Background
Viral infections are known to contribute to the development of several human cancers, the best known being the association of Human Papillomavirus (HPV) with cervical cancer [
1]. Tumorigenesis can be induced by infectious agents through the induction of chronic inflammation, cellular transformation by oncogene insertion, inhibition of tumour suppressors and induction of immunosuppression [
2]. While the consequences of inflammation on tumour initiation and progression are well studied [
3,
4], the relationship between inflammation and viral infections in carcinogenesis is much less understood.
The incidence of oesophageal cancer is highly variable depending on geographical and ethnic parameters. High risk areas include the so-called Asian oesophageal cancer belt from eastern Turkey, through Iraq, Iran and into Western and Northern China as well as Eastern-Southern Africa, South America and southern areas of France [
5]. Oesophageal cancer is the most common cancer among black South African men, and is second to cervical carcinoma in black women [
6]. There are two major types of oesophageal cancer: squamous cell carcinoma (OSCC) and adenocarcinoma, the former being the predominant form in developing countries. While chronic, frequent reflux of gastric acid into the distal oesophagus is considered as the primary factor underlying most cases of oesophageal adenocarcinoma affecting predominantly white populations [
7], oesophageal squamous-cell carcinomas are more frequent among Black and Asian populations and are most strongly associated with smoking, alcohol intake, lack of fresh fruit and vegetables in the diet, and infections [
8,
9]. Although still debated due to conflicting results, infection with HPV that was shown to be correlated to nasopharyngeal cancer [
10] is thought to be an aetiological factor in OSCC depending on the geographical region with prevalence ranging from 0% to 71% [
10‐
16]. This is in contrast to other types of cancer that are clearly related to HPV infection such as cervical cancers [
1]. It is assumed that only oesophageal cancers originating from high-incidence geographic areas are associated with HPV infection, particularly with the high-risk types 16 and 18 [
11,
17‐
20].
Previous studies from our laboratory revealed a correlation between HPV infection and oesophageal cancer in patients from the Transkei (Eastern Cape), a particularly high-risk area in South Africa: 46% were infected with HPV with the predominant type being HPV11 [
14,
21]. In the present study we investigated the role of HPV infection of OSCC patients from the Western Cape (South Africa) and specifically focussed on a possible correlation between inflammation and OSCC, the influence of HPV infection for tumour-associated inflammation, and the role of inflammation for virus uptake. To our knowledge, there is little information on the potential role of chronic inflammation in the aetiology of HPV-associated OSCC, with a possible connection being described for a subset of head and neck squamous cell carcinoma (HNSCC) [
22].
The genes that were selected in the present study for analysis of altered gene expression in the tumour samples were p16 as an HPV type-independent cellular correlate for increased HPV oncogene expression [
23], potential receptors for HPV (ITGA6, SDC) [
24] and representative genes of cancer-associated inflammation (IL6, IL8, IL12A, IFNGR1, TNFA1P1) [
25]. Additionally, the effects of benzo-α-pyrene (BαP), one of the most important tobacco smoke carcinogens [
26‐
29], as well as of peptidoglycan (PEP) and lipopolysaccharide (LPS), both known to induce IL6 and IL8 [
30‐
33], were tested.
We found that the presence of HPV DNA in OSCC tissues was rather low and appeared to be independent of inflammation or the presence of the potential HPV receptor ITGA6 [
34]. Virus uptake was only increased in vitro when the cells were treated with benzo-α-pyrene, one of the most abundant carcinogens in tobacco smoke.
Methods
Tissue collection
A total of 114 histopathologically-confirmed OSCC biopsies together with corresponding adjacent normal tissue samples were collected at both Groote Schuur Hospital and Tygerberg Hospital, Cape Town, South Africa, between 2008 and 2011. Patients were from different ethnic groups with 66% being black (Xhosa-speaking), 32% of mixed ancestry, and 2% whites. Informed consent was obtained from all participants, while ethical approval for this study was obtained from the Ethics Committee of the University of Cape Town. Samples were stored in RNAlater solution (Qiagen) at −80°C until used for nucleic acid extraction.
