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

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.

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 nuclear receptors, such as the androgen receptor (AR), estrogen receptor α (ESR1, also known as ERα), liver x receptor α (NR1H3, also known as LXRα), peroxisome proliferator-activated receptor gamma (PPARγ), RAR-related orphan receptor gamma (RORC, also known as RORγ) and retinoic acid receptors (RARs) are highlighted as examples; other nuclear receptors may also influence these processes.

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.