Open Access

The involvement of E6, p53, p16, MDM2 and Gal-3 in the clinical outcome of patients with cervical cancer

  • Authors:
    • Annika Stiasny
    • Christoph P. Freier
    • Christina Kuhn
    • Sandra Schulze
    • Doris Mayr
    • Christoph Alexiou
    • Christina Janko
    • Irmi Wiest
    • Christian Dannecker
    • Udo Jeschke
    • Bernd P. Kost
  • View Affiliations

  • Published online on: August 14, 2017     https://doi.org/10.3892/ol.2017.6752
  • Pages: 4467-4476
  • Copyright: © Stiasny et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

High-risk human papilloma virus (HPV) is the leading cause of cervical cancer. HPV oncogenes are responsible for the development of malignancy, and the E6 oncoprotein that HPV expresses induces the degradation of tumour suppressor protein p53 (p53). This degradation leads to the upregulation of p16; however, unidentified proteins may also serve a role in the development and progression of cervical cancer. Therefore, the aim of the present study was to analyse the expression levels of E6, p53, p16, MDM2 proto‑oncogene (MDM2) and galectin‑3 (gal‑3) in cervical cancer specimens. A total of 250 cervical cancer tissue slides were used. The expression of E6, p53, p16, MDM2 and gal‑3 was analysed with immunohistochemical methods and a semi‑quantitative scoring. SPSS software was used for the statistical evaluation of staining results and survival analysis of patients with cervical cancer. Cervical cancer specimens demonstrated significantly increased E6 staining with advanced T‑status and increased International Federation of Gynecology and Obstetrics classification. E6, p53 and p16 demonstrated significantly different expression levels in squamous epithelial tissue compared with adenocarcinomas. MDM2 and gal‑3 demonstrated positively correlated expression levels in cervical cancer. In addition, gal‑3 expression was correlated with poor prognosis in p16‑negative cases. A negative correlation between the expression of E6 and a mutated form of p53 was also identified in cervical cancer. p53 mutation was demonstrated to be common in cervical cancer, and gal‑3 and MDM2 appeared to act in a combined manner in this type of tumour. As gal‑3 is overexpressed in the cervical cancer tissue of patients with poor prognosis, the use of gal‑3 inhibitors should be investigated in future studies.

Introduction

Cervical cancer is the fourth most frequent cancer in women globally with ~530,000 new cases in 2012, accounting for 7.5% of all female cancer-associated mortalities (1). A major cause of cervical cancer is persistent infection with high-risk human papillomavirus (HR-HPV) (1). HPV subtypes 16 and 18 cause ~70% of all cases of HPV (1,2). At present, >170 HPV types have been identified (3). Infection with 15 subtypes of HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82) may lead to cancer, which is why these 15 types are known as carcinogenic or high-risk types (4). The genome of human papillomaviruses consists of ~8,000 base pairs and contains six early genes (E6, E7, E1, E2, E4 and E5) and two late genes (L1 and L2) (5). Upon replication of the viral gene E6, E6 oncoprotein is expressed, which alters the cell cycle (6). E6 oncoprotein and E6-associated protein (E6-AP) form a complex that binds to p53 and causes its proteolytic degradation (7).

The tumour suppressor protein p53 (p53) signalling pathways leads to cell cycle arrest or apoptosis in case of DNA damage (8). As E6 oncoprotein induces the degradation of p53, the function of this important cell cycle protein is disturbed following HPV infection (9). In addition, the cell cycle regulation protein p16 is expressed at high levels in HPV-infected epithelial cells, and thus acts as a marker for the diagnosis of HPV-associated carcinoma (9,10). In non-carcinoma tissues p53 is regulated by MDM2 proto-oncogene (MDM2) through a negative feedback mechanism. MDM2 promotes the ubiquitination and proteasome-dependent degradation of p53 (11). There is also an association between MDM2, p53 polymorphism and the progression of cervical carcinoma (12).

A protein previously demonstrated to be associated with cervical cancer is galectin-3 (gal-3) (5). Galectins are defined as lectins with a galactose-binding ability and a characteristic amino-acid sequence (13). Galectin is a name proposed by Hirabayashi and Kasai (14) for a family of animal lectins. Galectins are typically soluble and metal-independent in their activity (15). They have similar features to cytoplasmic proteins, including no disulfide bridges, no sugar chains, no signal sequences and, in most cases, their N-terminal amino acids are acetylated (16). It is possible to classify galectins into the following three types on the basis of their structural architecture: Proto, chimera and tandem-repeat types. Gal-3 is a chimera-type galectin (17).

