Glucose metabolic reprogramming from oxidative phosphorylation to glycolysis is one of the hallmarks of cancer development. Coenzyme Q10 (CoQ10) is essential for electron transport in the mitochondrial respiratory chain and for antioxidant defense. Here, we investigated the role of a key factor in CoQ10 synthesis, prenyldiphosphate synthase subunit 2 (PDSS2), in hepatocellular carcinoma (HCC) tumorigenesis. PDSS2 was frequently downregulated in HCC tissues and was significantly associated with poorer HCC prognosis (P = 0.027). PDSS2 downregulation was a prognostic factor independent of T status and stage (P = 0.028). Downregulation of CoQ10 was significantly correlated with downregulation of PDSS2 in HCC tumor tissues (R = 0.414; P < 0.001). Of the six different splicing isoforms of PDSS2, the five variants other than full-length PDSS2 showed loss of function in HCC. Reintroduction of full-length PDSS2 into HCC cells increased CoQ10 and mitochondrial electron transport complex I activity and subsequently induced a metabolic shift from aerobic glycolysis to mitochondrial respiration in cells. Reintroduction of PDSS2 also inhibited foci formation, colony formation in soft agar, and tumor formation in nude mice. Knockdown of PDSS2 induced chromosomal instability in the MIHA immortalized human liver cell line. Furthermore, knockdown of PDSS2 in MIHA induced malignant transformation. Overall, our findings indicate that PDSS2 deficiency might be a novel driving factor in HCC development.

Significance: Downregulation of PDSS2 is a driving factor in hepatocellular carcinoma tumorigenesis. Cancer Res; 78(16); 4471–81. ©2018 AACR.

Cancer cells reprogram their metabolism, which is characterized by a decrease in mitochondrial respiration and oxidative phosphorylation, together with strong enhancement of glycolysis, even in the presence of abundant oxygen (known as the Warburg effect; refs. 1–3). Rapid consumption of glucose in tumors has been associated with a poor prognosis in patients with oral squamous cell carcinoma (4), gastric cancer (5), esophageal cancer (6), pancreatic cancer (7), and other tumors (8). Lactate, once considered a waste product of glycolysis, has now been correlated with increased metastasis, tumor recurrence and poor outcome (9–11).

Initially, metabolic reprogramming was thought to be a consequence of rapid cell proliferation, but recent data have demonstrated that metabolic reprogramming can actually drive tumor development (12, 13). Inhibiting glucose flux by suppressing key enzymes such as PKM2 (14), LDH (15), and 6-phosphofructo-2-kinase (16) could effectively reduce tumorigenicity, implying that metabolic reprogramming from oxidative phosphorylation to glycolysis plays an important role in cancer development and progression (13). Recent studies showed that the oncogenes c-myc, Akt and NF-κB could upregulate many genes involved in glycolysis and glutaminolysis (1, 17). Similarly, loss of p53 function could lead to the Warburg effect through complex regulation of glycolysis, ROS levels and apoptosis (18–21).

In a previous study, we identified and characterized a tumor-suppressor PDSS2 (prenyldiphosphate synthase subunit 2), which was interrupted by the 6q21 breakpoint in the reciprocal-like chromosomal translocation t(6;17)(6q21:17p13) in the UACC930 melanoma cell line (22, 23). PDSS2 is important in determining the length of the side chain of ubiquinone in mammals (24). PDSS2 catalyzes the addition of isopentenyl diphosphate molecules to farnesyl diphosphate in multiple steps to produce the isoprenoid side chain of coenzyme Q10 (CoQ10). A decrease in the plasma CoQ10 level has been detected in patients with breast cancer (25), melanoma (26), and cervical cancer (27). 6q21 deletion is also one of the most frequently deleted regions in hepatocellular carcinoma (HCC; ref. 28). Although the introduction of PDSS2 into melanoma and gastric cancer cells could inhibit tumor growth (23, 29), the mechanism underlying the role of PDSS2 in tumor genesis is not well understood.

In the present study, we found that decreased expression of PDSS2 was frequently detected in HCC tumor tissues and was significantly associated with a poor outcome of HCC (P = 0.027). PDSS2 downregulation was an independent prognostic factor, independent of T status and stage (P = 0.028). Further study demonstrated that PDSS2 was able to increase mitochondrial electron transport complexes and then induce a metabolic shift from aerobic glycolysis to mitochondrial respiration in hepatocellular carcinoma tumor cells. Silencing PDSS2 in the MIHA immortalized human liver cell line promoted glycolytic flux and tumor cell growth. We also found PDSS2-Del2, a previously uncharacterized PDSS2 isoform, which was incapable of synthesizing CoQ10. The molecular mechanism of PDSS2 in HCC development was also evaluated.

Tissue microarray and staining for immunohistochemistry

Paraffin blocks from 295 patients with HCC and paired nontumorous tissues were collected from two hospitals (147 cases from the First Affiliated Hospital, Sun Yat-sen University, and 148 cases from Sun Yat-sen University Cancer Center). Written informed consent was obtained from the patients and the study was conducted in accordance with the Declaration of Helsinki. The study was approved by the Committees for Ethical Review of Research Involving Human Subjects in the Cancer Center and The First Affiliated Hospital, SunYat-sen University. A tissue microarray (TMA) was constructed according to the method previously described (30). The age of patients ranged from 9 to 83 years at the time of surgery (median age: 49.5 years) and the male/female ratio was 7.5:1. IHC was performed using the standard streptavidin–biotin–peroxidase complex method. After deparaffinization and endogenous peroxidase activity blockage, the section was incubated with antibodies against PDSS2 (1:300, Wolwo) or CoQ10B (1:800, Abcam). A staining score was obtained as the intensity of positive staining (negative=0, weak = 1, moderate = 2, strong = 3) multiplied by the proportion of immunopositive cells of interest (<25% = 1, 26–50% = 2, 51–75% = 3, >75% = 4). In the study, downregulation of PDSS2 was defined as Nscore-Tscore>2, and downregulation of CoQ10 was defined as Nscore-Tscore>3.

Tissue collection and CoQ10 analysis

Fresh HCC tumor tissues and the corresponding nontumorous tissues were collected at Sun Yat-sen University Cancer Center. The clinical specimens used in this study were approved by the Committees for Ethical Review of Research Involving Human Subjects at the Sun Yat-sen University Cancer Center. The tissue samples were frozen in liquid nitrogen, and then CoQ10 was analyzed. Ultra high-performance liquid chromatography tandem mass spectrometric analysis was carried out on a Waters ACQUITY UPLC/TQD system, which combines ultra high-performance liquid chromatography with triple quadrupole mass spectrometric (MS/MS) detection (31).

Measurement of glucose and lactate

The same number of cells from the experimental group and from the control group were plated in a 6-well plate. Fresh media were added, and analysis was performed after 24 hours or at the indicated time. The glucose and lactate levels in the culture media were measured using a glucose assay kit (glucose oxidase–peroxidase method) and a lactate assay kit (LDH catalysis method; Nanjing Jiancheng Technology), respectively. The cells were collected, and the protein content was quantified. The glucose and lactate levels were normalized by the protein content. Triplicate independent assays were performed.

