Breast carcinoma is one of the most frequent malignancy in females and poses a big threaten to women [1]. Breast carcinogenesis is a very complex progression with unknown etiology [2], knowledge of the genetic causes is still incomplete [3]. Among the genetic factors, mtDNA is involved in energy generation process. Normal cells use mitochondrial OXPHOS for energy production, whereas breast cancer cells depend on aerobic glycolysis to generate energy [4]. The altered metabolic activities can be linked to mitochondrial dysfunction that increases reactive oxygen species (ROS), promotes uncontrolled growth, and causes DNA damage in breast cancer [5].
The human mitochondrial genome is composed of 16569-bp in a double-chain structure characterized by high mutation rate and maternal inheritance [6,7]. Poorly protected mtDNA is sensitive to oxidative stress and other genotoxic damage [8]. mtDNA alternations are considered as the emerging factors which provoke breast cancer formation and progression [9,10]. For instance, mutations in the mitochondrial D-loop may alter the affinity of this region for transcripts involved in promotion of mtDNA replication, transcription, and protein production, leading to the development of breast cancer malignancy [11,12]. Furthermore, recent experimental studies revealed that mutations in OXPHOS genes, which cause impairments of respiration chain function, were involved in the metastasis of breast cancer [13,14].
Although human mt-tRNA is a relatively small molecule which accounts for only 4-10% of total RNAs, a large amount pathogenic mtDNA mutations are located in this region [15]. In fact, almost every tRNA has a well-conserved secondary structure including four stems and three loops, and plays important roles in mitochondrial translation [16]. Mutations in mt-tRNAs can affect the processes of transcription and translation, which subsequently leads to mitochondrial respiratory chain dysfunction, and are associated with a wide range of clinical diseases [17]. However, to date, no studies have been performed which assess the frequencies of mt-tRNA mutations and breast cancer.
With the purpose of understanding the relationship between mt-tRNA mutations and breast cancer, a total of 80 tissues that are derived from breast cancer patients and 80 matched normal tissues were enrolled for this mutational screening. After genetic amplifications and mtDNA sequence analysis, five possible pathogenic mt-tRNA mutations were identified. To see the contributions of these mutations to mitochondrial dysfunctions, the mtDNA copy number and ATP were analyzed.
Summary of clinico-pathological characteristics of breast cancer patients
Characteristics | Data (mean ± SD or n (frequency in %)) |
---|---|
Gender | |
Male | 2 (2.5) |
Female | 78 (97.5) |
Age | |
>50 years | 45 (56.25) |
≤50 years | 35 (43.75) |
Body mass index (kg/m2) | 25.4±3.3 |
Histological grade | |
I | 15 (18.75) |
II | 28 (35) |
III | 37 (46.25) |
TNM stage | |
I | 10 (12.5) |
II | 13 (16.25) |
III | 19 (23.75) |
IV | 38 (47.5) |
Cancer metastasis | |
Positive | 38 (47.5) |
Negative | 42 (52.5) |
Cloverleaf structures of five mt-tRNAs. Arrows indicate the locations of breast cancer-associated mt-tRNA mutations.
mt-tRNA mutations identified in this case-control study
Gene | Sequence alternation | CI (%)a | Homoplasmy /Heteroplasmy | Watson-Crick base pairingb | Nucleotide at tRNA | Location | Number of 80 breast cancer tissues (%) | Number of 80 matched normal adjacent tissues (%) | mtDNA haplogroup | Disease association |
---|---|---|---|---|---|---|---|---|---|---|
tRNAVal | G1606A | 100 | Heteroplasmy | G-C↓ | 5 | Acceptor arm | 1 (1.25) | 0 | N9a | Progressive ataxia, seizures, mental deterioration, mild myopathy, and hearing loss |
tRNAIle | A4300G | 100 | Heteroplasmy | C-G↓ | 42 | Anticodon stem | 1 (1.25) | 0 | C4c | Cardiomyopathy |
tRNASer(UCN) | T7505C | 100 | Homoplasmy | A-T↓ | 10 | D-arm | 2 (2.5) | 0 | F1 | Deafness |
tRNAGlu | A14693G | 100 | Homoplasmy | 54 | TΨC-loop | 2 (2.5) | 0 | Y2 | MELAS, deafness, LHON | |
tRNAThr | G15927A | 100 | Homoplasmy | C-G↓ | 42 | Anticodon stem | 1 (1.25) | 0 | B5b | Parkinson’s disease, LHON, deafness, CHD, |
CI: conservation index;
Classic Watson-Crick base pairing: created (↑) or abolished (↓)
Abbreviations: MELAS: mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode, LHON: Leber’s hereditary optic neuropathy, CHD: coronary heart disease
We further analyzed mitochondrial functions in patients carrying these mt-tRNA mutations, as shown in Figure 2. We found that patients with putative pathogenic mt-tRNA mutations had much lower levels of mtDNA copy number and ATP, as compared with the controls (
Analysis of mitochondrial functions. A. mtDNA copy number analysis in patients with and without mt-tRNA mutations. B. ATP qualification in patients with and without mt-tRNA mutations.
