Association of decreased mitochondrial DNA content with ovarian cancer progression

1Mitochondrion has its own genome, the mitochondrial DNA (mtDNA). Unlike nuclear genome, mtDNA exists in each cell with several hundreds to more than 10 000 copies. Generally, mtDNA content is tissue-specific and has a steady-state level in each type of tissue. Its maintenance depends mainly on nuclear-encoded factors which usually confers function through the Tfam pathway. Mitochondrial DNA copy number in cell is not under stringent control; and various internal or external factors associated with ATP demand may influence its level, such as: exercise, hypoxia, and steroid hormones stimulation. It is well known that carcinoma cells proliferate fast and survive in strict microenvironment, for example, under hypoxic condition. Either down- or upregulation of mtDNA content has been observed in a number of human malignancies. More importantly, mtDNA content change has also been found to be associated with histological types of gastric carcinoma.

2In our previous studies, we have demonstrated the occurrence of high frequencies of somatic mtDNA mutations in endometrial and ovarian carcinomas. In addition, we have also found that mtDNA copy number was significantly elevated in endometrial adenocarcinoma when compared with normal endometrial glandular epithelium. To further determine the mtDNA content change and its relationship with cancer, we therefore investigate the mtDNA copy number in primary ovarian carcinomas.

3Frozen samples of 38 cases of primary epithelial ovarian carcinomas and four borderline ovarian tumours diagnosed and treated from January 1991 to December 2004 were retrieved without specific selection for this study. The mean age is 52 (ranging from 26 to 83). The clinical and pathologic characteristics of the 42 patients with ovarian carcinomas are shown in Table 1. In addition, frozen samples of 17 normal ovarian tissues were also used for this study. Use of clinical samples in this study was approved by the Ethics Committee of the University of Hong Kong.

4In order to obtain pure tumour cell from cancerous tissues, laser capture microdissection (LCM) was employed. The LCM procedure and DNA isolation from LCM procured samples were previously described in detail. The recipes and conditions of quantitative PCR reaction were also described in our previous study.

5The raw data were processed using the software accompanying the ABI PRISM 7700 Sequencing Detection System. Linear regression was used to analyse the response of Ct (the cycle number at which fluorescence raises above the baseline level during real-time quantitative PCR) vs the logarithm of the DNA concentration. Pearson's correlation was used to test the relationship between patients' age and mtDNA copy numbers in tumour tissues. The copy number comparison between groups was performed by nonparametric test (Mann–Whitney or Kruskal–Wallis test). Statistical significance was set at P<0.05.

6Table 1 shows the mtDNA copy number per cell of the 42 ovarian cancer samples and the 17 normal ovarian tissues. Mitochondrial DNA content is significantly higher in ovarian tumours than in normal ovarian tissues (P<0.001) (Figure 1A). The mtDNA copy numbers in tumour tissues have no relationship with patients' ages.

7The levels of mtDNA in normal ovary was higher than that in endometrium (P=0.020) (Figure 1B). In ovarian tumours, an average of 3548±3421 copies of mtDNA was present in tumour cells. The highest and lowest copy numbers of mtDNA in cell were 16 772 and 444, respectively (Table 1). Compared with endometrial carcinomas (average mtDNA copy number=2012±2317 copies), a significantly higher level of mtDNA copy number was found in ovarian carcinomas (P=0.001) (Figure 1C).

8From the 42 patients, 20 were diagnosed as early-stage tumours, stage I or stage II; whereas 22 patients suffered from advanced carcinomas, stage III or stage IV. There is no significant difference of mtDNA copy numbers between the early-stage tumours (average at 3621±3974 copies per cell) and advanced stage carcinomas (average at 3482±2923 copies per cell) (Figure 1D).

9As two patients were uninformative on the grading, the analysis was carried out in 40 patients. No significant difference was observed between the four borderline tumours and other invasive carcinomas (Mann–Whitney test, Z=−1.328, P=0.199). In the 26 cases of Grade 3 carcinomas, the mean value of mtDNA content in tumour cells was 2361±1799 copies. In the other 14 patients with lower-grade tumours (including the four borderline tumours), the mean copy of mtDNA molecules in each cell were 5877±4659 copies, it is over two-folds and significantly higher (P=0.012) than that in the higher-grade carcinomas (Figure 1E).