Oxygen consumption rate analysis

Equal numbers of cells (5 × 106) were suspended in 1ml fresh medium pre-equilibrated with fresh air (21% oxygen and 5% CO2) at 37°C and transferred to a chamber equipped with a thermostat controller and a microstirring device (Oxytherm, Hansatech Instrument). The oxygen consumption rate is expressed as nanomoles of oxygen per milliliter per 5 × 106 cells.

SDH, IDH, and Complex I activity assays

Cells were plated in 6-well plates, and thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich) was added to the cells. The blue crystals were solubilized with DMSO and measured at 570 nm. The results were normalized to the cell protein. An isocitrate dehydrogenase activity assay was performed using an IDH activity assay kit (Biovision) according to the manufacturer's protocol. Complex I activity was determined by monitoring the oxidation of NADH to NAD+ and the simultaneous reduction of a dye, which led to increased absorbance (OD450 nm; Complex I activity kit, Abcam). Triplicate experiments were performed independently.

Measurement of cellular ATP

Cellular ATP was measured using an ATP assay kit (Beyotime) according to the manufacturer's protocol. Cells were seeded in a 6-well plate and lysed. The cellular ATP was normalized to the cell protein content. Triplicate experiments were performed independently.

Detection of micronuclei

The cells were fixed and stained with DAPI. For the analysis, 3 × 103 cells were examined for each sample, and the cells with micronuclei were counted. Data presented are the mean of three independent experiments.

Spectral karyotyping

Spectral karyotyping (SKY) analysis was performed on 1 month consecutive cultured cells. Metaphase spreads were prepared on glass slides, denatured and hybridized with denatured SKY probes (SkyPaint Probes, Applied Spectral Imaging) and then analyzed with SkyView according to the manufacturer's protocol (32). Fifty metaphase spreads from each cell group were analyzed.

Animal studies

All procedures involving mice were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University Cancer Center. Cells (enforced expression, knockdown and vector controls) derived from HCC and MIHA cell lines were inoculated subcutaneously into the flanks of 4- to 6-week-old BALB/c nude mice. Tumor formation in nude mice was monitored during the experimental period. Cells derived from the MIHA line (PDSS2-KD1 and MIHA-C) were mixed with Matrigel (10 vs. 100 μL cell suspension) before inoculation since the MIHA cell line is not tumorigenic. Tumor growth was examined by measuring the tumor size with calipers. The tumor volume was calculated (V = 0.5 × L × W2). After sacrifice, the tumors that formed in the tested mice were excised, fixed, and embedded in a paraffin block for hematoxylin and eosin staining or IHC study.

Statistical analysis

Results are presented as the average ± SEM. The correlation between PDSS2 or CoQ10 downregulation and the clinicopathologic features of patients with HCC was assessed by the Pearson's χ2 test. Survival curves were generated according to the Kaplan–Meier method and statistical analysis was performed by the log-rank test. Multivariate survival analysis was performed on all parameters that were found to be significant in univariate analysis using a Cox regression model. All statistical analysis was carried out using statistical software (SPSS 16.0 for Windows; SPSS, Inc.). Differences were considered significant if the P value was less than 0.05.

Supplementary Information about the Materials and Methods is provided in the Supplementary Information.

PDSS2 was frequently downregulated in HCC and significantly associated with poorer prognosis

The expression of PDSS2 was analyzed by immunohistochemistry on two sets of HCC tissue microarrays containing a total of 295 pairs of HCC cases (tumor vs. corresponding nontumor tissues). Informative data were obtained from 226 tumor tissues and 197 nontumor tissues. In 164 cases with both tumor and nontumor information, PDSS2 downregulation was observed in 57.32% (94/164) of the tumor tissues compared with that in the matched nontumor tissues (Fig. 1A). Although the correlation study demonstrated that PDSS2 downregulation was not significantly correlated with gender, age, T status, and stage (Supplementary Table S1), Kaplan–Meier survival analysis revealed that PDSS2 downregulation was significantly associated with a poorer overall survival (OS) rate in patients with HCC (log-rank test, P = 0.027; Fig. 1B). The mean OS time of the PDSS2 groups with and without downregulation was 41.366 months [95% confidence interval (CI), 33.997–48.736) and 52.197 months (95% CI, 43.956–60.438), respectively. The multivariate analysis result demonstrated that PDSS2 downregulation was an independent prognostic factor for a poorer OS (P = 0.028), independent of T status and stage (Table 1). Further analysis showed that downregulation of PDSS2 could be detected in early stages, suggesting that PDSS2 might play an important role in HCC tumor initiation (Fig. 1C). The expression of PDSS2 was also determined in four HCC cell lines (SMMC7721, QGY7703, Huh7, and PLC8024) and an immortalized human liver cell line, MIHA. The results demonstrated that PDSS2 expression was significantly decreased in HCC cell lines compared with that in the MIHA cells (Fig. 1D; Supplementary Fig. S1A).

Figure 1.

Downregulation of PDSS2 and CoQ10 was frequently detected in HCCs. A, Representative IHC images of PDSS2 staining in HCC tumor tissues and paired nontumor tissues (original magnification, ×200). B, Kaplan–Meier curve showing the overall survival rates in patients with HCC with or without PDSS2 downregulation. C, The percentage of PDSS2 downregulation in HCCs at different clinical stages determined by IHC. The numbers above the bars indicate the number of PDSS2 downregulated cases/total cases in each stage. D, PDSS2 expression was evaluated by IHC in HCC cell lines (SMMC7721, QGY7703, Huh7, and PLC8024) and an immortalized human liver cell line MIHA (original magnification, ×200). E, Representative IHC images of CoQ10 staining in HCC tumor and paired nontumor tissues. F, CoQ10 was measured in 13 pairs of HCC tumor tissues and paired nontumor tissues by ultra high-performance liquid chromatography tandem mass spectrometric analysis.

Figure 1.

Downregulation of PDSS2 and CoQ10 was frequently detected in HCCs. A, Representative IHC images of PDSS2 staining in HCC tumor tissues and paired nontumor tissues (original magnification, ×200). B, Kaplan–Meier curve showing the overall survival rates in patients with HCC with or without PDSS2 downregulation. C, The percentage of PDSS2 downregulation in HCCs at different clinical stages determined by IHC. The numbers above the bars indicate the number of PDSS2 downregulated cases/total cases in each stage. D, PDSS2 expression was evaluated by IHC in HCC cell lines (SMMC7721, QGY7703, Huh7, and PLC8024) and an immortalized human liver cell line MIHA (original magnification, ×200). E, Representative IHC images of CoQ10 staining in HCC tumor and paired nontumor tissues. F, CoQ10 was measured in 13 pairs of HCC tumor tissues and paired nontumor tissues by ultra high-performance liquid chromatography tandem mass spectrometric analysis.

Close modal
Table 1.

