The latent membrane protein 1 of EpsteinBarr virus and loss of the INK4a locus: paradoxes resolve to cooperation in carcinogenesis in vivo
Jennifer Macdiarmid,
David Stevenson,
Donald H. Campbell and
Joanna B. Wilson1
Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, 54 Dumbarton Road, Glasgow G11 6NU, UK
1 To whom correspondence should be addressed. Tel: +44 141 330 5108; Fax: +44 141 330 4878; Email: joanna.wilson{at}bio.gla.ac.uk
 |
Abstract
|
---|
Nasopharyngeal carcinoma (NPC) is the most tightly EpsteinBarr virus (EBV)-associated tumour. The EBV oncoprotein latent membrane protein 1 (LMP1) is frequently expressed in NPC tumours and may play a role in the genesis of the disease. NPC tumours often exhibit loss of expression (by deletion or methylation) of the INK4a locus, which encodes the tumour suppressor genes p16INK4a and p14ARF. To investigate the contribution of LMP1 and INK4a loss to tumourigenesis, skin chemical carcinogenesis was conducted using PyLMP1 and INK4a null mice. Surprisingly, INK4a null mice developed significantly fewer papillomas than wild-type mice, nevertheless, the papillomas that did develop grew faster and converted more rapidly to carcinoma than controls. This indicates that while loss of the INK4a locus plays an important role in the later stages of tumourigenesis, initially its loss inhibits papilloma formation. Conversely, LMP1 promoted papilloma formation but paradoxically inhibited papilloma growth. Using cross-breeds, it was found that LMP1 cooperates with loss of the INK4a locus during epithelial tumourigenesis. The expression of LMP1 overcame the inhibition of papilloma formation observed in INK4a null mice, whilst the loss of the INK4a locus counteracted the inhibition of papilloma growth rate found in PyLMP1 mice. This suggests that LMP1 mediates the inhibition of papilloma growth via one or both of the INK4a locus products. Intriguingly, mice heterozygous for INK4a loss showed lesion growth rates intermediate between wild-type and null, demonstrative of haploinsufficiency. We propose that LMP1 acts at the early stages in carcinogenesis to promote the development of benign tumours and that early reduction of INK4a locus expression allows these lesions to expand in size. In addition, loss of the INK4a locus accelerates the development of a more aggressive lesion. Conversely, complete loss of the INK4a locus in an otherwise normal cell might inhibit lesion formation.
Abbreviations: DMBA 7,12-dimethylbenz[a]anthracene; EBV, EpsteinBarr virus; LMP, latent membrane protein; NPC, nasopharyngeal carcinoma; Rb, retinoblastoma; TBST, 5% w/v dried milk in 20 mM Tris, pH 7.8, 140 mM NaCl, 0.1% Tween-20; TPA, 12-O-tetradecanoylphorbol-13-acetate
 |
Introduction
|
---|
EpsteinBarr virus (EBV) is a human herpesvirus associated with malignancies of both lymphocytic and epithelial origin, including Burkitt's lymphoma, Hodgkin's disease, T cell lymphoma, gastric carcinoma and nasopharyngeal carcinoma (NPC). NPC is a malignancy of the squamous epithelium of the post-nasal cavity and is the most consistently EBV-associated tumour. Within the tumour, EBV DNA is thought to be clonal, which would indicate that the tumour represents a proliferation of a single EBV-infected cell.
The incidence of NPC varies with geographical location and possibly ethnic origin, which is suggestive of both genetic and environmental factors contributing to tumourigenesis. NPC is endemic in Southeast Asia (812 cases/104/year) and has the highest incidence in the Guangzhou region of Southern China (3080/104/year), where it is the major cause of death from cancer. NPC also occurs at high frequencies in the native populations of the Arctic and in the North African countries of the Mediterranean basin. Throughout the rest of the world, NPC occurs at low frequency (0.52/104/year) (1). Further, though Chinese émigrés to low risk areas maintain a high rate of NPC incidence, this falls in subsequent generations (2). This suggests that environmental factors present in China contribute to the disease (3). One such factor may be salted fish, a common dietary component in Chinese and native Arctic populations, which has been shown to contain mutagenic volatile nitrosamines (4). Similar compounds have been found in the spicy foods of African Mediterranean countries (5).
An increased risk of developing NPC has been linked to specific HLA haplotypes; inheritance of the haplotypes HLA-A2, Bw46 and A19B17 confers double the risk of developing the cancer (6,7). Further to this, affected sibling pair analysis has identified a putative NPC susceptibility gene which is linked to the HLA locus. This gene codes for the cytochrome P450 2E1 enzyme (CYP2E1), which can activate nitrosamines and other carcinogens. Individuals homozygous for the susceptibility allele have a 21-fold increased risk for NPC (8,9).
A common feature of NPC tumours is loss of expression of the INK4a locus tumour suppressor genes, p16INK4a and p14ARF. Around 41% of NPC tumours have a homozygous deletion across chromosome 9p21 (10,11) which spans the INK4a tumour suppressor locus, whereas up to 85% of primary tumours show loss of heterozygosity in the region (12). A further 22% of NPC tumours show hypermethylation at the 5' CpG island upstream of the INK4a locus (13). This indicates that the inactivation of either one or both of the INK4a products may be important in the development of NPC (reviewed in 14).
Within NPC tissue, the virus expresses EBNA-1, latent membrane protein (LMP) 1, LMP2a and 2b, the EBERs and the BamA RNA transcripts. The proportion of tumours reported to show readily detectable LMP1 protein expression varies between 50 and 70%, while detection of LMP1 transcripts is closer to 100% (15,16). LMP1 expression in premalignant lesions of NPC has also been reported (17).