In addition, the classical pathogenicity scoring system [25] was then used to assess the scores of these mt-tRNA mutations. As a result, the total scores of
The predicted pathogenicity of breast cancer-associated mt-tRNA mutations
Scoring criteria | G1606A mutation | Score | A4300G mutation | Score | T7505C mutation | Score | A14693G mutation | Score | G15927A mutation | Score | Classification |
---|---|---|---|---|---|---|---|---|---|---|---|
Yes | 2 | Yes | 2 | Yes | 2 | Yes | 2 | Yes | 2 | ≤6 points: neutral polymorphisms; | |
No change | 2 | No change | 2 | No change | 2 | No change | 2 | No change | 2 | 7~10 points: possibly pathogenic; | |
No | 0 | No | 0 | No | 0 | No | 0 | No | 0 | ||
Yes | 2 | No | 0 | Yes | 2 | Yes | 2 | Yes | 2 | 11-13 points (not including evidence from single fiber, steady-state level or trans-mitochondrial cybrid studies): probably pathogenic | |
No evidence | 0 | No evidence | 0 | No evidence | 0 | No evidence | 0 | No evidence | 0 | ||
Yes | 2 | Yes | 2 | Yes | 2 | No | 0 | Yes | 2 | ||
No | 0 | No | 0 | No | 0 | No | 0 | No | 0 | ||
Strong evidence | 5 | Strong evidence | 5 | Strong evidence | 5 | Weak evidence | 3 | Strong evidence | 5 | ≥11 points (including evidence from single fiber, steady-state level or trans-mitochondrial cybrid studies): definitely pathogenic | |
Definitely pathogenic | 13 | Definitely pathogenic | 11 | Definitely pathogenic | 13 | Possibly pathogenic | 9 | Definitely pathogenic | 13 |
In this study, the frequencies of mt-tRNA mutations in tissue samples of 80 breast cancer patients and matched normal tissues were analyzed by direct sequencing. As a result, we identified five possibly pathogenic mutations:
In addition, the deafness-associated T7505C mutation was located at position 11 in the conserved base of the D-arm of
We next examined the mtDNA copy number and ATP levels in seven patients with mt-tRNA pathogenic/ likely pathogenic mutations and controls. As a result, we noticed that patients with these mutations had lower levels of mtDNA content and ATP when compared with the controls. In fact, the mtDNA copy number represented the number of mitochondria per cell and number of mitochondrial genomes per mitochondrion, being a biomarker of mitochondrial function [39]. Reductions in mtDNA copy number in cells can impair mitochondrial respiration and cause pathology including cancers [40]. Furthermore, reduction in mtDNA copy number will result an increasing in ROS production [41]. The over-production of ROS will lead to serious consequence such as increasing the oxidative stress in cells, damaging DNA; RNA; lipids and contributing to programmed cell death [42]. In addition, the respiratory chain of mitochondria was coupled with the phosphorylation of ADP in the process of electron transfer. Under the action of ATP synthase, ADP and 1‐molecule phosphate were combined to form ATP, providing energy for life activities. The activity of respiratory chain complex directly affected OXPHOS function of mitochondria and decreased the ATP production in breast cancer tissues with mt-tRNA mutations. The decreased in mtDNA copy number and ATP suggested the impairment of mitochondrial functions. Therefore, these mt-tRNA mutations caused the failures in tRNA metabolism and led to mitochondrial dysfunctions that were responsible for breast cancer.
In summary, this study suggested that mutations in mt-tRNAs are involved in breast carcinogenesis. Pathogenic mt-tRNA mutations may cause mitochondrial dysfunctions and play active roles in breast cancer. Mutational analysis of mt-tRNA genes were recommended, especially for those patients who had a family history of breast cancer.