Univariate and multivariate analyses of different prognostic variables in patients with HCC

Univariate analysisaMultivariate analysisa
VariableHR (95% CI)PHR (95% CI)P
Gender 1.208 (0.584–2.495) 0.611   
Age 0.778 (0.488–1.240) 0.291   
T status 
 T1-T2    
 T3-T4 2.796 (1.842–4.243) <0.001 2.104 (1.332–3.321) 0.001 
Stage 
 I–II    
 III–IV 3.098 (2.014–4.766) <0.001 2.327 (1.458–3.714) <0.001 
 PDSS2 downregulation in T 1.601 (1.045–2.454) 0.031 1.622 (1.054–2.495) 0.028 
Univariate analysisaMultivariate analysisa
VariableHR (95% CI)PHR (95% CI)P
Gender 1.208 (0.584–2.495) 0.611   
Age 0.778 (0.488–1.240) 0.291   
T status 
 T1-T2    
 T3-T4 2.796 (1.842–4.243) <0.001 2.104 (1.332–3.321) 0.001 
Stage 
 I–II    
 III–IV 3.098 (2.014–4.766) <0.001 2.327 (1.458–3.714) <0.001 
 PDSS2 downregulation in T 1.601 (1.045–2.454) 0.031 1.622 (1.054–2.495) 0.028 

Abbreviations: CI, confidence interval; HR, hazard ratio.

aCox regression model.

CoQ10 level decreased in HCC tumor tissues

As PDSS2 is important in CoQ10 synthesis and lower CoQ10 plasma level has been reported in many cancers (26, 27, 33, 34), we next measured the CoQ10 level in HCC tumor tissues by IHC. Informative IHC data were obtained from 211 tumor tissues and 165 nontumor tissues. In 144 cases with both tumorous and nontumorous information, CoQ10 downregulation was detected in 86/144 (59.72%) of tumor tissues compared with matched nontumor tissues (Fig. 1E). The correlation study showed that decreased expression of CoQ10 in HCCs was correlated with T status marginally (P = 0.053, Supplementary Table S2). In addition, CoQ10 was measured by Ultra high-performance liquid chromatography tandem mass spectrometric analysis in 13 pairs of freshly prepared HCC tumor tissues and corresponding nontumor tissues. The results showed that CoQ10 was decreased in tumor tissues compared with that in the corresponding nontumor tissues (P < 0.01, Fig. 1F). A CoQ10 decrease was also observed in HCC cells compared with that in MIHA cells (Supplementary Fig. S1A). Correlation analysis was also performed in 114 cases that had both PDSS2 and CoQ10 information. The results demonstrated that downregulation of CoQ10 was positively correlated with downregulation of PDSS2 in tumor tissues (R = 0.414, P < 0.001; Pearson correlation; Supplementary Table S3). IDH1 is an important enzyme in the krebs cycle. To confirm whether PDSS2 downregulation affects the enzymes in the krebs cycle, IDH1 expression was evaluated in HCC tumor tissues, and downregulation of IDH1 was positively correlated with downregulation of PDSS2 (R = 0.260, P = 0.017; Pearson correlation; Supplementary Tables S4–S5; Supplementary Fig. S1B).

PDSS2 introduction increased mitochondrial Complex I activity

The results demonstrated higher CoQ10 in PDSS2-transfected cells than in empty vector-transfected cells (Fig. 2A; Supplementary Fig. S1C). Because CoQ10 plays a key role in the electron transfer chain, and because CoQ10 addition has been reported to be associated with an increase in the Complex I-linked respiratory rate (35), we next examined the expression of the mitochondrial respiratory chain components by Western blot analysis. The results showed that the level of Complex I increased as expected, whereas the Complex II level increased slightly in PDSS2-transfected cells, compared with those in empty vector-transfected cells (Fig. 2B). For Complexes III and IV and ATP synthase (Complex V), no obvious change was observed (Fig. 2B). Complex I activity was then determined by measuring the oxidation of NADH to NAD+ using a Complex I enzyme activity assay kit. The result demonstrated that the activity of Complex I increased in PDSS2-transfected cells compared with that in empty vector-transfected cells (Fig. 2C).

Figure 2.

Reintroduction of PDSS2 enhanced mitochondrial respiration. A, Representative IHC images of CoQ10 in PDSS2- and vector-transfected cells (original magnification, ×200). B, The protein levels of mitochondrial complexes I∼V were determined by Western blotting in HCC cell line derivatives (PDSS2- and vector control). C, Complex I activity was compared between PDSS2- and vector-transfected cells (left) and the data are summarized (right). V, vector control; P, PDSS2. **, P < 0.01. D, Schematic diagram of key factors involved in glycolysis and the Krebs cycle. E, The molecules involved in glycolysis (TPI, GAPDH, and LDH), PDH, and molecules associated with the Krebs cycle (IDH, MDH2, and CS) were determined by Western blotting. F, SDH activity was compared between PDSS2- and vector-transfected cells. G, IDH activity was compared between PDSS2- and vector-transfected cells.

Figure 2.

Reintroduction of PDSS2 enhanced mitochondrial respiration. A, Representative IHC images of CoQ10 in PDSS2- and vector-transfected cells (original magnification, ×200). B, The protein levels of mitochondrial complexes I∼V were determined by Western blotting in HCC cell line derivatives (PDSS2- and vector control). C, Complex I activity was compared between PDSS2- and vector-transfected cells (left) and the data are summarized (right). V, vector control; P, PDSS2. **, P < 0.01. D, Schematic diagram of key factors involved in glycolysis and the Krebs cycle. E, The molecules involved in glycolysis (TPI, GAPDH, and LDH), PDH, and molecules associated with the Krebs cycle (IDH, MDH2, and CS) were determined by Western blotting. F, SDH activity was compared between PDSS2- and vector-transfected cells. G, IDH activity was compared between PDSS2- and vector-transfected cells.

Close modal

PDSS2 induced metabolic reprogramming from glycolysis to mitochondrial respiration

As the respiratory chain activity was upregulated in PDSS2-transfected cells, we proposed that PDSS2 could promote mitochondrial respiration. Western blot results demonstrated that the levels of pyruvate dehydrogenase (PDH), a key mitochondrial enzyme that converts pyruvate to acetyl-CoA for further metabolism through the Krebs cycle, and three molecules involved in the Krebs cycle (isocitrate dehydrogenase, IDH; malate dehydrogenase, MDH2; and citrate synthase, CS) increased in PDSS2-transfected cells compared with those in vector-transfected cells, except CS decreased in Huh7-PDSS2 cells (Fig. 2D–E and Supplementary Fig. S2A). The SDH and IDH activity also increased in PDSS2-overexpressing cells (Fig. 2F and G).

