Abstract
Cancer cells display an increased reliance on glycolysis despite the presence of sufficient oxygen levels to support mitochondrial functions. In this study, we asked whether ameliorating mitochondrial functions in cancer cells might limit their proliferative capacity. Specifically, we increased mitochondrial metabolism in a murine cellular model of ErbB2/Neu–induced breast cancer by ectopically expressing the transcriptional coactivator peroxisome proliferator–activated receptor γ coactivator 1α (PGC-1α), a master regulator of mitochondrial metabolism. As predicted, ErbB2/Neu cells ectopically expressing PGC-1α displayed an increased level of mitochondrial metabolism and reduced proliferative capacity in vitro, compared with controls. In contrast, ErbB2/Neu cells ectopically expressing PGC-1α formed larger tumors in vivo. These tumors exhibited increased concentrations of glucose and the angiogenic factor VEGF as well as higher expression of ErbB2/Neu compared with controls. We discovered that ErbB2/Neu levels were sensitive to nutrient availability, such that reduced glucose concentrations resulted in diminished ErbB2/Neu protein levels. Therefore, our data indicate that PGC-1α prevents the nutrient-mediated downregulation of ErbB2/Neu in tumors by increasing glucose supply. Mechanistic investigations revealed that the regulation of ErbB2/Neu levels by glucose was mediated by the unfolded protein response (UPR). Incubation of ErbB2/Neu–induced breast cancer cells in limited glucose concentrations or with drugs that activate the UPR led to significant reductions in ErbB2/Neu protein levels. Also, ErbB2/Neu–induced tumors ectopically expressing PGC-1α displayed lowered UPR activation compared with controls. Together, our findings uncover an unexpected link between PGC-1α–mediated nutrient availability, UPR, and ErbB2/Neu levels. Cancer Res; 72(6); 1538–46. ©2012 AACR.
Introduction
Cancer occurs when cells divide uncontrollably. To proliferate, cells need significant amount of energy to build all the components necessary for making new cells. It is therefore not surprising that cancer cells display metabolic reorganizations tailored for their high and rapid energy demands. The best known metabolic reorganization that cancer cells undergo is a switch from mitochondrial to glycolytic metabolism despite the presence of sufficient oxygen to support mitochondrial reactions (Warburg effect).
Metabolic reprogramming plays a central role in ErbB2/Neu–induced breast cancer. Indeed, ErbB2/Neu–induced breast cancer cells have increased reliance on glycolysis and decreased respiration (1, 2). Limiting the activity of the glycolytic pathway in ErbB2/Neu–induced breast cancer cells resulted in drastically smaller tumors (1), highlighting the glucose dependence of these cancer cells. Importantly, ErbB2/Neu–induced breast cancer cells with reduced glycolytic activity displayed significantly elevated mitochondrial respiration. Overall, these experiments in ErbB2/Neu–induced breast cancer cells show how central to tumor maintenance is the metabolic reprogramming of cancer cells and suggest that increasing the reliance of cancer cells on mitochondrial metabolism could limit tumor growth.
To modulate mitochondrial functions in cancer cells, we need to examine key regulators of mitochondrial metabolism. The peroxisome proliferator–activated receptor γ coactivators 1 (PGC-1), namely, PGC-1α (3) and PGC-1β (4), are master regulators of mitochondrial functions. PGC-1α and PGC-1β are expressed in highly oxidative tissues and both stimulate mitochondrial biogenesis and increase total respiration (5). Very little work has been done on the physiologic roles of the PGC-1 transcriptional coactivators in cancer, in particular breast cancer. Reports have shown that the expression of PGC-1 is reduced in breast cancer patients (6, 7) and that interference with PGC-1β activity through expression of miR-378* in breast cancer cells is accompanied by increased cell proliferation (2).