LMP1 shows oncogenic activity in a variety of cell types in culture (1821). Furthermore, genetic deletion studies have shown that LMP1 expression is essential for the EBV-induced transformation of B lymphocytes (22). The expression of LMP1 in the epidermis of transgenic mice leads to an increase in proliferation and hyperplasia (23,24). In epithelial cell culture, LMP1 has been demonstrated to up-regulate EGFR expression (25), correlating with the observation that EGFR is over-expressed in nasopharyngeal carcinoma (26). LMP1 has also been shown to up-regulate the anti-apoptotic gene A20 (25), the interleukins 6 and 8, which are involved in the acute inflammatory response (27,28), matrix metalloproteinase 9, which promotes tissue invasion (29,30), and, in transgenic mouse epidermis, the proliferative keratins K6 and K14 and (transiently) the differentiative keratins K1 and K10 (23,24).
In contrast to its role as an oncogene, LMP1 has also been found to have growth inhibitory effects. Transient expression of LMP1 in both carcinoma cell lines and normal primary epithelial cells leads to an inhibition of epithelial cell growth (31). This inhibition of cell growth is also observed in various B cell lines and in BALB/3T3 fibroblasts (3234). Further analysis revealed that LMP1-mediated growth inhibition results from cellular cytostasis and not apoptosis (35). Crucially, the expression of LMP1 in an EBV-negative NPC cell line, CNE2, results in growth inhibition and an increased sensitivity to cisplatin-induced cell death (36).
The mouse skin model of multistage carcinogenesis provides an accessible tool to study epithelial tumour progression in transgenic mice. The genetic events which occur during the tumourigenic process have been well characterized (37). The model uses two classes of chemicals, initiators and promoters (38). Tumour initiators are mutagens which can prime a cell for tumourigenesis. Tumour promoters activate proliferation pathways, together resulting in lesion formation.
Chemical carcinogen treatment of PyLMP1 transgenic mice has shown that LMP1 augments the action of chemical promoters, enhancing epidermal lesion formation (24). Following chemical carcinogenesis, PyLMP1 mice develop significantly more papillomas than their wild-type siblings. However, more papillomas from the wild-type mice grew to form larger lesions, suggesting that while LMP1 increases papilloma formation, it may also inhibit lesion growth. Therefore LMP1 could be involved in the early stages of carcinogenesis, but its role in the later stages is unclear.
Classical chemical carcinogen studies on wild-type mouse skin have shown that the INK4a locus is usually lost in the later stages of mouse skin tumourigenesis, at the time of conversion of squamous cell carcinoma to the more aggressive spindle cell phenotype (39). The INK4a locus encodes proteins involved in both the Retinoblastoma (Rb) and p53 tumour suppressor pathways (40). p16INK4a is a cyclin-dependent kinase inhibitor which responds to aberrant mitogenic signals and inhibits entry into S phase by preventing CDK4 or CDK6 from phosphorylating the Rb protein. p19ARF (the homologue of human p14ARF) activates the transcription factor p53 in response to aberrant growth signals by binding to and inhibiting Mdm2 (human Hdm2). INK4a null mice, which express low levels of a truncated p19ARF and do not express p16INK4a, are particularly susceptible to the spontaneous development of sarcomas and lymphomas, with 69% of mice developing tumours by 29 weeks of age (41).
The aim of this study was to further investigate the roles of both LMP1 and loss of the INK4a locus during the early and late stages of the genesis of NPC. PyLMP1 transgenic mice and INK4a null mice were used to model these genetic events. We show that LMP1 and loss of the INK4a locus cooperate strongly during epithelial tumourigenesis. LMP1 promotes the growth of small, benign tumours, whereas loss of the INK4a locus allows these tumours to expand in size and accelerates the development of a more aggressive, malignant phenotype.
 |
Materials and methods
|
---|
Transgenic mouse lines
In these experiments PyLMP1 line 53 mice, expressing LMP1 in the epidermis (23), were cross-bred with INK4a null mice generated by the targeted deletion of exons 2 and 3 of the INK4a locus (41). All of the mice used in this study were a minimum of back-cross three into the chemical carcinogen-sensitive FVB strain (average 87.5% FVB at back-cross three). All mice were housed in a conventional facility.
DNA preparation and genotyping
A standard phenol/chloroform method was used to extract genomic DNA from mouse tail segments as described (42). PyLMP1 transgene status was tested by slot blot or by Southern blot using Biodyne nylon membrane (ICN) as described (42). The blots were hybridized with [
-32P]dCTP-labelled, randomly primed DNA probe fragments (Prime It II kit; Stratagene) and washed under stringent conditions (0.1 x SSC, 0.1% SDS at 68°C) before autoradiography. For the LMP1 transgene probe, a 3.8 kb BamHIEcoRI fragment from the PyLMP1 transgene plasmid was used (23).
The INK4a status was tested by PCR. For each reaction, 300 ng of genomic DNA and 50 pmol of each oligonucleotide primer were added to 45 µl of PCR Reddymix (Abgene) to a final volume of 50 µl and amplified for 35 cycles of 91°C for 30 s, 53°C for 30 s and 72°C for 30 s, followed by 10 min at 72°C. Two PCR reactions were set up for each sample. (i) Neomycin, amplifying a 150 bp fragment indicative of the presence of the introduced neomycin resistance cassette in null and heterozygous animals; primers: Neo forward: tga atg aac tgc agg acg agg; Neo reverse: aag gtg aga tga cag gag atc. (ii) p16 exon 2, amplifying a 306 bp fragment of exon 2 of the wild-type locus present in wild-type and heterozygous mice; primers: p16 exon 2 forward: gtg atg atg atg ggc aac gt; p16 exon 2 reverse: ctg ggc gac gtt ccc agc gg.
Chemical carcinogen treatment
Chemical carcinogen treatment was initiated with 6-week-old mice following a 21 week protocol essentially as described (24,43). The chemical initiator used was the mutagen 7,12-dimethylbenz[a]anthracene (DMBA), dissolved in acetone to a working concentration of 125 µg/ml (4.8 x 10-4 M). The chemical promoter used was the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, an analogue of diacylglycerol and a potent stimulator of protein kinase C), dissolved in acetone to a working concentration of 31.25 µg/ml (5 x 10-5 M). The regime involved one application of 200 µl DMBA to shaved dorsal skin, followed a week later by twice weekly treatments with 200 µl TPA, for a further 20 weeks.