After reintroduction of PDSS2, both the glucose uptake and lactate level decreased (Fig. 3A). The oxygen consumption rate is another major biochemical parameter indicating glycolytic activity. As shown in Fig. 3B, reintroduction of PDSS2 in SMMC7721, QGY7703, and Huh7 cells led to a nearly 25% increase in the oxygen consumption rate compared with that in control cells. The maximal respiration also increased in 7721-PDSS2 cells compared with that in vector control cells (P < 0.01; Supplementary Fig. S2B). As glucose uptake, lactate production and oxygen consumption are three major biochemical parameters for estimating glycolytic activity (36), the ratio of glycolytic indexes upon reintroduction of PDSS2 was calculated. The ratio of glycolytic indexes in PDSS2-transfected cells relative to that in empty vector-transfected cells was 0.47:1 (SMMC7721), 0.45:1 (QGY7703), and 0.38:1 (Huh7), indicating that the glycolytic activity decreased in PDSS2-transfected cells. Taken together, these data suggest that the reintroduction of PDSS2 could upregulate mitochondrial respiration and down-regulate glycolysis. To examine whether the ATP level changed in the PDSS2-transfected cells after the glycolytic activity decreased in the cells, we compared the cellular ATP levels in cells between PDSS2- and vector-transfected cells. The reintroduction of PDSS2 increased the production of cellular ATP compared with that in vector-transfected cells (Fig. 3C). As the glycolytic index decreased to 30% to 50% in PDSS2-transfected cells, our data suggest that ATP was generated through mitochondrial oxidative phosphorylation rather than through glycolysis. We also detected an increased abundance of metabolic intermediates specific to the tricarboxylic acid (TCA) cycle and decreased pyruvate in PDSS2-transfected SMMC7721 cells (Supplementary Fig. S2C). This finding is consistent with the mitochondrial respiration activation results in 7721-PDSS2 cells.

Figure 3.

PDSS2 inhibited aerobic glycolysis and tumorigenicity. Reintroduction of PDSS2 could decrease glucose uptake and lactate secretion (A) and increase oxygen consumption (B) and the ATP level (C) compared with that in control cells. D, IHC staining confirmed the expression of ectopic PDSS2 in 7,721 cells (original magnification, ×200). E, The representative pictures and summary of the foci formation assay (left) and the colony formation assay in soft agar (right) demonstrated that the anchorage-dependent and anchorage-independent growth ability was inhibited in 7721-PDSS2 cells compared with those in control cells (7721-Vec). F–H, An in vivo tumor formation assay demonstrated that PDSS2 could inhibit tumor growth in nude mice. Compared with tumors induced by 7721-Vec cells (n = 6), tumors induced by 7721-PDSS2 cells (n = 6) showed a smaller size (F), slower growth rate and lower tumor weight (G) 5 weeks after subcutaneous injection. H, Representative images of xenograft sections stained with PDSS2 antibody (original magnification, ×200).

Figure 3.

PDSS2 inhibited aerobic glycolysis and tumorigenicity. Reintroduction of PDSS2 could decrease glucose uptake and lactate secretion (A) and increase oxygen consumption (B) and the ATP level (C) compared with that in control cells. D, IHC staining confirmed the expression of ectopic PDSS2 in 7,721 cells (original magnification, ×200). E, The representative pictures and summary of the foci formation assay (left) and the colony formation assay in soft agar (right) demonstrated that the anchorage-dependent and anchorage-independent growth ability was inhibited in 7721-PDSS2 cells compared with those in control cells (7721-Vec). F–H, An in vivo tumor formation assay demonstrated that PDSS2 could inhibit tumor growth in nude mice. Compared with tumors induced by 7721-Vec cells (n = 6), tumors induced by 7721-PDSS2 cells (n = 6) showed a smaller size (F), slower growth rate and lower tumor weight (G) 5 weeks after subcutaneous injection. H, Representative images of xenograft sections stained with PDSS2 antibody (original magnification, ×200).

Close modal

PDSS2 suppressed tumor growth in vitro and in vivo

Metabolic reprogramming was thought to be a consequence of rapid cell proliferation; however, recent findings have suggested that it might be a driving factor in tumorigenesis (12, 13). Therefore, both in vitro and in vivo functional assays were used to investigate the potential role of PDSS2 in HCC pathogenesis. The results showed that the reintroduction of PDSS2 (Fig. 3D; Supplementary Fig. S3A–S3B) could significantly inhibit foci formation (P < 0.01) and colony formation in soft agar (P < 0.01) in three tested cells (SMMC7721, QGY7703 and Huh7) compared with that in their control cells (Fig. 3E; Supplementary Fig. S3C–S3F). The cell-cycle distribution results indicated that compared with vector-transfected cells, 7721-PDSS2 cells were arrested at the G1–S checkpoint, manifested as an accumulation in G1-phase and a decrease in S-phase cells. The G2 distribution was also decreased significantly in 7721-PDSS2 cells (Supplementary Fig. S4A). The apoptotic index did not differ between 7721-PDSS2 cells and 7721-Vec cells (Supplementary Fig. S4B). The in vivo tumorigenic assay was performed by subcutaneously injecting PDSS2- and vector-transfected cells into the right and left flanks of nude mice (n = 6), respectively. Tumor formation in nude mice was monitored for 5 weeks, the mice were sacrificed, and the tumor volume and tumor weight were examined. The results showed that the reintroduction of PDSS2 could significantly inhibit tumor growth (P < 0.01) compared with that in control mice (Fig. 3F–G). IHC staining showed that the expression of PDSS2 was detected only in the tumors induced by PDSS2-transfected cells (Fig. 3H). The tumor formation assay was repeated in BEL7402 cells, and the results were consistent with the above referenced results (Supplementary Fig. S4C–S4D).

Silencing PDSS2 enhanced aerobic glycolysis and tumorigenicity in immortalized liver cells

To directly test whether PDSS2 is functionally important for HCC tumorigenesis, we used short-hairpin RNA (shRNA) to stably silence its expression in the MIHA immortalized liver cell line (Supplementary Fig. S5A–S5B). The knockdown efficiency was also confirmed in Huh7 cells (Supplementary Fig. S5C). Mitochondrial complex I decreased slightly in MIHA-KD cells compared with MIHA-C (Fig. 4A, Supplementary Fig. S5D). Compared with vector control cells, MIHA-KD cells displayed decreased PDH, IDH, and MDH2 and increased TPI1 protein levels (Fig. 4B; Supplementary Fig. S5E). SDH activity also decreased in the MIHA-KD cells compared with that in the vector control cells (P < 0.05; Fig. 4C). IDH activity decreased slightly in MIHA-KD cells compared with that in vector control cells (Fig. 4C).

Figure 4.

Silencing PDSS2 increased aerobic glycolysis and tumorigenicity. A, The protein levels of mitochondrial complexes I∼V were determined by Western blotting in MIHA-KD1 and MIHA-C cells. Actin was used as a loading control. B, The levels of PDH and the molecules associated with glycolysis and the Krebs cycle were determined by Western blotting. Actin was used as a loading control. C, SDH activity and IDH activity were compared between MIHA-KD1 and MIHA-C cells. D, Silencing PDSS2 expression in MIHA-KD1 cells significantly increased the glucose uptake and lactate secretion, decreased the oxygen consumption, and increased the ATP level compared with those in control cells. E and F, Knockdown of PDSS2 was able to significantly increase the cell growth rate (E) and foci formation frequency (F). G, MIHA-KD1 and control cells were subcutaneously injected into the left and right flanks of nude mice (n = 6), respectively. Tumor weight was compared 3 weeks after inoculation. H, Expression of PDSS2 was detected by IHC in xenograft sections (original magnification, ×400).

Figure 4.