In this study, we investigated the effects of increasing PGC-1α expression in ErbB2/Neu–induced breast cancer cells on their metabolism and proliferative capacity in vitro and in vivo. We uncovered an unexpected link between PGC-1α–mediated glucose supply, unfolded protein response (UPR) and ErbB2/Neu levels. We show that ErbB2/Neu–induced tumors ectopically expressing PGC-1α are larger than controls and display elevated levels of glucose and ErbB2/Neu. We discovered that ErbB2/Neu levels are reduced during glucose shortages. We conclude that PGC-1α–mediated glucose supply in tumors limits ErbB2/Neu downregulation, which in turn favors tumorigenesis.
Materials and Methods
Tissue culture and generation of stable cell lines
NMuMG and SK-BR-3 cells were obtained from the American Type Culture Collection. The NT2196 and TM15 cells are described in ref. 8. To generate NT2196-PGC-1α stable cell lines, NT2196 cells were transfected with a PGC-1α vector (Addgene #1026) and clones resistant to G418 were selected. NT2196-PGC-1α clones were independently derived twice to confirm reproducibility of data. NT2196-PGC-1α cells were cultured in Dulbecco's Modified Eagle's Medium, 10% FBS, 10 μg/mL insulin, 20 mmol/L HEPES, penicillin/streptomycin, 1 μg/mL puromycin, 400 μg/mL G418, pH 7.5, at 37°C and 5% CO2.
Gene expression
Total RNA was extracted with RNeasy Mini Kit and RNase-Free DNase Set (QIAGEN) or Aurum Total RNA Mini Kit (Biorad) and reverse transcribed with SuperScript II Reverse Transcriptase kit or iScript cDNA Synthesis kit (Invitrogen or Biorad). mRNA expression was assessed by real-time quantitative PCR using Brilliant SYBR Green QPCR Master Mix or iQ SYBR green Supermix (Stratagene or Biorad), gene-specific primers (Supplementary Table S1; ref. 8–11) and a Stratagene Mx3005P (Agilent Technologies) or MyiQ2 Real-Time Detection System (Biorad). Tbp was used as endogenous control gene.
Immunoblotting
Proteins were extracted with lysis buffer (50 mmol/L Tris-HCl pH7.4, 1% Triton X-100, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA) with inhibitors (2 μg/mL pepstatin, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 0.2 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovanadate) and quantified. The blots were incubated according to the manufacturer's instructions with the following primary antibodies: PGC-1α (Calbiochem; ST1202), Neu (Santa Cruz; sc-284), eIF2α (Cell Signaling; 9721,9722) and Actin (Santa Cruz; sc-1616) and with horseradish peroxidase–conjugated secondary antibodies (GE Healthcare or Santa Cruz). The results were visualized by Western Lightning Plus-ECL (Perkin Elmer). Densitometry analyses were conducted with ImageJ software (NIH).
Respiration
Cellular respiration was measured as previously published (1). The inhibitors were from Sigma.
In vitro cell proliferation assay
To determine proliferation, cells were grown in their respective medium, washed, trypsinized, and counted by a TC10 automated cell counter (Biorad). Viability was determined by exclusion of trypan blue dye.
In vivo tumorigenesis assays
Cells were resuspended at 5 × 104 cells per 30 μL in PBS and injected into the fourth mammary gland fat pad of 5 to 7-week-old athymic nude female mice (Taconic). For NT2196-derived clones, 3 groups of 5 mice were injected bilaterally with NT2196 empty vector Control-1 or Control-2 clones on the left side and NT2196-PGC-1α-1.1 (group 1), PGC-1α-1.2 (group 2), or PGC-1α-2 (group 3) cells on the right side. Tumor growth was monitored weekly with a caliper. Tumor volumes were calculated with the following formula: volume (mm3) = width2 × length/2. All animal studies were approved by the Animal Resource Centre at McGill University and comply with guidelines set by the Canadian Council of Animal Care. The transgenic MMTV/neu deletion mouse model (NDL2-5 strain) is described in ref. 12.
