Detection of Mitochondrial DNA Mutations in Nonmuscle Invasive Bladder Cancer

Abstract

Background: Mitochondrial DNA (mtDNA) mutations have been recently described in various tumors; however, data focusing on bladder cancer are scarce. To understand the significance of mtDNA mutations in bladder cancer development, we investigated the mtDNA alterations in bladder cancer cases. Methods: We studied the mtDNA in 38 bladder tumors and 21 microdissected normal bladder tissue samples. Mitochondrial genes ATPase6, CytB, ND1, and D310 region were amplified by polymerase chain reaction and then sequenced. Results: We detected 40 mutations in our patient population. Our findings indicate that G8697A, G14905A, C15452A, and A15607G mutations are frequent in bladder cancers (p<0.05). In addition, the incidence of A3480G, T4216C, T14798C, and G9055A mutations were higher in patients with bladder tumors. Conclusions: In conclusion, the high incidence of mtDNA mutations in bladder cancer suggests that mitochondria could play an important role in carcinogenesis and mtDNA could be a valuable marker for early bladder cancer diagnosis.

Introduction

The human cell contains hundreds to thousands of mitochondria, and each mitochondrion has 2-10 copies of mitochondrial DNA (mtDNA). mtDNA encodes 13 respiratory chain protein subunits, 22 tRNAs, 2 rRNAs and contains a noncoding region called D-loop that acts as a promoter for both heavy and light strands of mtDNA and contains essential transcription and replication elements (Taanman, 1999; Penta et al., 2001; Taylor and Turnbull, 2005).

Mitochondria are involved in important tasks and pathways in the cell such as energy production, reactive oxygen species (ROS) generation, and apoptosis, all of which have been associated with carcinogenesis (Warburg, 1956). Various studies in recent years have reported that mtDNA mutations are common in cancer, including colorectal, breast, cervical, ovarian, prostate, liver, pancreatic, esophagus, head, and neck cancers. Mutations of mtDNA identified in cancer cells include frameshift mutations, missense mutations, large deletions, and small insertions/deletions in repetitive regions of mtDNA (Carew and Huang, 2006; Chatterjee et al., 2006; Czarnecka et al., 2006; Kroemer, 2006). In recent years, a mononucleotide repeat between the nucleotides 303 and 315 (D310) has been detected, and this region has been accepted as a frequent hot-spot of deletion/insertion mutations in tumors (Sanchez-Cespedes et al., 2001; Parrella et al., 2003).

Bladder cancer is a common neoplasm of the urinary tract and a significant cause of morbidity and mortality (Hussain et al., 2005). It is the fourth and eighth most common malignant neoplasm in men and women, respectively. Urothelial carcinomas (Transitional Cell Carcinoma [TCC]) comprise the great majority of bladder tumors. Despite the relatively benign nature of nonmuscle invasive TCC, the recurrence rate can be as high as 70%, and 10%-15% of those will progress to a muscle invasive disease, which is lethal (Reznikoff et al., 2000; Halachmi et al., 2001). Although a number of genetic changes have been identified in bladder tumors, the majority of these studies focus on the nuclear genome and the cellular events (Reznikoff et al., 2000; Halachmi et al., 2001; Mitra et al., 2006). Until now, very few publications have been reported about the detection of mtDNA mutations in bladder cancer (Chen et al., 2004; Wada et al., 2006; Dasgupta et al., 2008).

In an attempt to provide further understanding of the status of mtDNA mutations in bladder cancer development, we analyzed the mtDNA by direct sequencing.

Materials and Methods

Clinical samples

Samples of bladder tumors (n=38) were collected by transurethral resection in cases diagnosed with clinical nonmuscle invasive bladder cancer. After pathological evaluation for histological grade and stage, DNA extraction was performed from the snap freezed samples. Additionally, samples of normal bladder tissues (n=21) were obtained during transurethral resection of the prostate and radical prostatectomy operations. The specimens were stored at−80°C until DNA extraction. Demographic, tumoral, and progression characteristics of patients are summarized in Table 1. The experimental protocol was approved by the ethics committee of Marmara University (MAR-SBY-2006-0146).