Cell culture
The immortalised human oesophageal epithelial cell line EPC2-hTERT [
35] was grown in Keratinocyte SFM medium (Invitrogen) supplemented with 50 μg/ml bovine pituitary extract (Invitrogen), 1 ng/ml human EGF (Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin. WHCO1 (human OSCC cell line) [
36], HaCaT (spontaneously immortalized human keratinocyte cell line and natural host for HPV [
37], ATCC), C33A (human HPV negative cervical cancer cell line, ATCC) and 293TT cells (virus packaging cell line established from primary embryonal human kidney cells transformed with modified human adenovirus) [
38,
39] were grown in DMEM (Gibco Life Technologies) supplemented with 10% heat-inactivated fetal calf serum (Gibco Life Technologies), 100U/ml penicillin and 100 μg/ml streptomycin. CHO-K1 (Chinese hamster epithelial-like cells, ATCC) and pgsD-677 cells (heparin sulfate deficient cells derived from CHO-K1, ATCC) were cultured in Ham`s F-12 K medium (Gibco Life Technologies) supplemented with 10% heat-inactivated fetal calf serum, 100U/ml penicillin and 100 μg/ml streptomycin.
Nucleic acid extraction and PCR
Frozen biopsies were cut in half for parallel RNA and genomic DNA extraction with the TRIzol reagent (Invitrogen) and the QIAamp Mini Kit (Qiagen), respectively. For PCR amplification of the HPV L1 gene, 100 ng DNA was used in a nested PCR reaction containing the degenerate consensus primers MY09/MY11 followed by PCR with the primer set GP5+/GP6+ as described previously [
14] using the FastStart Taq DNA polymerase (Roche). The presence of EBV DNA was detected similarly by targeting the virus capsid antigen p23 region by nested PCR using the primer set p23-1/p23-2 followed by PCR with the primer set p23-3/p23-4 [
40]. Amplification of human β-actin was used as an internal control (see Table
1 for all primer sequences and PCR product sizes). The integrity of the PCR products was visualised by agarose gel electrophoresis. The PCR products from all HPV positive samples were subjected to DNA sequence analysis to determine the HPV type. For quantitative RT-PCR, RNA extracted from the biopsies was reverse transcribed using the ImProm-II™ Reverse Transcription System (Promega). cDNA generated from 1 μg of total RNA was used for quantitative PCR with the KAPA SYBR®FAST qPCR Kit (Kapa Biosystems) on a LightCycler®480II System (Roche). Products were amplified with the primers listed in Table
1 under the following conditions: 1 min 95°C, 1 min 55°C, 30s 72°C. Results were analysed using the 2
-ΔΔCт method [
41], normalised to GAPDH expression and represented as x-fold increase in an individual tumour sample (T) compared to the corresponding non-cancerous normal tissue (N) from the same patient. Statistics were done to compare the significance of gene expression in T versus N using the 2-tailed non-parametric Mann–Whitney test. All primers were tested for amplification of the correct products by conventional PCR using appropriate tissue DNA.
Table 1
Primers used in this study
pGL3 forw | CGGGCGCGGTCGGTAAAGT | 380 |
pGL3 rev | AACAACGGCGGCGGGAAGT | |
p16 forw | GCCACTCTCACCCGACCCGT | 130 |
p16 rev | TCAGCCAGGTCCACGGGCAGA | |
ITGA6 forw | GCCAGCAAGGTGTAGCAGCTA | 101 |
ITGA6 rev | TTGCTCTACACGAACAATCCCTTT | |
SDC1 forw | CCCCGTTTCTGGTGGTCT | 175 |
SDC1 rev | TGTCTGAAGGCTGAGTCCC | |
CD21 forw | GCCGACACGACTACCAACC | 150 |
CD21 rev | AGCAAGTAACCAGATTCACAGC | |
IL8 forw | GAGAGTGATTGAGAGTGGACCAC | 111 |
IL8 rev | CACAACCCTCTGCACCCAGTTT | |
IL6 | SABiosciences (PPH00560B) | 160 |
IL12A | SABiosciences (PPH00544B) | 82 |
IFNGR1 | SABiosciences (PPH00871B) | 112 |
TNFA1P1 | SABiosciences (PPH01176A) | 101 |
GAPDH forw | GGCTCTCCAGAACATCATCC | 192 |