Silencing PDSS2 increased aerobic glycolysis and tumorigenicity. A, The protein levels of mitochondrial complexes I∼V were determined by Western blotting in MIHA-KD1 and MIHA-C cells. Actin was used as a loading control. B, The levels of PDH and the molecules associated with glycolysis and the Krebs cycle were determined by Western blotting. Actin was used as a loading control. C, SDH activity and IDH activity were compared between MIHA-KD1 and MIHA-C cells. D, Silencing PDSS2 expression in MIHA-KD1 cells significantly increased the glucose uptake and lactate secretion, decreased the oxygen consumption, and increased the ATP level compared with those in control cells. E and F, Knockdown of PDSS2 was able to significantly increase the cell growth rate (E) and foci formation frequency (F). G, MIHA-KD1 and control cells were subcutaneously injected into the left and right flanks of nude mice (n = 6), respectively. Tumor weight was compared 3 weeks after inoculation. H, Expression of PDSS2 was detected by IHC in xenograft sections (original magnification, ×400).

Close modal

MIHA-KD1 showed increased glucose uptake and lactate production and decreased oxygen consumption (Fig. 4D). The MIHA-KD1:MIHA-C ratio of glycolytic index was 4.75:1, indicating that PDSS2 knockdown cells had approximately 4-fold higher glycolytic activity than the vector control cells. We also noticed that the ATP level increased in MIHA-KD1 cells (Fig. 4D), which might be the result of increased glycolysis to produce more ATP to facilitate cell proliferation. Taken together, the results indicate that PDSS2 deficiency promotes a switch to a state of glucose addiction, a hallmark of cancers undergoing aerobic glycolysis.

Both in vitro and in vivo functional assays were performed to determine whether the metabolic shift toward aerobic glycolysis by PDSS2 knockdown could lead to tumorigenesis. Silencing PDSS2 in MIHA cells led to increased proliferation and greater colony formation in the foci formation assay (Fig. 4E–F). Because MIHA cells cannot form xenografts in vivo, we mixed the cells with Matrigel and injected into nude mice. Our results demonstrated that silencing PDSS2 in MIHA cells could promote tumor formation in nude mice compared with the tumor formation induced by control cells (Fig. 4G–H; Supplementary Fig. S6A). When CoQ10 was added to PDSS2-deficient MIHA (MIHA-KD1) cells, the glucose uptake ratio and lactate secretion ratio (CoQ10+/CoQ10) decreased in MIHA-KD1 cells compared with that in control cells (Supplementary Fig. S6B). Cell growth decreased more significantly in MIHA-KD1 cells than in control cells (Supplementary Fig. S6C). When cells were treated with a glycolysis inhibitor, 2-deoxy-D-glucose (2-DG), the cell growth was inhibited more significantly in MIHA-KD1 cells than in MIHA-C, indicating that MIHA-KD1 relied more on glycolysis than did control cells (Supplementary Fig. S6D). Taken together, these results indicate that the metabolic shift induced by PDSS2 knockdown results in a more malignant phenotype that favored tumor cell growth.

Silencing PDSS2 increased chromosomal instability

We also found that PDSS2 knockdown led to chromosomal instability. The frequency of the formation of micronuclei, which contain unstable extranuclear chromosomal fragments, was used to evaluate the chromosomal instability in MIHA-KD1 and vector control cells after stable cell lines were established. The frequency of micronuclei formation was significantly higher (10.56% ± 0.26%) in MIHA-KD1 cells than in vector cells (MIHA-C; 8.41% ± 0.78%, P < 0.01; Fig. 5A). SKY analysis was performed on MIHA-KD1, MIHA-C, and MIHA cells after 1 month consecutive culture. The results demonstrated that the chromosomal aberrations in the MIHA-KD1 cells were significantly higher than that in the vector and parental cells (P < 0.01; Fig. 5B and C).

Figure 5.

Silencing PDSS2 increased chromosomal instability. A, Representative images and summary of the frequency of micronuclei in MIHA-KD1 and MIHA-C cells. White arrows, micronuclei formed in the cells. B, Representative images of spectral karyotyping of one-month consecutive cultured cells (MIHA and MIHA-KD1). More chromosomal aberrations were observed in metaphase spreads of MIHA-KD1 cells than in those of MIHA cells. Green arrows, whole chromosome losses or gains; red arrows, novel structural aberrations. C, Summary of changes in chromosomal aberrations in 50 metaphase spreads of cells.

Figure 5.

Silencing PDSS2 increased chromosomal instability. A, Representative images and summary of the frequency of micronuclei in MIHA-KD1 and MIHA-C cells. White arrows, micronuclei formed in the cells. B, Representative images of spectral karyotyping of one-month consecutive cultured cells (MIHA and MIHA-KD1). More chromosomal aberrations were observed in metaphase spreads of MIHA-KD1 cells than in those of MIHA cells. Green arrows, whole chromosome losses or gains; red arrows, novel structural aberrations. C, Summary of changes in chromosomal aberrations in 50 metaphase spreads of cells.

Close modal

PDSS2-Del2 is a new variant of PDSS2

We next investigated whether there were other variants of PDSS2. The mRNA products of PDSS2 were cloned into the pMD 19-T vector, 30 clones were sequenced, and several different splicing isoforms were found, including PDSS2-Del2, in which alternative splicing deleted exon 2 (Supplementary Fig. S7A). This isoform is exclusively expressed in HCC cells, but not in MIHA cells (Fig. 6A). We sequenced the 5′ and 3′ flanking intron sequences of exon 2 in 8 HCC cases, and no SNV (single nucleoid variation) or mutation was observed. The antibody used to detect PDSS2 could only detect the full-length PDSS2 but not the PDSS2-Del2 (Fig. 6B). We synthesized an anti–PDSS2-Del2 antibody that could specifically stain PDSS2-Del2 (Fig. 6B). The polyprenyl synthesis domain of PDSS2 protein was disrupted in PDSS2-Del2 (Fig. 6A and C; Supplementary Fig. S7B). Consequently, PDSS2-Del2 was incapable of changing CoQ10 levels in cells with ectopic expression (Supplementary Fig. S7C). In addition, PDSS2-Del2 could not change the protein levels of the mitochondrial electron transport complexes in PDSS2-Del2 cells (Fig. 6D). Furthermore, Complex I activity decreased slightly in PDSS2-Del2 cells compared with that in vector control cells (Fig. 6D).

Figure 6.

PDSS2-Del2 was a new PDSS2 isoform. A, Different variants of PDSS2 were detected in three HCC cell lines (SMMC7721, QGY7703, and Huh7) and the MIHA immortalized liver cell line. Schematic showing the amino acid sequence of PDSS2 FL (full length) and PDSS2-Del2 (dark green, polyprenyl synthesis domain). B, The cell derivatives of 7721 (-Vec, -PDSS2 and -PDSS2-Del2) were detected by IHC with anti-PDSS2 and anti-Del2. IHC staining showed that Del2 protein could be detected by the anti-Del2 antibody but not by the anti-PDSS2 antibody (original magnification, ×200). C, 3-D structure of PDSS2 (exon 2, purple). D, Del2 overexpression in 7,721 cells did not affect the protein levels of mitochondrial complexes I∼V. The Complex I activity decreased slightly in PDSS2-Del2 cells compared with that in vector control cells. E, The levels of PDH and molecules associated with glycolysis and the Krebs cycle were determined by Western blotting in Del2-transfected 7721 cells and vector control cells. F, Glucose uptake, lactate secretion, and oxygen consumption were compared between Del2-transfected and vector-transfected cells or PDSS2-transfected cells. G, The tumor volume and tumor weight demonstrated that xenograft inhibition ability was absent in Del2-transfected cells, in contrast to PDSS2-transfected cells. H, Representative images of xenograft sections stained with anti-PDSS2 and anti-Del2 antibodies (original magnification, ×200).