Table 1.

Demographic, Tumoral, and Progression Characteristics of Patients

Patient number	Age	Gender	Stage	Grade	Recurrence	Progression
1	81	Male	T1	G3	Absent	Absent
2	82	Male	T1	G2	Absent	Absent
3	73	Female	T2	G3	Absent	Absent
4	68	Male	T1	G3	Absent	Absent
5	83	Male	T1	G2	Present	Absent
6	73	Male	T1	G2	Present	Absent
7	79	Female	T1	G2	Present	Absent
8	90	Female	T1	G2	Present	Present
9	78	Male	T1	G2	Present	Absent
10	63	Female	Ta	G1	Present	Absent
11	74	Female	T1	G1	Present	Absent
12	66	Male	T1	G3	Present	Present
13	76	Male	Ta	G2	Present	Present
14	82	Female	T1	G2	Present	Absent
15	79	Male	T1	G2	Absent	Absent
16	62	Male	T1	G2	Present	Absent
17	75	Female	T1	G3	Absent	Absent
18	63	Male	Ta	G2	Present	Absent
19	75	Male	Ta	G1	Present	Absent
20	68	Male	T1	G1	Present	Absent
21	69	Male	T1	G2	Present	Present
22	68	Female	T1	G1	Present	Present
23	57	Female	T1	G1	Present	Absent
24	65	Male	T1	G2	Present	Present
35	82	Female	T1	G2	Present	Absent
26	58	Male	Ta	G2	Present	Absent
27	78	Female	T1	G1	Present	Present
28	64	Male	Ta	G1	Absent	Absent
29	59	Male	Ta	G1	Absent	Absent
30	60	Male	T1	G2	Present	Absent
31	58	Male	T1	G2	Present	Absent
32	68	Male	Ta	G1	Present	Absent
33	74	Female	Ta	G1	Present	Absent
34	75	Male	Ta	G1	Present	Present
35	64	Male	Ta	G1	Present	Present
36	68	Male	T1	G2	Absent	Absent
37	69	Male	Ta	G1	Absent	Absent
38	65	Male	Ta	G1	Present	Absent

DNA extraction and polymerase chain reaction amplification of mtDNA

Genomic DNA was extracted from tissues by using a commercial kit (Invitek), according to the manufacturer's instructions. The final DNA pellet was dissolved in double-distilled water and frozen at−20°C until use. For detection of mtDNA mutations; the ATPase6, CytB, and ND1 genes, and the D310 region were amplified by polymerase chain reaction (PCR) using 10-20 ng of total DNA. Table 2 lists the sequences of primers specific for human mt genes and the D310 region.

Table 2.

The Primer Sequences Used for Polymerase Chain Reaction of Human Mitochondrial DNA Genes and D310 Region

mtDNA genes	Primer sequence (5′→3′)
ND1	5′-primer: 5′-CCA ACC TCC TAC TCC TCA TTG T-3′ (3318-3339)
	3′-primer: 5′-GGG AAT GCT GGA GAT TGT AAT G-3′ (4231-4252)
ATPASE6	5′-primer: 5′-AAC GAA AAT CTG TTC GCT TCA T-3′ (8531-8552)
	3′-primer: 5′-ATG TGT TGT CGT GCA GGT AGA G-3′ (9185-9206)
CYTB	5′-primer: 5′-ACC CCA ATA CGC AAA ATT AAC C-3′ (14751-14772)
	3′-primer: 5′-TAC GGA TGC TAC TTG TCC AAT G-3′ (15794-15815)
D310	5′-primer: 5′-ACA ATT GAA TGT CTG CAC AGC CAC TT-3′ (241-266)
	3′-primer: 5′ -GGC AGA GAT GTG TTT AAG TGC TG-3′ (327-349)

mtDNA, mitochondrial DNA.