GAPDH rev | GCCTGCTTCACCACCTTC | |
MY09a, b
| CGTCCMARRGGAWACTGATC | 452 |
MY11 | GCMCAGGGWCATAAYAATGG | |
GP5+ | TTTGTTACTGTGGTAGATACTAC | 150 |
GP6+ | GAAAAATAAACTGTAAATCATATTC | |
EBV-P23-1 | CAGCTCCACGCAAAGTCAGATTG | 482 |
EBV-P23-2 | ATCAGAAATTTGCACTTTCTTTGC | |
EBV-P23-3 | TTGACATGAGCATGGAAGAC | 363 |
EBV-P23-4 | CTCGTGGTCGTGTTCCCTCAC | |
β-actin forw | ATCATGTTTGAGACCTTCAA | 320 |
β-actin rev | CATCTCTTGCTCGAAGTCCA | |
HPV pseudovirion production, quantification and labelling
HPV16 and HPV18 pseudovirions encapsidating the luciferase reporter gene plasmid pGL3-control (Promega) were generated in 293TT cells as previously described [
38,
42]. The plasmid pXULL, coexpressing both codon-optimized HPV16 L1 and L2, was generously provided by Michelle Ozbun (University of New Mexico), while the plasmid HPV18-L1/L2 was kindly provided by Samuel K. Campos (University of Arizona). Purity and L1/L2 protein content were determined by SDS-PAGE and subsequent staining with the Pierce® Silver Stain Kit (Thermo Scientific). For PsVs labelling with a fluorochrome that is only activated after cell entry [
43], 20 μg of purified PsVs were mixed with 200 μM Oregon Green®488 carboxylic acid diacetate succinimidyl ester (Molecular Probes) in a final volume of 500 μl HSB (pH 7.5) and incubated for 24 h at room temperature in the dark rotating. Unincorporated fluorochrome was removed by washing and concentrating the labelled PsVs using Amicon Ultra-4 (100,000 kDa MWCO) filter devices (Millipore). To assess the amount of encapsidated pGL3 plasmid DNA (viral genome equivalent, vge), all purified pseudovirion preparations were subjected to quantitative Light Cycler PCR using the primers pGL3 forw/pGL3 rev (Table
1).
HPV pseudovirion infection assays
Cells were seeded in 12-well plates at a density of 5×104 per well, and the following day cells were infected with a vge of approximately 100 pseudovirion particles per cell. For neutralisation assays, PsVs were incubated with the neutralising antibodies H16.V5 and H18.J4, respectively (generously provided by Neil D. Christensen, Pennsylvania State University College of Medicine) for 1 h at 4°C before adding to the cells. Where indicated, cells were stimulated with either 200 ng/ml IFNγ (R&D systems), 100 ng/ml IL6 (R&D systems), 100 ng/ml IL8 (R&D systems), 50 ng/ml IL12 (R&D systems), 5 μg/ml LPS (lipopolysaccharide) from E. coli 0127: B8 (Sigma), 50 μg/ml PEP (peptidoglycan) from S.aureus (Sigma) or 10 μM BαP (benzo-α-pyrene) (Sigma) for 24 h before addition of the PsVs. 48 h after infection, cells were harvested and luciferase activity was measured using the Luciferase Assay System kit (Promega) with the Fluoroscan Ascent FL (Thermo Fisher Scientific) according to the manufacturer’s instructions. Luciferase data were normalized to cell numbers by fluorescently staining an independent set of samples with 5 μM CellTrace™ Oregon Green® 488 (Molecular Probes) in PBS for 5 min at room temperature. All experiments were performed in duplicates, repeated at least three times and calculated as means ± S.D. with the Student’s t test used for determination of statistical significances.
To assess the uptake of PsVs that were labelled with Oregon Green®488 carboxylic acid diacetate succinimidyl ester (Molecular Probes) as described above, cells were seeded on coverslips in 6-well plates at a density of 2×105 per well, grown overnight and infected with a vge of approximately 500 pseudovirion particles per cell for 2 h. Cells were washed, fixed with 4% paraformaldehyde and mounted in mowial®4-88 reagent (Calbiochem) on glass slides. Fluorescent cells were visualised using an Olympus IX81S8F fluorescent microscope at 40x magnification.
