PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of mconcolMolecular & Cellular Oncology
 
Mol Cell Oncol. 2015 Apr-Jun; 2(2): e980660.
Published online 2015 January 20. doi:  10.4161/23723556.2014.980660
PMCID: PMC4905022

Harnessing the nuclear receptor PPARγ to inhibit the growth of lung adenocarcinoma by rewiring metabolic circuitries

Paul Yenerall1,2 and Ralf Kittler1,3,4,5,*

Abstract

Altered metabolism and nuclear receptor activity have been reported in various cancer types. Here, we discuss our recent finding that the metabolic state of lung adenocarcinoma cells expressing the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) can be modulated by thiazolidinediones, culminating in accumulation of reactive oxygen species and decreased proliferation.

Keywords: lung cancer, metabolism, PPARγ, nuclear receptor, targeted therapy

Abbreviations

FDA
Food and Drug Administration
IDH
isocitrate dehydrogenase
PDK
pyruvate dehydrogenase kinase
PPARγ
peroxisome proliferator-activated receptor gamma
ROS
reactive oxygen species
siRNA
small interfering RNA
shRNA
small hairpin RNA

Initially described 90 years ago by Otto Warburg,1 the altered metabolism of cancer cells is a well-established phenomenon. However, until recently the mechanisms and clinical relevance of this phenomenon have remained enigmatic. In some cases, the altered metabolism of cancer cells may result in the production or removal of metabolites essential or detrimental to the growth and survival of cancer cells.2 In other cases, metabolites such as 2-hydroxyglutarate produced by enzymes expressed from mutant isocitrate dehydrogenase 1 and 2 genes (IDH1, IDH2) may perform non-canonical roles, such as inhibition of DNA and histone demethylases required for differentiation.3 Regardless of their function, these tumor-acquired metabolic alterations represent unique therapeutic opportunities.

Direct inhibition of aberrantly activated metabolic enzymes is a promising approach to modulating the altered metabolic state of cancer cells, as has been demonstrated for inhibitors of mutant IDH1.4 Another approach, however, is to indirectly alter the expression levels of metabolic enzymes. To this end, a subset of nuclear receptors may represent clinically actionable “handles” to alter the metabolic state of a cancer cell (reviewed in5). Nuclear receptors are liganded transcription factors that control diverse physiological processes such as metabolism, differentiation, and cellular growth (reviewed in6). As a result of the ligand dependency of their activation or repression, their transcriptional activity can generally be modulated by small molecules. This is directly evidenced by the fact that approximately 10–15% of all Food and Drug Administration (FDA)-approved drugs target fewer than 48 nuclear receptors.7 Thus, discovery of a nuclear receptor integral to a specific cancer type may lead to rapid clinical implementation as FDA-approved molecules against this nuclear receptor may already exist (Fig. 1).

Figure 1.
Nuclear receptor agonists and antagonists are FDA-approved drugs that may be harnessed to inhibit the growth of cancer cells. Top, pie chart for FDA-approved drug targets adapted from Overington et al.7. Bottom, the canonical roles of several ...

A previous study demonstrated that nuclear receptors are differentially expressed in non-small cell lung cancer. In addition, this study showed that for cell lines with high expression of peroxisome proliferator-activated receptor gamma (PPARγ), treatment with pioglitazone, an FDA-approved PPARγ agonist, significantly impeded cell growth.8 To better understand this phenomenon, we first undertook the unbiased functional genomics approach of identifying direct targets of PPARγ by integrating chromatin immunoprecipitation followed by sequencing (ChIP-seq) and gene expression analysis following pioglitazone treatment.9 We then analyzed how transcript abundance changed 12, 24, and 48 hours after pioglitazone treatment. At early time points (12 hours) there was a significant upregulation of genes involved in lipid metabolism. In contrast, at later time points, we saw a marked decrease in the transcript abundance of cell cycle-related genes, suggesting that pioglitazone treatment resulted in a metabolic change that ultimately culminated in cell cycle arrest.

To further investigate the mechanisms underlying these changes, we first examined whether there was a cell cycle defect following PPARγ activation. Treatment of PPARγ-expressing cells with the PPARγ agonists pioglitazone or troglitazone resulted in an increase in the percentage of cells in G1, hypophosphorylation of RB, and elevated reactive oxygen species (ROS) levels, as well as the absence of canonical apoptotic markers such as cleaved poly (ADP-ribose) polymerase 1 (PARP1) or cleaved caspase-3 (CASP3). These effects were mirrored in vivo, as xenograft tumors treated with pioglitazone were significantly smaller, had significantly lower Ki-67 positivity, and were negative for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) compared with controls. This phenotype was specific to PPARγ activation, as reduction of PPARγ levels by siRNAs in vitro or shRNAs in vivo alleviated the proliferation arrest following pioglitazone treatment. In addition, co-treatment with pioglitazone and antioxidants rescued cells from growth inhibition in vitro and in vivo, suggesting that ROS levels may be responsible for this cell cycle arrest.

ROS levels can increase in cells for a number of reasons, but the upregulation of genes involved in lipid metabolism and oxidative stress following pioglitazone treatment led us to believe that a metabolic switch may underlie this phenomenon. In accordance with this hypothesis, the most rapidly upregulated gene following pioglitazone treatment was pyruvate dehydrogenase kinase isozyme 4 (PDK4), which has been shown to inhibit glucose oxidation and increase β-oxidation of fatty acids.10 Consistent with this, pioglitazone treatment resulted in an increase in glucose utilization and lactate secretion, as well as a decrease in citric acid cycle intermediates. Inhibition of PDK4 by siRNAs in vitro or by the small molecule dichloroacetate, a known pyruvate dehydrogenase kinase inhibitor, in vitro and in vivo alleviated the ROS accumulation and cell cycle arrest observed following pioglitazone treatment, suggesting that PDK4 was a prominent player in the effects of pioglitazone treatment. In addition, treatment with the β-oxidation inhibitor trimetazidine prevented the increase in ROS levels and cell cycle arrest in vitro and in vivo, suggesting that a metabolic switch from glucose oxidation to β-oxidation mediated by PDK4 may underlie ROS accumulation and eventual cell cycle arrest.

