Cyclic phosphatidic acid decreases proliferation and survival of colon cancer cells by inhibiting peroxisome proliferator-activated receptor γ

Abstract

Cyclic phosphatidic acid (cPA), a structural analog of lysophosphatidic acid (LPA), is one of the simplest phospholipids found in every cell type. cPA is a specific, high-affinity antagonist of peroxisome proliferator-activated receptor gamma (PPARγ); however, the molecular mechanism by which cPA inhibits cellular proliferation remains to be clarified. In this study, we found that inhibition of PPARγ prevents proliferation of human colon cancer HT-29 cells. cPA suppressed cell growth, and this effect was reversed by the addition of a PPARγ agonist. These results indicate that the physiological effects of cPA are partly due to PPARγ inhibition. Our results identify PPARγ as a molecular mediator of cPA activity in HT-29 cells, and suggest that cPA and the PPARγ pathway might be therapeutic targets in the treatment of colon cancer.

Introduction

Globally, cancers of the colon and rectum are the third and fourth leading causes of cancer deaths in males and females, respectively [1]. Chemotherapeutic agents are the main tools for treating cancer. However, most of these drugs are nonspecific, or become less effective as tumor cells acquire multi-drug resistance. Therefore, numerous trials have been performed to enhance the therapeutic effectiveness and reduce the side effects of chemotherapeutic agents. Currently, the most common strategy for enhancing anti-cancer treatments is the use of sensitizers or drug combinations. If detected early, colon cancer is treatable; however, metastatic colon cancer is associated with high mortality. In combination with the first-choice chemotherapy agent 5-fluorouracil, new drugs, such as irinotecan, oxaliplatin, bevacizumab, and cetuximab, have improved the outcome of advanced colon cancer [2]. However, prognosis for metastatic colon cancer remains very poor. Therefore, novel therapeutic options are needed to reduce colon cancer mortality.

PPARγ is a nuclear receptor that plays an essential role in lipid and glucose homeostasis [3], cell proliferation [4], apoptosis [5], and inflammation [6]. Upon agonist binding, PPARγ becomes activated and forms a heterodimer with RXRα. The PPARγ–RXRα heterodimer translocates to the nucleus and binds to the peroxisome proliferator response element (PPRE; TGACCTnTGACCT) in the promoter region of target genes. Once bound, the heterodimer recruits the coactivators SRC-1, CBP, and TRAP220, and the corepressors NCoR and SMRT, to modulate gene transcription [7], [8], [9]. A variety of physiological and synthetic PPARγ agonists have been identified. Physiological agonists include 15d-PGJ₂ [10], selected forms of lysophospholipids, such as lysophosphatidic acid (LPA) [11] and alkyl glycerophosphate (AGP) [12], oxidized phospholipids [13], and nitrated fatty acids [14]. Thiazolidinedione (TZD) agonists, including ROSI and troglitazone (TRO), are widely used to ameliorate insulin sensitivity in type II diabetes mellitus [15]. PPARγ is overexpressed in many types of cancer, including colon, lung, breast, and stomach cancer, suggesting that regulation of PPARγ might affect cancer pathogenesis [4]. Several studies indicate that PPARγ agonists inhibit cancer cell proliferation, survival, and invasion in vitro and in vivo [16]. However, clinical trials with drugs that alter PPARγ function have yielded limited success in the treatment of advanced cancer [16]. Nonetheless, recent reports suggest that PPARγ inhibition might be advantageous in cancer treatment [17]. Recently, we reported that cyclic phosphatidic acid (cPA) is a physiological antagonist of PPARγ [18]. cPA is a naturally occurring LPA analog, containing an sn-2 hydroxy group that forms a 5-membered ring with the sn-3 phosphate [19]. cPA is generated by phospholipase D (PLD) catalyzed transphosphatidylation of lysophosphatidyl choline (LPC) [19], [20], [21]. LPA is a PPARγ agonist [11] that induces cellular proliferation and invasion (24), but cPA exerts the opposite effects in tumor cells, inhibiting proliferation and cancer cell invasion and metastasis in vitro and in vivo [19], [22], [23]. These findings suggest that cPA-mediated PPARγ inhibition could provide a novel strategy for treating advanced cancers. Furthermore, circulating cPA levels are lower in patients with ovarian cancer than in normal subjects [19].

Modulation of PPARγ in colon cancer remains controversial, because experimental evidence implies that PPARγ can either inhibit or stimulate cancer progression and tumorigenesis [16]. Some studies demonstrate that PPARγ agonists inhibit colon cancer cell proliferation, survival, and invasion in vitro and in vivo [24], [25]. However, others show that PPARγ agonists promote colon cancer progression [26], while PPARγ antagonists suppress it [27]. For example, PPARγ deficiency was shown to enhance colon tumorigenesis in Apc^Min/+ mice [28], while another study reports that ROSI induces colon tumors in normal mice [29]. It has been reported that HT-29 carry mutations in Apc [30] and p53 [31]. In contrast, HCT-116 colon cancer cells have normal Apc [32] and p53 [33]. Chiu et al. recently reported that p53-mutant HT-29 cells were more resistant to ROSI treatment (5–80 μM) than p53-wild type HCT-116 cells [34]. These results suggest that HT-29 cells were chemo-resistant in response to ROSI treatment. In this manuscript, we demonstrate the effects of PPARγ antagonist, cPA on cell growth inhibition in the human colon cancer cell line HT-29. We provide the first evidence that PPARγ is required in cell growth inhibition induced by cPA in HT-29 cells. Thus, cPA and its analogs could serve as new drug candidates for the treatment of colon cancer.