The peroxisome proliferator-activated receptor γ is an inhibitor of ErbBs activity in human breast cancer cells

Miguel Pignatelli 2 Marta Corts-Canteli 2 Cary Lai 1 Angel Santos 0 Ana Perez-Castillo 2 0 Departamento de Bioquimica y Biologia Molecular, Facultad de.Medicina, Universidad Complutense , Madrid 1 Department of Neuropharmacology, The Scripps Research Institute , La Jolla, California 92037 2 Instituto de Investigaciones Biomedicas, Consejo Superior de Investigaciones Cientificas-Universidad Autonoma , 28029-Madrid SUMMARY One of the most interesting recent developments in the nuclear receptor field has been the identification of natural and synthetic agonists of the peroxisome proliferatoractivated receptor (PPAR) family, coupled with a growing recognition that the g isoform (PPARg ) affects pathways important in a variety of human diseases. Here we show that the activation of PPARg through the 15-deoxy--12,14prostaglandin J2 (PG-J2) ligand causes a dramatic inhibition of ErbB-2 and ErbB-3 tyrosine phosphorylation caused by neuregulin 1 (NRG1) and neuregulin 2 (NRG2) in MCF-7 cells. This effect is accompanied by a very efficient blocking of ErbBs effects upon proliferation, differentiation and cell death in these cells. Preincubation of MCF-7 cells with PG-J2 before addition of NRG1 and NRG2 had a dramatic growth-suppressive effect accompanied by accumulation of cells in the G0/G1 Peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor gene superfamily (Mangelsdorf and Evans, 1995). The regulation of gene expression is exerted by the receptors upon heterodimerization with the 9-cis retinoic acid receptor and binding to specific response elements termed peroxisome proliferator-response elements (PPREs). Most PPREs identified to date reside in genes involved in lipid metabolism (Schoonjans et al., 1997). There are three members of the PPAR subfamily of nuclear receptors: a , d and g (Tontonoz et al., 1994a; Dreyer et al., 1992; Kliewer et al., 1994). PPARg is abundant in adipose tissue where it triggers adipocyte differentiation and lipid storage by regulating the expression of genes critical for adipogenesis (Tontonoz et al., 1994a; Tontonoz et al., 1994b). PPARs function as transcription factors regulating gene transcription in response to the binding of their ligands (Michalik and Wahli, 1999). There are several known ligands for PPARg , including the natural prostaglandin 15-deoxy--12,14-prostaglandin J2 (PG-J2, a prostaglandin D2 metabolite), the synthetic antidiabetic thiazolidinediones (Lehmann et al., 1995) and certain polyunsaturated fatty acids. One of the thiazolidenediones, troglitazone (TGZ), is currently used in some countries for the treatment of type II diabetes (Johnson et al., 1998). compartment of the cell cycle, and a marked increase in apoptosis. NRG1 and NRG2 induce G1 progression, which was associated with stimulation of the phosphatidylinositol3 kinase (PI 3-K) pathway, whereas survival was dependent on ERK1/ERK2 activation. Both pathways were inhibited by PG-J2. Furthermore, PG-J2 can abolish the NRG1 and NRG2-induced increase in anchorage-independent growth of these cells. PG-J2 also blocks phosphorylation of other receptor tyrosine kinases, such as IGF-IR, in MCF-7 cells, and suppress proliferation of other breast cancer cell lines. In summary, our data show a specific inhibitory action of PG-J2 on the activity of the ErbB receptors in breast cancer cells. Although PPARg is primarily expressed in adipose tissue, it is also expressed in many other tissues and cell types although its role is still poorly understood. Recent studies indicate that PPARg is expressed in cells of the monocyte/macrophage lineage and that ligand activation of this receptor powerfully regulates several aspects of monocyte biology such as the development of monocytes along the macrophage lineage, in particular in the conversion of monocytes to foam cells (cholesterol-engorged macrophages) (Spiegelman, 1998). Sarraf et al. have demonstrated that human colonic epithelium and colon cancer cell lines express PPARg and that growth of the cell lines is inhibited by diverse PPARg agonists, whereas an inactive metabolite of troglitazone and a selective PPARa agonist have no effect (Sarraf et al., 1998). In addition, Mueller et al. have shown that PPARg is expressed at significant levels in human primary and metastatic breast adenocarcinomas (Mueller et al., 1998). Ligand activation of this receptor in cultured breast cancer cells caused extensive lipid accumulation, changes in breast epithelial gene expression associated with a more differentiated, less malignant, state. Some effects upon breast cancer cells have also being observed by other groups (Elstner et al., 1998; Kilgore et al., 1997). Control of protein phosphorylation at tyrosine residues is a fundamental regulatory mechanism in signal transduction pathways involved in transformation and growth of breast cancer cells (Nguyen et al., 1995). Overexpression of type 1 receptor tyrosine kinases has been associated with several types of human cancers, including breast cancer and glioblastoma (Slamon et al., 1989; Kraus et al., 1987; Walker, 1998; Krisst and Yarden, 1996). This family of proteins consists of the epidermal growth factor receptor (EGFR/ErbB1), neu (ErbB2), ErbB3 and ErbB4 (Olayioye et al., 2000). Several studies have demonstrated that ErbB2 is amplified and overexpressed in 20-30% of primary breast cancers, a finding that correlates with poor patient prognosis (Paterson, 1991; Andrulis, 1998) and a more aggressive disease. An ErbB2-positive status may predict the likelihood of resistance to some conventional therapies. Furthermore, blockade and functional inhibition of c-erbB2 by monoclonal antibodies inhibits the growth of tumors that overexpress cerbB2 (Drebin et al., 1986). Breast tumor progression is also associated with elevated levels of ErbB3, and a survey of primary human breast tumors revealed frequent co-expression of both ErbB2 and ErbB3 transcripts (Siegel et al., 1999). The incidence of amplification of the neu and ErbB3 oncogeneencoded protein tyrosine kinases in human breast cancer strongly supports the concept that protein tyrosine phosphorylation and dephosphorylation are key regulatory mechanisms in the proliferation, differentiation and neoplastic transformation of breast epithelial cells. In view of all the evidence commented above, the ErbB2 and ErbB3 receptor proteins have become very important targets for novel and specific anticancer treatment. The neuregulins (NRGs) are a family of proteins that serve as ErbB ligands. They contain a region structurally related to EGF, the EGF-like domain, that can bind to and induce ErbB autophosphorylation. In addition to the NRGs and EGF, other molecules that contain an EGF-like domain and that can activate one or more ErbB receptors include transforming growth factor a (TGFa ), heparin-binding EGF (HB-EGF), amphiregulin, betacellulin, epiregulin and cripto (Riese and Stern, 1998; Alroy and Y (...truncated)