Background
Bladder cancer is the 9th most common cancer and a leading cause of cancer-related death worldwide. It has been estimated that around 550,000 new bladder cancer cases and 199,922 deaths occurred in the year 2018 worldwide and these numbers are expected to double in the upcoming years [
1]. The disease is highly recurring and do frequently progress to a muscle invasive phenotype which necessitate a vigilant and continuous monitoring protocol [
2]. Despite advances in diagnostic and treatment modalities, bladder cancer remains source of co-morbidity and continues to pose challenges for clinicians given that patients’ outcome being solely dependent on the grading and staging system [
3]. Therefore, a deeper understanding of the bladder cancer pathogenesis and associated mechanisms will undoubtedly improve patients’ outcome via prevention of disease progression and recurrence.
It is well documented that occupational exposure to chemical carcinogens including aromatic amines and polycyclic aromatic hydrocarbons is associated with the risk of bladder cancer development [
2,
4]. Kellen et al. reported an increased risk of developing bladder cancer associated with cumulative exposure to aromatic amines, but not to PAHs and diesel [
5]. In an independent study, Ferrís et al. concluded that bladder cancer is a result of the interaction between constitutional and environmental risk factors including aromatic amines and polycyclic aromatic hydrocarbons [
6]. The involvement of environmental factors such as cigarette smoking in bladder carcinogenesis has been extensively investigated [
7,
8]. Recent evidence supports the dynamic interplay between environmental factors and other co-factors, including genetic predisposition, in the pathogenesis of bladder cancer [
9].
Protecting against carcinogen-induced and chemotherapy-induced oxidative stress involves a series of event characterized by the activation of phase-II cellular detoxifying enzymes; Glutathione S-transferases (GSTs) or N-acetyltransferases (NATs) [
10]. GSTs enzymes superfamily consist of at least 16 genes located on more than 7 chromosomes [
11]. Although they are structurally different with distinct evolutionary origins, all GSTs isoenzymes are functionally similar in protection against electrophiles and oxidative stressors. The cytosolic sub-family of GST is found to be active in a homo- or heterodimeric state and is sub-divided into eight classes designated as follow: GST alpha (α), mu (μ), kappa (κ), omega (ω), pi (π), sigma (σ), theta (θ), and zeta (ζ) [
12]. GSTs play a critical protective anticancer role through glutathione conjugation with a range of potentially cytotoxic exogenous or endogenous molecules making them less toxic. Allelic polymorphisms in these genes elicit changes in enzyme activities leading to biotransformation and play important role in the development and progression of different cancers, such as lung, colorectal, leukemia, breast and bladder cancers. Furthermore, Sau et al. showed the contribution of GSTs overexpression in resistance against several anti-cancer drugs [
13].
GSTM1 gene is located on chromosome 1p13.3 and the most common polymorphic variant of
GSTM1 gene is the homozygous deletion (
GSTM1 null genotype) characterized by abolished enzyme activity [
14]. Many studies have investigated the relationship between the genetic polymorphism of
GSTM1 and the risk of cancer, but the association remains controversial among different populations. Previous epidemiological studies showed an association between the homozygous deletion of
GSTM1 and increased risk of lung, colorectal and head and neck cancers [
15‐
17]. However other studies failed to establish the association between
GSTM1 null and the risk of several types of cancers [
18‐
21].
GSTP1 is encoded by a single gene located on chromosome 11 [
22]. The common functional
GSTP1 polymorphism at codon 105 is an A to G substitution resulting in an amino acid switch from isoleucine to valine (Ile
105Val) and lowering the catalytic activity of GSTP1enzyme [
23]. The decreased detoxification capacity of the GSTP1 enzyme resulted in differences in chemotherapeutic responses. The increased expression of the
GSTP1 Val105 genotype was shown to be associated with a variety of tumors, such as ovarian, breast, colon, lymphoma, and pancreas [
24]. The hypothesis that
GSTP1 variants modulate the risk of urinary bladder cancer has also been investigated [
24,
25]. However, inconclusive results have been reported on the association between
GSTP1 gene polymorphisms and the risk of bladder cancer: while a number of studies identified an obvious association between
GSTP1 polymorphisms Ile
105Val and bladder carcinoma risk [
26‐
28], other studies illustrated that there are no association between
GSTP1 Ile
105Val polymorphism and bladder cancer [
29,
30].
