Abstract
Mitochondrial uncoupling (uncouples electron transport from ATP production) has recently been proposed as a novel survival mechanism for cancer cells, and reduction in free radical generation is the accepted mechanism of action. However, there is no direct evidence supporting that uncoupling proteins promote carcinogenesis. Herein, we examined whether mitochondrial uncoupling affects mouse skin carcinogenesis using uncoupling protein 2 (UCP2) homozygous knockout and wild-type mice. The results indicate that knockout of Ucp2 significantly reduced the formation of both benign (papilloma) and malignant (squamous cell carcinoma) tumors. UCP2 knockout did not cause increases in apoptosis during skin carcinogenesis. The rates of oxygen consumption were decreased only in the carcinogen-treated UCP2 knockout mice, whereas glycolysis was increased only in the carcinogen-treated wild-type mice. Finally, the levels of metabolites pyruvate, malate, and succinate showed different trends after carcinogen treatments between the wild-type and UCP2 knockout mice. Our study is the first to demonstrate that Ucp2 knockout suppresses carcinogenesis in vivo. Together with early studies showing that UCP2 is overexpressed in a number of human cancers, UCP2 could be a potential target for cancer prevention and/or therapy. Cancer Prev Res; 8(6); 487–91. ©2015 AACR.
Introduction
The mitochondrion is the major source of adenosine triphosphate (ATP) in eukaryotes, which is generated via a proton gradient across the mitochondrial inner membrane (Δψm). However, not all of the energy available in the electrochemical gradient is coupled to ATP synthesis. A portion of the energy is consumed by “proton leak” reactions. As a result, the energy derived from the metabolic oxidation reaction is dissipated as heat (1–4). This phenomenon is called “mitochondrial uncoupling.” Mitochondrial uncoupling is caused mainly by uncoupling proteins (UCP) at physiological conditions. Among the five isoforms of UCPs, UCP2 is ubiquitously expressed and has been found to be upregulated in human tumor tissues (5–7).
In cancer cells, it has been suggested that high levels of mitochondrial uncoupling provide a prosurvival advantage to these tumor cells (5, 8). This prosurvival advantage is accomplished via two mechanisms: attenuating ROS (reactive oxygen species) generation (9–11) and reprogramming of cancer cell metabolism (12). In cancer cells, ROS are involved in the regulation of many physiological processes, which promotes further genomic instability and in the upregulation of signaling pathways of cellular growth and proliferation. However, if produced excessively, ROS may also be harmful to the cell by initiating cell death pathways (13). Hence, a well-controlled ROS level in cancer cells is important for tumor cell physiology, growth, and survival (14, 15). Mitochondrial uncoupling has been suggested to have a natural antioxidant effect that increases respiratory rates and thus attenuates ROS generation. Cancer cells also change their metabolic behaviors toward glycolysis and lactate fermentation for ATP production even under ample supply of oxygen (Warburg effect). Because mitochondria are debilitated, upregulation of UCP2 forces the cell to undergo aerobic glycolysis, in order to produce sufficient energy. The Warburg effect produces ATP less efficiently than oxidative phosphorylation but at a faster rate, promoting cells to proliferate at an increased rate and keep its oversupply of UCP2.
All of these observations suggest that UCP2 can promote cancer development. However, to date, there is no direct evidence supporting this role of UCP2. Utilizing the well-characterized mouse skin carcinogenesis model, whether knockout (KO) of UCP2 suppresses skin carcinogenesis will be studied.
Materials and Methods
Reagents and animals
7,12-dimethylbenz[α]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma. Both reagents were dissolved in dimethyl sulfoxide (DMSO; Sigma).
Six- to 8-week-old female UCP2 homozygous KO mice (ucp2−/−) and wild-type (WT) C57BL/6 mice were purchased from the Jackson Laboratory (stock number: 005934, in C57BL/6 background).