Gal-3 may increase the invasiveness of cervical cancer by activating vascular endothelial growth factor receptor-3 (5). Therefore, the aim of the present study was to systematically analyse the expression and interactions of E6, p53, p16, MDM2 and gal-3 in cervical cancer specimens.

Materials and methods

Ethical approval

The present study was approved by the local Ethics Committee of the Ludwig-Maximilians-University of Munich (approval no. 259-16; Munich, Germany), and was performed in compliance with the guidelines of the Helsinki Declaration. Patient data were fully anonymised.

Specimens

Archived formalin-fixed paraffin-embedded (FFPE) sections from 250 cases of cervical cancer were used in the present study; it was possible to analyse 248 cases as there was no tumour tissue present on two sections (Table I). Cervical dysplasia [cervical intra-epithelial neoplasia (CIN) stage III] (18) and non-dysplastic cervical tissue (3 sections of each) was used for the E6 immunohistochemical staining, and breast cancer tissue was used for the mutated p53 immunohistochemistry. Specimens were obtained from the Department of Obstetrics and Gynecology of Ludwig-Maximilians-University of Munich, and were obtained from patients undergoing surgery there between 1993 and 2002. Follow-up data were received from the Munich Cancer Registry (Munich Tumour Centre, Munich, Germany).

Table I.

Clinical parameters of the patients included in the present study.

Table I.

Clinical parameters of the patients included in the present study.

Clinical parameterNo./total no.%
Age (years)
  ≤50143/24858
  >50105/24842
No. of metastasis positive lymph nodes
  0149/24860
  1–497/24839
  NA2/248  1
Tumour size (cm)
  <2111/24845
  2–4128/24852
  >49/248  3
Tumour grade
  I20/248  8
  II141/24857
  III78/24831
  NA9/248  4
Tumour subtype
  Squamous199/24880
  Adenocarcinoma49/24820
Progression (over 236 months)
  None190/24877
  ≥158/24823
Survival (over 236 months)
  Right censured210/24885
  Succumbed38/24815

[i] NA, not applicable as data not available.

Immunohistochemistry

The FFPE sections (3-µm-thick) were dewaxed in xylol, endogenous peroxidase was inhibited with 3% methanol/H2O2 and sections were rehydrated in a descending ethanol gradient. To stain for mutated p53, wild-type p53, E6, gal-3 and MDM2, the slides were pre-treated in citrate buffer (100°C; pH 6.0) for antigen retrieval. Following this, non-specific binding of the primary antibodies was blocked, and incubation with the primary antibodies followed (Tables II and III). Incubation with the secondary antibodies and the following steps of the detection system and colour development are illustrated in Tables II and III. For p16 detection, the specimens were automatically stained using the Ventana BenchMark XT Stainer (Ventana Medical Systems, Inc., Oro Valley, AZ, USA) and the CINtec Histology kit (cat. no. 9517; Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions, while all other antibodies were stained for manually. For wild-type p53, the slides were washed in PBS/0.05% Tween-20. All other slides were washed in PBS only. Finally, the slides were counterstained with hemalaun (Waldeck GmbH, Münster, Germany) for 2 min at room temperature, dehydrated in an ascending series of ethanol and stored.

Table II.

Procedures for gal-3 and MDM2 staining.

Table II.

Procedures for gal-3 and MDM2 staining.

ProtocolGal-3MDM2
Blocking methodHorse seruma, 20 min, RTGoat seruma, 20 min, RT
Primary antibody, dilution, incubation duration, incubation temperature, cat. no.Anti-galectin-3, 1:1,000 in PBS, 16 h, 4°C; NCL-GAL3bAnti-MDM2, 1:100 in PBS, 16 h, 4°C, NCL-MDM2b
Secondary antibody, dilution, incubation duration, incubation temperature, cat. no.Biotynilated anti-mouse IgGa, 30 min, RT; PK-6100Biotinylated goat anti-mouse IgM, 30 min, RT ZMB2020c
Detection of secondary antibody ABC-complexa, 30 min ABC-complexa, 30 min
Chromogen1 mg/ml DABd, 5 min1 mg/ml DABd, 1 min

a Vectastain ABC kit; Vector Laboratories, Inc., Burlingame, CA, USA.

b Novocastra; Leica Microsystems GmbH, Wetzlar, Germany.

c Linaris GmbH, Dossenheim, Germany.

d Dako, Glostrup, Denmark. Gal-3, galectin-3; MDM2, MDM2 proto-oncogene; Ig, immunoglobulin; DAB, 3,3′-diaminobenzidine; RT, room temperature.

