Mitochondria are the organelle responsible for ATP production in human cells [1
]. Each human cell contains several hundreds to one thousand mitochondria and each mitochondrion harbors 2–10 mitochondrial DNA (mtDNA) copies to form the mitochondrial network [3
]. The amount of ATP production is influenced by the number of mtDNA copies and the abundance of mitochondria under different cell types and various physiological conditions [2
Human mtDNA (Available online: http://www.mitomap.org/MITOMAP
) is a circular structure with 16,569 base-pair (bp) [2
]. It encodes 13 polypeptides that are essential for the assembly of respiratory enzyme Complexes I, III, IV, and V. The other ~90 polypeptides constituting the respiratory enzyme complexes are encoded in nuclear DNA (nDNA). The four subunits of respiratory enzyme Complex II are totally encoded in nDNA. The non-coding region, also called displacement loop (d
-loop), is the regulatory region for mtDNA replication and transcription [2
]. Several proteins are involved in the mtDNA replication or transcription, and mitochondrial transcriptional factor A (TFAM) plays dual roles in mtDNA replication and transcription through binding to d
-loop. TFAM plays a pivotal role in the regulation of mitochondrial biogenesis [6
Human cancers usually exhibit rapid tissue growth beyond the neo-vascularization with subsequent cancer central hypoxia [10
]. Mitochondrial alterations elicited by hypoxia in tumor microenvironment deserves appraisal. About 80 years ago, Dr. Otto Warburg found that human cancers displayed decreased mitochondrial respiration but increased glycolysis for ATP production during glucose metabolism. He contended that human cancer mitochondria are defective, impaired, or even destroyed. Such a glucose metabolic shift between mitochondrial respiration and glycolysis in human cancers has been coined as Warburg effect [12
Alterations of mtDNA copy numbers in human cancers have been extensively studied [15
]. An increase of the mtDNA copy number was noted in head and neck cancers, or esophageal squamous cell carcinoma, especially cigarette smokers [18
]. Such an increase was supposed to compensate for the damaged mtDNA to keep the supply of ATP by mitochondrial respiration above a threshold. A decrease of mtDNA copy number was noted in hepatic carcinoma, gastric carcinoma, lung cancer, or advanced lung cancer after neoadjuvant chemotherapy [23
]. Such a decrease of mtDNA copy number would cause the decline of mitochondrial function. Since human kidney harbors abundant blood supply, the newly-grown RCC would suffer from central hypoxia with reduced mtDNA content and impairment of mitochondrial function [10
In this study, we analyzed the alterations of mtDNA copy number in resected human RCC kidneys. We used the RCC cell line, the 786-O, to knockdown TFAM expression to decrease mtDNA replication and transcription, and to appraise the alterations of glucose metabolism, aggressiveness, and resistance to anticancer drugs. We have proposed a molecular mechanism to explain the role of mtDNA copy number alterations in the pathophysiology of human RCC.
During the past 20 years, alterations of mtDNA copy number in several human cancers had been extensively investigated [15
]. Simonnet et al.
and Meierhofer et al.
demonstrated a decrease of mtDNA copy number and a decline of mitochondrial enzyme activity in human RCC and such a decrease was associated with the aggressiveness of RCC [29
]. Similarly, we showed a significant decrease of mtDNA copy number among the five RCC samples in this study (Table 1
). Theoretically, the mitochondrial ATP production is positively related to the mtDNA copy number [2
]. RCC tissues with lower mtDNA copy number might display a lower mitochondrial ATP production. However, as proposed by Dr. Warburg, such a decline of mitochondrial respiration could be compensated for by an increased glycolysis [14
]. This scenario has been validated in several types of cancers. However, whether this is the case in RCC awaits further study.
Regarding the regulation of mtDNA copy number, DNA polymerase gamma and TFAM should be emphasized in cancers [6
]. Previous studies showed some mutations in the gene coding for DNA polymerase gamma or TFAM in breast cancers or colon cancers with low mtDNA copy numbers [39
]. Since TFAM plays the dual role of mtDNA replication and transcription [6
], we further appraised the role of decreased mtDNA copy number in RCC through the knockdown of TFAM.
