Selective activation of PPARγ in breast, colon, and lung cancer cell lines
Introduction
The American Cancer Society estimated that collectively cancers of the breast, colon and lung accounted for 42% of all cancer deaths in men and 50% of all cancer deaths in women in 2004. In fact, breast, lung, and colon cancer rank as the top three types of malignancies identified in women today and one out of every eight women will develop breast cancer. In men, lung cancer is the most prevalent cause of cancer related death with malignancies of the prostate and colon following as next most common. A wide variety of chemotherapeutic options are being explored to treat these diseases. Novel therapeutic targets are being developed in an effort to identify endogenous, hormonal targets to either suppress cancer cell growth or induce apoptosis. One of the emerging targets for such treatments is peroxisome proliferator-activated receptor gamma (PPARγ).
The orphan nuclear receptor, PPARγ, is one of three of a family of receptors (PPARα, β, and γ) (Dreyer et al., 1992, Issemann and Green, 1990, Kliewer et al., 1994). It is expressed in numerous cell types including adipocytes, epithelial cells of the breast, colon, and lung, and macrophages among others (Braissant et al., 1996, Kilgore et al., 1997, Lemberger et al., 1996, Nagy et al., 1998, Tontonoz et al., 1994). Several ligands of PPARγ have been identified including 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2), linoleic acid, lysophosphatidic acid, and the thiazolidinedione class of anti-diabetic drugs such as ciglitazone (Cig) and rosiglitazone (Ros) (Forman et al., 1995, Kliewer et al., 1997, Larsen et al., 2003, McIntyre et al., 2003, Thoennes et al., 2000). Transactivation of the receptor requires ligand binding, heterodimerization with retinoid X receptor alpha (RXRα), and binding of this complex to PPAR-specific response elements (PPREs) in the promoter regions of target genes (Kliewer et al., 1992, Tontonoz et al., 1994).
Recent evidence demonstrates that PPARγ is overexpressed in many different tumor types (DuBois et al., 1998, Tontonoz et al., 1997).In the breast, adenocarcinoma cells from patients expressed higher levels of PPARγ than normal epithelial cells from the surrounding mammary gland (Elstner et al., 1998). Similarly, in the colon, expression of PPARγ protein is significantly higher in human colon cancer sections when compared with non-tumor tissue (Chen et al., 2002). PPARγ has also been identified in both adenocaricoma and squamous cell carcinomas of the lung (Theocharis et al., 2002). Exposing cancer cells to PPARγ ligands produces physiological effects that may be exploited for treatment purposes. In culture, synthetic PPARγ ligands have been shown to inhibit growth of several tumor cell lines (Brockman et al., 1998, Elstner et al., 1998, Mueller et al., 1998). A number of studies have determined that PPARγ ligands induce cellular differentiation and/or apoptosis in breast, colon, and lung cancer cells (Chang and Szabo, 2002, Elstner et al., 1998, Mueller et al., 1998, Sarraf et al., 1998). The combination of receptor overexpression in tumors and known physiological effects of its ligands on cancer cells makes PPARγ a viable target of future chemotherapeutic agents.
The ability of individual ligands to selectively mediate the activity of a nuclear receptor dependent on the tissue type examined has been used to develop compounds that act as selective estrogen-receptor modulators (SERMs). Tamoxifen, which was originally described as an estrogen-receptor antagonist, has been found to act as an agonist in several different tissue types (Fisher et al., 1998, Jordan and Morrow, 1999, Levenson and Jordan, 1999). It has been proposed that individual ligands may be able to act as selective PPARγ modulators (SPARMs) in a manner similar to the way other compounds function as SERMs (Sporn et al., 2001). We previously demonstrated that individual fatty acids can selectively activate a PPRE-reporter assay in estrogen-dependent breast cancer (MCF-7) cells (Thoennes et al., 2000). Specifically, omega-3 fatty acids inhibited transactivation of PPARγ to levels below control while omega-6, monounsaturated and saturated fatty acids stimulated the activity of the PPRE reporter. These data demonstrated that individual compounds can selectively activate PPARγ within the context of a single breast cancer cell line. However, compounds have yet to be identified that act as PPARγ agonists in one tissue while functioning as antagonists of the receptor in other tissues.
In the studies presented here, we sought to determine if distinct ligands could selectively activate PPARγ across different cell lines of mammary, colon, and lung origin. To this end we have utilized a PPRE-reporter construct transfected into the cells prior to ligand treatment. Data from these experiments demonstrated that selective activation of PPARγ occurs in multiple ways. Distinct ligands selectively activate PPARγ dependent on the tissue type from which the cell line was derived. SPARM activity was also observed between different cell lines of the same tissue origin and individual ligands selectively activated the PPRE reporter within single cell lines. These data indicate that it may be possible to design PPARγ ligands that can be used to selectively mediate receptor activity and thus customize treatment regiments against specific cancers.
Section snippets
Reagents
All PPARγ ligands were purchased from Cayman Chemical Company (Ann Arbor, MI). Ciglitazone (Cig) and GW9662 (GW) were solubilized in ethanol purchased from Aaper Alcohol and Chemical Company (Shelbyville, KY). Rosiglitazone (Ros) was dissolved in dimethyl sulfoxide (DMSO) and PGJ2 was solubilized in methyl acetate purchased from Sigma (St. Louis, MO).
Cells and cell culture
Ten individual cell lines were used in these experiments. Four mammary cell lines including normal mammary epithelial (HMEC) and three breast
Effect of PPARγ ligands on reporter activation in breast cancer cells
Following transfection with a PPRE reporter plasmid, HMEC, T47-D, MDA-MB-231, and MCF-7 cells were treated with either vehicle control or PPARγ ligands for 18 h. For the four cell lines, differences in ligand activity were observed. In the HMEC, Ros and PGJ2 both significantly increased reporter activity over control (Fig. 1A). Interestingly, GW, a known antagonist of PPARγ, also significantly stimulated reporter activity. GW treatment did not change reporter activity compared to control in any
Discussion
Data from the present study demonstrate that individual PPARγ ligands have the ability to selectively activate a PPRE reporter in cancers of the breast, colon, and lung. Differences in PPRE reporter activation were observed between cells derived from different tissue types as well as between cell lines of the same cancer type. Also, within a single cell line, individual ligands selectively induced PPRE reporter activity. Expression of PPARγ and RXRα mRNA were measured in all cell lines in the
Acknowledgments
This work was supported by grants DAMD17-97-1-7248 from the US Department of Defense, R01-CA 95609-1, 5-K12-DA-14040-02 and NCRR-P20-RR15592 from the NIH to MWK, and W81XWH-04-1-0532 from the Department of Defense to CDA. We would like to thank Dr. Charlotte Kaetzel and Dr. David Kaetzel for providing cell lines used in these studies. We also thank Dr. Scott Diamond for his insight into the preparation of this manuscript and Dr. Arnold Stromberg for statistical support.
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