Somatic mitochondrial DNA (mtDNA) mutations have been increasingly observed in primary human cancers. As each cell contains many mitochondria with multiple copies of mtDNA, it is possible that wild-type and mutant mtDNA can co-exist in a state called heteroplasmy. During cell division, mitochondria are randomly distributed to daughter cells. Over time, the proportion of the mutant mtDNA within the cell can vary and may drift toward predominantly mutant or wild type to achieve homoplasmy. Thus, the biological impact of a given mutation may vary, depending on the proportion of mutant mtDNAs carried by the cell. This effect contributes to the various phenotypes observed among family members carrying the same pathogenic mtDNA mutation. Most mutations occur in the coding sequences but few result in substantial amino acid changes raising questions as to their biological consequence. Studies reveal that mtDNA play a crucial role in the development of cancer but further work is required to establish the functional significance of specific mitochondrial mutations in cancer and disease progression. The origin of somatic mtDNA mutations in human cancer and their potential diagnostic and therapeutic implications in cancer are discussed. This review article provides a detailed summary of mtDNA mutations that have been reported in various types of cancer. Furthermore, this review offers some perspective as to the origin of these of mutations, their functional consequences in cancer development, and possible therapeutic implications.
Each human cell contains a nuclear (nDNA) and mitochondria genome (mitochondrial DNA (mtDNA)). mitochondrial DNA has been shown to be exclusively maternally inherited (Giles et al., 1980). Mammalian cells typically contain 103–104 copies of mtDNA. mitochondrial DNA can replicate independently of nDNA (Lightowlers et al., 1997). Mitochondria possess a double-membrane structure and contain double-stranded genome capable of transcription, translation and protein assembly. Human mtDNA is a 16.6 kb circular double-stranded DNA molecule, which is present at a high copy number per cell, and the number varies widely with the cell type. Human mtDNA encodes 13 polypeptides involved in respiration and oxidative phosphorylation, 2 rRNAs and a set of 22 tRNAs that are essential for protein synthesis in the mitochondria (Attardi and Schatz, 1988). Mitochondria have also been shown to play an important role in apoptosis, a fundamental biological process by which cells die in a controlled manner (reviewed in (Wang, 2001)). Apoptosis plays a critical role in cancer development and in the cellular response to anticancer agents. Thus, it is important to understand the biology of mitochondria and its contribution to tumorigenesis. Mitochondria generate much of the cellular energy through the process of oxidative phosphorylation, a process in which hydrogen is oxidized to generate water and ATP. Reactive oxygen species (ROS) are produced as by-products of respiration and the oxidative phosphorylation process. The close proximity of mtDNA to the ROS production site makes it more vulnerable to oxidative injury and may account for a portion of the increased mtDNA mutations often observed in cancer.
Defects in mitochondrial function have long been suspected to contribute to the development and possibly progression of cancer. Over half a century ago, Warburg (Warburg, 1956a; Hockenbery et al., 2002) initiated research on mitochondrial alterations in cancer and proposed a mechanism to explain the differences in energy metabolism between normal and cancer cells. He suggested that mitochondrial alterations could provide unique therapeutic targets in various cancer types (Warburg, 1956a, 1956b, 1956c). As Warburg's proposal, several cancer-related mitochondrial alterations have been identified. These alterations include changes in mtDNA content, altered expression and activity of respiratory chain subunits, and mtDNA mutations. One recent report suggested that care should be taken when reporting mutations due to sample contamination and or mix up (Salas et al., 2005). This review article provides a detailed summary of mtDNA mutations reported in various types of cancer. We have focused on mutations, which cause an amino acid change in coding regions. Other references sited in this review predominantly deal with silent mutations in the control or non-coding regions. Furthermore, this review offers some perspective as to the origin of these mutations, their functional consequences in cancer development, and possible therapeutic implications.
Mitochondrial DNA mutations in human cancer
The human mitochondrial genome has been completely sequenced and all the mitochondrial encoded genes have been identified and characterized (Grivell, 1983; Blanchard and Schmidt, 1996). Several mtDNA mutations have been identified in various types of human cancer. Mutations have been found to be present in both the non-coding region and coding regions of the mtDNA and the majority of the mutations appeared to be homoplasmic in nature (Figure 1).
Several studies have examined the presence of mtDNA mutations in breast cancer. One of the most comprehensive studies is that by Tan et al. (2002) which sequenced the complete mitochondrial genome in 19 sets of paired normal and tumor tissues from the same patients. Tan's studies reported somatic mutations in 74% of the patients, with the majority of the mutations (81.5%) restricted to the control regions (D-loop) of the mtDNA. The remaining 18.5% of the mutations were detected in the 16S rRNA, ND2, and ATPase 6 genes (Tan et al., 2002). Of these mutations, five (42%) were deletions or insertions in a homopolymeric C-stretch between nucleotides 303–315 (D310) within the D-loop. The remaining seven mutations (58%) were single-base substitutions in the coding (ND1, ND4, ND5, and cytochrome b genes) or non-coding regions (D-loop) of the mitochondrial genome. We previously found that 61% (11/18) of the fine needle aspirates from primary breast tumors harbored mtDNA mutations that were not detected in matched lymphocytes from the same patient or in age-matched normal breast tissue. In that study, we reported that 42% of the mutations were present in the D310 region encompassed within the control region (D-loop) of the mtDNA (Parrella et al., 2001). Although the most common mtDNA mutations detected in breast cancer have been largely single base substitutions or insertions, a large deletion of 4977 bp has been detected in both the malignant and paired normal breast tissues of patients with breast cancer (Bianchi et al., 1995).
mtDNA mutations have been examined in human colorectal cancer cell lines as well as in human colon cancer. Studies by Polyak et al. (1998) analysed 70% of mtDNA sequence in primary samples. The mutations were found in regions encoding ND1, ND4L, ND5, Cytochrome b, COXI, COXII and COXIII as well as in the12S and 16S rRNA genes. The mutations in the cell lines also matched those in the primary tumors from which the cell lines were derived. Similar to the breast cancer mutations, mtDNA mutations in colorectal cancer were mostly homoplasmic in nature, and often transitions at purine sites, suggesting that the mutations may have occurred from ROS damage.