Table III.

Procedures for mutated p53, wild-type p53 and E6 staining.

Table III.

Procedures for mutated p53, wild-type p53 and E6 staining.

ProtocolMutated p53p53 wild-typeE6
Blocking methodReagent 1a; 5 min, RTReagent 1a; 5 min, RTReagent 1a; 5 min, RT
Primary antibody, dilution, incubation duration, incubation temperature, cat. no.Anti-p53, 1:100 in PBS, 16 h, 4°C, ab32049bAnti-p53, 1:200 in PBS, 16 h, 4°C, ab26bAnti-E6, 1:150 in PBS, 1 h, RT, ab70b
Post blocking methodReagent 2a; 20 min, RTReagent 2a; 20 min, RTReagent 2a; 20 min, RT
Secondary antibody, dilution, incubation duration, incubation temperature, cat. no.HRP-Polymer Reagent 3a, 30 min, RT POLHRP-100HRP-Polymer Reagent 3a, 30 min, RT POLHRP-100HRP-Polymer Reagent 3a; 30 min, RT POLHRP-100
Chromogen1 mg/ml DABc, 1 min1 mg/ml DABc, 1 min1 mg/ml DABc, 1 min

a From the ZytoChem-Plus HRP Polymer-kit; Zytomed Systems GmbH, Berlin, Germany.

b Abcam, Cambridge, UK.

c Dako, Glostrup, Denmark. RT, room temperature; HRP, horseradish peroxidase; DAB, 3,3′-diaminobenzidine.

Slides were examined with a Zeiss Axiophot light photomicroscope (Zeiss GmbH, Jena, Germany). Digital images were obtained with a digital-camera system (CF20DXC; KAPPA Messtechnik, Gleichen, Germany). All specimens were evaluated by a pathologist. The intensity and distribution patterns of the staining reaction was evaluated by two blinded, independent observers, including the gynecological pathologist, using the semi-quantitative immunoreactive (IRS)-score, as previously described (19), to asses steroid receptors (20) and cathepsin D (21) expression. The IRS score was calculated by multiplication of optical staining intensity (graded as 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining) and the percentage of positive stained cells (0, no staining; 1, ≤10% of the cells, 2, 11–50% of the cells, 3=51–80% of the cells and 4, ≥81% of the cells) and without knowing the pathological evaluation, the diagnosis or the standard performed hematoxylin reaction for 2 min at room temperature for each specimen.

Statistical analysis

Data were analysed using SPSS software (version 19.0; IBM SPSS, Armonk, NY, USA) for Microsoft Windows and visualised using Microsoft Office 7 (Microsoft Corporation, Redmond, WA, USA). Spearman coefficients were calculated to assess correlations, while the Mann-Whitney U test was applied to examine differences between groups. Differences in survival were assessed using the log-rank test and survival curves were plotted in accordance with Kaplan-Meier estimator. P<0.05 was considered to indicate a statistically significant difference and data were expressed as the mean ± standard error. Cox regression analysis was used to compare the risk of mortality in patients with and without gal-3 expression when the effects of further factors were accounted for. Independent variables included in the Cox regression model were gal-3 expression, age at the time of surgery, histological subtype, tumour size, lymph node status (pN), metastasis, tumour grade, International Federation of Gynecology and Obstetrics (FIGO) stage (22,23), and E6, mutated p53 and MDM2 expression status.

Results

Evaluation of E6 oncoprotein immunohistochemistry and the detection of mutated p53 on control slides

CIN III tissue slides were used for the evaluation of E6 oncoprotein staining. Moderate expression levels of E6 were observed in the CIN III sections (Fig. 1A). There was no expression of the E6 oncoprotein, and therefore no staining observed, in the non-dysplastic cervical tissue (Fig. 1B). Breast cancer tissue was used to evaluate the staining of mutated p53 (Fig. 1C), which exhibited nuclear and cytoplasmic staining.