Mice were removed from the study, as required, at a maximal accepted lesion load, when a single lesion became excessively large or ulcerated, when a lesion occurred at a site of irritation or due to reasons of general ill health. The decision to remove an animal from study because of these criteria was taken blind of transgenic status. Tissue samples were collected and snap frozen in liquid N2 prior to storage at -70°C.
Data collection and analyses
The number of lesions on each mouse was recorded on a weekly basis from the start of treatment until the mouse was removed from the study. Lesions were scored blind of transgenic status. Lesions were categorized by size, according to the following specifications: size 1, <0.2 cm; size 2, 0.2<0.5 cm; size 3, 0.5<1.0 cm; size 4,
1.0 cm diameter). Data were analysed using the Microsoft Excel and Minitab statistical analysis packages. Differences between the week of papilloma onset were analysed using the MannWhitney U-test; total papilloma numbers were compared using the two sample t-test. Regression analysis was used to model the rate of papilloma size expansion [independent variable (x) = number of size 1 lesions at week t; dependent variable (y) = number of new size 2+ lesions at week t + 1]. The rate of papilloma size expansion was statistically compared between different groups of mice using an F-test. Conversion of papillomas to carcinomas was compared by
2 analyses.
Protein sample preparation and western blotting
Protein was extracted from tumor specimens using protein lysis buffer (20 mM TrisHCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% v/v Triton X-100) containing phosphatase and protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 0.5 mM ß-glycerophosphate, 0.5 mM sodium pyrophosphate, 0.5 mM sodium fluoride, 0.5 mM sodium molybdate, 0.5 mM sodium orthovanadate, 1% Sigma protease inhibitor cocktail, catalogue no. P2714). Aliquots of 100 µg protein/sample were subjected to 12% PAGE and electroblotted onto Millipore Immobilon-P membranes at 1.5 A for 2.5 h in a Hoeffer electroblotting tank in blotting buffer (25 mM Tris, 192 mM glycine, 20% v/v methanol). The membranes were blocked for 1 h at room temperature in 100 ml of blocking buffer [5% w/v dried milk in 20 mM Tris, pH 7.8, 140 mM NaCl, 0.1% Tween-20 (TBST)], then incubated overnight at 4°C in primary antibody [1:1000 dilution of rabbit anti-p16INK4a polyclonal antibody (M-156; Santa Cruz) in blocking buffer]. The membranes were washed three times for 10 min each in TBST, followed by 1 h in blocking buffer, then incubated for 1 h at room temperature in secondary antibody [1:5000 anti-rabbit IgG HRP (sc-2006; Santa Cruz) in blocking buffer]. Antibody binding was visualized using the luminol system (
RPN2106, ECL; Amersham).
 |
Results
|
---|
LMP1 promotes papilloma formation but inhibits papilloma expansion
In order to investigate the role of LMP1 in inhibiting the expansion of small papillomas to larger sizes, 27 PyLMP1 and 31 wild-type, control mice were entered into a new study using a standard 21 week chemical carcinogen treatment regime as described. In agreement with our earlier studies (24), the PyLMP1 mice developed a higher number of total lesions than the wild-type mice. The combined data from both studies (PyLMP1 n = 59, controls n = 57) show a greater statistically significant difference between the two groups, validating this observation (Figure 1). The week of first papilloma onset was compared and no significant difference was found between PyLMP1 mice and the wild-type controls (P = 0.915). However, while the PyLMP1 mice developed an increased number of small papillomas (Figure 2A), they had a reduced number of large papillomas in comparison with their wild-type siblings (Figure 2B). This suggests that LMP1 inhibits the expansion in size of these papillomas.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1. Average number of lesions in PyLMP1 and wild-type mice. Graph showing the average (mean) lesion load (papillomas and carcinomas) per mouse in PyLMP1 (n = 59) and wild-type controls (n = 57) (data from two studies combined). All of the mice were treated with DMBA at the start of week 1; TPA treatment began at the start of week 2 and continued for 20 weeks. The data cover live mice remaining on study and as such the sample size (n) decreases over the study period. The two groups were compared using Student's two sample t-test and the P values are given in the chart for weeks 1028 (P < 0.05 indicates statistical significance, in bold).
|
|

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 2. Comparison of lesion sizes. (A and C) The average number of size 1 lesions (<2 mm) per mouse each week. (B and D) The average number of the larger size 2+ lesions ( 2 mm) per mouse each week. (A and B) PyLMP1 and wild-type control mice compared; (C and D) PyLMP1/INK4a-/- and INK4a-/- mice compared.
|
|
In order to quantify this observed difference and test it for significance, a regression model was applied to the data. The aim of the model was to calculate the rate of growth of the papillomas from small papillomas (<2 mm diameter) to larger sizes (
2 mm diameter). This rate is equivalent to the slope of the line (
) using the regression model:
where nL(t) is the number of large papillomas at week t, nL(t + 1) is the number of large papillomas at week t + 1, nS(t) is the number of small papillomas at week t,
is the rate of growth of small papillomas to large papillomas and
is the number of large papillomas at week t + 1 which have arisen since week t.
Analysis of the data clearly shows that papillomas grow at a slower rate in PyLMP1 mice than in wild-type mice (Figure 3A and Table I) and that this difference is statistically significant (using an F-test, F = 3.04, P = 0.049) (Table II).
