Co-expression of low-risk HPV E6/E7 and EBV LMP-1 leads to precancerous lesions by DNA damage

Abstract

Background Low-risk human papillomavirus (HPV), such as types 6 and 11, is considered non-oncogenic, but these types have been detected in oral cancer tissue samples, suggesting their possible involvement in oral carcinogenesis. 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. The cell lines were examined for indices of transformation activity such as proliferation, induction of DNA damage, resistance to apoptosis, anchorage-independent growth, and tumor formation in nude mice. To evaluate the signaling pathways involved in transformation, NF-κB and p53 activities were analyzed. We also assessed adhesion signaling molecules associated with anchorage-independent growth such as MMP-2, paxillin and Cat-1. Results Co-expression of low-risk HPV6 E6 and EBV LMP-1 showed increased cell proliferation, elevated NF-κB 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. However, it led to no malignant characteristics such as anchorage-independent growth, invasiveness and tumor formation in nude mice. 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.

A Review on the Role of Vascular Endothelial Growth Factor Receptors in Human Papillomavirus-related Oral Squamous Cell Carcinoma

“…By contrast, these proteins show a weaker binding ability in low-risk HPVs types, with no degradation of p53 or pRb. 34 As a tumour-suppressor gene, p53 functions as a nuclear transcription factor, transactivating many target genes in the induction of cell cycle arrest and, or apoptosis. 35 When DNA damage occurs, p53 expression increases, activating the downstream target p21 and signalling the cell to enter cell cycle arrest.…”

How Does Epstein–Barr Virus Interact With Other Microbiomes in EBV-Driven Cancers?

“…When co-expressed with low-risk HPV E6/E7 (HPV 6/11), EBV LMP-1 failed to induce the malignant transformation as that co-expressed with high-risk HPV E6/E7 (in primary mouse embryonic fibroblast (MEF) cells ( Shimabuku et al., 2014 ; Blanco et al., 2021 ). However, the co-expression was proved to lead to precancerous lesions ( Uehara et al., 2021 ) since it promoted the accumulation of DNA damage-related somatic mutations.…”

High-Risk Human Papillomavirus and Epstein–Barr Virus Coinfection: A Potential Role in Head and Neck Carcinogenesis

“…Moreover, a HPV16 E6 and LMP1 copresence reduces the expression of p53, RB, p27, and ChK1, suggesting a synergism between E6/LMP1 proteins and DDR [ 219 ]. Similarly, under genotoxic conditions, a reduction in DDR proteins, such as ATM, ATR, Chk1, and Chk2, as well as an increased resistance to apoptosis, was evidenced in HPV16 E6+/EBV LMP1+ cells compared to those expressing only the HPV16 E6 protein [ 220 ]. In fact, the expression of both HPV16 E6 and EBV LMP1 was able to increase the proliferation rates of MEFs cells compared to those cells expressing the HPV16 E6 oncoprotein alone, which was associated with NF-κB activation and p53 suppression [ 219 , 220 ].…”

“…Similarly, under genotoxic conditions, a reduction in DDR proteins, such as ATM, ATR, Chk1, and Chk2, as well as an increased resistance to apoptosis, was evidenced in HPV16 E6+/EBV LMP1+ cells compared to those expressing only the HPV16 E6 protein [ 220 ]. In fact, the expression of both HPV16 E6 and EBV LMP1 was able to increase the proliferation rates of MEFs cells compared to those cells expressing the HPV16 E6 oncoprotein alone, which was associated with NF-κB activation and p53 suppression [ 219 , 220 ]. In addition, increased soft agar colonies, as well as tumor formation in nude mice, were evidenced when HPV16 E6+/EBV LMP1+ cells were compared to mock cells.…”

“…In addition, increased soft agar colonies, as well as tumor formation in nude mice, were evidenced when HPV16 E6+/EBV LMP1+ cells were compared to mock cells. This finding was associated with increased levels of AKT and adhesion molecules (MMP2, CAT-1, and paxillin) in HPV16 E6 +/ EBV LMP1+ cells [ 220 ]. However, no effect in the proliferation rates was evidenced when MEFs expressing EBNA1 or EBNA1/E6 were analyzed [ 219 ].…”