E6 oncoprotein staining

A total of 81% of all cervical cancer tissue examined expressed E6 oncoprotein (data not shown). Cervical cancer specimens demonstrated significantly increased staining with a higher T stage (according to the Tumor-Node-Metastasis classification system) (24). T1 stage carcinomas (Fig. 2A) demonstrated E6 staining with a median IRS of 2, while T2 (Fig. 2B) and T3 (Fig. 2C) stage carcinoma tissues had a significantly higher median E6 expression of IRS 3 (P=0.017; Fig. 2D).

FIGO 1 carcinoma tissues had a median E6 expression of IRS 2 (Fig. 2E). FIGO 2 (Fig. 2F) and FIGO 3 (Fig. 2G) carcinoma tissues had a median IRS of 4. FIGO 4-classified cervical cancer tissue had a median E6 IRS score of 6 (Fig. 2H). E6 demonstrated a significant positive correlation with the FIGO classification (R=0.277, P<0.001; Fig. 2I).

E6 demonstrated significantly different expression levels in cervical cancer tissue dependent on the histological subtype. Squamous epithelial carcinomas (Fig. 2J) had a median expression of IRS 2. Adenocarcinoma tissue (Fig. 2K) had significantly increased staining with a median of IRS 5 (P=0.015 vs. squamous epithelial carcinoma; Fig. 2L).

Wild-type and mutated p53 expression

Expression of wild-type p53 was observed in the nucleus and cytoplasm of 60 and 66% of all cervical cancer specimens, respectively. Significantly different expression levels in cervical cancer tissue in different histological subtypes were also observed for p53 expression. Wild-type p53 demonstrated a median nuclear expression (Fig. 3A) of IRS 1 in squamous epithelial tissue, whereas in adenocarcinoma tissue (Fig. 3B) the median nuclear expression was significantly decreased in comparison (IRS 0, P=0.024; Fig. 3C).

In addition to nuclear expression, wild-type cytosolic p53 expression also demonstrated significant differences associated with the histological subtype. In squamous epithelial tissue (Fig. 3D) a median expression of IRS 3 was observed, whereas in comparison the median cytosolic expression of p53 was significantly decreased in adenocarcinoma tissue (Fig. 3E) to IRS 0 (P<0.001; Fig. 3F).

The monoclonal antibody that recognises a previously described mutated form of p53, (25) also revealed significant staining differences associated with the histological subtype of cervical cancer. In addition, 42% of all cervical cancer tissue slides demonstrated nuclear expression of mutated p53, and 67% of all cases demonstrated mutated p53 expression in the cytoplasm. Although the median expression of mutated p53 in squamous epithelial tissue (Fig. 3G) and adenocarcinoma tissue (Fig. 3H) was 0, differences between the subtypes were significant (P=0.011; Fig. 3I).

Expression of p16 oncoprotein in cervical cancer tissue

p16 overexpression is routinely used in the Pathology Department of the Ludwig-Maximilians-University of Munich as a marker for HPV-associated head and neck squamous carcinoma (11). A total of 94% of cervical carcinoma cases tested demonstrated p16 expression, and 61% of these cases were p16-overexpressing according to pathological evaluation. The cell cycle protein p16 demonstrated significant differences in expression between different histological subtypes of cervical cancer. Squamous epithelial tissue (Fig. 4A) had a median expression of IRS 6, while adenocarcinoma tissue (Fig. 4B) had a significantly lower expression in comparison, with an IRS of 4 (P<0.001; Fig. 4C). Notably, p16 demonstrated no significant correlation with E6 oncoprotein expression (data not shown).

Correlation analysis

A significant correlation was identified between MDM2 and gal-3 expression in cervical cancer tissue (R=0.181, P=0.005; data not shown). Cases of cervical cancer with low MDM2 expression (Fig. 5A) also demonstrated low gal-3 expression (Fig. 5B). Likewise, a case with high MDM2 expression (Fig. 5C) demonstrated high gal-3 expression (Fig. 5D). Cases of cervical cancer with low E6 oncoprotein expression (Fig. 5E) demonstrated enhanced staining of the mutated form of p53 in the same area of the tumour (Fig. 5F). However, cases with high expression of E6 (Fig. 5G) revealed low expression of mutated p53 (Fig. 5H). The statistical evaluation confirmed these results of serial section staining (R=−0.140, P=0.028; Table IV). A significant correlation was also identified between the expression of MDM2 and mutated p53 in cervical cancer tissue (R=0.144, P=0.025; Table IV). The correlation analyses and clinical parameters are summarised in Table IV.