VEGF quantification
VEGF was quantified by the VEGF ELISA Kit (Calbiochem) according to the manufacturer's instructions.
Glucose concentration
The samples were extracted from tumor tissues according to previously established protocols (13), dried and stored at −80°C until nuclear magnetic resonance (NMR) data collection. NMR data collection was carried out on a 500 MHz Inova NMR system (Agilent Technologies) equipped with a cryogenically cooled probe. One-dimensional NMR spectra of samples were collected using the first increment of the standard NOESY experiment supplied with the instrument. Glucose chemical shift assignments were confirmed by 2-dimensional 75 ms mixing time total correlation spectroscopy. Targeted profiling of glucose was achieved with a 500 MHz metabolite library from Chenomx NMR Suite 7.0 (Chenomx). The area fit for the glucose peaks was compared with that of the internal concentration standard (DSS) resulting in a concentration based on the Chenomx library glucose compound as described previously (14). The amount of glucose was normalized to the weight of the tumor.
Results
Neu-transformed NT2196 cells display the Warburg effect
Several ErbB2/Neu–initiated mammary tumor cell lines have been shown to display increased reliance on glycolysis even in the presence of normal levels of oxygen, a phenomenon called the Warburg effect (1, 2). We used NT2196 cells as ErbB2/Neu experimental cell line. These cells are ex vivo explants originating from tumors expressing an activated form of ErbB2/Neu (8). To determine the metabolic properties of NT2196 cells, we first examined the gene expression profile of various glycolytic and mitochondrial enzymes (Fig. 1A). The expression of the glycolytic genes hexokinase II (Hk2) and lactate dehydrogenase A (Ldha) was upregulated in NT2196 cells compared with parental controls. Conversely, the expression of genes involved in mitochondrial metabolism, such as cytochrome c oxidase subunit VIIa 1 (Cox7a1), uncoupling protein 2 (Ucp2), and NADH dehydrogenase (ubiquinone) 1 beta subcomplex 5 (Ndufb5), was downregulated in NT2196 cells compared with controls. In addition, the expression of PGC-1α was significantly decreased in NT2196 cells. To determine the physiologic relevance of these changes in gene expression, we quantified the activity of the glycolytic and mitochondrial pathways by measuring respectively lactate production and cellular respiration. NT2196 cells showed increased lactate production and decreased respiration compared with parental control cells (Fig. 1B) illustrating that NT2196 cells display the Warburg effect and are thus a good experimental model to study the impact of increasing mitochondrial metabolism on tumor growth.
Neu-induced mammary tumors cells NT2196 display the Warburg effect. A, NT2196 cells display increased expression of glycolytic genes hexokinase 2 (Hk2) and lactate dehydrogenase A (Ldha) and decreased expression of mitochondrial metabolism genes cytochrome c oxidase subunit 7A1 (Cox7a1), uncoupling protein 2 (Ucp2), and NADH dehydrogenase (ubiquinone) 1 beta subcomplex 5 (Ndufb5) compared with parental controls. PGC-1α mRNA levels are lower in NT2196 cells compared with parental controls, paralleling the reduced expression of mitochondrial metabolism genes. B, uncoupled respiration and lactate production of NT2196 cells and their parental controls. Data are presented as means ± SEM. *, P < 0.05, Student's t test, n = 3 to 6 for mRNA expression, n = 4 for respiration, n = 9 for lactate production.
Ectopic expression of PGC-1α in NT2196 cells increases mitochondrial metabolism and lowers proliferative capacities in vitro
To ameliorate mitochondrial functions in NT2196 cells, we created stable NT2196 cell lines ectopically expressing PGC-1α. We independently derived 2 sets of clones to ensure reproducibility in the results. In the first set, we isolated 2 PGC-1α clones called PGC-1α-1.1 and PGC-1α-1.2 and one control clone called Control-1. In the second set, we isolated one PGC-1α clone called PGC-1α-2 and one control clone called Control-2.