Multistage skin carcinogenesis
The animal study was performed under the approved animal protocol by the Institutional Animal Care and Use Committee of the LSU Health Sciences Center in Shreveport (Shreveport, LA). UCP2 and WT C57BL/6 mice were separated into four groups: (i) WT + vehicle (DMSO): 8 mice; (ii) UCP2 KO + vehicle (DMSO): 8 mice; (iii) WT + DMBA/TPA: 13 mice; (iv) UCP2 KO + DMBA/TPA: 18 mice. Hair on the back was shaven 2 days before treatments. For groups (i) and (ii), a single topical application of 200 μL of DMSO was performed once per day, five times a week for 40 weeks. For groups (iii) and (iv), a single dose of 400 nmol/L of DMBA was topically applied. Two weeks later, a single topical application of 5 μg TPA was performed once per day, 5 days per week for 40 weeks. At the end of the study, mice were euthanized by injecting overdosed pentobarbital (150 mg/kg). The skin tissues were removed, and the following steps were conducted: several small pieces of the non-tumor epidermal tissues were fixed; the remaining non-tumor epidermal tissues were stripped off and frozen for biochemical studies; the remaining skin tissues with tumors were fixed in Karnovsky's fixative for pathologic examinations. A pathologist (X. Gu) examined skin tumor formation of these tumor-bearing tissues.
Counting apoptotic and mitotic cells
Skin epidermal tissues were fixed in 4% buffered formaldehyde, embedded in paraffin, and processed for hematoxylin and eosin (H&E) stain. Mitotic and apoptotic cells were counted via light microscopic evaluation. Morphologic features, such as cell shrinkage, nuclear condensation, and formation of cytoplasmic vacuoles, were used to identify apoptosis. All phases of mitosis were included. Morphologic analysis was supervised by a certified pathologist (X. Gu).
Measurements of oxygen consumption of skin cells stripped from mouse skin epidermal tissues
Skin epidermal cells were collected as described previously (16). Stripped skin cells were resuspended in mitochondrial isolation buffer (0.225 mol/L mannitol, 0.075 mol/L sucrose, 1 mmol/L EGTA, pH 7.4) and transferred into a thermostated closed vessel at 37°C. Oxygen consumption was measured polarographically using a Clark-type O2 electrode (Yellow Spring Instruments). The rate of mitochondrial O2 consumption was determined as the cyanide-sensitive rate.
Preparation of whole cell lysate
Collected skin cells were suspended in 250 μL of PBS containing a proteinase inhibitor cocktail (Calbiochem). Cells were sonicated on ice for two strokes (10 seconds per stroke) using a Fisher Sonic Dismembrator (model 100, scale 4). After incubating on ice for 30 minutes, cell lysate was centrifuged at 18,000 × g for 20 minutes, and the supernatant was collected and designated as whole cell lysate.
Determination of pyruvate, malate, and succinate by high-performance liquid chromatography
The Shimadzu high-performance liquid chromatography (HPLC) system consisted of a binary, high-pressure gradient solvent delivery pump (model LC 20AB), an autosampler equipped with a cooling sample device (model SIL-20AC HT), a UV-visible absorbance detector (model SPD-20A), and data processing software (LCsolution version 1.23). The settings of the machine and sample preparation have been described previously (16).
Determination of ATP levels
The levels of ATP were determined using the ATP Assay Kit (BioVison, K354-100) following the instructions provided by the manufacturer. Whole cell lysate from mouse skin tissues was diluted to 2 μg/μL in PBS and deproteinized by passing through a 10-kD cutoff membrane (VWR, 82031-348). For each sample, 50 μL of the whole cell lysate filtrate was used.
Detection of lactate levels
The levels of lactate were determined using the Lactate Assay Kit (BioVison, K607-100) following the instructions provided by the manufacturer. Whole cell lysate was diluted to 2 μg/μL in PBS and deproteinized by passing through a 10-kD cutoff membrane (VWR, 82031-348). For each sample, 50 μL of the whole cell lysate filtrate was used.
Statistical analysis
Incidence expressed as percent was computed as the number of mice with tumor divided by the number of mice in that group. Data were presented as mean and SD. The nonparametric method exact Wilcoxon test was used to compare the tumor multiplicity. The Fisher exact test was used to compare the tumor incidence. Statistical software SAS 9.4 (SAS Institute Inc.) was used for all data analysis, and two-sided P < 0.05 was considered statistically significant.
