- Co-expression of low-risk HPV E6/E7 and EBV LMP-1 leads to precancerous lesions by DNA damage. - Because double infection of high-risk HPV and Epstein-Barr virus (EBV) is known to be involved in oral carcinogenesis, we hypothesized that low-risk HPV and EBV co-infection can transform the oral cells. - To verify our hypothesis, we evaluated the transformation activity of cell lines expressing both low-risk HPV E6/E7 and EBV LMP-1.. - Methods: We transduced HPV6, 11 and 16 E6/E7 genes and EBV LMP-1 gene into primary mouse embryonic fibroblasts. - Results: Co-expression of low-risk HPV6 E6 and EBV LMP-1 showed increased cell proliferation, elevated NF- κ B activity and reduced p53 induction. - Moreover, co-expression of low-risk HPV6 E6 and EBV LMP-1 induced DNA damage, escaped from apoptosis under genotoxic condition and suppression of DNA damage response (DDR). - Co-expression of low-risk HPV11 E6/E7 and EBV LMP-1 demonstrated similar results. - Compared with the cells co-expressing high-risk HPV16 E6 and EBV LMP-1 that induce. - transformation, co-expression of low-risk HPV6 E6 and EBV LMP-1 was associated with low MMP-2, paxillin and Cat-1 expression.. - Conclusions: The co-expression of low-risk HPV E6/E7 and EBV LMP-1 does not induce malignant transformation, but it allows accumulation of somatic mutations secondary to increased DNA damage and suppression of DDR. - Thus, double infection of low-risk HPV and EBV could lead to precancerous lesions.. - Keywords: Low-risk HPV, High-risk HPV, EBV LMP-1, Co-expression, Precancerous lesion. - By contrast, low-risk HPV E6 and E7 gene ex- pression only weakly inactivates p53 [3] and pRb [4].. - Consequently, low-risk HPV is considered to have a lower transformation activity.. - However, it remains uncertain whether low-risk HPV actually causes oral cancer.. - We previously reported that co-expression of high-risk HPV16 E6 and EBV LMP-1 transforms primary mouse embryonic fibroblasts (MEFs) [15]. - Since double infec- tion of high-risk HPV and EBV is involved in oral car- cinogenesis, we hypothesized that low-risk HPV could lead to changes in oral cells if the cells are co-infected with EBV. - In this study, we examined transformation activity of MEFs co- expressing low-risk HPV6/11 E6/E7 and EBV LMP-1, comparing with those co-expressing high-risk HPV16 E6 and EBV LMP-1. - Plas- mids expressing EBV LMP-1, (i.e. - Co-expression of low-risk HPV6 E6 and EBV LMP-1 increased cell proliferation via NF- κ B activation and reduced p53 induction. - To evaluate the transformation activity by low-risk HPV6 E6 and EBV LMP-1 co-expression, we firstly ex- amined cell proliferation rates using MTT assay. - In particular, the cells co-expressing both HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) and HPV16 E6 + EBV LMP-1. - (16E6 + LMP-1) showed significantly higher proliferation rates regardless of whether the E6 was from high-risk or low-risk HPV (*p <. - HPV6 E7 + EBV LMP-1 (6E7 + LMP-1) also demonstrated elevated prolif- eration compared with mock cells, as seen in Supple- mental Figure S2A.. - It has been reported that EBV LMP-1 activates nu- clear factor-kappa B (NF-κB) pathways [17], while HPV E6 and E7 suppress or degrade p53 and pRb [1]. - To examine the relationship in the cells express- ing low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) between high proliferative activity and NF-κB activ- ity, NF-κB-related proteins and its transactivation ac- tivity were measured by Western blotting and luciferase reporter assay, respectively. - On Western blotting analyses, low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) showed phosphorylation of IκB, pro- cessing of p105 to p50, and high RelB expression (Fig. - On reporter assay for NF-κB, low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) had higher activity than cells expressing only HPV6 E6 (6E6) (*p <. - As EBV LMP-1 suppresses transcription of p53 [18], we performed a reporter assay to examine p53 induction in each cell line. - The cells co-expressing low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) re- duced p53 induction significantly more than those expressing HPV6 E6 alone (6E6) (Fig. - However, both NF-κB activation and p53 suppression of low- risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) were lower than those resulting from high-risk HPV16 + EBV LMP-1 (16E6 + LMP-1) (Fig. - Experiments with HPV11 E6/E7 + EBV LMP-1 (11E6 + LMP-1 and 11E7 + LMP-1) yielded similar findings, as seen in Supplemental Figure. - Co-expression of low-risk HPV6 E6 and EBV LMP-1 induced DNA damage, escape from apoptosis and suppression of DNA damage response (DDR) under genotoxic condition. - In particular, the cells ex- pressing low-risk HPV6/11 E6/E7 + EBV LMP-1 (6E6 + LMP-1, 6E7 + LMP-1, 11E6 + LMP-1 and 11E7 + LMP-1) induced greater DNA damage than the cells expressing low-risk HPV6/11 E6 or E7 alone (6E6, 6E7, 11E6 and 11E7) (Fig. - A comet assay also showed that low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) induced longer comet tails than mock cells and increased the comet positivity rate (Fig.. - Low-risk HPV6/11 E6/7 + EBV LMP-1 (6E6 + LMP-1, 6E7 + LMP-1, 11E6 + LMP-1 and 11E7 + LMP-1) had a lower percentage of TUNEL-positive cells than MEFs expressing a single viral protein (6E6, 6E7, 11E6 and 11E7) (Fig. - The cells co- expressing low- or high-risk HPV E6 + EBV LMP-1 (6E6 + LMP-1 and 16E6 + LMP-1) grew faster than those expressing single viral protein and mock cells. - B Low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) showed phosphorylation of I κ B, processing of p105 to p50, and high RelB expression which were comparable to those seen in high-risk HPV16 E6 and EBV LMP-1 (16E6 + LMP-1). - Although high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) showed increased ratios of both pI κ B/I κ B and p50/. - p105, low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) had a high pI κ B/I κ B ratio but a p105/p50 ratio comparable to mock cells. - E A luciferase assay for NF- κ B activity indicated that low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) had higher activity than cells expressing HPV6 E6 alone (6E6).. - However, high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) showed more than eight-fold increase in activity over low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1). - p53 expression decreased more in low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) than those expressing HPV6 E6 alone (6E6). - However, p53 suppression in low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) was lower than that in high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1). - However, these DDR proteins were suppressed in cells expressing low- risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) suggesting inhibition of DDR (Fig. - Taken together, low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) caused greater DNA damage and suppression of DDR, leading to muta- genesis identical to that seen with high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1).. - No malignant phenotype was seen in the cells co- expressing low-risk HPV6 E6 and EBV LMP-1. - To assess the malignant features associated with low- risk HPV6 E6 and EBV LMP-1 co-expression, we per- formed soft agar colony formation and cell invasion as- says. - In the former, high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) cells formed abundant colonies of vari- ous sizes in the agar medium, but low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) cells formed no colony (Fig. - In cell invasion assay, high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) cells displayed higher invasive capacity than other clones, while low-risk HPV6/11 E6/E7 + EBV LMP-1 (6E6 + LMP-1, 6E7 + LMP-1, 11E6 + LMP-1 and 11E7 + LMP-1) exhibited only slightly more invasion than mock cells, a result comparable to that with clones expressing only one of the viral proteins (Fig. - Although all the mice injected with high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) cells developed tumors, no tumor was found in mice injected with low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) cells, low-risk HPV11 E6/E7 + EBV LMP-1 (11E6 + LMP-1 and 11E7 + LMP-1) cells or mock cells (Table 2 and Supplemental Table S4).. - Anchorage-independent growth was suppressed in the cells co-expressing low-risk HPV6 E6 and EBV LMP-1 through downregulation of adhesion signaling. - Despite the mutagenetic effects of low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) cells and low-risk HPV11 E6/E7 + EBV LMP-1 (11E6 + LMP-1 and 11E7 + LMP-1), they were unable to induce transformation (Table 2 and Supplemental Table S4). - The most significant difference between low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) and high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) cells was invasiveness and anchorage-independent growth. - Conversely, it suppressed the effect of LMP-1 on MMP-2 induction, which was seen in low- risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) (Fig. - However, low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) induced higher rates of DNA damage than cells expressing HPV6 E6 alone (6E6). - Both low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) and high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) showed significantly increased γ -H2AX signals compared with the cells expressing a single viral protein. - More comet signals and longer comet tails were observed in the cells expressing HPV6 E6 + EBV LMP-1 (6E6 + LMP-1). - 0.01), but there was no significant difference between low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) and high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1). - E Low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) showed less TUNEL signals than mock cells and single low-risk HPV6 E6 (6E6) expressing cells under genotoxic conditions. - Under genotoxic conditions, low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) and high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) showed suppression in both the ATR-ChK1 and ATM- ChK2 pathways. - risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) and high- risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) using im- munocytochemistry and Western blotting. - Although some of the viral proteins demonstrated increased levels of paxillin, low-risk HPV6 E6 + EBV LMP-1 (6E6. - Elevated Cat-1 expression was only seen in cells with high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) (Fig. - We confirmed similar cytoplasmic expres- sion of MMP-2, paxillin and Cat-1 in the tumors arising in the nude mice injected with high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) (Supplemental Figure S5).. - High-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) in- duced greater Akt signaling, whereas low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) showed low Akt activity (Fig. - These results suggest that co-infection with a low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) is. - A High-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) formed many colonies on DMEM with agar, whereas low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) was unable to form any colonies. - High-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) formed about 40 colonies of various sizes, but