The sizes of the fragments after amplification were 675 bp, 1064 bp, and 934 bp for the ATPase6, CytB, and ND1 genes, respectively. PCR amplifications were performed in a total volume of 50 μL containing 50-100 ng DNA template in 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl₂, 100 mM each of dNTPs, 1.0 U Taq DNA polymerase, and 1.0 mM of each primer. The conditions of PCR amplification were as follows: a denaturation step at 94°C for 5 min followed by 35 cycles at 94°C for 1 min, 59°C for 1 min, 72°C for 1 min, a final extension at 72°C for 5 min, and a stop at 4°C.¹⁵ PCR amplifications for the D310 region were performed in a total volume of 50 μL containing 50-100 ng DNA template in 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.25 mM MgCl₂, 100 mM each of dNTPs, 1.0 U Taq DNA polymerase, and 10 pmol of each primer. The condition of PCR amplification of D310 region was as follows: a denaturation step at 95°C for 2 min followed by 35 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, a final extension at 72°C for 5 min, and a stop at 4°C.⁹ The size of the fragment containing the D310 repeat was 109 bp. All PCR products were fractionated by electrophoresis on a 2% agarose gel, and products showing appropriate sizes were purified by using a commercial kit (Roche).

Purification of PCR products and direct sequencing of mtDNA

All PCR products were purificated from agarose gel by using a commercial kit (Roche), according to the manufacturer's instructions before direct sequencing. Then, purified PCR products were sequenced by using the ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Table 3 lists the primers used for PCR direct sequencing of different mtDNA genes.

Table 3.

The Primer Sequences Used for Polymerase Chain Reaction Direct Sequencing of Human Mitochondrial DNA Genes and D310 Region

mtDNA genes	Primer sequence (5′→3′)
ND1	5′-primer: 5′-CCA ACC TCC TAC TCC TCA TTG T-3′
	5′-primer: 5′-TGA TCA GGG TGA GCA TCA AA-3′
ATPASE6	5′-primer: 5′-AAC GAA AAT CTG TTC GCT TCA T-3′
	3′-primer: 5′-ATG TGT TGT CGT GCA GGT AGA G-3′
CYTB	5′-primer: 5′-TAT CCG CCA TCC CAT ACA TT-3′
	3′-primer: 5′-GGT GAT TCC TAG GGG GTT GT-3′
D310	5′-primer: 5′-ACA ATT GAA TGT CTG CAC AGC CAC TT-3′
	3′-primer: 5′ -GGC AGA GAT GTG TTT AAG TGC TG-3′

Statistical analysis

SPSS 16.0 was performed for statistical analysis. The chi-square test was used to determine the relationship between each categorical variable and mtDNA mutation.

Results

The results of DNA sequencing demonstrated many point mutations in the mt genome of the human tumor samples. A total of 40 polymorphisms were identified in the bladder cancer patients, of which 33 were previously recorded. Seventeen of the previously recorded mutations resulted in an amino acid substitution. In this study also, seven novel polymorphisms were found. These are A8742G, T15541C, T15654C, T15783C, C3741T, C4029T, and G4048A polymorphisms, which are found in the ATPase6, Cytb, and ND1 genes. In these novel polymorphisms, T15654C, which is found in the Cytb gene, and G4048A, which is found in the ND1 gene, cause amino acid changes. The polymorphism results are summarized in Table 4. DNA sequencing chromatograms of novel polymorphisms in ATPase6, Cytb, and ND1 genes are shown in Figure 1, and representative chromatograms of the D310 region are shown in Figure 2.

FIG. 1.

DNA sequencing chromatograms showing novel polymorphisms in ATPase6, CytB, and ND1 genes.

FIG. 2.

Representative chromatograms of two D310 sequence somatic mutations. (A) 9C/10C heteroplasmic insertion of cytosine and (B) 10C/11C heteroplasmic insertion of cytosine.

Table 4.