In addition, despite the increased lesion formation compared with controls, PyLMP1 mice were not more susceptible to carcinoma formation than wild-type mice. Conversely, on average the PyLMP1 mice developed their first carcinoma later, with fewer mice developing carcinomas and with a lower papilloma to carcinoma conversion rate compared with controls (Figure 4A and Table III). However, a higher percentage of PyLMP1 mice were removed from the study due to causes other than carcinoma formation, and this lowered the total overall percentage of PyLMP1 mice which could succumb to carcinoma. A
2 analysis did not reveal a statistically significant difference in the proportion of mice which developed carcinomas between the two groups from weeks 15 to 30 of the study (data not shown).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4. Percentage of mice with carcinomas. The week of first carcinoma development for each mouse is shown as a proportion of each group. The percentage (carc) was calculated from the total number of mice in each group. The graph also shows the percentage of mice which had been removed from the study (rem) throughout the experiment due to causes other than carcinoma size or load. This factor places a limit on the percentage of the total group which can develop carcinomas. By the end of the study, the percentage of mice with carcinomas + the percentage of mice otherwise removed from study = 100%. (A) PyLMP1 (n = 27) and wild-type control mice (n = 31); (B) INK4a-/- (n = 22) and wild-type mice; (C) PyLMP1/INK4a-/- (n = 20) and INK4a-/- mice compared.
|
|
These data reveal a paradox in the action of LMP1. Expression in the transgenic skin leads to an increase in lesion formation and we have previously shown that LMP1 augments the tumour promotion stages (24). However, once formed, lesion growth is inhibited by LMP1 and conversion to carcinoma may also be reduced.
Loss of the INK4a locus inhibits papilloma formation but promotes lesion progression
In order to explore the role of the INK4a locus in chemical carcinogenesis and evaluate this with respect to LMP1 expression, PyLMP1 transgenic mice were cross-bred with mice which carry a targeted deletion at the INK4a locus. This targeted deletion completely eliminates the function of p16INK4a, however, the mice express low levels of a transcript containing exon 1ß, which encodes a truncated p19ARF. It is possible that these mice may be partially functional for p19ARF, although the phenotypic similarities between these mice and p19ARF null mice suggest that this is unlikely (41,44). Mice treated were either heterozygous (+/), null (/) or wild-type (+/+) for the INK4a locus and either PyLMP1 transgenic or not [PyLMP1/INK4a+/-, n = 13; PyLMP1/INK4a-/-, n = 20; PyLMP1/INK4a+/+, n = 20; INK4a+/-, n = 12; INK4a-/-, n = 22; wild-type (INK4a+/+), n = 30]. Mice were treated using the standard 21 week chemical carcinogen regime (Figure 5).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5. Average number of lesions in all genetic groups in the study. Graph showing the average (mean) lesion load per mouse in all six groups in the study: PyLMP1/INK4a-/-, INK4a-/-, PyLMP1/INK4a+/-, INK4a+/-, PyLMP1 and wild-type controls. All mice were treated with DMBA at the start of week 1; TPA treatment began at the start of week 2 and continued for 20 weeks. The data cover live mice remaining on study (up to week 23 shown) and as such the sample size (n) decreases over the study period.
|
|
Surprisingly, the INK4a-/- mice developed significantly fewer papillomas than the wild-type mice (INK4a+/+) (Figure 5 and Table IV). This suggests that loss of the INK4a locus, which encodes two tumour suppressor genes, paradoxically inhibits the development of papillomas during chemical carcinogenesis.
Conversely, loss of the INK4a locus was found to play an important role in the growth of lesions (Figure 3B) and at the later stage of papilloma conversion to carcinoma. The INK4a-/- mice were more susceptible to carcinoma formation than wild-type mice. On average, the INK4a-/- mice developed carcinomas much sooner than wild-type, a higher percentage of the group developed carcinomas and the conversion rate from papilloma to carcinoma was double that of the wild-type mice (Figure 4B and Table III).
2 analysis showed that the proportion of mice which had developed carcinomas was significantly higher in the INK4a-/- mice than the wild-type mice from week 16 through to week 21 of the study (data not shown). These data indicate that loss of the INK4a locus products promotes lesion progression to carcinoma.
Expression of p16INK4a was examined during each of the stages of chemical carcinogenesis, in small and large papillomas and in carcinomas from wild-type mice (Figure 6). p16INK4a was expressed in all of the small and large papillomas, but was down-regulated in the carcinoma samples. These data are in accordance with our observation that loss of the INK4a locus promotes conversion to carcinoma.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 6. p16INK4a expression in lesions of wild-type mice. A western blot of proteins extracted from four small papillomas, four large papillomas and three carcinomas taken from wild-type control mice. A papilloma from an INK4a-/- mouse was used as a negative control. Proteins were separated by 12% SDSPAGE and electroblotted. Gels were stained post-transfer with Coomassie blue to confirm sample loading equivalence and examine transfer efficiency. The blot was probed with an anti-p16INK4a antibody. A band of 16 kDa is evident in the papilloma sample lanes, expressed at lower levels in two of the carcinoma samples and absent in the third. The negative control does not have a band at 16 kDa, however, the antibody does react with a lower molecular weight protein, the identity of which is not known. The anti-p16 antibody (M-156) recognizes the first 167 residues at the N-terminus of the p16INK4a protein. Exon 1a of the p16INK4a gene is still intact in the INK4a-/- mice and the antibody may be detecting expression of this exon.
|
|
LMP1 and loss of the INK4a locus in papilloma formation
Whilst the loss of the INK4a locus leads to an inhibition of papilloma formation, the expression of LMP1 appears to overcome this inhibition. PyLMP1/INK4a-/- mice developed significantly more lesions than INK4a-/- mice (Table V) and, on average, also developed more lesions than wild-type mice (Figure 5). Furthermore, papillomas first appeared significantly sooner in the PyLMP1/INK4a-/- mice compared with the INK4a-/- mice (MannWhitney U-test, P = 0.0028).
Loss of the INK4a locus, LMP1 and the inhibition of papilloma expansion
Analysis of papilloma size expansion in an INK4a-/- background revealed that PyLMP1/INK4a-/- mice developed more small papillomas than INK4a-/- mice (Figure 2C), as was also observed for LMP1 expression in a wild-type (INK4a+/+) background (Figure 2A). However, the LMP1-induced inhibition of papilloma size expansion seen in a wild-type background was not found in the INK4a-/- background. In this case, PyLMP1/INK4a-/- mice developed slightly more large papillomas than the INK4a-/- mice (Figure 2D).