First, we confirmed that PGC-1α expression was increased in the PGC-1α clones. All 3 PGC-1α clones displayed increased expression of PGC-1α at the mRNA and protein levels compared with their respective controls (Fig. 2A and B). Importantly, the expression of PGC-1β, another member of the PGC-1 family, remained unchanged (Fig. 2A). The expression of the mitochondrial electron transport chain gene Cox7a1, which is a known target of PGC-1α, was increased in all 3 clones (Fig. 2A). The expression of the glycolytic gene Ldha was unchanged (Fig. 2A). It is important to mention that the clones from the first set (Control-1, PGC-1α-1.1, and PGC-1α-1.2) had lower levels of Neu compared with those from the second set (Control-2 and PGC-1α-2; Fig. 2B). To evaluate the physiologic impact of PGC-1α in NT2196 cells, we carried out bioenergetics analyses of mitochondrial functions. All PGC-1α clones displayed significantly higher rates of total and uncoupled respiration compared with controls (Fig. 2C). Finally, we assessed the impact of PGC-1α ectopic expression on the growth properties of NT2196 cells in vitro. All 3 PGC-1α clones displayed reduced proliferation rates compared with controls (Fig. 2D). Together, these results show that elevated PGC-1α levels in NT2196 cells enhance mitochondrial functions and limit their proliferative capacity in vitro.
Ectopic expression of PGC-1α in NT2196 cells ameliorates mitochondrial functions. A, PGC-1α and Cox7a1 mRNA levels are increased in clones of NT2196 ectopically expressing PGC-1α, while the expression of PGC-1β and Ldha remains unchanged. B, protein levels for PGC-1α and Neu in NT2196-PGC-1α clones. Note that we independently derived 2 sets of clones to ensure reproducibility in the results. C, total and uncoupled respiration of NT2196-PGC-1α clones normalized to their controls. D, proliferation curves for NT2196-PGC-1α clones and their controls. Data are presented as means ± SEM. *, P < 0.05, Student's t test, n = 3 for mRNA expression, n = 6 to 7 for total and uncoupled respiration, n = 3 to 5 for proliferation.
PGC-1α increases Neu-mediated tumorigenesis in vivo
To determine whether the PGC-1α–mediated improved mitochondrial functions in NT2196 cells impacted tumor growth in vivo, PGC-1α clones and controls were injected in the mammary fat pad of female nude mice. Each mouse was injected on the left side with a control clone and on the right side with a PGC-1α clone. Contrary to the decreased proliferation of PGC-1α clones in vitro, Neu-initiated mammary tumors ectopically expressing PGC-1α grew faster and were larger than controls (Fig. 3A). The growth rates of the PGC-1α-2 and Control-2 tumors were higher than those of PGC-1α-1 and Control-1 tumors (Fig. 3A), consistent with their higher Neu protein levels (Fig. 2B). Given the difference between the in vitro and in vivo results, we first confirmed that PGC-1α endpoint tumors displayed higher PGC-1α mRNA levels than control tumors (Fig. 3B). Furthermore, the expression of Cox7a1, a PGC-1α target gene, was higher in PGC-1α endpoint tumors compared with controls (Fig. 3B). The expression of PGC-1β and LdhA was not different between PGC-1α and control endpoint tumors (Fig. 3B). Together, these gene expression data illustrate that PGC-1α and control endpoint tumors display the same expression profile as the PGC-1α and control clones (Figs. 2A and 3B).