Figure 6.

PDSS2-Del2 was a new PDSS2 isoform. A, Different variants of PDSS2 were detected in three HCC cell lines (SMMC7721, QGY7703, and Huh7) and the MIHA immortalized liver cell line. Schematic showing the amino acid sequence of PDSS2 FL (full length) and PDSS2-Del2 (dark green, polyprenyl synthesis domain). B, The cell derivatives of 7721 (-Vec, -PDSS2 and -PDSS2-Del2) were detected by IHC with anti-PDSS2 and anti-Del2. IHC staining showed that Del2 protein could be detected by the anti-Del2 antibody but not by the anti-PDSS2 antibody (original magnification, ×200). C, 3-D structure of PDSS2 (exon 2, purple). D, Del2 overexpression in 7,721 cells did not affect the protein levels of mitochondrial complexes I∼V. The Complex I activity decreased slightly in PDSS2-Del2 cells compared with that in vector control cells. E, The levels of PDH and molecules associated with glycolysis and the Krebs cycle were determined by Western blotting in Del2-transfected 7721 cells and vector control cells. F, Glucose uptake, lactate secretion, and oxygen consumption were compared between Del2-transfected and vector-transfected cells or PDSS2-transfected cells. G, The tumor volume and tumor weight demonstrated that xenograft inhibition ability was absent in Del2-transfected cells, in contrast to PDSS2-transfected cells. H, Representative images of xenograft sections stained with anti-PDSS2 and anti-Del2 antibodies (original magnification, ×200).

Close modal

Considering that many proteins involved in glycolysis and the mitochondrial respiratory chain were changed in the PDSS2-overexpressing cells, we next studied whether proteins were affected by PDSS2-Del2. Similar to PDSS2-overexpressing cells, cells overexpressing PDSS2-Del2 had increased PDH and MDH2 protein levels but no significant changes in the IDH and CS protein levels (Fig. 6E). Unlike that in PDSS2-overexpressing cells, LDH increased significantly in PDSS2-Del2–overexpressing cells (Fig. 6E).

We next evaluated whether PDSS2-Del2 could reduce glycolysis and upregulate the mitochondrial respiratory chain as PDSS2 did. As shown in Fig. 6F, glucose uptake showed no difference between PDSS2-Del2 cells and control cells. However, lactate production was higher in PDSS2-Del2 cells than in control cells. Oxygen consumption decreased in PDSS2-Del2 cells compared with that in PDSS2-transfected cells (Fig. 6F). To determine whether PDSS2-Del2 had similar effects on tumor cell growth to those of full-length PDSS2, SMMC7721 cells transfected with PDSS2-Del2 and PDSS2 were inoculated into the left and right flanks of nude mice, respectively. The results showed that the tumors induced by 7721-PDSS2-Del2 grew much faster than the tumors induced by 7721-PDSS2 cells (Fig. 6G–H), suggesting that the tumor-suppressive ability was absent in 7721-PDSS2-Del2 cells.

Metabolic dysregulation is one of the hallmarks of tumor cells to support rapid cell proliferation (37). In proliferative tumor cells, changes occur in the metabolism of carbohydrates, lipids and peptides to meet increased energy demands and support nucleic acid and protein biosynthesis and membrane biogenesis (38, 39). Here, we demonstrated that the downregulation of PDSS2 might be a driving factor in HCC development by reprogramming glucose metabolism from aerobic oxidation to aerobic glycolysis and increasing chromosomal instability. Sequencing analysis found that PDSS2 has at least 6 different splicing isoforms. No coding proteins could be predicted for four variants due to loss of the start codon. Only two transcripts, the full-length PDSS2 and a variant with exon 2 deleted, could encode proteins: PDSS2 and PDSS2-Del2, respectively. Sequencing and IHC data showed that the downregulation of PDSS2 mainly affected the full-length isoform.

As PDSS2 is one of the key enzymes in CoQ10 synthesis, we next studied the effect of PDSS2 on CoQ10 synthesis. Our data demonstrated that CoQ10 downregulation was significantly correlated with PDSS2 downregulation in clinical HCC samples, indicating that the downregulation of PDSS2 in HCC could negatively affect CoQ10 biosynthesis. CoQ10 deficiency has been reported in many solid tumors, and a recent long-term clinical study demonstrated that patients with breast cancer who received adjuvant CoQ10 therapy showed a better outcome (40). Because the PDSS2 knockout mouse reveals mitochondrial defects and CoQ10 is essential for electron transport in the mitochondrial respiratory chain and antioxidant defense (24, 41), the effect of PDSS2 reintroduction on the expression level and bioactivity of mitochondrial respiratory chain components was investigated. Our results suggested that Complex I might have a greater contribution to mitochondrial respiration changes in cells with ectopic PDSS2 expression.

Our next question was what will happen when Complex I activity decreases? Our results showed that reintroduction of PDSS2 in HCC cell lines could increase the expression of the Krebs cycle-related proteins PDH, IDH, MDH2 and CS, as well as SDH and IDH activity. This finding suggests that the PDSS2 deficiency might inhibit TCA activity because the decreased activity of Complex I blocked the electron transport in the mitochondrial respiratory chain, which subsequently inhibited TCA cycle activity. Therefore, we hypothesized that the PDSS2 deficiency might switch glucose metabolism from mitochondrial respiration to glycolysis. A further study demonstrated that PDSS2 was able to decrease glucose uptake and lactate secretion and increase the oxygen consumption rate, indicating that the PDSS2 deficiency could indeed increase glycolysis, which is beneficial for biomass production and tumor cell growth. All these findings support the possibility that PDSS2 deficiency is a driving factor in HCC carcinogenesis through reprogramming glucose metabolism from mitochondrial respiration and oxidative phosphorylation to aerobic glycolysis. If this hypothesis is true, PDSS2 deficiency should also affect cell behavior besides metabolic reprogram of glucose. Indeed, our functional studies demonstrated that reintroduction of PDSS2 into HCC cell lines could significantly suppress tumor cell growth in vitro and in vivo.

Genomic instability is one of the most common hallmarks of cancer and can be responsible for the accumulation of mutations affecting tumor cell malignant properties and response to therapies (42). Chromosomal alterations are frequently detected in HCCs, including loss of 1p, 4q, 6p, 16q, and 17p, and gain of 1q, 8q, and 20q (28, 43–45). Another interesting finding is that knockdown of PDSS2 in MIHA cells could increase chromosomal instability. The formation of micronuclei and frequency of chromosomal rearrangements both increased significantly in PDSS2 knockdown cells. In addition, we found that PDSS2 deficiency not only increased genomic instability but also transformed immortalized hepatocytes (MIHA) into malignant cells, which has been demonstrated by both in vitro and in vivo functional assays. In summary, we found that PDSS2 deficiency might be a driving factor in hepatocarcinogenesis through reprograming energy metabolism from mitochondrial respiration to aerobic glycolysis, increasing chromosomal instability, and finally transforming a normal cell into a malignant cell.

