1 Introduction
In recent years, bladder cancer risk loci have been identified through genome-wide association studies (GWAS) [
1]. Until now, 14 bladder cancer risk loci, including
TP63,
c-MYC [
2],
TERT/CLPTM1L [
3],
FGFR3/TACC3 [
4],
PSCA [
5],
APOBEC3A/CBX6,
CCNE1,
UGT1A [
6],
SLC14A1 [
7,
8],
JAG1 [
9,
10],
TERC,
LSP1 [
10] and
MCF2L [
11] have been described. The GWAS-identified variants are unlikely functional by themselves. Instead, identified single-nucleotide polymorphisms (SNPs) are often correlated to the actual (unmeasured) causal variants, and hence further fine-mapping is required. Moreover, they mostly map to non-coding intronic and intergenic regions [
12]. Finding the causative SNPs proved to be challenging and only four of the GWAS-identified bladder risk loci, including
PSCA,
UGT1A,
CCNE1 and
APOBEC3B, have been functionally characterized, showing allele-specific regulation of gene expression [
5,
13‐
16].
The importance of non-coding regions in the regulation of gene expression has been demonstrated by the Encyclopedia of DNA Elements (ENCODE) initiative. ENCODE data revealed that GWAS-identified risk SNPs are significantly enriched in active regulatory regions, which are often cell type and disease specific [
17,
18]. The presence of a SNP in a non-coding region can alter gene expression levels both
in cis (locally) and
in trans (distally) via multiple mechanisms [
19]. For example, one of the most studied colorectal cancer risk SNPs (rs6983267), located in a 8q24 gene desert [
20], was found to enhance binding of the TCF4 transcription factor in an enhancer region [
21]. This enhancer interacts with the
c-MYC promoter and modulates its activity in an allele-specific manner [
22,
23], leading to increased
c-MYC expression in the presence of the risk allele [
24]. Later it was found that rs6983267 lies within a long non-coding RNA,
CCAT2, and that colorectal cancer cell lines express significantly higher levels of
CCAT2 transcripts containing the risk G allele.
CCAT2 was found to positively regulate
c-MYC transcription levels and thereby to affect the growth, metastasis and energy metabolism of colorectal cancer cells in an allele-specific manner [
25].
One of the bladder cancer risk SNPs, rs710521 (A > G variant, A is risk-increasing allele) is located in an intergenic region between
TP63 and
LEPREL1 on chromosome 3q28, in a linkage disequilibrium (LD) block encompassing the
TP63 gene [
2], which codes for a member of the p53 family of transcription factors.
TP63 encodes two isoforms:
TATP63 and
ΔNTP63, which have opposite effects on cell cycle regulation and apoptosis. Furthermore, it has been found that p63 expression plays a role in epithelial development and the formation of squamous epithelium. It has also been found to play a role in cancer development [
26]. In non-muscle invasive bladder cancer (NMIBC), altered p63 expression has been found to be inversely correlated with pathological grade, whereas in muscle invasive tumors (MIBC) p63 expression has been found to be frequently down-regulated [
27]. The second gene within the 3q28 locus,
LEPREL1, encodes a member of the Leprecan family of proteoglycans, involved in posttranslational modification of collagen, leading to protein stability [
28]. The expression of
LEPREL1 has been found to be down-regulated in breast cancer [
29] and hepatocellular carcinoma [
30] and to be up-regulated in thyroid cancer [
31].
In our study, we aimed at fine-mapping the original GWAS signal, rs710521, within the TP63/LEPREL1 gene locus and, subsequently, at a functional characterization of genetic variants within this locus that are associated with bladder cancer risk. Using publicly available regulatory data an enhancer region containing the fine-mapped bladder cancer risk SNPs was identified. We found that the presence of the identified enhancer increased ΔNTP63 promoter activity and affected cell viability. From our data we conclude that the bladder cancer-associated SNPs at the 3q28 locus map to an enhancer element that contributes to bladder cancer development by modulating gene expression levels.
4 Discussion
Susceptibility to bladder cancer has been shown to be modified by complex interactions between genetic and environmental factors [
51]. Several genetic variants have been identified that are associated with bladder cancer risk, which point to novel genes and mechanisms involved in bladder cancer development [
12]. Here, we fine-mapped a GWAS signal at the 3q28 locus and showed that a region containing the bladder cancer-associated SNPs regulate gene expression levels and modulate cell survival.
Using publicly available data for regulatory elements, we identified an enhancer region that significantly increased
ΔNTP63 promoter activity in bladder cancer-derived cells, although not in an allele-specific manner. Genetic variants identified by GWAS are usually associated with small risks and their effects on gene expression levels may be modest [
12]. Therefore, the sensitivity of the current assays may not be sufficient to reveal allele-specific effects. Furthermore, an enhancer region may regulate multiple target genes and a single gene may be regulated by multiple enhancer elements. CRISPR/Cas9 genome editing allows for assessment of the impact of a single genetic variant within particular genomic and/or cell type contexts [
52]. Improvements in the efficiency of CRISPR/Cas9-mediated homology directed repair (HDR) [
53] may facilitate functional studies of GWAS-identified variants in the future. The expression of transcription factors is tightly controlled and hence no significant, allele-specific effect on the promoter activity and expression of transcription factors (TFs) may be observed. However, allele-specific differences could be manifested through expression of TF-target genes [
54]. We indeed found that the expression levels of the
ΔNTP63 target gene
NOTCH1 significantly correlated with the risk SNP genotype. The Notch signaling pathway has been shown to be involved in bladder cancer development, although it is not clear yet whether Notch signaling is oncogenic or tumor suppressive. For example, high expression of Jagged2 (a NOTCH ligand) has been shown to be associated with tumor aggressiveness and the formation of metastases [
55]. Other studies have shown that
NOTCH inactivating mutations in vivo accelerated the development of bladder cancer and promoted the formation of squamous cell carcinoma [
56,
57].