HER2 is a trans-membrane glycoprotein receptor tyrosine kinase of the epidermal growth factor receptor family EGFR/ErbB. It plays an important role in the development and progression of many tumor types including breast, gastric and bladder cancers [
31]. Recent sequencing efforts to uncover the complex genomic landscape of bladder cancer identified six distinct molecular subtypes. HER2-like is one of the main subtypes characterise by higher
ERBB2 amplification and signalling [
32]. HER2 is considered one of the most important prognostic biomarkers that play an important role in the patho-physiology of bladder cancers and a potential therapeutic target in bladder cancer [
31,
33,
34]. Also, interactions between GST gene family and other genes including
HER2 may be involved in cancer susceptibility and clinical management of cancer patients. In the present study, we aim to investigate the prognostic value of
GSTM1 and
GSTP1 genetic polymorphisms in patients with bladder cancer and evaluate their association with patients’ clinicopathological parameters. We also attempted to evaluate the clinical significance of HER2 status in cases confirmed to have GSTM1/ GSTP1 variants with bladder cancer prognosis.
Discussion
Globally, bladder cancer is a leading cause of mortality [
37,
38]. It has long been perceived that bladder cancer is a result of occupational and environmental exposure to carcinogens and tobacco smoking, however, the exact mechanisms of bladder carcinogenesis remain unclear. Recent findings suggested that genetic factors contribute potentially, through mutations in key genes, in the etiology and pathogenesis of bladder cancer [
7,
8,
39]. Glutathione S-Transferases (
GSTs) are members of a large gene family of cytosolic phase II xenobiotic metabolizing enzymes involved in catalyzing and detoxifying a variety of carcinogens including reactive electrophilic compounds [
11]. Members of the GST family play an important role in cellular defense through conjugation of xenobiotics with sulfhydryl group and promoting their excretion at later stage [
11,
40]. It has been proposed that polymorphisms in members of
GST of carcinogen-detoxifying gene family as well as in
NAT2 confer increased risk of bladder cancer [
39]. Moreover,
increased expression of GST family members, especially GSTP1 and GSTM1
, was reported in several human solid tumors and is believed to confer resistance to various platinum-base chemotherapy drugs and metformin through regulation of many genes and molecular pathways [
41,
42]. Mechanistically, it is believed that polymorphisms in genes involved in drug-metabolizing enzymes may result in drastic changes in carcinogens biotransformation leading to increased cancer susceptibility [
2].
In our investigation we examined the frequency of GSTP1 and GSTM1 variants in a cohort of 93 bladder cancer patient from Saudi Arabia. We also evaluated the association between GSTP1 and GSTM1 gene polymorphisms with a set of clinical and pathological parameters as well as the prognostic value of both genes polymorphisms in bladder cancer patients.
The frequency and distribution of
GSTM1 and
GSTP1 gene variants was represented in Table
2. In our study, the ratio of
GSTM1 present and null is equally distributed in our cohort 48.38 and 47.31% respectively. This data is in agreement with previous report on the frequency of the
GSTM1 null genotype in the Caucasian population [
43]. In an independent study, Kang et al, revealed that the frequency of the
GSTM1 null genotype was 59.1% in patients with muscle invasive bladder cancer (MIBC) [
44]. Nonetheless, it is well documented that the prevalence of
GSTM1 null genotype varies significantly among populations from different ethnic groups [
45]. As for
GSTP1 gene polymorphism when we considered patients holding at least one copy of the dominant allele, data indicated that the frequency of AA and AG genotypes were found to be significantly high in our study group with a combined ratio of 77.4% for both genotypes compared to the GG genotype (6.45%). The reported frequency of
GSTP1 AA/AG genotypes is around 67% of the Iranian patients [
26] and Indian patients [
46]. However, a slight high frequency, approximately 80%, of
GSTP1 AA/AG variants was observed in in the Caucasian population with bladder cancer [
47].