Using a combination of heteroduplex analysis and single-stranded conformation polymorphism (SSCP) analysis, Alonso et al. (1997) examined mtDNA mutations in the non-coding D-loop region in paired normal and tumor DNA from 13 colorectal patients and reported the presence of somatic mutations in three of 13 (23%) cases. These mutations were A → T and G → C transitions, one base pair deletions and 2 bp insertions. Using PCR-SSCP, Habano et al. (1998, 1999) reported that 44% (20/45) of sporadic human colorectal carcinomas harbored mutations in the D310 region of the D-loop. Habano's studies also identified frame shift mutations in the regions encoding ND1 and ND5, however there was no report of large-scale deletions such as those observed in breast cancer (Habano et al., 1998, 1999). Some of the colorectal cancer harbored only homoplasmic mutations, while others harbored both normal and mutated mtDNA (heteroplasmy).
Liu et al. (2001) sequenced the D-loop region of mtDNA of 15 primary ovarian carcinomas and matched normal control tissues. Their study revealed that 20% of tumor samples carried single or multiple somatic mtDNA mutations. In the same study, a complete sequence analysis of the mtDNA genomes of another 10 pairs of primary ovarian carcinomas and control tissues showed a high incidence (60%) of somatic mtDNA mutation. A majority of the mutations were homoplasmic, and most were T → C or G → A transitions. The four regions of the mitochondrial genome primarily affected by these mutations were the D-loop, 12S rRNA, 16S rRNA and cytochrome b, suggesting that these regions may be mutational hotspots in ovarian cancer.
The mtDNA D-loop contains the initial site of heavy chain replication and the promoters for heavy and light chain transcription. PCR amplification of the D-loop region by Zhao et al. (2005) revealed 18 gene mutations in the cancerous tissue from seven patients. Four of these alterations appeared to arise from microsatellite instability in the D-loop region.
Using polymerase chain reaction and direct sequencing Wu et al. (2005) reported that 48% (15/31) gastric carcinomas displayed somatic mutations in the D-loop region. Interestingly in the same study they showed that 10 (67%) cancers with the somatic mutations in the D-loop had insertion or deletion mutations in nucleotide position (np) 303–309 of the mononucleotide repeat region (poly-C track). In a separate study of the mtDNA D-loop region alone by Alonso et al. (1997)), three of eight gastric tumors showed similar D-loop alterations.
Mutational variations in conserved 12S rRNA regions have also been found, specifically np652 G insertion and np716 T-G transversion in gastric tissues. A higher frequency of these changes were observed in intestinal type (12/17, 70.59%) compared to the diffuse type of gastric carcinoma (5/17, 29.41%) (Han et al., 2005). Burgart et al. (1995) analysed 77 gastric adenocarcinomas and found a 50 bp deletion in the mitochondrial D-loop region (4/32, 12.5%). The deletion included the CSB2 region and was flanked by 9-bp direct repeats. The deletion was present only in the adenocarcinomas arising from the gastroesophageal junction but was reported absent in the distal tumors. All of the above studies suggest that somatic mtDNA mutations and mtDNA deletion occur commonly in gastric cancers.
Hepatocellular carcinoma (HCC) is a highly malignant tumor prone to multicentric occurrence. Mitochondrial DNA mutations within the D-loop control region are a frequent event in HCC. Nomoto et al. (2002a, 2002b) identified mitochondrial mutations in HCC and found that 13 of 19 patients (68%) harbored a D-loop mtDNA mutation in at least one tumor. They also observed C-tract deletion/insertion mutations in eight of 19 cases (42%) and five other missense and deletion/insertion mutations in an additional five cases (26%).
A separate study testing malignant and non-malignant liver tissue of HCC patients found a high frequency of mutations in the D-loop region, especially in the region between nucleotides 100 and 600. A G → A transition at nucleotide 263 was found in all mtDNA samples and a T → C transition at nucleotide 489 as well as a C insertion between nucleotides 311 and 312 (Nishikawa et al., 2001). The frequency of these mutations correlated with the degree of malignancy. Most of the mutations detected were homoplasmic in nature, which indicates that the mutated mtDNA had become dominant in both HCC tissue and hepatocytes in non-cancerous regions of the liver. Further this suggests that the repeated destruction and regeneration of liver tissue associated with chronic viral hepatitis lead to the accumulation of mtDNA mutations.
In recent studies, mutations and deletion of specific areas of the mitochondrial genome in tumor and matched normal tissue of 62 patients with HBV infection were identified. The frequency of subjects harboring a D-loop mutation, was significantly greater in the liver tissue of subjects with HCC compared with normal control cases (59 vs 11%) while the incidence of deletion in mtDNA was significantly lower in patients with HCC (Wheelhouse et al., 2005). In an earlier study involving direct sequencing of mtDNA in 54 hepatocellular carcinomas (HCCs), mtDNA alterations were found in the D-loop region both in the HCC and the non-cancerous liver tissue. Twelve of 52 mutation sites in the D-loop region of mtDNA were claimed to be specific for HCC. No mtDNA mutation was detected in normal liver without chronic inflammation (Tamori et al., 2004). It was pointed out that frequent D-loop mutations in HCC could help determine the clonality of multiple HCCs. These mutations could thus serve to distinguish metastatic disease from the occurrence of multiple independent primary tumors.
Recently, the mitochondrial genome in 15 pancreatic cancer cell lines and five ductal adenocarcinoma xenografts was sequenced and revealed somatic mutations in virtually all cancers examined. Homoplasmic mutations were found within coding sequences or regulatory sequences. The mutations were found in mitochondrial rRNA genes, NADH dehydrogenase genes coding for complex I proteins (mtND1, mtND2, mtND3, mtND4, mtND4L and mtND5). Mutations were also found in complex III, mitochondrial cytochrome c oxidoreductase gene (mtCytB), complex IV mitochondrial cytochrome c oxidase genes (mtCOX1, mtCOX2, and mtCO3) and complex V mitochondrial ATP synthase genes (mtATP6 and mtATP8) and the D-loop regulatory region (Jones et al., 2001).