NT2196-PGC-1α clones form larger tumors during in vivo tumorigenesis assays compared with controls. A, tumor growth curves for NT2196-PGC-1α and control clones injected in mammary fat pad of nude mice. B, PGC-1α and Cox7a1 mRNA levels are increased in NT2196-PGC-1α tumors, while the expression of PGC-1β and Ldha remains unchanged. C, Neu protein levels in NT2196-PGC-1α and control tumors. Each mouse was injected on the left with control clone and on the right with NT2196-PGC-1α clone. D, densitometry analyses for (C). Fold changes were calculated by dividing the relative Neu signal values for the NT2196-PGC-1α clones by those for the control clones. Note that 11 of 15 of NT2196-PGC-1α tumors display higher Neu levels compared with their paired controls. E, correlation between tumor size and Neu protein levels across NT2196-PGC-1α and control tumors (n = 30). Relative Neu levels were calculated by dividing the signal of each band by that of the loading control. The correlation analysis was conducted by Spearman test. F, comparison of Neu protein levels between NT2196-PGC-1α clones and their derived tumors, contrasting the identical Neu levels between NT2196-PGC-1α and control clones with the different Neu levels between NT2196-PGC-1α and control tumors. A and B, data are presented as means ± SEM. *, P < 0.05, Student's paired t test. D, the horizontal bars represent the averages. A–D, n = 5 mice per group. E, n = 15 mice.
Next, we quantified Neu protein levels in endpoint tumors (Fig. 3C and D). Strikingly, we noticed that a large fraction (11 of 15) of PGC-1α endpoint tumors displayed higher levels of Neu compared with their paired control (P < 0.05). Indeed, all mice of the group injected with the PGC-1α-1.1 clones, 2 mice of the group injected with the PGC-1α-1.2 clones, and 4 out of 5 mice of the group injected with PGC-1α-2 clones displayed higher levels of Neu compared with their paired controls. Importantly, there was no difference in the protein levels of Neu between controls and PGC-1α clones in vitro (Fig. 2B). To further highlight the differences between the in vitro and in vivo conditions, we loaded side by side on 1 gel the controls and PGC-1α clones next to the controls and PGC-1α tumors (Fig. 3F). As shown in Fig. 2B, the protein levels of Neu for the clones were similar between PGC-1α and controls, whereas for the tumors the levels of Neu were higher in the PGC-1α compared with controls (Fig. 3F). Furthermore, the Neu protein levels for both PGC-1α and controls were lower in the tumors compared with the clones (Fig. 3F).
To determine whether there was a correlation between Neu levels and tumor size, we graphed the relative Neu protein levels of all control and PGC-1α tumors in relation with their tumor size and carried out a correlation analysis (Fig. 3E). There was a significant positive correlation between Neu protein levels and tumor volumes across all tumors (r = 0.685, P < 0.0001). Furthermore, for each group of mice, the mice with the highest fold changes in tumor volume were the ones with the highest fold changes in Neu levels (data not shown). Together, these data show that a large fraction of PGC-1α tumors displayed higher protein levels of Neu compared with controls, and that there was a link between Neu levels and tumor volumes.
PGC-1α regulates Neu levels indirectly by increasing nutrient availability
One key difference between in vitro and in vivo conditions is the control of nutrient availability through blood supply. Given that PGC-1α impacted Neu levels in vivo but not in vitro and that PGC-1α is a powerful regulator of VEGF expression and angiogenesis (15), we hypothesized that PGC-1α regulates Neu levels in vivo by controlling nutrient availability through angiogenesis.
To assess the ability of PGC-1α to regulate VEGF expression, we measured the amount of VEGF secreted in culture medium for all experimental clones (Fig. 4A). All PGC-1α clones secreted larger amounts of VEGF in culture medium compared with controls. Furthermore, PGC-1α tumors displayed higher levels of VEGF compared with their paired controls (Fig. 4B). Specifically, 12 of the 15 PGC-1α tumors had higher levels of VEGF compared with their paired control. In support of the increased VEGF levels in the PGC-1α tumors, they also displayed elevated concentration of glucose compared with their paired controls (Fig. 4C). Indeed, 11 out of the 15 PGC-1α tumors displayed higher glucose levels compared with their paired controls.