Flow cytometry
Cells were prepared for FACS analysis as previously described [
44]. Rat anti-human ITGA6 (clone NKI-GoH3, AbD Serotec) antibody together with R-Phycoerythrin-conjugated donkey anti-rat IgG were used to detect ITGA6 cell surface expression using a FACSCalibur (Becton Dickinson) together with the software CellQuest.
Discussion
A number of human cancers have an infectious aetiology that have been extensively studied in the past, the best known being the association of HPV with cervical cancer [
1]. However, recent studies suggest that viruses may play a more significant role in several other malignancies than the ones with a known viral association. HPV and EBV for example, although widely debated, have been implicated in oesophageal and nasopharyngeal cancers, respectively [
10‐
12,
14‐
16]. While the consequences of viral oncogenes and infection-related inflammation for tumorigenesis have been extensively studied, the impact of tumour-associated inflammation on viral infection is much less understood.
In the present study we chose oesophageal squamous cell carcinoma as an example as this type of cancer is both associated with chronic irritation and inflammation as well as viral infections. Although the role of HPV in the carcinogenesis of OSCC is still debated, a previous study from our laboratory found a high association of HPV11 with OSCC from patients of the Eastern Cape, South Africa [
14]. Conducting a similar study with OSCC samples from patients of the Western Cape in South Africa, we here present evidence that underlying infection with HPV plays a minor role as only 9% of the studied patient cohort was infected with HPV with type 18 being the predominant one. Parallel analysis for underlying infection with EBV that served as a technical control for the applied PCR-based detection of viral DNA revealed a slightly higher infection rate of approximately 26%. While the low HPV infection rate was rather unexpected, the number of OSCC patients that were infected with EBV correlates well with the percentage of EBV DNA found in oral squamous cell carcinoma from South African patients ranging from 24% to 27% [
52,
53]. However, neither virus infection had any significant influence on the expression of the tested inflammatory genes or potential uptake receptors, although statistical analysis was rather limited due to the low number of HPV positive samples. The observed upregulation of IL6 and IL8 and downregulation of IL12A were therefore probably due to tumour-specific immunological responses that were independent of the presence of HPV. The high variability observed between individual samples might be explained by the complexity of the patient cohort and the fact that no personal parameters (such as age, sex, smoking and drinking status, ethnical and social background, stage of tumour etc.) were taken into account in the analysis. However, the large number of patients in the studied cohort allowed us to derive some general conclusion on the significance of the expression profile of the selected genes.
From a technical point of view, detection of HPV sequences in the host genome is rather challenging [
54,
55]. While various PCR approaches targeting HPV-type specific and HPV-type unspecific mRNA or inserted genomic DNA were tested, including commercially available kits (data not shown), only the widely described nested PCR approach using the primer combination MY09/MY11 followed by GP5+/GP6+ targeting L1, the most conserved protein among papillomaviruses [
14,
54‐
57], revealed highest sensitivity and reproducibility. Indeed, only 60-80% of our positive samples could be verified by a given alternative technique while only the combination of all data derived from the various tested HPV detection methods would confirm our (MY09/MY11//GP5+/GP6+)-nested PCR-based results, therefore supporting our data as not being false positives. The primer set MY09/MY11 that uses degenerate primers to detect a wide range of HPV types was not sufficient to pick up any HPV sequences in our patient cohort (data not shown), while a subsequent nested PCR with the GP5+/GP6+ primer set that uses consensus primers with a low annealing temperature to detect a similar range of HPV types resulted in HPV positive tissue samples. However, this approach could still have underestimated HPV prevalence as discussed before [
55]. We conclude that both the infection rate as well as the HPV copy number in our patient cohort were very low and probably under the detection level of the particular assay used. The low infection rate indicates that HPV is not a major contributor to the carcinogenesis of OSCC in our patient cohort. This is in agreement with a previous publication that suggests that low HPV copy numbers and infection rates are unlikely to play an essential role in OSCC when compared to cervical cancer [
18]. The low number of HPV infected OSCC biopsies was not expected from our previous study [
14] but might be explained in part by ethnical parameters and the different living circumstances of the patients in the Eastern versus the Western Cape [
58,
59]. The Eastern Cape of South Africa is a predominantly poor and underdeveloped province consisting mainly of tribal and informal urban areas with limited access to health services. In contrast to the Western Cape, it has particularly high rates of oesophageal cancer that are attributed to risk factors such as poverty, underdevelopment, poor nutrition and exposure to indoor smoke from combustion of solid fuels [
58,
60,
61]. Although still debated as being the causative agent, it is assumed that HPV infection is only associated with oesophageal cancers originating from high-incidence geographic areas [
11,
17‐
20]. This might account for the significantly higher HPV infection rate observed in our previous study [
14]; however, infection with the non-oncogenic HPV type 11 might just be interpreted as an indirect measure of poorer environmental conditions.