We applied the regression model outlined above to these data to calculate the rate of size expansion of the papillomas (equivalent to the slope of the line,
). This revealed that loss of the INK4a locus leads to a dramatic increase in lesion growth. Intriguingly, this effect is not recessive since the INK4a heterozygotes showed intermediate lesion growth rates (Figure 3B and Tables I and II). Moreover, loss of INK4a released the LMP1-induced inhibition of papilloma size expansion (Table II).
LMP1, loss of the INK4a locus and carcinoma formation
Carcinomas appear significantly sooner in the PyLMP1/INK4a-/- mice than in the INK4a-/- mice (Figure 4C) (MannWhitney U-test, P = 0.0103). However, this difference reflects the fact that the PyLMP1/INK4a-/- mice develop significantly more papillomas and sooner than the INK4a-/- mice (Figures 2B and 6). Ultimately, very similar percentages of the two groups develop carcinomas (Figure 4C and Table III). Indeed, the PyLMP1/INK4a-/- mice show a lower conversion rate of papillomas to carcinomas than the INK4a-/- mice (Table III). This accords with the comparison above of PyLMP1 mice and controls in that while the expression of LMP1 promotes the formation of papillomas, it may inhibit their subsequent conversion to carcinoma.
 |
Discussion
|
---|
The viral oncoprotein LMP1 plays a paradoxical role during mouse skin tumourigenesis. LMP1 is involved in the early stages of tumour promotion and augments the action of the tumour promoter TPA (24). However, we have shown that whilst the expression of LMP1 in the mouse skin increases susceptibility to papilloma formation, it inhibits further papilloma growth. Loss of the INK4a locus shows the reverse paradox. The locus encodes two tumour suppressor proteins, p16INK4a and p19ARF, which act in the Rb and p53 pathways respectively, and its loss might therefore be predicted to increase susceptibility to skin tumour formation. On the contrary, we have shown that loss of the INK4a locus inhibits papilloma formation whilst subsequently promoting conversion of papillomas to carcinomas.
Expression of LMP1 in an INK4a null background shows a cooperation that overcomes both paradoxes. First, LMP1 acts to increase lesion formation and overcomes the inhibition of papilloma formation observed in the INK4a null background. Second, in a wild-type background LMP1 inhibits lesion growth, while in an INK4a null background, no inhibition of lesion growth by LMP1 is observed. Logically, this would suggest that this action of LMP1 is mediated by products of the INK4a locus. However, in rat embryonic fibroblasts, LMP1 over-expression was found to inhibit p16 expression and cellular senescence (45). Oncogenic activation of several genes (such as ras) can lead to apparently opposite actions, proliferation versus senescence and apoptosis or differentiation, and LMP1 may similarly cause different effects in different cell types or between culture and in vivo expression. Expression of both INK4a products in this context requires exploration to test this hypothesis.
Lesion growth in the INK4a heterozygotes was intermediate between wild-type and null, both in the presence and absence of LMP1. This further demonstrates that INK4a products have an inhibitory effect upon lesion growth and that heterozygous loss reveals haploinsufficiency in this property. This is the first report to demonstrate that heterozygous loss of the INK4a locus is not recessive.
The epidermis has a remarkable capacity for homeostatic regulation and it might be expected that several safeguards are in place to control growth. A stimulus that can lead to an increase in proliferation may trigger a regulatory response to balance this, such as an increase in differentiation. It is possible that expression of LMP1 continuously up-regulates both proliferation and differentiation. As such, the increase in proliferation would explain the tumour promoting activity of LMP1 and the observation of increased lesion formation, while the simultaneous increase in terminal differentiation (or other cell cycle exit, such as arrest or apoptosis) could have the effect of counteracting lesion expansion. The data presented here indicate that the growth inhibitory effect induced by LMP1 is mediated via the INK4a locus.
Conversely, a stimulus which inhibits terminal differentiation may lead to a reduction in proliferation as a homeostatic response and, consequently, inhibit lesion formation. However, once an aberrant growth signal is supplied in conjunction with the inhibition of cell cycle exit, then enhanced lesion progression would result. Loss of Rb can inhibit terminal differentiation (46) and it is therefore probable that loss of p16INK4a can do the same. There are other examples of events which should lead to an inhibition of cell death, stasis or differentiation which have been found to inhibit papilloma formation. Transgenic mice over-expressing Bcl-2 in the basal epidermal layer of skin develop fewer papillomas than wild-type mice, following either UVB irradiation or classical two stage chemical carcinogenesis, but once formed these papillomas grow to larger sizes (47). Similarly, chemical carcinogen-treated p53 null mice develop fewer papillomas than wild-type controls, but the papillomas which do develop have a high rate of conversion to malignancy (48,49). It is tempting to speculate that loss of the INK4a locus might mirror the loss of p53, since p19ARF activates p53 in response to aberrant mitogenic signals (such as the constitutive H-ras signalling which occurs in a DMBA-initiated cell).
Thus, in this model, LMP1 supplies the aberrant proliferative signal leading to papilloma formation and in the absence of the INK4a locus LMP1-mediated growth inhibition does not occur. Moreover, loss of the tumour supressors expressed from the INK4a locus then facilitates tumour progression.
Expression of LMP1 in the EBV-negative NPC cell line CNE2 results in growth inhibition and an increased sensitivity to cisplatin-induced cell death (36). With conflicting reports using epithelial cell lines concerning LMP1 expression and cellular differentiation (20,21,50), it would be informative to examine the status and expression of the INK4a locus and other genes in the Rb and p53 pathways in the different cell lines, as the response to LMP1 may hinge on whether these pathways are functional.