Previous studies of mtDNA have identified larger mtDNA deletions in prostate cancer (Jessie et al., 2001). In one study, the authors isolated DNA from 34 radical prostatectomy specimens, and the entire mitochondrial genome (16.5 kb) was amplified using long-range PCR (LXPC) (Jessie et al., 2001). Gel electrophoresis was performed to visualize the presence of low molecular weight (<16 kb) bands due to mtDNA deletions. In one of our studies, partial mitochondrial genome sequence analysis of 16 matched premalignant prostate lesions and primary prostate cancers revealed 20 mtDNA mutations in the tumor tissue of three of 16 patients. Identical mutations were also identified in the PIN lesion from one patient (Jeronimo et al., 2001). Petros et al. (2005) showed that 11–12% of all prostate cancer patients harbored cytochrome oxidase subunit I (COX I) mutations that altered conserved amino acids.
An investigation of mtDNA in 27 primary lung cancers identified somatic variants in nine of 27 tumors (Sanchez-Cespedes et al., 2001). Two of these tumors harbored a C-to-G transversion in nucleotide position 16 114 (inside the D-loop) and an A-to-G transition in position 10 448 (inside tRNAArg), respectively. The remaining seven tumors showed deletions or insertions in the mononucleotide C repeat sequence in the D-loop (Sanchez-Cespedes et al., 2001). Recent direct sequencing of the D-loop region of 12 SCLC cell lines and 16 NSCLC cell lines showed that either homopolymeric C tract or single base substitutions were present in 17 (61%) of the cell lines. In the same study sequence analysis of the D-loop region of 55 primary tumor cases of NSCLC showed D-loop changes in 11 cases (20%) (Suzuki et al., 2003).
Renal cell carcinoma
A study involving end-stage renal disease (ESRD) revealed 94 mtDNA polymorphisms (4–27 per individual) in the kidneys of the six patients. A few heteroplasmic variations were found of which 19/69 (56%) resulted in alteration of the amino acid sequences (Nagy et al., 2003). In another study involving eight chromophobe renal cell carcinomas (RCCs) and corresponding kidney renal parenchymal cells the entire mitochondrial genome was sequenced. One-third (28%) of the sequence variants were reported in the D-loop region. (Nagy et al., 2002). Loss of mtDNA and the mRNA coding for NADH dehydrogenase subunit 3 was reported in eight of 13 tumor kidney tissues in another study (Selvanayagam and Rajaraman, 1996). Moreover, PCR was used to study 39 human renal cell carcinomas (RCC) and matched normal kidney tissue removed during radical nephrectomy. Sequence analysis of one tumor specimen revealed a 264 bp deletion in the first subunit NDI (complex I) of the electron transport chain (Horton et al., 1996). Other studies involving mtDNA content measurement and functional mitochondrial enzyme measurement showed that both differed significantly from normal tissue. With increasing aggressiveness of RCCs, there was an increase in mitochondrial impairment, with decreased content of oxidative phosphorylation (OXPHOS) (complexes II, III and IV of the respiratory chain, and ATPase/ATP synthase) rather than to the mitochondrial content (citrate synthase and mitochondrial DNA). All renal cell carcinomas (RCCs) of clear cell type (CCRCCs) and chromophilic tumors studied exhibited a low content of complex V protein. But F(1)-ATPase activity was not decreased and its impairment was associated with increased aggressiveness in CCRCCs (Simonnet et al., 2002). These findings support the hypothesis that a decreased oxidative phosphorylation favors increased tumor growth or invasiveness. The same authors reported decreased protein content and NADH dehydrogenase activity in renal oncocytomas (Simonnet et al., 2003).
The role of mtDNA mutation in thyroid cancer is yet to be established, though it has been known for a long time that thyroid tumors contain abnormally high numbers of mitochondria (Stefaneanu and Tasca, 1979). In a recent study involving 24 thyroid tumor specimens (19 primary papillary thyroid carcinomas (PTC), one follicular thyroid carcinoma, and four multinodular hyperplasias), mtDNA mutations were analysed by sequencing the entire coding region of the mitochondrial genome. Seven of 19 PTC had somatic mutation and one of four multinodular hyperplasias also showed somatic mutation. The majority of the mutations were found in complex I and a severe defect in complex I activity was detected in thyroid cancer cell lines using flow cytometry (Abu-Amero et al., 2005). Studies by Maximo et al. (2002) involving 79 benign and malignant thyroid tumors reported 57 somatic mutations. Unlike many other cancer types the incidence of D310 C-tract mutation was reported as relatively low (Tong et al., 2003). Previous studies using real-time PCR, involving adult patients possibly exposed to radioactive fallout from Chernobyl reported high mtDNA content in thyroid tumor tissue. Increased large-scale deletions were found mainly in tumor tissues of the radiation-associated group and correlated with the level of radio pollutant in PTC (Rogounovitch et al., 2002).
Mitochondrial alterations have been described in malignant gliomas for some time now. Earlier studies on gliomas described changes in the copy number of mtDNA (Liang and Hays, 1996). In another study, 100% of the low-grade tumors studied revealed an increase in copy number when compared to a normal brain control (Liang, 1996). Recent studies of 42 cases of malignant gliomas showed alterations in 36% of the cases in the D-loop region. However, MRI and clinical follow-up of these patients suggested that these mutations were not associated with increased aggressiveness (Montanini et al., 2005). In another study by Wong et al. (2003) mitochondrial mutations were found in medulloblastoma. The entire mitochondrial genome of the 15 cases of medulloblastoma and the corresponding cerebrospinal fluid (CSF) of eight of 15 cases was analysed using temporal temperature gradient gel electrophoresis. Results indicated that 40% of the cases studied had at least one mitochondrial mutation in each of the tumors studied. Remarkably, seven of eight of the CSF samples studied also showed the presence of mtDNA mutation (Wong et al., 2003).
Other solid tumors
The frequency of mitochondrial mutation in other forms of cancer has been studied to a lesser extent. Previous studies from our lab involving bladder and head and neck cancer revealed base transitions from T to C and G to A in ND3, ND4, mitochondrial Cytochrome b, 16S rRNA and D-loop region (Fliss et al., 2000). Another study involving 56 tumors (16 transitional cell carcinoma of the bladder, 20 breast cancer, 14 squamous cell carcinomas of the cervix and six endometrial tumors) samples for deletion and insertion in the D310 region, revealed 13 mutations, with the highest frequency of mutations in cervical cancer (Parrella et al., 2003). Mitochondrial mutations have also been reported in the D-loop control region of esophageal carcinomas (Abnet et al., 2004). Earlier studies involving adenocarcinomas arising in Barrett's esophagus revealed alterations in the D-loop region in 40% of the patients examined (Miyazono et al., 2002). Another study showed that 2/37 primary esophageal cancers (5%) contained somatic mutations in the D-loop region of mtDNA (Hibi et al., 2001). These correlative finding reveal that mtDNA play a crucial role in the development of cancer but further work is required to establish the functional significance of specific mitochondrial mutations in cancer and disease progression.
