Epstein-Barr virus-encoded LMP2A induces an epithelial-mesenchymal transition and increases the number of side population stem-like cancer cells in nasopharyngeal carcinoma

Abstract

It has been recently reported that a side population of cells in nasopharyngeal carcinoma (NPC) displayed characteristics of stem-like cancer cells. However, the molecular mechanisms underlying the modulation of such stem-like cell populations in NPC remain unclear. Epstein-Barr virus was the first identified human tumor virus to be associated with various malignancies, most notably NPC. LMP2A, the Epstein-Barr virus encoded latent protein, has been reported to play roles in oncogenic processes. We report by immunostaining in our current study that LMP2A is overexpressed in 57.6% of the nasopharyngeal carcinoma tumors sampled and is mainly localized at the tumor invasive front. We found also in NPC cells that the exogenous expression of LMP2A greatly increases their invasive/migratory ability, induces epithelial-mesenchymal transition (EMT)-like cellular marker alterations, and stimulates stem cell side populations and the expression of stem cell markers. In addition, LMP2A enhances the transforming ability of cancer cells in both colony formation and soft agar assays, as well as the self-renewal ability of stem-like cancer cells in a spherical culture assay. Additionally, LMP2A increases the number of cancer initiating cells in a xenograft tumor formation assay. More importantly, the endogenous expression of LMP2A positively correlates with the expression of ABCG2 in NPC samples. Finally, we demonstrate that Akt inhibitor (V) greatly decreases the size of the stem cell side populations in LMP2A-expressing cells. Taken together, our data indicate that LMP2A induces EMT and stem-like cell self-renewal in NPC, suggesting a novel mechanism by which Epstein-Barr virus induces the initiation, metastasis and recurrence of NPC.

Figure 1. Endogenous and exogenous expression of LMP2A detected using monoclonal antibodies.

A. Stable expression of LMP2A in NPC cells. The left and right panels reveal the stable ectogenic expression of LMP2A in both CNE2 and SUNE1 NPC cell lines. The upper and lower panels show mRNA and protein levels, respectively. B. Immunofluorescence staining clearly showing the membrane localization of LMP2A in the CNE2 and SUNE1 cell lines. C. Immunohistochemical analysis of LMP2A expression in a nude mice xenograft of LMP2A-expressing CNE2 cells using MoAb 4A11B3A3 or control IgG. D. Immunohistochemical analysis of endogenous LMP2A expression in NPC biopsies using MoAb 4A11B3A3 or control IgG. Representative moderate (left panel) and strong (middle panel) staining results are shown. E. Immunohistochemical analysis (left panel) with the MoAb 4A11B3A3 shows specific staining of LMP2A in NPC tumor nests (indicated by black arrows) but not in adjacent normal tissues (indicated by red arrow). The middle and right panels show that LMP2A is usually expressed in the invasive tumor front cells and is mainly localized on the cell membrane.

Figure 2. The overexpression of LMP2A in NPC cell lines induces EMT-like cellular marker alterations.

A. Western blotting of E-cadherin, α-catenin, vimentin, fibronectin and snail in the vector control and LMP2A-expressing cells. B. Immunofluorescence staining reveals a reduced expression of E-cadherin, α-catenin and an increased expression of vimentin, fibronectin and snail in LMP2A-expressing cells in comparison with the vector controls.

Figure 3. LMP2A induces stem-like properties in NPC cells.

A. The right and left panels show up-regulated expression of the stem cell markers ABCG2, Bmi1, Nanog, SOX2 at both the mRNA and protein levels, respectively. B. LMP2A increases the size of the side population (SP) cells. Flow cytometric profiles of SP cells among the CNE2 and SUNE1 NPC cell lines after stable expression of LMP2A. SP cell profiles in the presence of verapamil are shown in the bottom panels. The percentages of SP cells are indicated. C. LMP2A induces stem cell-like self-renewal properties. Sphere sizes are shown in the left panels, and the numbers of spheres in both LMP2A-positive and -negative cells are shown in the right panels.

Figure 4. LMP2A enhances the transforming ability of NPC cells.

A. Colony formation assay of the CNE2 and SUNE1 cell lines. Upon the stable expression of LMP2A, these cells form bigger (left panel) and more colonies (right panel) compared with the vector control cells. Error bar = SD. B. The anchorage-independent growth in soft agar of CNE2 and SUNE1 cells with or without exogenous LMP2A expression. Error bar = SD; scale bar = 200 µm.

Figure 5. LMP2A enhances the initial tumor cell population in CNE2 cells.

A. Tumor growth curves after injection of nude mice with LMP2A or vector control expressing NPC CNE2 cells. Once they become palpable, the LMP2A tumor (red) cells grow at a higher rate than the vector control (blue) cells in all cases. This difference becomes more pronounced when the injected cell number is 10⁴ and 10³. B. Tumors become palpable at an earlier timepoint and grow at a faster rate in LMP2A-expressing cells compared with vector control cells. Tumor formation was monitored for 20 days after injection of nude mice. Differences were not evident when the injected cell number was above 10⁵. However, at 10³ and 10⁴ cells, the differences were significant. In the case of injections with 10⁴ cells followed by monitoring for 17 days, 9 tumors arose in 10 mice for LMP2A-expressing cells, whereas only 6 of 10 mice infected with vector control cells formed tumors. When 10³ cells were injected followed by monitoring for 20 days, all mice formed tumors in the LMP2A-expressing cell group, but only half of the mice did so in the vector control group.

Figure 6. The expression of LMP2A correlates with the expression of EMT and stem cell-related markers in NPC.

A. The mRNA level of LMP2A, ABCG2, Bmi-1, E-cadherin and Fibronectin in NPC biopsy samples and control inflammatory samples measured by real time RT-PCR. T = tumor, N = normal. B. LMP2A correlates positively with ABCG2, Bmi-1, Fibronectin, and negatively with E-cadherin in tissue samples. T = tumor, N = normal. C. The expression of LMP2A, Bmi-1 and E-cadherin in a cohort of NPC biopsy using immunohistochemistry assay. Final magnification 40X, scale bar = 50 µm. D. LMP2A significantly correlates with Bmi-1and E-cadherin in NPC tissues. The coordinate axis equals to the percentage of positive cells in whole cancer cells.

Figure 7. Akt contributes to the enhancement of the SP by LMP2A.

A. Phospho-Akt (Thr308) and phospho-GSK3β were analyzed by Western blotting. B. The suppression of phospho-Akt (Thr308) in LMP2A-expressing and vector control cells after Akt inhibitor treatment determined by western blot analysis. C. The reduction in the SP after Akt inhibitor treatment detected by flow cytometry.