Neu levels are regulated by glucose availability. A, amount of VEGF secreted in the medium for the NT2196-PGC-1α clones normalized to controls. B, fold change in tumor VEGF content. For each mouse, the content of VEGF in the NT2196-PGC-1α tumor was divided by the content of VEGF in the paired control tumor. C, glucose levels in NT2196-PGC-1α tumors compared with their paired controls. Glucose levels were expressed as fold changes representing glucose concentration values in NT2196-PGC-1α tumors divided by those of their paired control tumors. D, Neu protein levels are regulated by glucose availability. NT2196-PGC-1α and control clones were grown for 5 days in 0.5, 2.5, or 25 mmol/L glucose. A, data are presented as means ± SEM. n = 9. *, P < 0.05, Student's t test. B and C, the horizontal bars represent the averages. B and C, n = 5 mice per group.
To assess whether there was a link between the elevated levels of Neu, VEGF, and glucose in PGC-1α tumors, we determined whether Neu levels are regulated by nutrient availability. PGC-1α clones and controls were incubated in various glucose concentrations (25, 2.5, and 0.5 mmol/L) for 5 days to mimic the low nutrient levels that can be found in solid tumors. Neu protein levels were drastically reduced in PGC-1α and control clones incubated in 2.5 and 0.5 mmol/L glucose compared with those incubated in 25 mmol/L glucose (Fig. 4D). Together, these data illustrate that PGC-1α positively regulates the angiogenic factor VEGF and glucose levels in tumors, and that Neu levels are regulated by glucose availability.
Regulation of ErbB2/Neu levels by the UPR
To gain further insight into the regulation of Neu protein levels upon glucose availability, we studied the impact of UPR. The UPR is a response to endoplasmic reticulum (ER) stress that can be initiated by various stress stimuli, notably glucose deprivation (16). Acute glucose removal drastically reduced Neu levels in PGC-1α and control clones (Fig. 5A). The reduction in Neu levels occurred at the posttranslational level as neuNT mRNA levels remained unchanged after glucose withdrawal (Supplementary Fig. S1). Decreased ErbB2/Neu protein levels upon glucose withdrawal were also observed in another mouse ErbB2/Neu–induced breast cancer cell line (TM15; ref. 8) as well as in the human ErbB2-positive SKBR3 breast cancer cell line (Fig. 5A), indicating the generality of ErbB2/Neu regulation by glucose levels.
Regulation of ErbB2/Neu levels by the UPR. A, acute glucose deprivation reduces Neu protein levels. NT2196-PGC-1α and control clones as well as TM15 cells were deprived of glucose overnight. SK-BR-3 cells were deprived of glucose for 45 hours. B, the ratio of spliced XBP-1 mRNA is increased upon glucose withdrawal in all control and NT2196-PGC-1α clones. The fold change in XBP-1 splicing was calculated by dividing the ratio of spliced XBP-1/total XBP-1 mRNA in no glucose condition by that of the control condition. C, Neu protein levels are reduced upon induction of the UPR by glucose withdrawal, DTT, or thapsigargin. D, phosphorylated eIF2α (eIF2α-P) and total eIF2α (eIF2α-T) protein levels in NT2196-PGC-1α and control tumors. Each mouse was injected on the left with control clone and on the right with NT2196-PGC-1α clone. E, densitometry analyses for (D). The data represent the average of 4 independent loadings and quantifications. Fold changes were calculated by dividing the ratio eIF2α-P/eIF2α-T for the NT2196-PGC-1α tumors by that of their paired control tumors. Note that 13 of 15 of NT2196-PGC-1α tumors display lower ratios eIF2α-P/eIF2α-T compared with their paired controls. B, data are presented as means ± SEM, n = 7. *, P < 0.05; ***, P < 0.001, Student's t test. D and E, n = 5 mice per group. E, the horizontal bars represent the averages.