Altered mitochondrial metabolism in cancer
Somatic mutations, in the homoplasmic or heteroplasmic state occurs in all kinds of tissues and body fluids of cancer patients (Fliss et al., 2000; Chinnery et al., 2002). In the above writing, we focused on the mutations reported to date in various cancer types. In addition several metabolic alterations associated with mitochondrial function have been observed in cancer: Increased gluconeogenesis (Leij-Halfwerk et al., 2000) and high rate of glycolysis (Bando et al., 2005; Chowdhury et al., 2005). Increased rates of glucose transport in malignant cells have been associated with increased and deregulated expression of glucose transporter proteins in cancer (Cooper et al., 2003; Rudlowski et al., 2004; Macheda et al., 2005). Other defects in biochemical pathway include reduced pyruvate oxidation and increased lactic acid production (Lopez-Alarcon and Eboli, 1986; Mazurek et al., 1997; Eigenbrodt et al., 1998; Basso et al., 2004; Wenzel et al., 2005). Reduced fatty acid oxidation (Ockner et al., 1993; Hardy et al., 2003) increased glycerol production (Shaw and Wolfe, 1987; Beck and Tisdale, 2004) modified amino acid metabolism (Peluso et al., 2000; Denda et al., 2002; Maxwell and Rivera, 2003) and increased pentose phosphate pathway activity (Ferretti et al., 1993; Boros et al., 1998, 2000) are some common alterations associated with cancer cells.
Mitochondrial mutations were also reported in normal subjects, especially with advancing age. In particular the A189G age-associated mutation was found only in older individuals and prevalently in ragged red fibers in muscle (Cormio et al., 2005). Sequence analysis of substantia nigra of normal human subjects also revealed mitochondrial somatic mutations in both neurons and glia (Cantuti-Castelvetri et al., 2005). Other reports have shown the existence of two point mutations (A189G and T408A) within the D-loop region in skeletal muscles from aged individuals (Del Bo et al., 2002). Other somatic mutations in apparently normal subjects have been reported (http://www.mitomap.org/cgi-bin/mitomap/tbl14gen.pl), but most were associated with aging or one of the known mitochondrial diseases, the discussion of which is beyond the scope of this review article.
The activity of other enzymes involved in oxidative phosphorylation is known to be decreased in cancer cells. The alpha subunit of mitochondrial F(1)F(0)-ATP synthase has been shown to be downregulated in colorectal carcinomas (Sakai et al., 2004; Shin et al., 2005). Studies have also revealed reduced expression of the beta 1-subunit of Na-K-ATPase in poorly differentiated carcinoma cell lines derived from colon, breast, kidney and pancreas (Espineda et al., 2004). Significant reduction in the expression of beta-F1-ATPase has also been observed in breast and gastric adenocarcinomas, as well as in squamous esophageal and lung carcinomas (Isidoro et al., 2004). Decreased respiratory ATP synthesis (OXPHOS) and decreased ATPase activity has been observed in mitochondria of human hepatocellular carcinoma (Capuano et al., 1997). Decreased content of oxidative phosphorylation complexes (complexes II, III and IV of the respiratory chain, and ATPase/ATP synthase) has been associated with renal cell carcinoma (Simonnet et al., 2002). Defects in respiratory enzymes due to mtDNA mutations and/or nuclear mutations or directly due to oxidative damage of the enzyme proteins and associated lipids can lead to enhanced ROS production. Cytochrome c oxidase may also contribute to decrease the level of ROS due to its antioxidant effects. Thus, a defect in cytochrome-c oxidase can cause enhanced ROS levels and a subsequent increase in mtDNA damage. Studies have shown a decreased activity of cytochrome oxidase in colon tumors as compared to normal mucosa (Cavelier et al., 1995). Biopsies from human colon carcinoma have revealed lower expression of mitochondrial COX III, compared to normal mucosa (Heerdt et al., 1990). A similar decrease in cytochrome c oxidase activity has been reported in human colonic adenocarcinoma (Sun et al., 1981; Cavelier et al., 1995). Further studies involving rat hepatoma cells have revealed three times lower activity of cytochrome c oxidase and succinate dehydrogenase in tumors (Sun and Cederbaum, 1980). In contrast to the above studies, two to four times higher amounts in the poly(A)-rich RNA of COX IV and five times higher amounts in COX I and COX II have been observed in rat hepatoma (Luciakova and Kuzela, 1992).
Altered metabolism in cancer cells has been directly or indirectly linked to mitochondria. Cancer cells are metabolically adapted for rapid growth and proliferation under hypoxic conditions, a condition in which normal cells would not grow at all or only poorly (Griffiths, 2001). Other notable differences in the mitochondria of transformed versus non-transformed cells have been observed. Differences in the ultra structure of mitochondria (Hoberman, 1975; Springer, 1980), and depletion cellular mitochondrial numbers have been reported in liver carcinogenesis (Cuezva et al., 2002). Also, as discussed above, differences in content and composition of all oxidative phosphorylation complexes (Irwin et al., 1978; Cuezva et al., 2002; Simonnet et al., 2002) respiratory chain activity (Boitier et al., 1995; Rossignol et al., 2004) expression of oxidative phosphorylation genes (Weber et al., 2002) and levels of mitochondrial DNA (Simonnet et al., 2002; Meierhofer et al., 2004; Mambo et al., 2005) were reported relative to normal controls.