No potential conflicts of interest were disclosed.

Conception and design: Y. Li, Y. Hu, X.-Y. Guan

Development of methodology: S. Lin, Z. Cai

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Li, L. Li, Z. Tang, X. Ban, T. Zeng, Y. Zhou, Y. Zhu, W. Deng, X. Zhang, D. Xie, Y. Yuan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Li, Z. Tang, S. Gao, P. Huang

Writing, review, and/or revision of the manuscript: Y. Li, X.-Y. Guan

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Li

Study supervision: Y. Li, X.-Y. Guan

We thank Dr. Tao Wang (Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences) for his technical support. This work was supported by National Key R&D Program of China [(2017YFC1309000 to X.Y. Guan and Y. Li), NSFC (81472255 to Y. Li), 81472250 (to X.Y. Guan)], National Key Sci-Tech Special Project of Infectious Diseases (2018ZX10723204-006-005 to X.Y. Guan), GDNSF (2014A030313071 to Y. Li), GECI (M201511 to Y. Li), GDSTP (2016A020214008 to Y. Li), SYSUIP (16ykjc34 to Y. Li), Hong Kong RGC Collaborative Research Funds (C7038-14G, HKBU5/CRF/10 to X.Y. Guan), RGC GRF Funds (767313 and 17143716 to X.Y. Guan), Theme-based Research Scheme Fund (T12-704/16-R to X.Y. Guan). X.-Y. Guan is a Sophie YM Chan Professor in Cancer Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Levine
AJ
,
Puzio-Kuter
AM
. 
The control of the metabolic switch in cancers by oncogenes and tumor suppressor genes
.
Science
2010
;
330
:
1340
4
.
2.
Warburg
O
. 
On the origin of cancer cells
.
Science
1956
;
123
:
309
14
.
3.
Gasparre
G
,
Porcelli
AM
,
Lenaz
G
,
Romeo
G
. 
Relevance of mitochondrial genetics and metabolism in cancer development
.
Cold Spring Harbor Perspect Biol
2013
;
5
. pii: a011411.
4.
Kunkel
M
,
Reichert
TE
,
Benz
P
,
Lehr
HA
,
Jeong
JH
,
Wieand
S
, et al
Overexpression of Glut-1 and increased glucose metabolism in tumors are associated with a poor prognosis in patients with oral squamous cell carcinoma
.
Cancer
2003
;
97
:
1015
24
.
5.
Mochiki
E
,
Kuwano
H
,
Katoh
H
,
Asao
T
,
Oriuchi
N
,
Endo
K
. 
Evaluation of 18F-2-deoxy-2-fluoro-D-glucose positron emission tomography for gastric cancer
.
World J Surg
2004
;
28
:
247
53
.
6.
Schreurs
LM
,
Smit
JK
,
Pavlov
K
,
Pultrum
BB
,
Pruim
J
,
Groen
H
, et al
Prognostic impact of clinicopathological features and expression of biomarkers related to (18)F-FDG uptake in esophageal cancer
.
Ann Surg Oncol
2014
;
21
:
3751
7
.
7.
Xi
Y
,
Guo
R
,
Hu
J
,
Zhang
M
,
Zhang
X
,
Li
B
. 
18F-fluoro-2-deoxy-D-glucose retention index as a prognostic parameter in patients with pancreatic cancer
.
Nucl Med Commun
2014
;
35
:
1112
8
.
8.
Podoloff
DA
,
Advani
RH
,
Allred
C
,
Benson
AB
 3rd
,
Brown
E
,
Burstein
HJ
, et al
NCCN task force report: positron emission tomography (PET)/computed tomography (CT) scanning in cancer
.
J Nat Compr Cancer Network
2007
;
5
:
S1
22
.
9.
Doherty
JR
,
Cleveland
JL
. 
Targeting lactate metabolism for cancer therapeutics
.
J Clin Invest
2013
;
123
:
3685
92
.
10.
Walenta
S
,
Wetterling
M
,
Lehrke
M
,
Schwickert
G
,
Sundfor
K
,
Rofstad
EK
, et al
High lactate levels predict likelihood of metastases, tumor recurrence, and restricted patient survival in human cervical cancers
.
Cancer Res
2000
;
60
:
916
21
.
11.
Brizel
DM
,
Schroeder
T
,
Scher
RL
,
Walenta
S
,
Clough
RW
,
Dewhirst
MW
, et al
Elevated tumor lactate concentrations predict for an increased risk of metastases in head-and-neck cancer
.
Int J Radiat Oncol Biol Phys
2001
;
51
:
349
53
.
12.
Sebastian
C.
Tracking down the origin of cancer: metabolic reprogramming as a driver of stemness and tumorigenesis
.
Crit Rev Oncog
2014
;
19
:
363
82
.
13.
De Miguel
MP
,
Alcaina
Y
,
de la Maza
DS
,
Lopez-Iglesias
P
. 
Cell metabolism under microenvironmental low oxygen tension levels in stemness, proliferation and pluripotency
.
Curr Mol Med
2015
;
15
:
343
59
.
14.
Goldberg
MS
,
Sharp
PA
. 
Pyruvate kinase M2-specific siRNA induces apoptosis and tumor regression
.
J Exp Med
2012
;
209
:
217
24
.
15.
Fantin
VR
,
St-Pierre
J
,
Leder
P
. 
Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance
.
Cancer Cell
2006
;
9
:
425
34
.
16.
Telang
S
,
Yalcin
A
,
Clem
AL
,
Bucala
R
,
Lane
AN
,
Eaton
JW
, et al
Ras transformation requires metabolic control by 6-phosphofructo-2-kinase
.
Oncogene
2006
;
25
:
7225
34
.
17.
Miller
DM
,
Thomas
SD
,
Islam
A
,
Muench
D
,
Sedoris
K
. 
c-Myc and cancer metabolism
.
Clin Cancer Res
2012
;
18
:
5546
53
.
18.
Bensaad
K
,
Tsuruta
A
,
Selak
MA
,
Vidal
MN
,
Nakano
K
,
Bartrons
R
, et al
TIGAR, a p53-inducible regulator of glycolysis and apoptosis
.
Cell
2006
;
126
:
107
20
.
19.
Feng
Z
,
Hu
W
,
de Stanchina
E
,
Teresky
AK
,
Jin
S
,
Lowe
S
, et al
The regulation of AMPK beta1, TSC2, and PTEN expression by p53: stress, cell and tissue specificity, and the role of these gene products in modulating the IGF-1-AKT-mTOR pathways
.
Cancer Res
2007
;
67
:
3043
53
.
20.
Hu
W
,
Zhang
C
,
Wu
R
,
Sun
Y
,
Levine
A
,
Feng
Z
. 
Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant function
.
PNAS
2010
;
107
:
7455
60
.
21.
Matoba
S
,
Kang
JG
,