10The mtDNA copy numbers among different histological subtypes of carcinomas was not significantly different (Kruskal–Wallis test, χ2=8.397, P=0.136, and df=5). Nevertheless, the mucinous tumours seem to have relatively higher level of mtDNA content than other subtypes. In the 42 cases, except two subjects of serous carcinoma uninformative on grading, 19 cases were classified as type I tumours and the other 21 cases were classified as type II tumour (for tumour classification, please see footnotes of Table 1). An average of 4998±4279 copies of mtDNA was detected in type I tumours; whereas, the mean copy number of mtDNA in type II tumours is 2318±1930. It is remarkable lower than that in the type I tumours (P=0.019) (Figure 1F).

11In this study, mtDNA content in ovarian carcinoma cells was found significantly higher than that in normal ovary. Except for a few reports, the change in mtDNA content does not associate with clinicopathological characteristics. Interestingly, in present study, we found that the mtDNA copy number in high-grade tumours is significantly lower than that in low-grade tumour. As the grade of tumour is a crucial prognostic factor, therefore, mtDNA content change might be an important genetic event in the progression of ovarian carcinoma.

12Mitochondrial DNA copy number change was also found related with other human diseases. A significantly lower level of mtDNA content was observed in oocytes from women with ovarian insufficiency. In addition, increased sperm mtDNA content was observed in male infertility. The findings suggested that the change of mtDNA copy number was related with the impairment of cellular physiologic function. A probable explanation is that the change in mtDNA content would likely cause or be caused by the deficiencies in oxidative phosphorylation activity. Reactive oxygen species generation in mitochondria, which lead to DNA oxidative damage in cells, could thus impair cell function.

13The significantly different levels of mtDNA content in different grades of tumour may be accounted for either by the downregulation in mtDNA replication in the high-grade tumours, or upregulation in mtDNA replication in low-grade tumours. Detection of many mtDNA alterations in the premalignant or preinvasive lesions indicated that mtDNA alterations could be an early genetic event in tumorigenesis. Mitochondrial DNA copy number was suggested to be increased by a feedback mechanism that compensates for the defective respiratory system owing to mutated mtDNA. So, it is possible that mtDNA copy number increased in early- and lower-grade malignancies.

14On the other hand, long-term exposure to severe environmental insult such as hypoxia decreases the mitochondrial content of muscle fibres. Undoubtedly, mitochondria are oxygen sensitive and the mtDNA content decrease might account for hypoxia. So, an alternative explanation of the decrease of mtDNA copy number in high-grade tumour is due to the fact that such tumour has rapid proliferation rate, and thus, survives in more severe hypoxia microenvironment leading to the downregulation of the mtDNA replication.

15The most important finding in this study is the association between decreased mtDNA copy number with high grade and histological subtype of ovarian carcinoma. The histopathological phenotypes of ovarian carcinoma are complex. The epithelial-derived tumours including serous, mucinous, endometrioid, clear cell, transitional, squamous, mixed, and undifferentiated types comprise the majority of malignant tumours. Each of these histological subtypes is associated with distinct molecular genetic alterations. Based on recent clinicopathological and molecular studies, it has been proposed that surface epithelial tumours could be divided into two broad categories, type I and type II tumours. Mutations in KRAS and BRAF have been found to be common in low-grade serous and mucinous ovarian carcinomas. KRAS belongs to RAS families which coding proto-oncogene that functions as a relay switch that transduces various growth signals in the cell surface to the nucleus through activation of the RAS–RAF–MEK–ERK–MAP kinase signalling pathway. Till now, no report connected the regulation of mtDNA replication with the genetic alterations in RAS–RAF pathway. As shown above, type I tumour had a significantly higher mtDNA copy number than that in type II tumour. So, whether or not a potential relationship between the genetic alterations in RAS–RAF pathway and mtDNA content change would be an interesting field to be explored.

16On the other hand, mutations in the tumour suppressor molecule p53 were detected in type II tumours frequently. Recent studies provided the potential mechanistic explanation. p53 binding sequence was identified in mtDNA suggested that p53 might be involved in the regulation of mitochondrial transcription and replication. In addition, p53 might also enhance the DNA replication function of mtDNA polymerase γ through their interaction. So, the loss of p53 owing to the mutations in the type II tumours could result in the decrease of mtDNA replication.

17Taken together, the finding of grade and histological associated change in mtDNA copy number provide a novel insight of the role of mtDNA alterations in cancer progression. Mitochondrial DNA content in cell may be potentially used as a tool to predict prognosis. Mechanisms of mtDNA maintenance in carcinoma cells warrant further investigation.