In MIBC tissue specimens, the levels of
ΔNTP63 do correlate with the risk SNP genotype. Previously, it was found that in response to genotoxic stress ΔN-p63 is recruited to a p53-binding element in its own promoter leading to silencing of
ΔNTP63 expression [
58]. Smoking is the most important risk factor for developing bladder cancer, accounting for 50% of the tumors [
51]. Therefore, it is plausible that the modest allele-specific differences in
ΔNTP63 expression are manifested upon (smoking-induced) DNA damage. Moreover, eQTL discovery in tumor tissue has been shown to be challenging due to frequent genetic and epigenetic alterations affecting transcript levels [
54].
In our study, the deletion of the enhancer region in the 5637 bladder cancer-derived cell line led to decreases in
ΔNTP63 and p63 target gene mRNA expression levels, subsequently affecting cell viability. Similarly, it has previously been shown that shRNA-mediated silencing of
ΔNTP63 expression in 5637 bladder cancer cells resulted in cell cycle arrest, a decreased proliferation rate and Cyclin D1 downregulation. Moreover, it has been reported that also in a 5637 xenograft model in mice, knockdown of
ΔNTP63 inhibited tumor growth through induction of apoptosis [
59]. In another study, knockdown of
ΔNTP63 was found to increase adhesion and to decrease migration of 5637 cells by increasing F-actin levels [
60]. Interestingly, reduced
ΔNTP63 expression has been found to sensitize 5637 bladder cancer cells to DNA damage-induced apoptosis independent of p53 [
61]. In contrast, Fukushima et al. showed that
ΔNTP63 knockdown led to upregulation of N-cadherin and increased motility and invasion of 5637 cells [
62].
We found that deletion of the identified enhancer region E1 led to a decrease in both
ΔNTP63 and
LEPREL1 expression levels. In thyroid carcinoma,
LEPREL1 has been found to serve as a target of the TWIST1 transcription factor (associated with epithelial-to-mesenchymal transition, metastasis formation and a poor prognosis). In addition, it has been reported that down-regulation of
LEPREL1 significantly reduced the growth of thyroid cancer cells [
31]. In hepatocellular and breast cancer overexpression of
LEPREL1 has been found to inhibit cell proliferation and colony formation [
29,
30]. Until now, however, the role of
LEPREL1 has not been studied in bladder cancer.
Luminal and basal subtypes of MIBC have been identified by several groups [
43,
63‐
66]. The basal subtype has been shown to be associated with a shorter overall survival and to be characterized by deregulation of p63 target genes [
65]. High expression of basal keratins, such as KRT5 and KRT6A, is the most characteristic feature of the basal subtype of bladder cancer [
63]. ΔNp63 has been shown to regulate
KRT5 and
KRT6A gene expression in several tissues [
65,
67,
68]. The rs4687103 non-risk allele was found to be significantly correlated with increased
KRT5, but not
KRT6A, expression, suggesting that the risk locus on 3q28 stimulates basal/squamous differentiation of the tumors, with a shorter overall survival.
Multiple transcription factor binding sites have been shown to overlap the rs4687103 and rs4687104 SNPs in the
TP63 E1 enhancer region, including proteins known to be involved in basal/squamous bladder cancer subtype development, like STAT3 and c-MYC [
65,
66]. Moreover, STAT3, FOS, c-MYC and CEBPB transcription factors have been found to bind to the promoter regions of the
KRT5 and
KRT6A genes [
66]. Further studies are required to evaluate which transcription factors can bind to the E1 enhancer in an allele-specific manner, both in normal urothelium and in bladder cancer cells.
Genome-wide association studies have successfully led to the identification of genetic variants that modulate gene expression levels in bladder cancer. The presence of rs2294008, a missense variant in the
PSCA gene, has been found to lead to formation of a truncated PSCA protein, due to alteration of the start codon. The presence of the variant allele has been found to be associated with reduced
PSCA promoter activity [
5]. The protective T allele of rs17863783 within the
UGT1A locus has been found to be associated with increased
UGT1A6.1 expression, leading to a higher clearance of carcinogens from the urothelium and a decreased bladder cancer risk [
15]. In addition, urine concentration was found to be significantly decreased in carriers of the risk T allele of rs10775480 (intron of
SLC14A1) [
69]. In the
CCNE1 locus, the original GWAS-identified SNP rs8102137 and a
CCNE1 promoter variant rs7257330 were found to be associated with bladder cancer aggressiveness, marked by increased CCNE1 protein expression [
14].
APOBEC3B expression and enrichment in
APOBEC3B-signature mutations have been shown to correlate with rs1014971. Moreover, the presence of a SNP within the non-coding region upstream of
APOBEC3A has been found to affect binding of proteins in an allele-specific manner [
16]. Recently, Wang et al. identified a bladder cancer risk variant within the 3’-UTR of the
TP63 gene. The presence of this variant was shown to disrupt
miR-140-5p binding leading to allele-specific TP63 expression [
70]. Additional functional studies are warranted to uncover the molecular mechanisms by which this and other GWAS-identified SNPs influence bladder cancer risk.
In conclusion, we identified an enhancer region within the TP63/LEPREL1 intergenic locus containing bladder cancer risk SNPs that regulate gene expression levels in cis and, subsequently, tumor cell viability. Our study underlines the importance of GWAS-identified signals in non-coding regions for bladder cancer development. Further characterization of the identified region may unravel novel allele-specific pathways involved in the modulation of bladder cancer susceptibility.