In an important insight, investigators focused on the ATP6 (Mt ATP6, a mitochondrial DNA encoded gene for ATPase 6 of complex V of OXPHOS chain) gene in prostate cancer to assess its functional significance to tumor formation through mutation. To determine whether mutant tumors harbored a growth advantage, they introduced a pathogenic mtDNA ATP6 T8993G mutation into the PC3 prostate cancer cell line through cybrid transfer and tested for tumor growth in nude mice. The resulting mutant (T8993G) cybrids (cytoplasmic hybrids) were found to generate tumors that were seven times larger than the wild-type (T8993T) cybrids, with wild-type cybrids having little perceptible growth in mice. The T8993G mutation causes impaired mitochondrial ATPase synthesis. Importantly, these investigators found that the mutant tumors also generated significantly more ROS and postulated that an increase in ROS may lead to an increase in DNA damage and hence tumor growth. Thus, this ATP6 mutation actively contributes to tumor progression. However, most somatic mitochondrial changes do not produce altered amino acids and their biologic functional contribution remains unclear (Petros et al., 2005).
Towards mitochondrial therapeutics
No successful method has been established for clinically complementing mitochondrial dysfunctions in human mitochondrial disorders. However, a promising therapeutic approach to patients with mtDNA mutation is based on allotopic gene expression (allotopic gene expression is expressing a mitochondrially encoded gene from nucleus transfected constructs as fusion with an N-terminus mitochondrial target sequence). This concept was first developed by Phillip Nagley in Australia in the late 1980s, in Saccharomyces cerevisiae genes (Gearing and Nagley, 1986; Farrell et al., 1988; Law et al., 1988, 1990). The first successful mammalian allotopic expression of a mtDNA-encoded polypeptide was shown in 2002 by Manfredi et al. (2002). In this study, wild-type ATPase 6 protein was allotopically expressed from nucleus-transfected constructs as a fusion protein with an amino-terminal mitochondrial targeting signal peptide. Its expression in homoplasmic mutant T8993G mtATP6 cybrids corrected the ATP synthesis defect. Using a similar approach, Guy et al. (2002) have shown the allotopic expression of a synthetic ND4 subunit compatible with the ‘universal’ genetic code, into the mitochondria of cells harboring the G11778A mtND4 mutation that causes Leber's hereditary optic neuropathy (LHON). A different approach has shown the possibility of developing a tRNA mitochondrial import systems in human cells. Yeast cytosolic tRNAs with altered aminoacylation identities can be selectively targeted into human mitochondria, where they are functional in mitochondrial translation and can cure respiratory defects due to nonsense mutations in mtDNA-coded protein genes. But this transport is dependent on both yeast cytosolic import-directing factors (ScIDPs), and human cytosolic extracts (HmIDPs) (Kolesnikova et al., 2000). In a recent study, the same author demonstrated that expression of yeast tRNALys derivatives are partially imported into mitochondria and are able to participate in mitochondrial translation. The import of this tRNALys was accompanied by partial rescue of the mitochondrial function affected by the mutation A8344G, which occurs in myoclonic epilepsy with ragged-red fibers (MERRF) syndrome (Kolesnikova et al., 2004).
Targeted restriction endonucleases have been used as a tool for treatment of mitochondrial dysfunction, The SmaI gene with a mitochondrial targeting sequence, when expressed in cybrids carrying mutant mtDNA, resulted in elimination of the mutant mtDNA, followed by progression of the wild-type mtDNA, which led to the restoration of normal intracellular ATP level and normal mitochondrial membrane potential (Tanaka et al., 2002). Earlier studies have also demonstrated the targeting of endonucleases (PstI) to mitochondria as a potential tool for mitochondrial gene therapy (Srivastava and Moraes, 2001). Other therapeutic approaches included the use of Oligomycin which showed an increase of wild-type cells over cells harboring the mutation T8993G of mtATP6 gene (Manfredi et al., 1999).
Non-small-cell bronchial–carcinoma cell lines treated with Clofazimine, a known inhibitor of respiratory function, inhibiting mitochondrial OXPHOS and hence energy metabolism, in combination with Oligomycin resulted in inhibition of mitochondrial function. Administration of Clofazimine to athymic mice bearing these non-small cell lung cancer cell lines as a subcutaneous xenograft, showed significant reduction in tumor growth rate (Sri-Pathmanathan et al., 1994). Studies have also demonstrated the successful use of co-enzyme Q in several mitochondrial dysfunctional diseases (Luft, 1994). Recent studies with arsenic trioxide shows that it inhibits mitochondrial respiratory function, increases free radical generation, and enhances the activity of another super oxide generating agent against cultured leukemia cells and primary leukemia cells isolated from patients (Pelicano et al., 2003). Moreover Xu et al. (2005) reported that inhibition of glycolysis effectively kills colon cancer cells and lymphoma cells.
Delocalized lipophilic cations (DLCs) have been used as selective mitochondria-toxic chemotherapeutic agent for cancer cells. Owing to higher plasma and mitochondrial membrane potential of cancer cells compared to normal cells, these compounds are concentrated in mitochondria of cancer cells. A number of DCLs have been shown to selectively kill cancer cells both in vivo and in vitro as discussed in the review (Modica-Napolitano and Aprille, 2001). To increase the efficacy of DCLs as chemotherapeutic agent, they have also been used in photochemotherapy (PCT), reviewed in (Modica-Napolitano and Aprille, 2001; Modica-Napolitano and Singh, 2004). Other treatment procedures describe the use of peptide nucleic acids (PNA) for delivery of oligonucleotides into the mitochondrial matrix. Studies have shown that PNAs conjugated with mitochondrial localizing peptides are imported into the mitochondria of intact cells from the outside (Chinnery et al., 1999; Flierl et al., 2003).
Mitochondrial diagnostics of tumors
Over the past decade, a variety of approaches have been developed to improve the results of conventional cancer screening by detecting molecular markers in clinical samples (Sidransky, 2002) Although nuclear genetic and epigenetic changes have been the cornerstone of such studies, mitochondrial cellular content and mutations are also emerging as new molecular markers. We have shown that damaged mtDNA in cell lines led to the rapid evolution of homoplasmic mutations (Mambo et al., 2003). These types of homoplasmic mutations have been confirmed to occur in vivo in early preneoplastic and cancerous lesions (Ha et al., 2002). Recent refinements in techniques for the detection of mtDNA content combined with rapid high throughput methods of mutation detection, have spurred interest in clinical studies of various tissues and bodily fluids (Nomoto et al., 2002a). Examination of human bladder, head and neck, and lung primary tumors revealed a high frequency of mtDNA mutations (Fliss et al., 2000) The majority of these somatic mutations were homoplasmic in nature, indicating that the mutant mtDNA became dominant in tumor cells. The mutated mtDNA was readily detectable in cancer-paired bodily fluids (including urine, saliva and sputum) from each type of cancer and was 19–22 times more abundant than mutated nuclear p53 DNA (Fliss et al., 2000).