Patino
WD
,
Wragg
A
,
Boehm
M
,
Gavrilova
O
, et al
p53 regulates mitochondrial respiration
.
Science
2006
;
312
:
1650
3
.
22.
Guan
XY
,
Zhang
HE
,
Zhou
H
,
Sham
JS
,
Fung
JM
,
Trent
JM
. 
Characterization of a complex chromosome rearrangement involving 6q in a melanoma cell line by chromosome microdissection
.
Cancer Genet Cytogenet
2002
;
134
:
65
70
.
23.
Fung
JM
,
Smith
R
,
Brown
MA
,
Lau
SH
,
Xie
D
,
Lau
GK
, et al
Identification and characterization of a novel melanoma tumor suppressor gene on human chromosome 6q21
.
Clin Cancer Res
2009
;
15
:
797
803
.
24.
Quinzii
CM
,
Lopez
LC
,
Von-Moltke
J
,
Naini
A
,
Krishna
S
,
Schuelke
M
, et al
Respiratory chain dysfunction and oxidative stress correlate with severity of primary CoQ10 deficiency
.
FASEB J
2008
;
22
:
1874
85
.
25.
Jolliet
P
,
Simon
N
,
Barre
J
,
Pons
JY
,
Boukef
M
,
Paniel
BJ
, et al
Plasma coenzyme Q10 concentrations in breast cancer: prognosis and therapeutic consequences
.
Int J Clin Pharmacol Ther
1998
;
36
:
506
9
.
26.
Rusciani
L
,
Proietti
I
,
Rusciani
A
,
Paradisi
A
,
Sbordoni
G
,
Alfano
C
, et al
Low plasma coenzyme Q10 levels as an independent prognostic factor for melanoma progression
.
J Am Acad Dermatol
2006
;
54
:
234
41
.
27.
Palan
PR
,
Mikhail
MS
,
Shaban
DW
,
Romney
SL
. 
Plasma concentrations of coenzyme Q10 and tocopherols in cervical intraepithelial neoplasia and cervical cancer
.
Eur J Cancer Prev
2003
;
12
:
321
6
.
28.
Huang
SF
,
Hsu
HC
,
Fletcher
JA
. 
Investigation of chromosomal aberrations in hepatocellular carcinoma by fluorescence in situ hybridization
.
Cancer Genet Cytogenet
1999
;
111
:
21
7
.
29.
Chen
P
,
Zhao
SH
,
Chu
YL
,
Xu
K
,
Zhu
L
,
Wu
Y
, et al
Anticancer activity of PDSS2, prenyl diphosphate synthase, subunit 2, in gastric cancer tissue and the SGC7901 cell line
.
Anticancer Drugs
2009
;
20
:
141
8
.
30.
Wang
Y
,
Wu
MC
,
Sham
JS
,
Zhang
W
,
Wu
WQ
,
Guan
XY
. 
Prognostic significance of c-myc and AIB1 amplification in hepatocellular carcinoma. A broad survey using high-throughput tissue microarray
.
Cancer
2002
;
95
:
2346
52
.
31.
Tang
Z
,
Li
S
,
Guan
X
,
Schmitt-Kopplin
P
,
Lin
S
,
Cai
Z
. 
Rapid assessment of the coenzyme Q10 redox state using ultrahigh performance liquid chromatography tandem mass spectrometry
.
Analyst
2014
;
139
:
5600
4
.
32.
Deng
W
,
Tsao
SW
,
Guan
XY
,
Lucas
JN
,
Si
HX
,
Leung
CS
, et al
Distinct profiles of critically short telomeres are a key determinant of different chromosome aberrations in immortalized human cells: whole-genome evidence from multiple cell lines
.
Oncogene
2004
;
23
:
9090
101
.
33.
Cobanoglu
U
,
Demir
H
,
Cebi
A
,
Sayir
F
,
Alp
HH
,
Akan
Z
, et al
Lipid peroxidation, DNA damage and coenzyme Q10 in lung cancer patients–markers for risk assessment?
Asian Pacific J Cancer Prev
2011
;
12
:
1399
403
.
34.
Cooney
RV
,
Dai
Q
,
Gao
YT
,
Chow
WH
,
Franke
AA
,
Shu
XO
, et al
Low plasma coenzyme Q(10) levels and breast cancer risk in Chinese women
.
Cancer Epidemiol Biomark Prev
2011
;
20
:
1124
30
.
35.
Fisar
Z
,
Hroudova
J
,
Singh
N
,
Koprivova
A
,
Maceckova
D
. 
Effect of simvastatin, coenzyme Q10, resveratrol, acetylcysteine and acetylcarnitine on mitochondrial respiration
.
Folia Biol
2016
;
62
:
53
66
.
36.
Xu
RH
,
Pelicano
H
,
Zhou
Y
,
Carew
JS
,
Feng
L
,
Bhalla
KN
, et al
Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia
.
Cancer Res
2005
;
65
:
613
21
.
37.
Hanahan
D
,
Weinberg
RA
. 
Hallmarks of cancer: the next generation
.
Cell
2011
;
144
:
646
74
.
38.
Robey
RB
,
Weisz
J
,
Kuemmerle
NB
,
Salzberg
AC
,
Berg
A
,
Brown
DG
, et al
Metabolic reprogramming and dysregulated metabolism: cause, consequence and/or enabler of environmental carcinogenesis?
Carcinogenesis
2015
;
36
:
S203
31
.
39.
DeBerardinis
RJ
,
Lum
JJ
,
Hatzivassiliou
G
,
Thompson
CB
. 
The biology of cancer: metabolic reprogramming fuels cell growth and proliferation
.
Cell Metab
2008
;
7
:
11
20
.
40.
Bjorklund
G.
The adjuvant nutritional intervention in cancer (ANICA) Trial
.
Nutr Cancer
2015
:
1
4
.
41.
Lu
S
,
Lu
LY
,
Liu
MF
,
Yuan
QJ
,
Sham
MH
,
Guan
XY
, et al
Cerebellar defects in Pdss2 conditional knockout mice during embryonic development and in adulthood
.
Neurobiol Dis
2012
;
45
:
219
33
.
42.
Nieborowska-Skorska
M
,
Kopinski
PK
,
Ray
R
,
Hoser
G
,
Ngaba
D
,
Flis
S
, et al
Rac2-MRC-cIII-generated ROS cause genomic instability in chronic myeloid leukemia stem cells and primitive progenitors
.
Blood
2012
;
119
:
4253
63
.
43.
Hertz
S
,
Rothamel
T
,
Skawran
B
,
Giere
C
,
Steinemann
D
,
Flemming
P
, et al
Losses of chromosome arms 4q, 8p, 13q and gain of 8q are correlated with increasing chromosomal instability in hepatocellular carcinoma
.
Pathobiology
2008
;
75
:
312
22
.
44.
Nishida
N
,
Nishimura
T
,
Ito
T
,
Komeda
T
,
Fukuda
Y
,
Nakao
K
. 
Chromosomal instability and human hepatocarcinogenesis
.
Histol Histopathol
2003
;
18
:
897
909
.
45.
Guan
XY
,
Fang
Y
,
Sham
JS
,
Kwong
DL
,
Zhang
Y
,
Liang
Q
, et al
Recurrent chromosome alterations in hepatocellular carcinoma detected by comparative genomic hybridization
.
Gen Chromosom Cancer
2000
;
29
:
110
6
.