Although total ROS levels were elevated following pioglitazone treatment, whether this was due to an increase in free radical generation or a decrease in the activity of cellular detoxification systems remained unclear. We found that treatment with pioglitazone reduced the conversion rate of glutamine to glutamate, which is required for the synthesis of glutathione, a central component of the cellular ROS detoxification machinery. Not surprisingly, this resulted in a reduced level of glutathione, which could be prevented by inhibiting fatty acid oxidation with trimetazidine. Treatment with a glutaminase inhibitor, BPTES, phenocopied the effects of pioglitazone treatment, indicating that decreased glutathione levels following pioglitazone treatment were responsible for the ROS-mediated cell cycle arrest.

The dysregulation of metabolism in cancer cells has begun to shift from a poorly understood phenomenon to an understood adaptation to cellular stresses, external stimuli, and growth requirements. Thus, modulation of aberrant metabolism represents a novel therapeutic opportunity for cancer treatment. Nuclear receptors, well known for their oncogenic roles in breast and prostate cancer, may represent an attractive and so far poorly utilized “handle” to modulate cancer metabolism, particularly in cancer types where they remain understudied. Importantly, identification of nuclear receptors whose actions may affect cancer-relevant pathways represents a unique opportunity for rapid bench-to-bedside translation because of the large number of FDA-approved drugs that target nuclear receptors.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Caroline Humphries for proofreading this manuscript.

Funding

This work was supported by grants RP101251-P06 and RP120732-P3 of the Cancer Prevention and Research Institute of Texas (CPRIT). P.Y. is supported by a Green Center Trainee Fellowship. R.K. is a John L. Roach Scholar in Biomedical Research and a CPRIT Scholar in Cancer Research.

References

1. Warburg O., Posener K., Negelein E. On the metabolism of carcinoma cells. Biochemische Zeitschrift 1924; 152:309-44.
2. DeBerardinis RJ., Mancuso A., Daikhin E., Nissim I., Yudkoff M., Wehrli S., Thompson CB.. Beyond aerobic glycolysis: transformed cells can engage in glutamine metabolism that exceeds the requirement for protein and nucleotide synthesis. Proc Natl Acad Sci U S A 2007; 104:19345-50; PMID:18032601; http://dx.doi.org/10.1073/pnas.0709747104 [PubMed] [Cross Ref]
3. Figueroa ME., Abdel-Wahab O., Lu C., Ward PS., Patel J., Shih A., Li Y., Bhagwat N., Vasanthakumar A., Fernandez HF, et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell 2010; 18:553-67; PMID:21130701; http://dx.doi.org/10.1016/j.ccr.2010.11.015 [PMC free article] [PubMed] [Cross Ref]
4. Rohle D., Popovici-Muller J., Palaskas N., Turcan S., Grommes C., Campos C., Tsoi J., Clark O., Oldrini B., Komisopoulou E, et al. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells. Science 2013; 340:626-30; PMID:23558169; http://dx.doi.org/10.1126/science.1236062 [PMC free article] [PubMed] [Cross Ref]
5. Sonoda J., Pei L., Evans RM.. Nuclear receptors: decoding metabolic disease. FEBS Lett 2008; 582:2-9; PMID:18023286; http://dx.doi.org/10.1016/j.febslet.2007.11.016 [PMC free article] [PubMed] [Cross Ref]
6. Aranda A., Pascual A.. Nuclear hormone receptors and gene expression. Physiol Rev 2001; 81:1269-304; PMID:11427696 [PubMed]
7. Overington JP., Al-Lazikani B., Hopkins AL.. How many drug targets are there? Nat Rev Drug Discov 2006; 5:993-6; PMID:17139284; http://dx.doi.org/10.1038/nrd2199 [PubMed] [Cross Ref]
8. Jeong Y., Xie Y., Lee W., Bookout AL., Girard L., Raso G., Behrens C., Wistuba II., Gadzar AF., Minna JD, et al. Research resource: Diagnostic and therapeutic potential of nuclear receptor expression in lung cancer. Mol Endocrinol 2012; 26:1443-54; PMID:22700587; http://dx.doi.org/10.1210/me.2011-1382 [PMC free article] [PubMed] [Cross Ref]
9. Srivastava N., Kollipara RK., Singh DK., Sudderth J., Hu Z., Nguyen H., Wang S., Humphries CG., Carstens R., Huffman KE, et al. Inhibition of Cancer Cell Proliferation by PPARgamma Is Mediated by a Metabolic Switch that Increases Reactive Oxygen Species Levels. Cell Metab 2014; 20:650-61; PMID:25264247; http://dx.doi.org/10.1016/j.cmet.2014.08.003 [PMC free article] [PubMed] [Cross Ref]
10. Holness MJ., Kraus A., Harris RA., Sugden MC.. Targeted upregulation of pyruvate dehydrogenase kinase (PDK)-4 in slow-twitch skeletal muscle underlies the stable modification of the regulatory characteristics of PDK induced by high-fat feeding. Diabetes 2000; 49:775-81; PMID:10905486; http://dx.doi.org/10.2337/diabetes.49.5.775 [PubMed] [Cross Ref]

Articles from Molecular & Cellular Oncology are provided here courtesy of Taylor & Francis