The activity of LMP1 in the context of INK4a locus products has been modelled here in the mouse epidermis. Murine/human differences as well as mucosal epithelial/epidermal differences may well lead to activation or loss of different but parallel pathways, for example activating mutations in H-ras are a feature of DMBA treatment of mouse skin, while ras mutations are not observed in NPC. Nevertheless, loss of expression or deletion of RASSF1A (on chromosome 3p21.3) in NPC tissues (reviewed in 14) could suggest that a ras pathway is active, since RASSF1A is thought to mediate the apoptotic effects of oncogenic ras (51). Indeed, high level LMP1 expression may achieve this (21). LMP1 may act at the early stages of NPC tumourigenesis, promoting the development of small benign lesions. There is a strong association between EBV infection and NPC, but there are also likely to be genetic and environmental contributory factors. The nasopharynx is commonly exposed to a wide variety of dietary carcinogens and it has been postulated that NPC risk is associated with the high level of nitrosamines in the diet of susceptible populations. These contributory factors alone are unlikely to be sufficient to induce lesion formation in the nasopharynx, but the expression of LMP1 in the epithelial cells, together with these other factors, may be sufficient to promote the development of benign tumours. The role of LMP1 as a tumour promoter has previously been modelled in PyLMP1 mice, where using a minimal chemical carcinogen regime was found to be sufficient to induce papilloma formation in the transgenic but not in wild-type mice (24). Our model would suggest that loss of the INK4a locus would promote the growth of benign tumours and increase the conversion rate to carcinoma. Importantly, INK4a heterozygous mice in this study showed an intermediate, haploinsufficient effect with respect to lesion growth. As such, it is possible that this locus also plays a part in NPC predisposition, where a weak allele might provide a growth advantage within a benign lesion. Indeed, the Balb/c p16INK4A allele has been implicated in plasmacytoma susceptibility in this strain of mice (52) and p16INK4A and p14ARF (the human homologue of p19ARF) in melanoma susceptibility (53,54). Interestingly, no susceptibility p16INK4A alleles have been found in patients of squamous cell cancer of the head and neck (55), which raises the possibility that a potential susceptibility at this locus in NPC might be exclusively tied to EBV association.
In malignant NPC tumour cells the INK4 locus is frequently deleted (10,11) or its products not expressed (13). INK4a loss has also been associated with the later stages of tumourigenesis (carcinoma progression) in chemical carcinogenesis of mouse skin (39). However, recent studies indicate that loss of the INK4 locus may be an early event in the genesis of NPC, with the detection of chromosome 9p alterations in histologically normal nasopharyngeal epithelial samples and pre-invasive lesions (14). Our data suggest that heterozygous loss of INK4a locus expression would give a growth advantage to a benign lesion, where EBV infection and specifically LMP1 expression may contribute to formation of the lesion. Subsequent loss of expression of the second INK4a allele would dramatically increase the chances of lesion progression to carcinoma. Thus the action of chemical mutagens, loss of the INK4a locus and EBV LMP1 expression may be tightly linked factors in the genesis of NPC.
 |
Acknowledgments
|
---|
We thank D.Beach and M.Serrano for the INK4a-/- mice. We are very grateful to J.Cuthbert for advice on statistical methodology. This work was supported by grants from the Association for International Cancer Research (97-44) and the Carnegie Trust.
 |
References
|
---|
- Le Roux,F. and Joab,I. (1998) Epstein-Barr virus and nasopharyngeal carcinoma. Epstein-Barr Virus Rep., 5, 5357.
- IARC (1997) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, no. 70, Epstein-Barr Virus and Kaposi's Sarcoma Herpesvirus/Human Herpesvirus 8. IARC, Lyon.
- Buell,P. (1974) The effect of migration on the risk of nasopharyngeal cancer among Chinese. Cancer Res., 34, 11891191.[ISI][Medline]
- Huang,D.P., Ho,J.H., Saw,D. and Teoh,T.B. (1978) Carcinoma of the nasal and paranasal regions in rats fed Cantonese salted marine fish. IARC Sci. Publ., 20, 315328.[Medline]
- Poirier,S., Ohshima,H., de-The,G., Hubert,A., Bourgade,M.C. and Bartsch,H. (1987) Volatile nitrosamine levels in common foods from Tunisia, south China and Greenland, high-risk areas for nasopharyngeal carcinoma (NPC). Int. J. Cancer, 39, 293296.[ISI][Medline]
- Simons,M.J., Wee,G.B., Chan,S.H. and Shanmugaratnam,K. (1975) Probable identification of an HL-A second-locus antigen associated with a high risk of nasopharyngeal carcinoma. Lancet, 1, 142143.[Medline]
- Liebowitz,D. (1994) Nasopharyngeal carcinoma: the Epstein-Barr virus association. Semin. Oncol., 21, 376381.[ISI][Medline]
- Lu,S.J., Day,N.E., Degos,L. et al. (1990) Linkage of a nasopharyngeal carcinoma susceptibility locus to the HLA region. Nature, 346, 470471.[CrossRef][ISI][Medline]
- Hildesheim,A., Anderson,L.M., Chen,C.J. et al. (1997) CYP2E1 genetic polymorphisms and risk of nasopharyngeal carcinoma in Taiwan. J. Natl Cancer Inst., 89, 12071212.[Abstract/Free Full Text]
- Chen,Y.J., Ko,J.Y., Chen,P.J., Shu,C.H., Hsu,M.T., Tsai,S.F. and Lin,C.H. (1999) Chromosomal aberrations in nasopharyngeal carcinoma analyzed by comparative genomic hybridization. Genes Chromosomes Cancer, 25, 169175.[CrossRef][ISI][Medline]
- Lo,K.W., Huang,D.P. and Lau,K.M. (1995) p16 gene alterations in nasopharyngeal carcinoma. Cancer Res., 55, 20392043.[Abstract]
- Lo,K.W., Teo,P.M., Hui,A.B., To,K.F., Tsang,Y.S., Chan,S.Y., Mak,K.F., Lee,J.C. and Huang,D.P. (2000) High resolution allelotype of microdissected primary nasopharyngeal carcinoma. Cancer Res., 60, 33483353.[Abstract/Free Full Text]
- Lo,K.W., Cheung,S.T., Leung,S.F. et al. (1996) Hypermethylation of the p16 gene in nasopharyngeal carcinoma. Cancer Res., 56, 27212725.[Abstract]
- Huang,D.P., To,K.F. and Lo,K.W. (2002) Molecular pathogenesis of nasopharyngeal carcinoma. Epstein-Barr Virus Rep., 9, 4354.