Table IV.

Correlation analyses of clinical parameters and immunohistochemical staining parameters.

Table IV.

Correlation analyses of clinical parameters and immunohistochemical staining parameters.

Clinical parameterStatisticAgeHistologypTpNpMGradeFIGOE6p53-mutatedMDM2Galectin-3
Age (years)Correlation coefficient−.019.004−.115−.065−.140*.354b.133a.169b.107.026.050
Sig. (2-tailed).766.945.070.307.028.000.037.008.095.686.438
N250250250250246250245244246241240
HistologyCorrelation coefficient.0281.000.000−.073−.108−.084.045.156a−.162a.075.029
Sig. (2-tailed).664..994.249.089.185.477.015.010.242.652
N245249249249249249249245249244243
pTCorrelation coefficient.276b.0001.000.359b−.202b.182b.380b.174b.055−.019−.039
Sig. (2-tailed).000.994..000.001.004.000.006.384.762.540
N246249250250250250250246250245244
pNCorrelation coefficient.037−.073.359b1.000−.172b.212b.240b.033−.050−.058−.033
Sig. (2-tailed).559.249.000..007.001.000.610.435.370.608
N246249250250250250250246250245244
pMCorrelation coefficient−.081−.108−.202b−.172b1.000−.146*−.150a−.037.004.032−.074
Sig. (2-tailed).206.089.001.007..021.018.560.951.619.252
N246249250250250250250246250245244
GradeCorrelation coefficient−.081−.084.182b.212b−.146a1.000.093.084−.065−.175b−.047
Sig. (2-tailed).203.185.004.001.021..142.191.302.006.461
N246249250250250250250246250245244
FIGOCorrelation coefficient.076.045.380b.240b−.150a.0931.000.227b−.099.021.067
Sig. (2-tailed).233.477.000.000.018.142..000.120.748.296
N246249250250250250250246250245244
E6Correlation coefficient.088.156a.174b.033−.037.084.227b1.000.093.001.009
Sig. (2-tailed).173.015.006.610.560.191.000..147.986.893
N242245246246246246246246246244243
p53-mutatedCorrelation coefficient.092−.290b.016−.019.004−.115−.065−.140a1.000.133a.169b
Sig. (2-tailed).148.000.796.766.945.070.307.028..037.008
N246249250250250250250246250245244
MDM2Correlation coefficient.026.075−.019−.058.032−.175b.021.001−.0421.000.181b
Sig. (2-tailed).686.242.762.370.619.006.748.986.511..005
N241244245245245245245244245245243
Galectin-3Correlation coefficient.050.029−.039−.033−.074−.047.067.009−.020.181b1.000
Sig. (2-tailed).438.652.540.608.252.461.296.893.760.005.
N240243244244244244244243244243244

a P<0.05

b P<0.01. pT, pathological tumour stage; pN, pathological node stage; pM, pathological metastasis stage; FIGO, International Federation of Gynecology and Obstetrics stage; MDM2, MDM2 proto-oncogene.

Gal-3 is a negative prognosticator in p16-negative patients with cervical cancer

In patients with cervical cancer with no or very low p16 expression, gal-3 expression was correlated with a poor prognosis in overall survival analyses (P=0.0313; Fig. 6). Multivariate Cox regression analysis was performed to test which histopathological variables were independent prognosticators for survival rate in the tested breast cancer collective. It was demonstrated that the histological subtype (P=0.02), tumour size (P=0.011) and pN (P=0.045) were independent prognosticators for overall survival (Table V). No significant effect was demonstrated for the other histopathological variables.

Table V.

Cox regression of overall survival on cervical cancer variables.

Table V.

Cox regression of overall survival on cervical cancer variables.