In 46 primary breast tumors, poly-C alterations in D310 were found in seven cancers by PCR. Using D310 mutations as clonal marker, investigators detected identical changes in five of five matched fine-needle aspirates and in four of four metastases-positive lymph nodes (Parrella et al., 2001). In another study, identical changes were detected in four of four urine sediments from patients with bladder cancer and in three of three fine needle aspirates of patients with breast cancer (Parrella et al., 2003). Nipple aspirate fluid has also yielded mitochondrial C-tract alterations in breast cancer patients (Isaacs et al., 2004) In another study, urine from five patients with bladder cancer and duodenal aspirates from four patients with pancreatic cancer were tested using the Mitochip array (Maitra et al., 2004). At least one cancer associated mitochondrial mutation was found in six of the nine body fluid samples. In prostate cancer, identical mitochondrial mutations were detected in all (three of three) matched urine and plasma samples (Jeronimo et al., 2001). In hepatocellular carcinoma, identical mitochondrial mutations were detected in eight of 10 tested paired serum samples (Nomoto et al., 2002b). In all of these studies, mitochondrial mutations were detected in clinical samples from early stage patients. Thus, by virtue of their clonal nature and high copy number, mitochondrial mutations provide a powerful molecular marker for noninvasive early detection of cancer. With the advent of high throughput approaches such as the Mitochip (Maitra et al., 2004), larger more prospective studies will be carried out to determine the value of these mutations in early detection approaches.
Mitochondrial DNA mutations and or insertions/deletions have been observed in many types of human cancer. Mitochondrial functional defects have also been observed due to abnormal expression of mtDNA encoded proteins due to defective oxidative phosphorylation. The clinical phenotypic variability of the mitochondrial mutations and polymorphic alterations in the mitochondrial genome generates technical challenges for applying clinical samples for early detection of cancer. Future studies are required to access the functional role of the various mitochondrial mutations in initiation and progression of cancer. Studies on the defects in oxidative phosphorylation and their inhibition or reversal, may also help in developing therapeutic strategies in cancer.
Abnet CC, Huppi K, Carrera A, Armistead D, McKenney K, Hu N et al. (2004). BMC Cancer 4: 30.
Abu-Amero KK, Alzahrani AS, Zou M, Shi Y . (2005). Oncogene 24: 1455–1460.
Alonso A, Martin P, Albarran C, Aquilera B, Garcia O, Guzman A et al. (1997). Electrophoresis 18: 682–685.
Attardi G, Schatz G . (1988). Annu Rev Cell Biol 4: 289–333.
Bando H, Atsumi T, Nishio T, Niwa H, Mishima S, Shimizu C et al. (2005). Clin Cancer Res 11: 5784–5792.
Basso D, Millino C, Greco E, Romualdi C, Fogar P, Valerio A et al. (2004). Gut 53: 1159–1166.
Beck SA, Tisdale MJ . (2004). Lipids 39: 1187–1189.
Bianchi MS, Bianchi NO, Bailliet G . (1995). Cytogenet Cell Genet 71: 99–103.
Blanchard JL, Schmidt GW . (1996). Mol Biol Evol 13: 537–548.
Boitier E, Merad-Boudia M, Guguen-Guillouzo C, Defer N, Ceballos-Picot I, Leroux JP et al. (1995). Cancer Res 55: 3028–3035.
Boros LG, Brandes JL, Yusuf FI, Cascante M, Williams RD, Schirmer WJ . (1998). Med Hypotheses 50: 501–506.
Boros LG, Torday JS, Lim S, Bassilian S, Cascante M, Lee WN . (2000). Cancer Res 60: 1183–1185.
Burgart LJ, Zheng J, Shu Q, Strickler JG, Shibata D . (1995). Am J Pathol 147: 1105–1111.
Cantuti-Castelvetri I, Lin MT, Zheng K, Keller-McGandy CE, Betensky RA, Johns DR et al. (2005). Neurobiol Aging 26: 1343–1355.
Capuano F, Guerrieri F, Papa S . (1997). J Bioenerg Biomembr 29: 379–384.
Cavelier L, Jazin EE, Eriksson I, Prince J, Bave U, Oreland L et al. (1995). Genomics 29: 217–224.
Chinnery PF, Samuels DC, Elson J, Turnbull DM . (2002). Lancet 360: 1323–1325.
Chinnery PF, Taylor RW, Diekert K, Lill R, Turnbull DM, Lightowlers RN . (1999). Gene Therapy 6: 1919–1928.
Chowdhury SK, Gemin A, Singh G . (2005). Biochem Biophys Res Commun 333: 1139–1145.
Cooper R, Sarioglu S, Sokmen S, Fuzun M, Kupelioglu A, Valentine H et al. (2003). Br J Cancer 89: 870–876.
Cormio A, Milella F, Vecchiet J, Felzani G, Gadaleta MN, Cantatore P . (2005). Neurobiol Aging 26: 655–664.
Cuezva JM, Krajewska M, de Heredia ML, Krajewski S, Santamaria G, Kim H et al. (2002). Cancer Res 62: 6674–6681.
Del Bo R, Bordoni A, Martinelli Boneschi F, Crimi M, Sciacco M, Bresolin N et al. (2002). J Neurol Sci 202: 85–91.
Denda A, Kitayama W, Murata A, Kishida H, Sasaki Y, Kusuoka O et al. (2002). Carcinogenesis 23: 245–256.
Eigenbrodt E, Kallinowski F, Ott M, Mazurek S, Vaupel P . (1998). Anticancer Res 18: 3267–3274.
Espineda CE, Chang JH, Twiss J, Rajasekaran SA, Rajasekaran AK . (2004). Mol Biol Cell 15: 1364–1373.
Farrell LB, Gearing DP, Nagley P . (1988). Eur J Biochem 173: 131–137.
Ferretti A, Chen LL, Di Vito M, Barca S, Tombesi M, Cianfriglia M et al. (1993). Anticancer Res 13: 867–872.
Flierl A, Jackson C, Cottrell B, Murdock D, Seibel P, Wallace DC . (2003). Mol Ther 7: 550–557.