The Mitochondrial DNA Mutations Identified in the D310, ATPase6, CytB, and ND1 Regions

Nucleotide position	Nucleotide change	Amino acid change	Mutation frequency (% of cases)	p-Value	Reported in Mitomap and other tumors
D310 region
	7C→9C/10C	—	1/38	0.644	x
	7C→10C/11C	—	1/38	0.644	x
ATPase6 gene
8557	G→A	Ala→Thr	1/38 (3)	0.644	x
8573	G→A	Gly→Asp	1/38 (3)	0.644	x
8584	G→A	Ala→Thr	2/38 (5)	0.714	x
8684	C→T	Thr→Ile	2/38 (5)	0.714	x
8697	G→A	Silent	9/38 (24)	0.013	x
8701	A→G	Thr→Ala	3/38 (8)	0.551	x
8730	A→G	Silent	1/38 (3)	0.411	x
8742	A→G	Silent	2/38 (5)	0.356	Novel
8848	T→C	Silent	1/38 (3)	0.356	x
8950	G→A	Val→Ile	2/38 (5)	0.123	x
9055	G→A	Ala→Thr	6/38 (18)	0.356	x
9110	T→C	Ile→Thr	1/38 (3)	0.644	x
CytB gene
14783	T→C	Leu→Ile	3/38 (8)	0.551	x
14793	A→G	His→Arg	1/38 (3)	0.644	x
14798	T→C	Phe→Leu	7/38 (18)	0.306	x
14905	G→A	Silent	9/38 (24)	0.013	x
15043	G→A	Silent	7/38 (18)	0.306	x
15218	A→G	Thr→Ala	3/38 (8)	0.259	x
15301	G→A	Silent	3/38 (8)	0.551	x
15323	G→A	Ala→Thr	1/38 (3)	0.644	x
15452	C→A	leu→Ile	13/38	0.034	x
15454	T→C	Silent	3/38 (8)	0.551	x
15498	G→A	Gly→Asp	3/38 (8)	0.259	x
15541	T→C	Silent	1/38 (3)	0.644	Novel
15607	A→G	Silent	9/38 (24)	0.013	x
15622	T→C	Silent	1/38 (3)	0.644	x
15654	T→C	Met→Thr	1/38 (3)	0.644	Novel
15783	T→C	Silent	1/38 (3)	0.644	Novel
ND1 gene
3480	A→G	Silent	6/38 (18)	0.356	x
3507	C→T	Silent	3/38 (8)	0.259	x
3546	C→A	Silent	2/38 (5)	0.411	x
3703	C→T	Leu→Trp	1/38 (3)	0.644	x
3741	C→T	Silent	2/38 (5)	0.714	Novel
3930	C→T	Silent	1/38 (3)	0.644	x
4029	C→T	Silent	1/38 (3)	0.589	Novel
4048	G→A	Asp→Asn	1/38 (3)	0.644	Novel
4071	C→T	Silent	1/38 (3)	0.644	x
4164	A→G	Silent	1/38 (3)	0.644	x
4188	A→G	Silent	4/38 (11)	0.410	x
4216	T→C	Tyr→His	11/38 (29)	0.078	x

G8697A, G14905A, C15452A, and A15607G polymorphisms were also found to be statistically higher in patients than in controls (p<0.05). In addition, the incidence of A3480G, T4216C, T14798C, and G9055A mutations were found to be higher in patients with bladder tumors. The association between mtDNA mutations and gender, age, tumor stage and grade, recurrence and/or progression were not found (p>0.05). The numbers and percentages of mutations in different stages and grades are summarized in Table 5. As shown in Table 6, there was also no significant difference in terms of mutation profiles between the risk groups constituted according to European Organisation for Research and Treatment of Cancer (EORTC) risk tables as suggested by the EAU guidelines, version 2011 (Babjuk et al., 2011).

Table 5.

Frequency of Mutations and Tumor Stages

Classification of tumors	ATPASE6 mutations	CYTB mutations	ND1 mutations	D310 mutations
Ta	9/13 (69.2%)	11/13 (84.6%)	8/13 (61.5%)	3/13 (23.1%)
T1	18/24 (75%)	20/24 (83.3%)	18/24 (75%)	12/24 (50%)
T2	0/1	0/1	1/1	0/1
G1	9/15 (60%)	14/15 (93.3%)	9/15 (60%)	3/15 (20%)
G2	11/18 (61.1%)	14/18 (50%)	15/18 (83.3%)	10/18 (55.6%)
G3	3/5 (60%)	3/5 (60%)	3/5 (60%)	2/5 (40%)

Table 6.