As HPV infection had no significant influence on inflammation in OSCC we next asked whether the opposite effect played a role, i.e. whether tumour-related inflammation affected the uptake of virus particles. Using HPV as a model, we set up an in vitro infection assay and infected a panel of representative cell lines with HPV-PsVs that were composed of the L1/L2 envelope proteins of HPV18 (as the majority of the HPV positive OSCC biopsies were infected with HPV18) as well as HPV16 (being the most common carcinogenic HPV type worldwide [
62,
63] and have been reported to be associated with oesophageal cancer [
11,
17,
18]). Surprisingly, we observed no or relatively low HPV pseudovirion infectivity of the immortalised normal oesophageal epithelial cell line EPC2-hTERT and the OSCC cell line WHCO1, respectively, compared to HaCaT or C33A control cells. Although the measured reporter gene activities in EPC2-hTERT cells were negligible, we found that these cells did take up the PsVs. We therefore conclude that these cells might display a non-infectious entry pathway with the virions possibly getting stuck in endocytic vesicles. Unproductive internalization with the hallmark of a stabilised capsid phenotype has been described before [
50,
64] but was not further investigated in this study as non-infectious uptake routes would not lead to viral oncogene expression, therefore having no relevance for the development of OSCC.
Interestingly, the observed differences in cell line infectivity did not correlate with the presence of ITGA6. Although questioned as a general HPV entry receptor component [
24,
65], ITGA6 was found to play a role in the uptake of HPV6b L1 pseudovirions into HaCaT cells [
34,
48] as well as HPV16 L1-VLPs into various cell lines [
49]. Moreover, increased expression of ITGA6 was shown to be correlated with the development of oesophageal cancer [
66]; and increased aggressiveness, drug resistance and poor prognosis has been correlated with the presence of ITGA6 in leukemia [
67]. However, the observed upregulation of ITGA6 in the OSCC tissues as well as its high expression on the surface of the tested cell lines did not correlate with the extent of HPV infectivity. Although we cannot exclude that ITGA6 is involved in HPV uptake, our data indicate that its presence and upregulation in the tumour samples do not play a rate limiting role in the establishment of HPV infection. When the cell lines were stimulated with various inflammatory agents or irritants known to be associated with tumorigenesis of the oesophagus [
9], only benzo-α-pyrene facilitated HPV18 pseudovirion (and to a lesser extent HPV16) uptake. BαP, one of the most important carcinogens in tobacco smoke, has long been known to exert a wide range of carcinogenic and pro-inflammatory effects [
28,
29]. Indeed, both epidemiological evidence as well as in vitro data exist to implicate interactions of HPV and cigarette smoke carcinogens in the progression of cervical cancer [
26,
27,
68]. It was found that BαP increased the infectivity of the oncogenic HPV type 31b but did not directly affect viral gene expression on a transcriptional level [
27]. The observed increase in luciferase activity upon BαP treatment might therefore be a result of increased pseudovirion uptake due to altered gene expression of BαP-induced DNA adducts in the host cells [
9]. It is unlikely that the BαP-mediated HPV pseudovirion uptake was due to an inflammatory response as none of the tested substances associated with inflammation increased HPV pseudovirion infectivity.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
GS and IMP conceived and designed the study. GS, BvR, SK and MBM performed the experiments. GS analysed the data. GS, LB and IMP contributed reagents and materials. GS wrote the paper. All authors read and approved the final manuscript.