Fliss MS, Usadel H, Caballero OL, Wu L, Buta MR, Eleff SM et al. (2000). Science 287: 2017–2019.
Gearing DP, Nagley P . (1986). EMBO J 5: 3651–3655.
Giles RE, Blanc H, Cann HM, Wallace DC . (1980). Proc Natl Acad Sci USA 77: 6715–6719.
Griffiths JR . (2001). Bioessays 23: 295–296.
Grivell LA . (1983). Sci Am 248: 78–89.
Guy J, Qi X, Pallotti F, Schon EA, Manfredi G, Carelli V et al. (2002). Ann Neurol 52: 534–542.
Ha PK, Tong BC, Westra WH, Sanchez-Cespedes M, Parrella P, Zahurak M et al. (2002). Clin Cancer Res 8: 2260–2265.
Habano W, Nakamura S, Sugai T . (1998). Oncogene 17: 1931–1937.
Habano W, Sugai T, Yoshida T, Nakamura S . (1999). Int J Cancer 83: 625–629.
Han CB, Ma JM, Xin Y, Mao XY, Zhao YJ, Wu DY et al. (2005). World J Gastroenterol 11: 31–35.
Hardy S, El-Assaad W, Przybytkowski E, Joly E, Prentki M, Langelier Y . (2003). J Biol Chem 278: 31861–31870.
Heerdt BG, Halsey HK, Lipkin M, Augenlicht LH . (1990). Cancer Res 50: 1596–1600.
Hibi K, Nakayama H, Yamazaki T, Takase T, Taguchi M, Kasai Y et al. (2001). Int J Cancer 92: 319–321.
Hoberman HD . (1975). Cancer Res 35: 3332–3335.
Hockenbery DM, Giedt CD, O’Neill JW, Manion MK, Banker DE . (2002). Adv Cancer Res 85: 203–242.
Horton TM, Petros JA, Heddi A, Shoffner J, Kaufman AE, Graham Jr SD et al. (1996). Genes Chromosomes Cancer 15: 95–101.
Irwin CC, Malkin LI, Morris HP . (1978). Cancer Res 38: 1584–1588.
Isaacs C, Cavalli LR, Cohen Y, Pennanen M, Shankar LK, Freedman M et al. (2004). Breast Cancer Res Treat 84: 99–105.
Isidoro A, Martinez M, Fernandez PL, Ortega AD, Santamaria G, Chamorro M et al. (2004). Biochem J 378: 17–20.
Jeronimo C, Nomoto S, Caballero OL, Usadel H, Henrique R, Varzim G et al. (2001). Oncogene 20: 5195–5198.
Jessie BC, Sun CQ, Irons HR, Marshall FF, Wallace DC, Petros JA . (2001). Exp Gerontol 37: 169–174.
Jones JB, Song JJ, Hempen PM, Parmigiani G, Hruban RH, Kern SE . (2001). Cancer Res 61: 1299–1304.
Kolesnikova OA, Entelis NS, Jacquin-Becker C, Goltzene F, Chrzanowska-Lightowlers ZM, Lightowlers RN et al. (2004). Hum Mol Genet 13: 2519–2534.
Kolesnikova OA, Entelis NS, Mireau H, Fox TD, Martin RP, Tarassov IA . (2000). Science 289: 1931–1933.
Law RH, Devenish RJ, Nagley P . (1990). Eur J Biochem 188: 421–429.
Law RH, Farrell LB, Nero D, Devenish RJ, Nagley P . (1988). FEBS Lett 236: 501–505.
Leij-Halfwerk S, van den Berg JW, Sijens PE, Wilson JH, Oudkerk M, Dagnelie PC . (2000). Cancer Res 60: 618–623.
Liang BC, Hays L . (1996). Cancer Lett 105: 167–173.
Liang BC . (1996). Mutat Res 354: 27–33.
Lightowlers RN, Chinnery PF, Turnbull DM, Howell N . (1997). Trends Genet 13: 450–455.
Liu VW, Shi HH, Cheung AN, Chiu PM, Leung TW, Nagley P et al. (2001). Cancer Res 61: 5998–6001.
Lopez-Alarcon L, Eboli ML . (1986). Cancer Res 46: 5589–5591.
Luciakova K, Kuzela S . (1992). Eur J Biochem 205: 1187–1193.
Luft R . (1994). Proc Natl Acad Sci USA 91: 8731–8738.
Macheda ML, Rogers S, Best JD . (2005). J Cell Physiol 202: 654–662.
Maitra A, Cohen Y, Gillespie SE, Mambo E, Fukushima N, Hoque MO et al. (2004). Genome Res 14: 812–819.
Mambo E, Chatterjee A, Xing M, Tallini G, Haugen BR, Yeung SC et al. (2005). Int J Cancer 116: 920–924.
Mambo E, Gao X, Cohen Y, Guo Z, Talalay P, Sidransky D . (2003). Proc Natl Acad Sci USA 100: 1838–1843.
Manfredi G, Fu J, Ojaimi J, Sadlock JE, Kwong JQ, Guy J et al. (2002). Nat Genet 30: 394–399.
Manfredi G, Gupta N, Vazquez-Memije ME, Sadlock JE, Spinazzola A, De Vivo DC et al. (1999). J Biol Chem 274: 9386–9391.
Maximo V, Soares P, Lima J, Cameselle-Teijeiro J, Sobrinho-Simoes M . (2002). Am J Pathol 160: 1857–1865.
Maxwell SA, Rivera A . (2003). J Biol Chem 278: 9784–9789.
Mazurek S, Boschek CB, Eigenbrodt E . (1997). J Bioenerg Biomembr 29: 315–330.
Meierhofer D, Mayr JA, Foetschl U, Berger A, Fink K, Schmeller N et al. (2004). Carcinogenesis 25: 1005–1010.
Miyazono F, Schneider PM, Metzger R, Warnecke-Eberz U, Baldus SE, Dienes HP et al. (2002). Oncogene 21: 3780–3783.
Modica-Napolitano JS, Aprille JR . (2001). Adv Drug Deliv Rev 49: 63–70.
Modica-Napolitano JS, Singh KK . (2004). Mitochondrion 4: 755–762.
Montanini L, Regna-Gladin C, Eoli M, Albarosa R, Carrara F, Zeviani M et al. (2005). J Neurooncol 74: 87–89.