To assess whether the UPR was activated upon glucose withdrawal in our experimental conditions, we measured the splicing of X-box binding protein1 (XBP-1) mRNA, which is an indicator of ER stress. The ratio of spliced XBP-1/total XBP-1 mRNA was significantly increased after glucose withdrawal in all PGC-1α and control clones (Fig. 5B). In addition to glucose withdrawal, various drugs can be used to stimulate the UPR, in particular DTT and thapsigargin. Incubation of PGC-1α clones and controls with either DTT or thapsigargin led to significant reduction in Neu protein levels (Fig. 5C and Supplementary Fig. S2). There was similar reduction in Neu levels upon DTT or thapsigargin treatment between the PGC-1α clones and controls. These data illustrate that Neu levels are regulated by the UPR. Given that Neu is regulated at the protein level during glucose withdrawal-induced ER stress and that a central way by which the UPR halts protein synthesis is through phosphorylation of the eukaryotic initiation factor 2α (eIF-2α), we quantified the phosphorylation of eIF-2α in PGC-1α tumors and their paired controls to assess whether UPR activation was lowered in PGC-1α tumors in accordance with their higher Neu and glucose levels. Indeed, the PGC-1α tumors displayed significantly less phosphorylation of eIF-2α compared with their paired controls (P = 0.0012; Fig. 5D and E). Specifically, 13 out of 15 PGC-1α tumors showed this effect, illustrating lowered UPR activation in these tumors. Furthermore, 11 out of 15 PGC-1α tumors displayed reduced XBP-1 mRNA splicing (P = 0.076; Supplementary Fig. S3). Together, the results presented here suggest that PGC-1α, by regulating glucose availability, would reduce ER stress, thereby alleviating the UPR, preventing Neu downregulation, and favoring tumorigenesis.
Discussion
In this article, we examined the role of PGC-1α in ErbB2/Neu-induced breast cancer. There is currently much interest to investigate whether ameliorating mitochondrial functions in cancer could limit tumor growth (17). The PGC-1α transcriptional coactivator is a well known master regulator of mitochondrial metabolism (5). However, it is important to appreciate that in addition to increasing mitochondrial functions, PGC-1α can stimulate VEGF expression and angiogenesis (15). These 2 functions of PGC-1α can have opposing consequences on tumorigenesis. Elevated mitochondrial metabolism could in theory reduce tumor growth, while increased angiogenesis would support tumorigenesis. Therefore, PGC-1α may play a positive or negative role in cancer depending on the balance between these 2 processes in a given tumor.
Reports on the expression of PGC-1 in cancer show that it is dependent on tumor types. The expression of PGC-1 is reduced in breast (6, 7), colon (18), ovarian (19), and liver (20) tumors, and low levels of PGC-1 are associated with poor clinical outcome in breast cancer patients (6). Overexpression of PGC-1α in ovarian and intestinal cell lines stimulated apoptosis (19), while interference with PGC-1β activity through expression of miR-378* in breast cancer cells was accompanied by increased cell proliferation (2). On the contrary, PGC-1α expression is increased in endometrial cancer (21) and renal tumors (22), and PGC-1 activity has been shown to promote the growth of prostate cancer cells (23). A recent paper by Tiraby and collaborators (24) showed no impact of PGC-1α on the growth of the MDA-MB-231 breast cancer cell line in vitro and in xenograft experiments. Given that MDA-MB-231 cells do not overexpress ErbB2, these results support our conclusion that PGC-1α promotes the growth of ErbB2/Neu tumors by preventing ErbB2/Neu levels downregulation through increasing nutrient supply. Importantly, there are in vivo situations in which increased ErbB2/Neu signaling is associated with elevated levels of PGC-1α. Indeed, mammary tumors from transgenic mice expressing an activated form of ErbB2/Neu (NDL2-5) displayed elevated mRNA levels of PGC-1α compared with adjacent mammary gland (Supplementary Fig. S4). However, it is also possible that PGC-1α promotes the growth of ErbB2/Neu tumors independently of its impact on ErbB2/Neu levels. In our study, 4 out of the 15 PGC-1α tumors were larger than their paired controls, but did not display increased levels of ErbB2/Neu. It is important to appreciate that PGC-1α levels are highly sensitive to many environmental factors and this could explain the reported variability in PGC-1α expression in different tumor types. In agreement with this, PGC-1α levels were found to be regulated by oxygen and nutrient levels (15), which can fluctuate greatly during tumor growth.