Results and Discussion
To explore whether mitochondrial uncoupling affects skin carcinogenesis, we performed the animal study by using uncoupling protein 2 (UCP2) homozygous KO and WT mice. The animal bodyweights were recorded at the end of the study, and the results were as follows: WT/DMSO group: 22.4 ± 2.5 g (mean ± SD); WT/TPA group: 22.6 ± 1.7 g; UCP2-KO/DMSO group: 25.9 ± 2.0 g; and UCP2-KO/TPA group: 25.9 ± 2.5 g. The UCP2 mice weighed more than the WT mice in both DMSO-treated (P = 0.01) and TPA-treated (P = 0.0002) groups; however, TPA treatment did not significantly affect bodyweights. Because the skin carcinogenesis study has been performed for 40 weeks, both benign (papilloma) and malignant (squamous cell carcinoma) tumors have developed. Representative images of H&E-stained skin epidermal tissues/tumors are shown in Fig. 1. The data on tumor incidence and multiplicity are summarized in Table 1. Using the two-sided Fisher exact test, the difference in the incidences of papillomas between WT and UCP2 KO mice was not significant (P = 0.3679), whereas the difference in the incidences of carcinomas between WT and KO was significant (P = 0.0040). Using the exact Wilcoxon test to compare tumor multiplicity between WT and KO groups, both the differences in papillomas and carcinomas were significant (P = 0.0084 and 0.0023, respectively).
Representative H&E-stained mouse skin epidermal tissues/tumors at the end of the skin carcinogenesis study. Each sample represented one treatment group. A, WT/DMSO, amplification 10×; B, WT/TPA, amplification 2×; C, UCP2 KO/DMSO, amplification 10×; D, UCP2 KO/TPA, amplification 2×. A squamous cell carcinoma is shown in B and a papilloma is shown in D.
Skin tumor formation in the multistage carcinogenesis model
Next, the mitotic and apoptotic index in mouse skin epidermal was assessed by morphologic determination. As summarized in Table 2, both mitosis and apoptosis were increased in WT mice by carcinogen treatments. Mitosis was marginally increased in UCP2 KO mice (P = 0.076), and apoptosis was not significantly changed in these mice after carcinogen treatments. Similar results were obtained from immunohistochemical staining of the skin epidermal tissues (Supplementary Fig. S1). The WT/TPA group showed higher Ki-67 and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining, whereas the rest three groups (WT/DMSO, UCP2 KO/DMSO, and UCP2 KO/TPA) showed similar and lower levels of Ki-67 and TUNEL staining.
Mitosis and apoptosis index in the multistage carcinogenesis model
The levels of mitochondrial oxygen consumption and ATP production were measured using stripped skin cells collected at the end of the skin carcinogenesis study. As shown in Fig. 2A, although there appeared a slight increase in the levels of oxygen consumption in the vehicle-treated UCP2 group compared with the WT group similarly treated, the difference was not significant (P = 0.81). Carcinogen treatments did not cause a significant change in oxygen consumption levels in the WT mice (P = 0.77), but induced a significant decrease in the UCP2 KO group. ATP production (Fig. 2B) was not significantly different between WT and UCP2 KO groups, and between vehicle- and carcinogen-treated groups. It has been demonstrated that upregulated UCP2 promotes aerobic glycolysis of cancer cells, which is accompanied by an increase in lactate production (16). Thereafter, lactic acid levels were measured using mouse skin epidermal tissues mentioned above. The results (Fig. 2C) showed that lactate generation was significantly increased in the carcinogen-treated WT mice, but there was no difference between carcinogen-treated and vehicle-treated UCP2 KO mice.
The levels of mitochondrial oxygen consumption, not ATP production, were significantly decreased only in UCP2 KO mice after carcinogen treatment; lactate generation was significantly increased only in the WT mice after carcinogen treatment. A, measurement of oxygen consumption using stripped skin epidermal cells. B, the levels of ATP and lactate (C) were determined using whole cell lysate from stripped skin epidermal cells. KO, UCP2 KO. n = 8 per group; data, mean ± SEM. *, P < 0.05, compared with the DMSO group.