We next sought to evaluate the association between polymorphism of the
GSTP1 and
GSTM1 genes and patients’ outcome. Our results indicated a significant association between the null
GSTM1 genotype and poor overall survival among bladder cancer patients. The association between
GSTs and poor survival was previously highlighted in many cancer types including bladder cancer [
48‐
50]. As for
GSTP1 genotypes, our data show trend for better survival for patients with the wild allele homozygote AA in comparison to heterozygote AG and variant allele homozygote GG genotypes or to GG/AG combined though data are not significant. When
GSTP1 GG/AG and
GSTM1 null genotypes were present together, poor overall survival increased in comparison to
GSTP1 alone.
The accumulating data suggested that genetic polymorphism of
GSTs leads to reduced detoxification potential which may result in increased DNA adduct levels in the target tissues and eventual mutations in the driver genes leading carcinogenesis. Therefore, the association of
GSTP1/
GSTM1 variants with highly malignant disease and poor prognosis in cancer patients was suggested [
50].
Previous studies on patients from different ethnic origins revealed that individuals with the null
GSTM1 were at high risk of developing bladder cancer [
26,
51‐
54]. This association was also seen between
GSTM1 null and other cancers such as breast [
50], lung [
55] and colorectal cancers [
35]. Anwar et al. showed significantly higher
GSTM1 null distribution in bladder cancer patients than in healthy individuals [
51]. The distribution of the null
GSTM1 in our cohort did not show any significant difference in comparison to the wild-type allele which may indicate that the null genotype is not the only factor in determining the increased risk and aggressiveness of bladder cancer but is certainly one of many combined genetic factors that contribute to the pathogenesis of the disease. To-Figueras et al. suggested a relation between
GSTM1 null genotype and
p53 mutation in increasing the risk of lung cancer susceptibility among smokers [
55]. In an early observation by Ryk et al. the investigators demonstrated that the carriers of the variant allele of the
GSTP1 Ile
105Val polymorphism were characterized by frequent mutations in the tumor suppressor gene
p53 and high-grade/ high stage tumors in bladder cancer [
56]. In an independent investigation we performed high throughput mutational analysis of 50 oncogenes and tumor suppressor genes using cancer hotspot panel (CHP, v.2). Our data indicated that high proportion (~ 82%) of our bladder cancer cohort harbor
p53 mutation (data not published) which may suggest the involvement of
p53 mutation in association with GSTP1 in the risk of bladder cancer development and drug resistance. This suggestion is valid knowing that
GSTP1 gene contains a functional canonical p53 binding motif and the capacity of p53 to transcriptionally activate the human
GSTP1 gene [
57].
In the same context and for the first time we investigated the relationship between different GSTP1/GSTM1 variants and Human Epidermal growth factor Receptor 2 (HER2) gene/ protein status in bladder cancer patients. Our data indicated that patients with high HER2 protein expression/ gene amplification and null GSTM1 genotype had significant poor survival compared to patients with low HER2 expression and null GSTM1 genotype, suggesting that combining HER2 status with GSTM1 genotype may have a prognostic value for bladder cancer patients. The exact mechanism of the influence of GSTM1 and HER2 on bladder cancer is yet to be elucidated. Together, our data showed that GSTM1 gene deletion either alone or in combination with HER2 may serve as markers for bladder cancer prognosis.
We observed no association between the
GSTP1 Ile
105Val genotype,
GSTM1 genotype alone or in combination with HER2 status and patients’ clinicopathological features. This is consistent with previous published reports [
29,
58], and disagree with Safarinejad et al [
26] who found a significant increase in tumor grade and stage of bladder cancer patients carrying
GSTP1 Val/Val genotype and
GSTM1/
GSTT1 double null genotypes.
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