Frequency and Percentage of Mutations Between the Risk Groups Constituted According to European Organisation for Research and Treatment of Cancer Risk Tables

	High grade group		Intermediate and low grade group
Mutations	Frequency	Percentage	Frequency	Percentage
ATPase
8557 (G→A)	1/13	8%	0/25	0%
9055 (G→A)	1/13	8%	5/25	20%
8697 (G→A)	5/13	38%	4/25	16%
8701 (A→G)	2/13	15%	1/25	4%
Cytb
14798 (T→C)	1/13	8%	6/25	24%
14905 (G→A)	5/13	38%	4/25	16%
15452 (C→A)	5/13	38%	8/25	32%
15607 (A→G)	5/13	38%	5/25	20%
15043 (G→A)	3/13	23%	4/25	16%
15323 (G→A)	1/13	8%	0/25	0%
15541 (T→C)	1/13	8%	0/25	0%
14783 (T→C)	2/13	15%	1/25	4%
15301 (G→A)	2/13	15%	1/25	4%
ND1
3480 (A→G)	1/13	8%	5/25	20%
4216 (T→C)	5/13	38%	6/25	24%
4048 (G→A)	1/13	8%	0/25	0%
4071 (C→T)	1/13	8%	0/25	0%
4164 (A→G)	1/13	8%	0/25	0%

Discussion

Mitochondria play a fundamental role in energy production and oxidative phosphorylation. The mutation rate of somatic mtDNA is approximately 10-100 times higher than that of the nuclear DNA. Instability of mtDNA has been detected in neurodegenerative diseases, degenerative diseases, aging, longevity, and sudden infant death syndrome. The effect of mitochondria in energy metabolism, aging, the generation of ROS, and the initiation of apoptosis are important factors in tumorigenesis. In recent years, mtDNA mutations have been identified in several types of cancer in humans. Somatic mtDNA mutations have been identified in colon, bladder, head and neck, lung, breast, kidney, liver, stomach, esophagus, pancreas, and in hematological malignancies, lymphoma and leukemia (Carew and Huang, 2006; Chatterjee et al., 2006; Czarnecka et al., 2006; Kroemer, 2006). In this study, mtDNA mutations have been investigated in patients with bladder cancer. Although many genetic factors have been found in the nuclear DNA of bladder cancer, the data on genetic changes in the mtDNA are scant. In a study by Chen et al., high rates of point mutations and deletions were detected in human and rat bladder cancers. These changes were mostly base substitutions, single base insertions, and D-loop deletions (Chen et al., 2004).

We detected numerous base-pair substitutions in human bladder cancer samples. At least one substitution was observed in 27/38 (71%) of the tumors in the ATPase6 gene, 31/38 (81%) of the tumors in the CytB gene, and 28/38 (73%) of the tumors in the ND1 gene. Of the 40 polymorphisms identified, 7 were new polymorphisms. In our patient population, 12 mutations have been identified in the ATPase6 gene. A statistically significant difference was detected (p<0.05) between patients and control groups in terms of the G8697A mutation. The newly identifed A8742G mutation was found in two patients. Changes in the function of ATPase genes are important for ATP synthase enzyme function and, thus, for ATP production (Ruiz-Pesini et al., 2007). Shidara et al. (2005) showed that pathogenic mutations in the mitochondrial ATPase6 gene contribute to promotion of cancer using a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham).

Sequencing reactions were performed on both strands of the PCR fragments, and the results were compared with the human mtDNA revised Cambridge reference sequence (Ruiz-Pesini et al., 2007). Sequence variations found in both healthy and tumor tissue mtDNA were recorded as germ-line polymorphisms, whereas those not found in healthy tissues and in the database were categorized by prevention of apoptosis (Shidara et al., 2005). The role of ATP synthase genes in mtDNA maintenance may help explain our findings. Sixteen mutations have been identified in the CytB gene in which G14905A, C15452A, and A15607G mutations were statistically significant (p<0.05). Moreover, three new mutations have been found. In various cancers, a large number of CytB mutations has been reported. Dasgupta et al. (2008) investigated the effects of CytB mutations on cell metabolism and growth. They discovered a significant increase in tumor growth in the CytB mutant model. Twelve mutations have been found in the ND1 gene, of which the A3480G mutation had been previously identified in prostate cancer patients (Jeronimo et al., 2001).