Consistent with our expectation, the mtDNA copy number, protein levels of mtDNA-encoded polypeptides (ND1, ND6 and COX-2) and the rates of oxygen consumption (mOCRB
) of the TFAM-KD clone were lower than those of the NT clone (Table 2
and Table 3
; Figure 1
). Undoubtedly, the TFAM-KD clone had significant lower expression levels of proteins involved in mitochondrial biogenesis. Interestingly, the TFAM-KD clone showed higher expression levels of glycolytic enzymes, including HK-II, PFK (the rate-limiting step of glycolysis), and LDHA (Table 2
and Figure 1
]. This indicates an increase of glycolysis to compensate for the impairment of mitochondrial biogenesis. However, the difference in the LDHA mRNA expression between the NT and TFAM-KD clones was not as obvious as protein expression (Table 2
). As a result, additional information is needed to validate the upregulation of glycolysis in the TFAM-KD clone. Since an increase of glycolysis would contribute to the accumulation of lactate, the TFAM-KD clone did exhibit a higher ECARB
value (Table 3
). We confirmed that RCC cells had a lower mtDNA copy number, suggesting a decrease of mitochondrial biogenesis, which would be compensated for by an increase of glycolysis. However, the underlying mechanism for the upregulation of glycolysis in RCC warrants further investigation [13
Dr. Dang and coworkers have made great efforts to establish the associations among HIF-1α, Warburg effect, and glycolysis in human cancers [10
]. HIF-1α can suppress mitochondrial biogenesis and respiration, and concurrently enhance glycolysis [13
]. However, in the literature, the 786-O RCC cell line is negative for HIF-1α protein expression [45
]. Consistently, TFAM-KD or NT clones derived from 786-O RCC cells were negative for HIF-1α protein expression as revealed in this study (Figure 1
). It has been established that HIF-1α, PDK1 and PDH play an important role to regulate mitochondrial biogenesis during hypoxia [13
]. Without the signaling from the upstream HIF-1α, it is reasonable that we observed no obvious difference in the mRNA expression levels of PDK1 and PDHA1 between the NT and TFAM-KD 786-O clones. Interestingly, the TFAM-KD clone had a higher protein expression level of HIF-2α (Figure 1
). Although HIF-1α and HIF-2α have some overlapping effects, they regulate distinct cellular functions [47
]. HIF-1α primarily participates in the upregulation of glycolysis [48
]. HIF-2α is mainly involved in the regulation of tumor growth and cell cycle progression (Figure 1
and Figure 2
]. As a result, the higher expression of HIF-2α in the TFAM-KD clone might confer its higher invasive activity (Table 3
, trans-well migration activity). However, the signaling pathways that lead to the upregulation of glycolysis in the TFAM-KD clone has remained unknown.
Several oncogenes have been implicated in the upregulation of glycolysis of human cancers, including AKT
]. AKT can mobilize glucose transporters to the cell surface and activate HK-II to enhance glycolysis [51
]. MYC can activate most of the genes coding for glycolytic enzymes and directly binds to numerous glycolytic genes, including those encoding HK-II, enolase, and LDHA [42
]. Both AKT and MYC may enhance the Warburg effect in human cancers. Interestingly, we observed that the TFAM-KD clone not only expressed higher levels of AKT and MYC, but also showed higher expression levels of glycolytic enzymes including HK-II, PFK, and LDHA (Figure 1
). These findings led us to conclude that AKT and MYC, in addition to HIF-1α, play important roles to upregulate glycolysis in RCC 786-O cells.
Recently, the role of mechanistic target of rapamycin (mTOR) signaling through mTOR complex 1 (mTORC1) or mTORC2 in regulating human protein translation and ribosome biogenesis have been extensively investigated [52
], and their roles in the TFAM-KD 786-O RCC cells with mitochondrial dysfunction deserved discussion. Xu et al.
reported that stimulation of mTORC1 with l
-leucine increased the efficiency of mitochondrial transcription and translation to improve the mitochondrial function in Robert syndrome, a human developmental disorder [53
]. Morita et al.
further demonstrated that mTORC1 could stimulate the function and biogenesis of mitochondria through the upregulation of TFAM translation [55
]. Additionally, Morita et al.
and Masui et al.
also showed that mTORC2 could stimulate glycolysis though activation of AKT and MYC [55
]. In this study, we found that the expression levels of AKT and MYC were increased in the TFAM-KD 786-O RCC cells. We thus speculate that the mTOR signaling could orchestrate the metabolic shift in the TFAM-knockdown 786-O cells examined in this study [57
]. However, further studies are warranted to establish the signaling cascade.
The role of glycolysis in the resistance to anticancer drug, which is an important characteristic of malignancies, has been investigated [58
]. Since an increase of glycolysis not only provides ATP production, the off-shoot pentose phosphate pathway also offers ribose and NADPH for the biosynthesis of nucleotides and proteins, cell proliferation and protection from oxidative damage [60
]. It is reasonable to explain that the increased glycolysis might render the TFAM-KD clone higher proliferation rate and higher resistance to oxidative damages caused by doxorubicin [61
Cancer stem cells have a highly invasive activity and are highly resistant to chemotherapeutic agents, and the surface markers identification are the standard methods to select cancer stem cells [62
]. Recently, the association between the cancer cell stemness and Warburg effect has been discussed and that the glucose metabolic reprogramming is considered an important biological hallmark of cancer stemness [63
]. Chen et al.
reported that hypoxia might confer cancer cells to gain stemness and Shen et al.
further demonstrated that a decrease of OCR/ECAR ratio, indicating that a metabolic shift, might render cancer cells the specific feature of stemness [30
]. Furthermore, Guha et al.
reported that mtDNA reduction may drive the generation of breast cancer stem cells [66
]. It is of interest to note the effect of TFAM knockdown on the stemness of cancer cells. Interestingly, the TFAM-KD clone expressed a decrease in mOCRB
(indicating conditions that mimic hypoxia) and mOCRB
ratio (indicating metabolic reprogramming), and displayed a higher trans-well migration activity, higher resistance to doxorubicin, and a higher level of vimentin expression. These findings and other preliminary results suggest that TFAM knockdown could induce the stemness of human renal cancer cells. However, we did not evaluate the surface markers of the TFAM-KD clone, further studies have been designed to validate these observations.