95.0% CI

ParametersP-valueHazard ratioLowerUpper
Age (years)0.0711.0290.9971.062
Histology0.0023.5761.5868.063
pT0.0111.2701.0571.525
pN0.0452.1131.0164.395
pM0.7021.3050.3355.085
Tumour grade0.0651.7170.9683.048
FIGO0.8750.9940.9261.068
E6CytIRS0.4750.9610.8631.071
p16CytIRS0.6961.0260.9031.165
p53IRS0.2670.8920.7291.092
p53mutIRS0.9750.9960.7651.296
MDM2IRS0.4601.0500.9221.196
Gal-3 IRS score0.4520.9370.7921.109

[i] CI, confidence interval; pT, pathological tumour stage; pN, pathological node stage; pM, pathological metastasis stage; FIGO, International Federation of Gynecology and Obstetrics stage; IRS, immunoreactive score; Cyt, cytoplasmic; mut, mutated; MDM2, MDM2 proto-oncogene; Gal-3, galectin-3.

Discussion

Within the present study, immunohistochemical evaluation of E6 oncoprotein expression was conducted. In addition, E6 expression levels were demonstrated to be associated with the histological subtype. The expression of wild-type p53 and a mutated form of p53 were identified in the cervical cancer specimens tested. Finally, correlation analyses revealed a combined positive expression pattern for galectin-3 and MDM2, and a negative correlation between E6 and mutated p53 expression.

Although the early era of HPV research identified that ≤99.5% of cervical cancer cases are HPV-associated (26), it remains controversial in the literature whether viral load and disease severity are positively correlated (12). Therefore, the present study investigated a number of markers that are associated with HPV-driven changes in cell cycle proteins. Using these markers permitted a comparative analysis of the influence of HPV on the progression of cervical cancer for >10 years following surgery.

The replication of the viral genes E6 and E7 results in the cellular expression of E6 and E7 oncoproteins, which interfere with the cell cycle (6). E6 oncoprotein binds to E6-AP, forming a complex that selectively binds to p53 and leads to its ubiquitin-dependent proteolytic degradation (7). The present study demonstrated that E6 immunohistochemistry was a fast and simple method for the detection of the HPV-associated oncoprotein E6 in cervical cancer tissues. As a routine practice, E6 and E7 are detected using either in situ hybridisation (11) or polymerase chain reaction (27) methodology due to the non-specific immunohistochemical staining results of antibodies used in former studies (28).

In the present study a well-tested antibody, and specific antigen retrieval and staining protocol was used, resulting in the establishment of a useful immunohistochemical evaluation protocol for the detection of the HPV E6 oncoprotein. The optimal results were obtained with the E6 antibody supplied by Abcam (Cambridge, UK). The advantage of immunohistochemical evaluation is that it is easier to apply and less expensive compared with mRNA in situ hybridisation. mRNA in situ hybridisation may be the optimal way to detect HPV; however, this method is more complicated for routine detection compared with immunohistochemistry (26). Evaluation of E6 immunohistochemical staining in cervical cancer tissue has previously revealed positive correlations with advanced T staging and FIGO classification (22). Although specific studies have indicated correlations between E6/E7 gene expression and the clinicopathological parameters of cervical cancer (26), such a correlation was not demonstrated in the present study.

An additional finding of the present study is the negative correlation between E6 and mutated p53 expression. Mutations of the gene encoding p53 (TP53) are the most frequent alterations in multiple human malignancies (2931). In total, >50% of human tumours contain a mutation/deletion of TP53, ranging from 5–80% depending on the type, stage and etiology of the tumours (32). A number of previous studies have investigated a potential genetic link between these variations and cancer susceptibility, but the results have been controversial. A previous meta-analysis study from 49 pooled studies failed to demonstrate a link between a common TP53 mutation (25) and cervical cancer susceptibility (33). Later on, the same mutation was revealed to be associated with higher pancreatic cancer risk among males; however, results also indicated that it may protect Arab women against the development of breast cancer (34,35). Multiple other mutations of TP53 have since been described. Mutations that deactivate p53 in cancer are primarily located in the central DNA binding domain. These mutations typically ablate the ability of the protein to bind to its target DNA sequences, prevent the transcriptional activation of p53 target genes. In total, ~80% of the most common p53 mutants demonstrate the capacity to exert dominant-negative effects over wild-type p53 and thus prevent the activation of transcription. In contrast, only 45% of the less frequent mutants studied have this capacity (36).