Because UCP2 has been recently shown to be able to transport C4 tricarboxylic acid (TCA) cycle intermediates (17), the skin epidermal tissue levels of pyruvate, malate, and succinate were detected, and the results are summarized in Table 3. In WT animals, carcinogen treatments increased the tissue levels of pyruvate and malate but decreased the levels of succinate, whereas in UCP2 KO animals, the levels of these metabolites had little or no change. These results were associated with the expression levels of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) in each treatment group (Supplementary Fig. S2). PFKFB2 belongs to a family of the bifunctional enzyme PFKFB that controls the levels of fructose 2,6-bisphosphate (F2,6BP). F2,6BP in turn activates the rate-limiting enzyme of glycolysis: phosphofructokinase-1 (PFK-1). PFKFB2 has been shown to be highly expressed in human liver (18), thyroid (19), and leukemia (20) tumor tissues, and PFKFB has become a molecular target in the development of anticancer drugs (21). Future studies will be performed to examine whether UCP2-transported TCA cycle intermediates regulate PFKFB2 expression and/or activity.
Levels of pyruvate, malate, and succinate in skin epidermal tissues (μg/mL)
Inflammation also contributes to skin carcinogenesis. Does UCP2 affect cutaneous inflammatory response? As shown in Supplementary Fig. S3, neutrophils infiltration around the invaded tumors was identified in the WT mice treated by carcinogens, which was not clearly observed in skin tumors from the UCP2 KO group. This result suggests that UCP2 may contribute to cutaneous inflammation, which provides another potential mechanism of how UCP2 promotes carcinogenesis.
As an anion carrier, UCP2 has also been found to play a role in several metabolic processes, including insulin secretion (22) and lipid/glucose metabolism (23). Not surprising, UCP2 has been suggested to be involved in a few metabolic diseases, such as obesity, diabetes, and cancer. Upregulation of UCP2 has been reported in human colon (5) and breast (7) cancers. Our recent studies also reveal that UCP2 is overexpressed in human skin, head and neck, pancreatic, and prostate tumor tissues compared with the adjacent normal tissues (6). It has been proposed that upregulation of UCP2 may serve as a novel survival mechanism for cancer cells, which is thought to be mediated by lowering ROS generation (24). However, there is no direct evidence supporting that UCP2 promotes carcinogenesis.
This article provides the first in vivo evidence using UCP2 mice supporting that UCP2 promotes carcinogenesis. Although the current data are unable to answer the question of how UCP2 KO suppresses skin cancer formation, the apoptosis index study suggests that UCP2 KO does not promote apoptosis during skin carcinogenesis. An interesting and important finding is the adverse changes of the three metabolites during carcinogenesis, which provides a clue for future studies.
Disclosure of Potential Conflicts of Interest
C. Kevil reports receiving commercial research support from Faraday Pharmaceutical and has ownership interest (including patents) in Theravasc, Inc. and Innolyzer LLC. No potential conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: C. Zhang, R. Shi, Y. Zhao
Development of methodology: W. Li, X. Shen, C.G. Kevil, Y. Zhao
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Jackson, X. Shen, C.G. Kevil, X. Gu, Y. Zhao
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): W. Li, X. Shen, G. Li, X. Gu, R. Shi, Y. Zhao
Writing, review, and/or revision of the manuscript: W. Li, X. Shen, R. Shi, Y. Zhao
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): W. Li, R. Jin, G. Li
Study supervision: Y. Zhao
Grant Support
This study was supported by NIH grant number 1R03CA167689 (Y. Zhao) NS088719 (G. Li), HL113303 (C.G. Kevil) and R21CA164218 (Y. Zhao).
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.
Acknowledgments
The authors thank Paul Polk at the Research Core Facility of their institution for technical support.
Footnotes
Note: Supplementary data for this article are available at Cancer Prevention Research Online (http://cancerprevres.aacrjournals.org/).
- Received September 9, 2014.
- Revision received January 30, 2015.
- Accepted March 9, 2015.
- ©2015 American Association for Cancer Research.
References
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