In this study, relative mtCN was determined in cervical exfoliated cells from 591 cervical cancer patients and 373 cancer-free controls. We found that median mtCN in the patients was significantly higher than that in the controls. After adjustment for age and HPV types, a higher level of mtCN remained associated with increased odds of having cervical cancer, and there was a dose-response effect of mtCN on cervical cancer. The results suggest that mtCN alterations may be implicated in cervical carcinogenesis and might represent a potential biomarker for this malignancy.
To date, few studies have investigated the association between mtDNA abundance and cervical cancer. Based on tissue samples, Warowicka et al. showed that total mtCN was cumulatively increased in high-grade squamous intraepithelial lesion (n = 30) and cervical cancer (n = 29), compared to that in low-grade lesion samples (n = 29) [29], whereas Kabekkodu et al. reported a lower level of mtCN in cervical cancer tissues (n = 20) than in cervicitis samples (n = 10) [30]. Small sample sizes and different cancer stages might partly explain the inconsistency. Moreover, neither of the studies detected HPV infection, which is a pre-requisite for cervical cancer. In fact, cervical exfoliated cells are collected in cytology-based cervical screening and may also be a source of molecular biomarkers indicative of neoplastic changes in the underlying tissue. In the current study, for the first time we determined HPV types and mtCN in cervical exfoliated cells from cervical cancer patients and healthy controls. We found that mtCN in cervical cells was positively associated with cervical cancer after adjustment for age and HPV types. However, given the relatively weak association with cervical cancer, a combination with other biomarkers, such as E6/E7 mRNA, p16INK4a-Ki-67, and HPV integration [31, 32], may improve the prediction of cervical cancer risk for HPV-positive women. Furthermore, many epidemiological studies have explored the association of mtCN in peripheral blood leukocytes with different types of cancer, leading to quite conflicting results [33]. It is also unclear whether the variation of mtCN in blood can really reflect the etiology of a specific cancer.
High-risk HPV infection may cause a series of mitochondrial dysfunction by accelerating the production of ROS [34, 35]. Warowicka et al. observed that both mtCN and ROS were increased during cervical cancer development [29]. Although HPV infection usually does not mount an inflammatory response, viral oncogenes can induce a chronic ROS response. In vitro studies showed that the expression of E6*, a truncated isoform of HPV16 E6 protein, increased ROS levels in cervical cancer cells [24]. HPV16 E6 and E7 proteins can also evoke a ROS response via NOX2 oxidase activation [25]. Additionally, HPV18 E2 protein has been demonstrated to localize to mitochondrial membranes and augment mitochondrial ROS production without cell death, whereas low-risk HPV6 E2 exhibits very low interaction to mitochondria [36]. Increased ROS are thought to cause mtDNA injuries and initiate mtDNA replication to counterbalance functional defects in impaired mitochondria [37, 38]. Another possibility is that specific genetic events in the D-loop region, i.e., the non-coding mtDNA region which contains crucial elements for replication, may lead to the up-regulation of mtDNA replication [16]. Due to major roles of normal mitochondria in energy production, metabolism, and apoptosis, the accumulation of mtDNA alterations may contribute to the pathogenesis of cervical cancer.
Furthermore, several studies point out that tumor suppressor p53 can regulate mtCN and mitochondrial biogenesis [39, 40], inhibit the mitochondrial damage induced by ROS [41], and participate in the regulation of mitochondrial respiration [42]. High-risk HPV E6 protein has the ability to induce p53 degradation via the ubiquitin-proteasome pathway. [43, 44]. Consequently, high-risk HPV E6 may disorder the p53 functions and thereby cause mtCN alterations. The interplay of HPV, p53, ROS, and mitochondria warrants further investigations to uncover the mechanism underlying cervical cancer.
Although this study suggests a potential role of mtCN in cervical carcinogenesis, several limitations should be addressed. First, due to the retrospective nature of a case-control study, a causal relationship remained to be established. Second, among the controls, only those with HPV16/18 positivity underwent colposcopy and showed normal findings. A possibility of cervical precursor lesions in the remaining controls cannot be completely excluded. Finally, although the interaction between mtCN and HPV types was statistically nonsignificant, our interaction analysis was possibly underpowered.