We also investigated a specific and highly polymorphic homopolymeric C stretch (D310), located within the displacement (D) loop, a mutational hotspot in primary tumors. This mononucleotide repeat between np 303 and 309 is among the most unstable microsatellite regions in the mtDNA. It is involved in the formation of a persistent RNA-DNA hybrid that leads to the initiation of mtDNA heavy-strand replication (Sanchez-Cespedes et al., 2001; Parrella et al., 2003). Sanchez-Cespedes et al. (2001) found insertions at the D310 region in the stomach, breast, colon, lung, bladder, and prostate cancer at 62.5%, 29%, 28%, 16%, 20%, and 0%, respectively. In another study, Wada et al. found somatic mutations in 7/31 (23%) patients with bladder cancer in the D-loop region (Dasgupta et al., 2008). We found an insertion at the np 303-309 poly C tract region in tumors, but this was not statistically significant.

It was identified that the ATPase6, CytB, and ND1 gene mutations were in the homoplasmic state. In previous studies with cancer patients, the majority of mutations in mtDNA had also been shown as homoplasmic. Several hypotheses include selective advantage and random segregation of mutant genomes to explain the formation of homoplasmic mtDNA mutations in tumors.

However, the mechanism by which mtDNA mutations in cancer cells become homoplasmic remains unclear (Polyak et al., 1998; Fliss et al., 2000; Tan et al., 2002).

We found that T4216C, G8697A, G14905A, C15452A, and A15607G mutations have also been observed in the same patients. These polymorphisms belong to mitochondrial haplogroup T. Mutations in mtDNA have accumulated sequentially, and maternal lineages have diverged to form population specific genotypes. These specific combinations are called mitochondrial haplogroups (Penta et al., 2001). Mitochondrial haplogroups and polymorphisms have been found to be linked with carcinogenesis. Booker et al. (2006) demonstrated that the inheritance of mitochondrial haplogroup U is associated with an approximately 2-fold increased risk of prostate cancer and a 2.5-fold increased risk of renal cancer in white North American individuals. In addition, Bai et al. (2007) showed that European-American women with haplogroup U have significantly decreased risk of breast cancer. Another group reported that haplogrup X was associated with endometrial cancer (Darvishi et al., 2007). However, such a study has not been done for bladder cancer.

The statistically significant relationship could not be found between tumor stages, grades, and mutations. Due to the heterogenity of groups and the small number of patients in some groups, the relationship between genetic and phenotypic data is difficult to evaluate. Since ND1 mutations are increasing in relation to stage and grade, this mutation requires elucidation. CytB mutations seem to be closely associated with every stage and every grade of the cases, thus reflecting a mutation that occurs during early carcinogenesis (Table 4).

As mentioned in the results, we have also stratified our patients according to risk groups using EORTC risk tables as suggested by the EAU guidelines (version 2011). However, there was no significant difference in terms of mutation profiles between the risk groups (Table 5). The relationship between genetic and phenotypic data is difficult to evaluate due to the heterogeneity of groups and the small number of patients.

Conclusion

In conclusion, our data suggest that mtDNA mutations can be considered as a bladder cancer biomarker. They can also be examined in body fluids such as urine and saliva. Specifically, the mtDNA haplogroup T may be important to detect bladder cancer risk. More extensive biochemical and molecular studies and further studies in larger groups are necessary to determine the clinical significance of the mtDNA mutations in bladder cancer. Urinary mtDNA would be the practical choice, and we plan to concentrate our future research on this issue.

Footnotes

Acknowledgments

The authors thank Prof. Turgay Isbir for recommendations, Assoc. Prof. Nural Bekiroğlu for statistical analysis, and Mustafa Ozyurek for technical assistance. This study was supported by Marmara University Scientific Research Projects Commission (Project Number: SAG-DKR-150107-0006).

Author Disclosure Statement

No competing financial interests exist.

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