- Brooks,L., Yao,Q.Y., Rickinson,A.B. and Young,L.S. (1992) Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP1 and LMP2 transcripts. J. Virol., 66, 26892697.[Abstract]
- Sheen,T.S., Huang,Y.T., Chang,Y.L., Ko,J.Y., Wu,C.S., Yu,Y.C., Tsai,C.H. and Hsu,M.M. (1999) Epstein-Barr virus-encoded latent membrane protein 1 co-expresses with epidermal growth factor receptor in nasopharyngeal carcinoma. Jpn. J. Cancer Res., 90, 12851292.[ISI][Medline]
- Pathmanathan,R., Prasad,U., Sadler,R., Flynn,K. and Raab-Traub,N. (1995) Clonal proliferations of cells infected with Epstein-Barr virus in preinvasive lesions related to nasopharyngeal carcinoma. N. Engl. J. Med., 333, 693698.[Abstract/Free Full Text]
- Wang,D., Liebowitz,D. and Kieff,E. (1985) An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell, 43, 831840.[ISI][Medline]
- Baichwal,V.R. and Sugden,B. (1988) Transformation of Balb 3T3 cells by the BNLF-1 gene of Epstein-Barr virus. Oncogene, 2, 461467.[ISI][Medline]
- Fåhraeus,R., Rymo,L., Rhim,J.S. and Klein,G. (1990) Morphological transformation of human keratinocytes expressing the LMP gene of Epstein-Barr virus. Nature, 345, 447449.[CrossRef][ISI][Medline]
- Dawson,C.W., Rickinson,A.B. and Young,L.S. (1990) Epstein-Barr virus latent membrane protein inhibits human epithelial cell differentiation. Nature, 344, 777780.[CrossRef][ISI][Medline]
- Kaye,K.M., Izumi,K.M. and Kieff,E. (1993) Epstein-Barr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation. Proc. Natl Acad. Sci. USA, 90, 91509154.[Abstract]
- Wilson,J.B., Weinberg,W., Johnson,R., Yuspa,S. and Levine,A.J. (1990) Expression of the BNLF-1 oncogene of Epstein-Barr virus in the skin of transgenic mice induces hyperplasia and aberrant expression of keratin 6. Cell, 61, 13151327.[ISI][Medline]
- Curran,J.A., Laverty,F.S., Campbell,D., Macdiarmid,J. and Wilson,J.B. (2001) Epstein-Barr virus encoded latent membrane protein-1 induces epithelial cell proliferation and sensitizes transgenic mice to chemical carcinogenesis. Cancer Res., 61, 67306738.[Abstract/Free Full Text]
- Miller,W.E., Earp,H.S. and Raab-Traub,N. (1995) The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor. J. Virol., 69, 43904398.[Abstract]
- Zheng,X., Hu,L., Chen,F. and Christensson,B. (1994) Expression of Ki67 antigen, epidermal growth factor receptor and Epstein-Barr virus-encoded latent membrane protein (LMP1) in nasopharyngeal carcinoma. Eur. J. Cancer B Oral Oncol., 30B, 290295.[CrossRef]
- Eliopoulos,A.G., Stack,M., Dawson,C.W., Kaye,K.M., Hodgkin,L., Sihota,S., Rowe,M. and Young,L.S. (1997) Epstein-Barr virus-encoded LMP1 and CD40 mediate IL-6 production in epithelial cells via an NF-
B pathway involving TNF receptor-associated factors. Oncogene, 14, 28992916.[CrossRef][ISI][Medline]
- Eliopoulos,A.G., Gallagher,N.J., Blake,S.M., Dawson,C.W. and Young,L.S. (1999) Activation of the p38 mitogen-activated protein kinase pathway by Epstein-Barr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production. J. Biol. Chem., 274, 1608516096.[Abstract/Free Full Text]
- Yoshizaki,T., Sato,H., Furukawa,M. and Pagano,J.S. (1998) The expression of matrix metalloproteinase 9 is enhanced by Epstein-Barr virus latent membrane protein 1. Proc. Natl Acad. Sci. USA, 95, 36213626.[Abstract/Free Full Text]
- Horikawa,T., Yoshizaki,T., Sheen,T.S., Lee,S.Y. and Furukawa,M. (2000) Association of latent membrane protein 1 and matrix metalloproteinase 9 with metastasis in nasopharyngeal carcinoma. Cancer, 89, 715723.[CrossRef][ISI][Medline]
- Eliopoulos,A.G., Dawson,C.W., Mosialos,G. et al. (1996) CD40-induced growth inhibition in epithelial cells is mimicked by Epstein-Barr Virus-encoded LMP1: involvement of TRAF3 as a common mediator. Oncogene, 13, 22432254.[ISI][Medline]
- Hammerschmidt,W., Sugden,B. and Baichwal,V.R. (1989) The transforming domain alone of the latent membrane protein of Epstein-Barr virus is toxic to cells when expressed at high levels. J. Virol., 63, 24692475.[ISI][Medline]
- Cuomo,L., Ramquist,T., Trivedi,P., Wang,F., Klein,G. and Masucci,M.G. (1992) Expression of the Epstein-Barr virus (EBV)-encoded membrane protein LMP1 impairs the in vitro growth, clonability and tumorigenicity of an EBV-negative Burkitt lymphoma line. Int. J. Cancer, 51, 949955.[ISI][Medline]
- Floettmann,J.E., Ward,K., Rickinson,A.B. and Rowe,M. (1996) Cytostatic effect of Epstein-Barr virus latent membrane protein-1 analyzed using tetracycline-regulated expression in B cell lines. Virology, 223, 2940.[CrossRef][ISI][Medline]
