The Role Of Histone H3K27 In Urinary Bladder Carcinoma: the Overview of Clinical Project

Introduction

Bladder cancer (BC) is the seventh most common cancer worldwide and the second most common cause of death in patients suffering from genitourinary tract malignancies. The relative frequency of BC in Egypt is 10. 7% among males and 3% among females at the National Cancer Registry of Egypt in 2014. Urothelial carcinomas (UC), arising from superficial cell layers in the urogenital tract, account for more than 90% of BC cases with the remainder of squamous cell carcinomas (SCC) and adenocarcinomas.

Tobacco smoking and occupational exposure to aromatic amines being the main environmental risk factors for BC occurrence in Western countries. However, Schistosoma haematobium (SH) infection is the most accused in developing countries, particularly in Egypt.

Histones are basic protein molecules of the chromatin, where coiled DNA wrapped around. Each of histone has a tail extension that can be modified by a number of histone post-translational modifying enzymes resulting in methylation, acetylation, phosphorylation, and ubiquitination. These modifications usually alters the binding affinity of the tails to DNA, resulting in the alteration of gene transcription, DNA replication, DNA repair, in addition to organization of chromosomes. Histone methylation is the addition of one, two, or three methyl groups mostly at lysines(K) and arginines (R) amino acids.

Lysines could be mono, di, or tri-methylated, while arginines are only mono or di- methylated by histone-methyltransferases(HMTs) and demethylases(HDMs). Methylation of lysine residue 27 of histone H3 (H3K27) may induce the heterochromatin formation by recruiting polycomb group (PcG) proteins. On the other hand, demethylation of trimethylated histone H3K27 (H3K27me3) depressed and promoted gene expression to change heterochromatin into euchromatin.

Consequently, H3K27 is one of the histone methylation targets that implicated in transcription repression of neighbouring genes. Several studies indicated that global levels of histone modifications are suitable cancer biomarkers, However, H3K27 methylation levels were inadequately investigated in BC patients. The aim of this study is to determine the global level of histone H3K27 di-methylation (H3K27me2) as a potential biomarker for early detection of BC.

Materials and Methods

Patients

This is a case-control study included 90 participants. They were classified into two groups: group I: included 45 (stage 1 and 2) bladder cancer patients recruited from the Surgical Oncology Department, South Egypt Cancer Institute from November 2016 to October 2017. Group IІ: Included 45 apparently healthy, age and sex matched volunteers. Smokers included in this study are defined as heavy smokers, who are consuming ≥1 pack daily (20 Egyptian cigarettes). The study protocol was approved by the Ethics Committee of Assiut University and according to The Code of Ethics of the World Medical Association (Declaration of Helsinki). All the participants gave written informed consents before their participation in the study.

Sample size calculation was conducted using G Power program in order to detect significant difference in proportion of histone H3K27 di- methylation levels between two independent groups included in this study with power 0. 95 in a hypothetical effect size 0. 33. Each group is 45 with total sample size 90. Patients were subjected to complete medical history taking, physical examination, MRI, CT, cystoscopy and pathological examination of the excised tumour. The practical part was done in the Medical Research Centre, Faculty of Medicine, Assiut University, Egypt.

Sample collection: Two ml of venous blood were collected from both patients and controls into EDTA tubes and stored in -80°C until the histone extraction procedure.

Total histone extraction from the samples:Total histone extraction was done using the EpiQuik™ Total Histone Extraction Kit (Catalog # OP-0006, made in USA). Histone extraction was done according to the kit protocol, with some modifications where whole blood was used instead of isolated blood cells. Briefly, blood samples were treated by equal volume of pre-lysis buffer, centrifuged and the supernatant were removed. Pellets were re-suspended in lysis buffer, incubated then centrifuged where the supernatant fraction (containing acid-soluble proteins) were transferred into a new vial. Lastly, balance-DTT buffer was added to the supernatant immediately. The protein concentration was measured with an OD reading. Bovine serum albumin (BSA) was used as a standard. This extracted histone was used for detection of global histone H3-K27 di-methylation level.

Determination of the global histone H3-K27 di-methylation levels: Global histone H3-K27 di-methylation levels were determined using the EpiQuik™ Global Di-Methyl Histone H3-K27 Quantification Colorimetric Kit (Catalog # P-3040, made in USA). Briefly, the di-methylated histone H3 at lysine 27 is captured to the wells coated with an anti-dimethyl H3-K27 antibody. Then, H3-K27 di-methylated histone can be detected with a labelled antibody, followed by a colour development reagent. Statistical Analysis of Data:Data was collected and analysed those using SPSS (Statistical Package for the Social Science, version 20, IBM, and Armonk, New York). Continuous data was expressed as mean ± standard deviation (SD), median and range while nominal data was expressed in form of frequency (percentage).

Chi²-test was used to compare the nominal data of both studied groups while Student t-test or one way analysis of variance (ANOVA) test were used to compare means of continuous variables between both study and control groups. A probability (P-value) of < 0. 05 was considered statistically significant. The threshold value for optimal sensitivity and specificity of the histone H3-K27 di-methylation level were determined by the receiver operating characteristics (ROC) curve.