low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) demonstrated no colony formation. - C High-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) displayed higher invasive capacity than other clones, whereas low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) exhibited only slightly more invasion than mock cells. - High-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) showed a two-fold higher invasive capacity than mock cells. - However, low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) demonstrated a slightly increased invasive capacity compared with the single viral protein expressing clones. - incapable of causing invasion and anchorage- independent growth because such an expression would not affect adhesion signaling by MMP-2, paxillin, and Cat-1 in the same way that high-risk HPV16 E6 and EBV LMP-1 co-expression does.. - Our study demonstrated that co-expression of low-risk HPV6/11 E6/E7 and EBV LMP-1 (HPV6 E6 + LMP-1, HPV6 E7 + LMP-1, HPV11 E6 + LMP-1 and HPV11 E7 + LMP-1) led to increased cell proliferation, elevated NF-κB activity, and reduced induction of p53. - However, in contrast to finding with high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1), low-risk HPV6 E6 + EBV LMP- 1 (6E6 + LMP-1) did not induce anchorage-independent growth, invasiveness and tumor formation in nude mice.. - These findings suggest that a co-infection with a low- risk HPV and EBV increases mutagenicity but does not cause malignant transformation.. - Compared with high-risk HPV E6 and E7, low-risk HPV genes had little effect on p53 and pRb, eliciting weak degradation of those tumor suppressor genes [1].. - This may explain why low-risk HPVs evoke neither immortalization nor transformation.. - In previous reports, we demonstrated that dual expres- sion of high-risk HPV16 E6 and EBV LMP-1 in primary MEFs induced transformation, whereas either viral protein alone did not [15]. - In addition, transformation of cells co- expressing high-risk HPV16 E6 and EBV LMP-1 is associ- ated with suppression of DDR and increased NF-κB activ- ity [15]. - In the present study, low-risk HPV6/11 E6/E7 + EBV LMP-1 (6E6 + LMP-1, 11E6 + LMP-1 and 11E7 + LMP- 1) activated the NF-κB pathway and suppressed p53 transcriptional activity, an effect not seen in cells ex- pressing low-risk HPV6/11 E6 or E7 alone (6E6, 11E6 and 11E7). - However, we found that low-risk HPV6 E6 and EBV LMP-1 co-expression was unable to cause transform- ation. - One striking difference between co-expression of EBV LMP-1 with E6 or E7 from low- versus high-risk HPV was the effects on anchorage-independent growth. - Tong and Howley reported that paxillin interacts with high-risk HPV E6 to cause transformation, but these effects are not seen with low-risk HPV E6 [28]. - Our data showed that low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) induced less expression of paxillin and Cat-1 than high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1). - Probably, the insufficient anchorage-independent growth with low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP- 1) may be explained by downregulation of cell signaling via paxillin and Cat-1. - Similar to high-risk HPV E6 and E7, EBV LMP-1 pro- tein isolated from nasopharyngeal carcinoma also induces MMPs [31]. - As with our other findings, MMP-2 expression with low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) was lower than with high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1). - Clones only expressing high-risk HPV16 E6 or EBV LMP-1 (LMP-1, 16E6 and 16E6 + LMP-1) showed increased MMP-2 expression compared with low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1). - Some of the viral protein expressing clones demonstrated increased levels of paxillin, but low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) displayed comparable levels with mock cells. - Elevated Cat- 1 expression was only seen in high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1). - Low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) expressed low levels of Cat-1 that are comparable to those of mock cells. - High-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1) demonstrated an overall increase in the expression of adhesion molecules and higher Akt expression, whereas low-risk HPV6 E6 + EBV LMP-1 (6E6 + LMP-1) showed low expression and activity. - As summarized in Table 3, our findings with regard to the ability of HPV to affect various factors involved oncogenesis indicate that low-risk HPV proteins alone cause little effect but that low-risk HPV E6 or E7 along with EBV LMP-1 leads to significant mutagenesis with- out malignant transformation seen with high-risk HPV16 E6 + EBV LMP-1 (16E6 + LMP-1). - Because low-risk HPV E6/E7 + EBV LMP-1 (6E6 + LMP-1, 6E7 + LMP-1, 11E6 + LMP-1 and 11E7 + LMP-1) induces more muta- genesis than infection with low-risk HPV E6/E7 alone (6E6, 6E7, 11E6 and 11E7), co-infection of low-risk HPV and EBV may therefore induce precancerous lesions that could be more easily transformed if they are subse- quently subjected to further mutagenic effects.. - In summary, our research demonstrated that the co- expression of low-risk HPV6/11 E6/E7 and EBV LMP-1 does not induce malignant transformation, but it allows accumulation of somatic mutations secondary to in- creased DNA damage and suppression of DDR. - Low-risk HPV. - Low-risk HPV + EBV. - Increasing rates of low-risk human papillomavirus infections in patients with oral cavity squamous cell carcinoma: association with clinical outcomes. - Decreased expression of E- cadherin and increased invasive capacity in EBV-LMP-transfected human epithelial and murine adenocarcinoma cells
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