Nagy A, Wilhelm M, Kovacs G . (2003). J Pathol 199: 237–242.
Nagy A, Wilhelm M, Sukosd F, Ljungberg B, Kovacs G . (2002). Genes Chromosomes Cancer 35: 256–260.
Nishikawa M, Nishiguchi S, Shiomi S, Tamori A, Koh N, Takeda T et al. (2001). Cancer Res 61: 1843–1845.
Nomoto S, Sanchez-Cespedes M, Sidransky D . (2002a). Methods Mol Biol 197: 107–117.
Nomoto S, Yamashita K, Koshikawa K, Nakao A, Sidransky D . (2002b). Clin Cancer Res 8: 481–487.
Ockner RK, Kaikaus RM, Bass NM . (1993). Hepatology 18: 669–676.
Parrella P, Seripa D, Matera MG, Rabitti C, Rinaldi M, Mazzarelli P et al. (2003). Cancer Lett 190: 73–77.
Parrella P, Xiao Y, Fliss M, Sanchez-Cespedes M, Mazzarelli P, Rinaldi M et al. (2001). Cancer Res 61: 7623–7626.
Pelicano H, Feng L, Zhou Y, Carew JS, Hileman EO, Plunkett W et al. (2003). J Biol Chem 278: 37832–37839.
Peluso G, Nicolai R, Reda E, Benatti P, Barbarisi A, Calvani M . (2000). J Cell Physiol 182: 339–350.
Petros JA, Baumann AK, Ruiz-Pesini E, Amin MB, Sun CQ, Hall J et al. (2005). Proc Natl Acad Sci USA 102: 719–724.
Polyak K, Li Y, Zhu H, Lengauer C, Willson JK, Markowitz SD et al. (1998). Nat Genet 20: 291–293.
Rogounovitch TI, Saenko VA, Shimizu-Yoshida Y, Abrosimov AY, Lushnikov EF, Roumiantsev PO et al. (2002). Cancer Res 62: 7031–7041.
Rossignol R, Gilkerson R, Aggeler R, Yamagata K, Remington SJ, Capaldi RA . (2004). Cancer Res 64: 985–993.
Rudlowski C, Moser M, Becker AJ, Rath W, Buttner R, Schroder W et al. (2004). Oncology 66: 404–410.
Sakai H, Suzuki T, Maeda M, Takahashi Y, Horikawa N, Minamimura T et al. (2004). FEBS Lett 563: 151–154.
Salas A, Yao YG, Macaulay V, Vega A, Carracedo A, Bandelt HJ . (2005). PLoS Med 2: e296.
Sanchez-Cespedes M, Parrella P, Nomoto S, Cohen D, Xiao Y, Esteller M et al. (2001). Cancer Res 61: 7015–7019.
Selvanayagam P, Rajaraman S . (1996). Lab Invest 74: 592–599.
Shaw JH, Wolfe RR . (1987). Ann Surg 205: 368–376.
Shin YK, Yoo BC, Chang HJ, Jeon E, Hong SH, Jung MS et al. (2005). Cancer Res 65: 3162–3170.
Sidransky D . (2002). Nat Rev Cancer 2: 210–219.
Simonnet H, Alazard N, Pfeiffer K, Gallou C, Beroud C, Demont J et al. (2002). Carcinogenesis 23: 759–768.
Simonnet H, Demont J, Pfeiffer K, Guenaneche L, Bouvier R, Brandt U et al. (2003). Carcinogenesis 24: 1461–1466.
Springer EL . (1980). Cancer Res 40: 803–817.
Sri-Pathmanathan RM, Plumb JA, Fearon KC . (1994). Int J Cancer 56: 900–905.
Srivastava S, Moraes CT . (2001). Hum Mol Genet 10: 3093–3099.
Stefaneanu L, Tasca C . (1979). Endocrinologie 17: 233–239.
Sun AS, Cederbaum AI . (1980). Cancer Res 40: 4677–4681.
Sun AS, Sepkowitz K, Geller SA . (1981). Lab Invest 44: 13–17.
Suzuki M, Toyooka S, Miyajima K, Iizasa T, Fujisawa T, Bekele NB et al. (2003). Clin Cancer Res 9: 5636–5641.
Tamori A, Nishiguchi S, Nishikawa M, Kubo S, Koh N, Hirohashi K et al. (2004). J Gastroenterol 39: 1063–1068.
Tan DJ, Bai RK, Wong LJ . (2002). Cancer Res 62: 972–976.
Tanaka M, Borgeld HJ, Zhang J, Muramatsu S, Gong JS, Yoneda M et al. (2002). J Biomed Sci 9: 534–541.
Tong BC, Ha PK, Dhir K, Xing M, Westra WH, Sidransky D et al. (2003). J Surg Oncol 82: 170–173.
Wang X . (2001). Genes Dev 15: 2922–2933.
Warburg O . (1956a). Science 124: 269–270.
Warburg O . (1956b). Science 123: 309–314.
Warburg O . (1956c). Oncologia 9: 75–83.
Weber K, Ridderskamp D, Alfert M, Hoyer S, Wiesner RJ . (2002). Biol Chem 383: 283–290.
Wenzel U, Schoberl K, Lohner K, Daniel H . (2005). J Cell Physiol 202: 379–390.
Wheelhouse NM, Lai PB, Wigmore SJ, Ross JA, Harrison DJ . (2005). Br J Cancer 92: 1268–1272.
Wong LJ, Lueth M, Li XN, Lau CC, Vogel H . (2003). Cancer Res 63: 3866–3871.
Wu CW, Yin PH, Hung WY, Li AF, Li SH, Chi CW et al. (2005). Genes Chromosomes Cancer 44: 19–28.
Xu RH, Pelicano H, Zhou Y, Carew JS, Feng L, Bhalla KN et al. (2005). Cancer Res 65: 613–621.
Zhao YB, Yang HY, Zhang XW, Chen GY . (2005). World J Gastroenterol 11: 3304–3306.
This work was supported by NCI Grant CA-P01-77664.
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Chatterjee, A., Mambo, E. & Sidransky, D. Mitochondrial DNA mutations in human cancer. Oncogene 25, 4663–4674 (2006) doi:10.1038/sj.onc.1209604
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