Results

The main presenting symptom in the studied bladder cancer patients was hematuria that existing in 37 (82. 2%) of patients while hematuria and dysuria presented in 8 (17. 8%)of patients. Most of the patients had high grade of carcinogenesis 43(95. 6%) andonly 2 (4. 4%) had low grade of malignancy. Patients with bladder cancer had significantly lower level of histone H3K27me2 in comparison to control group. It was noticed that smokers whatever BC patient or apparently healthy control had lower level of histone H3K27me2 in comparison to those who were non-smokers in all subjects with significant difference (76. 55 ± 17. 88 versus 175. 03 ± 57. 09; P = 0. 02). In addition, there was no significant difference in the histone H3K27me2 level of smokers patients as regard histological types (P = 0. 7).

Bladder cancer patients with history of bilharziasis had higher level of histone H3K27me2 in comparison to those withouthistory ofbilharziasis, but P value was insignificant (56. 9 ± 10. 7 versus 53. 1 ± 13. 1; P = 0. 6). Receiver Operating Characteristic (ROC) curve showed that histone H3K27me2 level at cut off point < 49. 68ng/μlhad 69% sensitivity and 65 % specificity for prediction of bladder cancer with the Area under Curve (AUR) = 0. 67 (P= 0. 001).

Discussion

Alteration in the cancer cells global histone modification level and its prognostic efficacy was demonstrated for numerous human malignancies. The H3K27 methylation allowed differentiation of malignant tissue from normal one in various urological malignancies such as prostate cancer ; penile carcinoma ; renal cell carcinoma. However, H3K27 methylation levels have not been widely investigated in BC patients. In the current study, we clarified that the H3K27 di-methylation level was significantly decreased (P = 0. 001) in BC patients compared to the healthy control. Additionally, there is a significant difference in the H3K27 di-methylation levels between smokers and non-smokers, as the smokers (both patients and controls) had lower level of histone H3K27 di-methylation in comparison to those were non-smokers (P=0. 02). However, there was no correlation between histone H3K27 di-methylation levels and history of Schistosoma haematobium infection, histopathological type and the stage (first and second stage) of disease. These findings are in accordance with other studies that illustrated a decrease in the histone methylation levels of many cancers. Ellinger et al. documented that global levels of H3K9 and H3K27 methylation were significantly higher in healthy control than in BC, and in non-muscle invasive bladder cancer compared to muscle invasive bladder cancer. Moreover, Rogenhofer et al. found an inverse correlation of the H3K27me1, H3K27me2, H3K27me3 levels with Fuhrman grading and pathological T-stages in renal cell carcinomas. Similarly, global levels of H3K4me1, H3K9me1, H3K9me2, H3K27me2 and H3K27me3 were reported to be decreased in penile squamous cell carcinoma. Additionally, the level of tri-methylated H3K27 was lower in renal cell cancer tissues compared to normal tissues. Pellakuru et al. established that global levels of H3K27me3 were decreased in prostatic intraepithelial neoplasia and invasive adenocarcinoma lesions, that correlate with increased markers of disease aggressiveness (e. g. , Gleason score and pathological stage). Also, global levels of H3K4 and H4K20 methylation were decreased in BC compared with normal urothelium tissue, in metastatic tumour than in the primary tumour. As well, Ellinger et al. reported that H3K4me1, H3K9me2, H3K9me3 were significantly reduced in prostate carcinoma.

In addition to its diagnostic role, several studies have shown that global levels of histone modifications are of predictive value for the outcome, as the lower the histone methylation levels, the worse the prognosis and the outcome. H3K27me2 levels were found to be considerably lower in liver metastases than in the corresponding primary colorectal cancer, while increased H3K27me3 levels was associated with better prognosis and survival in numerous breast tumour subtypes.

Furthermore, Rogenhofer et al. recognized that decreased H3K9me1 levels indicate poor prognosis in patients with renal cell carcinoma. Moreover, Ellinger et al. (23)reported that H3K27me1 is also of prognostic value as its levels were correlated with pathological T-stage, capsular infiltration, seminal vesicle penetration and Gleason score in localized prostate carcinomas. On the contrary of our results, some studies reported an association between elevation of histone methylation levels and bad prognosis in different cancers. WhileHe et al. correlated the high expression of H3K27me3 in oesophageal squamous cell carcinoma with poor prognosis, Tzao et al. correlated it with nodal status and stage of the carcinoma. Besides, high expression ofH3K27me3 in hepatocellular carcinoma was positively correlated with large tumour size, multiplicity, poor differentiation, advanced stage, vascular invasion and shortsurvival. Moreover, high level of H3K9me3 in gastric adenocarcinoma was associated with higher T stage, lympho-vascular invasion, high recurrence rate and a poor survival rate. Finally, Benard et al. explored a high expression of H3K4me3 in early stage colon cancer that correlated with shorter patient survival and higher recurrence rate.

In conclusion, this study demonstrated that histone H3K27me2 level was decreased in bladder cancer patients compared to apparently healthy control, therefore assessment of histone H3K27 di-methylation level could be clinically helpful in diagnosis of both bladder carcinomas and its recurrence. In addition, the assessment of histone H3K27me2levels could be used to exclude advanced stage bladder cancer if histone H3K27 di-methylation level is above 50ng/μl. Besides, it may be considered as a non-invasive blood based marker for follow up of BC patients after cystectomy as it could exclude recurrence as well as the histone H3K27me2 level is more than 50 ng/μl. Hence, estimation of histone H3K27me2 level may be considered a good negative biomarker could exclude recurrent case. Moreover, the decrease in the histone H3K27me2 level in smoker patients and in all smokers (both patients and controls), could be one possible mechanism that explain smoking as risk factor for BC. The most important result is that H3K27me2 level will be used as potential biomarkers from blood samples in different tumours.