-ray film but in nonadjacent columns were put adjacent, which was displayed by a solid line. B. The expression profiles of diverse NPC cell lines were evaluated by western blot assay. GAPDH was used as an internal control and the expression of LMP1 and TLR3 were normalized to that of GAPDH. Correlation plots demonstrating LMP1 expression plotted against TLR3 in diverse NPC cell lines, and a tight correlation was observed (R²=0.796).

Figure 3

LMP1-transfection and poly (I:C) stimulation were able to induce TLR3 expression and NF-κB p65 activation (A). The immunoblotting assay was carried out to visualize the expression patterns of LMP1, TLR3, and p65 in CNE1 and CNE1-LMP1 cells with or without poly (I:C) stimulation. The expression alterations of LMP1 (B), TLR3 (C), and p65 (D) were presented in a histogram. All experiments were performed in triplicate, and data are presented as the mean ± SD.

Figure 4

LMP1 rescued the impaired NF-κB p65 activation and subsequent pro-inflammatory gene production in the TLR3-knockdown cells. CNE1-745 and CNE1-LMP1-745 were generated through TLR3 knockdown in WT CNE1 and CNE1-LMP1 cell lines. The expression levels of TLR3 and p65-regulated pro-inflammatory genes including iNOS, MMP-9, and c-Myc in all these cell lines were assessed by immunoblotting assay with corresponding antibodies, with or without pretreatment by 50 μg/mL of poly (I:C) for 16 h. GAPDH was used as an internal control to ensure equal loading.

Figure 5

TLR3 silencing counteracted the inhibitory effects of poly (I:C) on the proliferation of CNE1 and CNE1-LMP1 cell lines. Blank: cells without RNA interference; scr: scramble siRNA, another negative control that has the same nucleotide composition, but not the same sequence. *P<0.05 compared to the negative controls in the presence of poly (I:C).