- Kaykas,A. and Sugden,B. (2000) The amino-terminus and membrane-spanning domains of LMP-1 inhibit cell proliferation. Oncogene, 19, 14001410.[CrossRef][ISI][Medline]
- Liu,Y., Wang,X., Lo,A.K., Wong,Y.C., Cheung,A.L. and Tsao,S.W. (2002) Latent membrane protein-1 of Epstein-Barr virus inhibits cell growth and induces sensitivity to cisplatin in nasopharyngeal carcinoma cells. J. Med. Virol., 66, 6369.[CrossRef][ISI][Medline]
- Akhurst,R.J. and Balmain,A. (1999) Genetic events and the role of TGFß in epithelial tumour progression. J. Pathol., 187, 8290.[CrossRef][ISI][Medline]
- Berenblum,I. (1978) Established principles and unresolved problems in carcinogenesis. J. Natl Cancer Inst., 60, 723726.[ISI][Medline]
- Linardopoulos,S., Street,A.J., Quelle,D.E., Parry,D., Peters,G., Sherr,C.J. and Balmain,A. (1995) Deletion and altered regulation of p16INK4a and p15INK4b in undifferentiated mouse skin tumors. Cancer Res., 55, 51685172.[Abstract]
- Sherr,C.J. (2001) The INK4a/ARF network in tumour suppression. Nature Rev. Mol. Cell. Biol., 2, 731737.[CrossRef][ISI][Medline]
- Serrano,M., Lee,H., Chin,L., Cordon-Cardo,C., Beach,D. and DePinho,R.A. (1996) Role of the INK4a locus in tumor suppression and cell mortality. Cell, 85, 2737.[ISI][Medline]
- Wilson,J.B. and Drotar,M.E. (2001) Considerations in generating transgenic mice: DNA, RNA and protein extractions from tissuesrapid and effective blotting. In Wilson,J.B. and May,G.H.W. (eds), Methods in Molecular Biology, Vol. 174, Epstein-Barr Virus Protocols. Humana Press, Totowa, NJ, pp. 361377.
- Curran,J. (2001) Topical chemical carcinogen treatment in mice. In Wilson,J.B. and May,G.H.W. (eds), Methods in Molecular Biology, Vol. 174, Epstein-Barr Virus Protocols. Humana Press, Totowa, NJ, pp. 391399.
- Serrano,M. (2000) The INK4a/ARF locus in murine tumorigenesis. Carcinogenesis, 21, 865869.[Abstract/Free Full Text]
- Yang,X., He,Z., Xin,B. and Cao,L. (2000) LMP1 of Epstein-Barr virus suppresses cellular senescence associated with the inhibition of p16INK4a expression. Oncogene, 19, 20022013.[CrossRef][ISI][Medline]
- Macleod,K. (1999) pRb and E2f-1 in mouse development and tumorigenesis. Curr. Opin. Genet. Dev., 9, 3139.[CrossRef][ISI][Medline]
- Rossiter,H., Beissert,S., Mayer,C., Schon,M.P., Wienrich,B.G., Tschachler,E. and Kupper,T.S. (2001) Targeted expression of bcl-2 to murine basal epidermal keratinocytes results in paradoxical retardation of ultraviolet- and chemical-induced tumorigenesis. Cancer Res., 61, 36193626.[Abstract/Free Full Text]
- Kemp,C.J., Donehower,L.A., Bradley,A. and Balmain,A. (1993) Reduction of p53 gene dosage does not increase initiation or promotion but enhances malignant progression of chemically induced skin tumors. Cell, 74, 813822.[ISI][Medline]
- Greenhalgh,D.A., Wang,X.J., Donehower,L.A. and Roop,D.R. (1996) Paradoxical tumor inhibitory effect of p53 loss in transgenic mice expressing epidermal-targeted v-rasHa, v-fos, or human transforming growth factor-
. Cancer Res., 56, 44134423.[Abstract]
- Nicholson,L.J., Hopwood,P., Johannessen,I., Salisbury,J.R., Codd,J., Thorley-Lawson,D. and Crawford,D.H. (1997) Epstein-Barr virus latent membrane protein does not inhibit differentiation and induces tumorigenicity of human epithelial cells. Oncogene, 15, 275283.[CrossRef][ISI][Medline]
- Vos,M.D., Ellis,C.A., Bell,A., Birrer,M.J. and Clark,G.J. (2000) Ras uses the novel tumor suppressor RASSF1 as an effector to mediate apoptosis. J. Biol. Chem., 275, 3566935672.[Abstract/Free Full Text]
- Zhang,S.L., DuBois,W., Ramsay,E.S., Bliskovski,V., Morse,H.C.,III, Taddesse-Heath,L., Vass,W.C., DePinho,R.A. and Mock,B.A. (2001) Efficiency alleles of the Pctr1 modifier locus for plasmacytoma susceptibility. Mol. Cell. Biol., 21, 310318.[Abstract/Free Full Text]
- Rizos,H., Darmanian,A.P., Holland,E.A., Mann,G.J. and Kefford,R.F. (2001) Mutations in the INK4a/ARF melanoma susceptibility locus functionally impair p14ARF. J. Biol. Chem., 276, 4142441434.[Abstract/Free Full Text]
- Mantelli,M., Barile,M., Ciotti,P. et al. (2002) High prevalence of the G101W germline mutation in the CDKN2A (P16(ink4a)) gene in 62 Italian malignant melanoma families. Am. J. Med. Genet., 107, 214221.[CrossRef][ISI][Medline]
- Jefferies,S., Edwards,S.M., Hamoudi,R.A., A'Hern,R., Foulkes,W., Goldgar,D. and Eeles,R. (2001) No germline mutations in CDKN2A (p16) in patients with squamous cell cancer of the head and neck and second primary tumours. Br. J. Cancer, 85, 13831386.[ISI][Medline]
Received December 24, 2002;
revised March 20, 2003;
accepted April 16, 2003.