A central discovery of this study is that ErbB2/Neu levels are regulated by glucose availability and the UPR. The UPR is activated when misfolded proteins accumulate in the ER upon stressful cellular environmental conditions like glucose limitations. The UPR is particularly relevant for solid tumors, which experience nutrient stress due to poor vascularization. Upon an environmental insult, the UPR initially halts translation and activates signaling cascades aiming at restoring proper protein folding. If the insult persists or proper folding is not achieved, the UPR leads to activation of apoptosis. Therefore, the UPR can have prosurvival and prodeath functions depending on the severity of the environmental insult (16). It is of paramount importance to understand how modulation of the UPR in cancer affects the balance between its prosurvival and prodeath functions. Indeed, activation of the prosurvival functions of the UPR would promote tumorigenesis, while activation of the prodeath functions of the UPR would limit tumorigenesis. So far, reports have shown both increased and decreased UPR in different tumor types (16).
PGC-1α has been shown to mediate the UPR in skeletal muscle, a response particularly important during exercise training (11). The UPR is activated during exercise, and muscle tissues from MCK-PGC-1α transgenic mice that have increased expression of PGC-1α display less ER stress after exercise than wild-type mice (11). In this study, we did not detect any difference in the downregulation of ErbB2/Neu protein levels upon activation of the UPR during glucose withdrawal between ErbB2/Neu–induced breast cancer cells ectopically expressing PGC-1α and controls (Fig. 5A and C; Supplementary Fig. S2). However, ErbB2/Neu–induced mammary tumors ectopically expressing PGC-1α displayed less ER stress than their paired controls (Fig. 5D and E; Supplementary Fig. S3).
ErbB2-positive breast tumors display a large increase in ErbB2 expression (25). It is important to appreciate that cells spend approximately 30% of their energy budget for protein translation, with mRNA processing also making a significant contribution (26). Strictly from an energy budget standpoint, it would make sense under harsh cellular conditions to halt the translation of a protein that is significantly overexpressed and hence costly to maintain. Therefore, reducing the supply of nutrients in solid growing tumors would be an efficient way to limit ErbB2 levels. In support of this point, VEGF inhibitors are currently showing promise in breast cancer patients in combination therapy with trastuzumab, a humanized her2 antibody (27). Clearly, the results presented in this article provide a strong rationale for reduction of angiogenesis in ErbB2/Neu breast tumors.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant Support
This work was supported by grants from the Canadian Institutes of Health Research to J. St-Pierre (MOP-106603) and W.J. Muller (MOP-93525). E. Klimcakova was supported by a Tomlinson fellowship (McGill University) and a fellowship from the McGill Integrated Cancer Research Training Program. S. McGuirk was supported by a studentship from the McGill Integrated Cancer Research Training Program (McGill University). NMR experiments were recorded at the Québec/Eastern Canada High Field NMR Facility, supported by the Natural Sciences and Engineering Research Council of Canada and Canada Foundation for Innovation. J. St-Pierre is an FRSQ research scholar and W.J. Muller holds a Canada Research Chair in Molecular Oncology.
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 Drs Vincent Giguère, John Bergeron, Peter Siegel, Nicole Beauchemin, and Damien D'Amours for helpful discussion. The authors also thank Erzsebet Nagy Kovacs and Vasilios Papavasiliou for technical assistance.
Footnotes
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received September 1, 2011.
- Revision received January 6, 2012.
- Accepted January 11, 2012.
- ©2012 American Association for Cancer Research.
References
Volume 72, Issue 6