The mutation detected by the antibody used in the present study is an mutation at position 20 (serine to aspartic acid), which abolishes the phosphorylation site on p53. The phosphorylation of this serine when DNA damage is detected weakens the interaction between p53 and MDM2, thereby stabilising p53 (3739). Thus, this mutation maintains increased protein levels of p53 following DNA damage. The analysis of the immunohistochemical detection of mutated p53 revealed that cervical cancer specimens derived from squamous epithelial tissue demonstrated significantly higher expression levels compared with adenocarcinoma tissue. In addition, a positive correlation between the expression of mutated p53 and MDM2, and a negative correlation between the expression of mutated p53 and E6, were identified. Therefore, it is possible to speculate that E6 also degrades the mutated form of p53. In a previously published study, this mutation was demonstrated to be associated with the improved survival of patients with cervical cancer (25).

Finally, a positive correlation between MDM2 and gal-3 expression was demonstrated in cervical cancer tissue. Little information concerning the involvement of galectins in cervical cancer exists at present. Research has primarily focused on gal-1 (40,41), gal-7 (42,43) and gal-9 (44). A previous publication described the influence of gal-3 on vascular endothelial growth factor C expression and its influence on the enhancement of cervical cancer cell invasiveness (5). The present study demonstrated that gal-3 was a negative independent prognosticator for the overall survival of patients with p16-negative cervical cancer. In this group of patients, gal-3 may be responsible for the aggressiveness of cervical cancer, whereas in p16-positive carcinomas different factors/signal transduction pathways may be responsible.

In the present study, a total of 250 cervical cancer cases were systematically analysed for the expression and interaction of E6, p53, p16, MDM2 and gal-3 in FFPE tumour tissue. Significantly increased levels of E6 staining were correlated with an advanced T stage and FIGO classification. Furthermore, MDM2 and gal-3 expression levels were positively correlated in cervical cancer. In addition, gal-3 expression levels were negatively correlated with prognosis in p16-negative cases. As gal-3 is overexpressed in the cervical cancer tissue of patients with a worse prognosis, the investigation of gal-3 inhibiting compounds is an additional task for the development of alternative treatments for this tumour type. In addition, a negative correlation between E6 and a mutated form of p53 in cervical cancer was identified. In conclusion, the results of the present study indicate that immunohistochemical staining may be a useful method for the detection the HPV E6 oncoprotein.

Acknowledgements

The present study was supported by the German Research Foundation. The authors would like to thank Professor Jutta Engel and Mr. Max Wiedemann (The Munich Cancer Registry, Munich Tumour Centre, Munich, Germany) for providing the follow-up data.

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October-2017
Volume 14 Issue 4

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Spandidos Publications style
Stiasny A, Freier CP, Kuhn C, Schulze S, Mayr D, Alexiou C, Janko C, Wiest I, Dannecker C, Jeschke U, Jeschke U, et al: The involvement of E6, p53, p16, MDM2 and Gal-3 in the clinical outcome of patients with cervical cancer. Oncol Lett 14: 4467-4476, 2017
APA
Stiasny, A., Freier, C.P., Kuhn, C., Schulze, S., Mayr, D., Alexiou, C. ... Kost, B.P. (2017). The involvement of E6, p53, p16, MDM2 and Gal-3 in the clinical outcome of patients with cervical cancer. Oncology Letters, 14, 4467-4476. https://doi.org/10.3892/ol.2017.6752
MLA
Stiasny, A., Freier, C. P., Kuhn, C., Schulze, S., Mayr, D., Alexiou, C., Janko, C., Wiest, I., Dannecker, C., Jeschke, U., Kost, B. P."The involvement of E6, p53, p16, MDM2 and Gal-3 in the clinical outcome of patients with cervical cancer". Oncology Letters 14.4 (2017): 4467-4476.
Chicago
Stiasny, A., Freier, C. P., Kuhn, C., Schulze, S., Mayr, D., Alexiou, C., Janko, C., Wiest, I., Dannecker, C., Jeschke, U., Kost, B. P."The involvement of E6, p53, p16, MDM2 and Gal-3 in the clinical outcome of patients with cervical cancer". Oncology Letters 14, no. 4 (2017): 4467-4476. https://doi.org/10.3892/ol.2017.6752