1 Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas-Universidad Autónoma, 28029-Madrid
2 Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037
3 Departamento de Bioquímica y Biología Molecular, Facultad de.Medicina, Universidad Complutense, Madrid
*Authors for correspondence (aperez{at}iib.uam.es; piedras3{at}eucmax.sim.ucm.es)
Accepted July 31, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Breast cancer, ErbBs, Phosphorylation, PPAR, Transformation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although PPAR 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 PPAR
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 PPAR
and that growth of the cell lines is inhibited by diverse PPAR
agonists, whereas an inactive metabolite of troglitazone and a selective PPAR
agonist have no effect (Sarraf et al., 1998). In addition, Mueller et al. have shown that PPAR
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 c-erbB2 (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 oncogene-encoded 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 (TGF
), heparin-binding EGF (HB-EGF), amphiregulin, betacellulin, epiregulin and cripto (Riese and Stern, 1998; Alroy and Yarden, 1997). Four distinct neuregulin genes (NRG1, NRG2, NRG3 and NRG4) have been described (Carraway et al., 1997; Zhang et al., 1997; Harari et al., 1999). NRG1 was first cloned as neu differentiation factor (NDF) and heregulin (Burden and Yarden, 1997; Lemke, 1996), but is best known for its roles as the acetylcholine receptor inducing activity (ARIA), and as the potent Schwann cell mitogen, glial growth factor (GGF). Mice that lack NRG1 die at E10.5 from a heart defect and have virtually no Schwann cell precursors. NRG1 has been implicated in multiple cellular processes, including proliferation, differentiation, survival and migration (Burden and Yarden, 1997; Lemke, 1996). The neuregulin 1 gene encodes multiple isoforms that each contain an EGF-like domain. Splicing appears to give rise to alternative extracellular regions including Ig-domain-containing forms, which are believed to serve in the function described as ARIA, and a cysteine-rich domain (CRD)-containing form, which is thought to serve as GGF. Recombinant neuregulin protein forms that contain the EGF-like domain have been shown to induce receptor activation in vitro (Olayioye et al., 2000).
We have investigated the ability of PG-J2, a natural specific ligand of PPAR, to affect the induction by NRG1 and NRG2 of tyrosine phosphorylation/activation of ErbB2 and ErbB3 receptors expressed in MCF-7 cells, as well as its capacity to overcome the cellular responses elicited by the activation of these receptors. Our results provide evidence that PPAR
effectively blocks ErbB phosphorylation and interferes with ErbB signaling pathways. It therefore appears to play a suppressive regulatory role in the tumor growth of human breast carcinoma cells that express c-erbB2/neu and ErbB3 protein tyrosine kinases.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunoprecipitation and immunoblotting
For immunoprecipitation assays, cells were seeded at a density of 20,000/cm2 and grown for 24 hours in complete medium, switched to the serum-treated medium and exposed for 10 hours to 10 µM PG-J2 (Calbiochem-Novabiochem Corp.). Cells were then stimulated with the growth factors NRG-1, NRG-2, EGF (30 nM) or IGF-I (100 nM) for 5 minutes. Cells were washed twice with ice-cold TD buffer (20 mM Tris-HCL pH 7.4, 130 mM NaCl, 5 mM KCl, 1 mM Na2HPO4), lysed in 500 µl of PBS containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS and protease and phosphatase inhibitors, and spun at 12,000 g for 20 minutes. Supernatants were precleared with pansorbin (Calbiochem-Novabiochem Corp.) and incubated for 12 hours at 4°C with the appropriate antibodies. Protein-A-Sepharose was added to each sample and additionally incubated for 5 hours at 4°C. Beads were collected by microcentrifugation and washed five times with lysis buffer.
The immunocomplexes were eluted by boiling for 3 minutes in SDS sample buffer (100 mM Tris, pH 6.8, 36% glycerol, 4% SDS, 0.01% bromophenol blue and 200 mM DTT) and subjected to SDS-PAGE. Proteins were then transferred to nitrocellulose membranes. The blots were blocked with 3% BSA in TBST buffer (20 mM Tris-HCL pH 7.6, 130 mM NaCl, 0.1% Tween-20) for 1 hour at room temperature and incubated with the horseradish peroxidase-coupled antiphosphotyrosine antibody RC-20 (Transduction Laboratories, Lexington, KY) used at 1:2500 dilution in TBST with 3% BSA for 2 hours. After several washes, immunoreactive bands were visualized using Amershams ECL detection kit according to the manufacturers instructions. Membranes were stripped and incubated with the corresponding antibodies for loading control.
MAPK activation was determined with an antiphospho-MAPK (p42 and p44)-specific mouse monoclonal antibody (New England Biolabs, Beverly, MA), and activated Akt was measured using a polyclonal antibody specific to phosphoserine 473 (New England Biolabs). After stripping, the membranes were incubated with anti-MAPK and anti-Akt polyclonal antibodies (Santa Cruz Biotechnology) for loading control.
Proliferation assay and cell cycle studies
To monitor proliferation, cells were seeded in triplicate onto 96-well plates at a density of 7000 cells/well. After 24 hours of growth in normal medium, the cells were switched to serum-free medium and stimulated with NRG1 or NRG2 for 24 hours in the presence or absence of PG-J2. Radiolabeled [3H]thymidine (0.5 µCi) was then added and the cells were grown for an additional 24 hours. Cells were harvested and [3H] radioactivity was measured in a solid scintillation counter. For cell cycle analysis, cells were treated with the NRGs for 24 hours and some of the cultures were preincubated for 10 hours with PG-J2. Cells were then fixed in 70% ethanol/PBS, pelleted and resuspended in buffer containing 100 µg/ml RNAse A and 0.01 mg/ml propidium iodide. Cell cycle distribution was determined by cytofluorometry using the ModFit LT program.
Soft agar colony assays
Anchorage-independent growth was determined by first suspending 10,000 cells in 0.3% agar in tissue culture medium containing 5% stripped serum in 60 mm plates over a bottom layer of 0.5% agar in medium. The cells were allowed to grow for 20 days with weekly refeeding. NRGs and/or PG-J2 were added every 3-4 days. Colonies were stained with p-iodotetrazolium violet. Experiments were carried out three times in duplicate.
Nile red staining
Nile red staining was performed essentially as described (Greenspan et al., 1985). Briefly, cells were seeded at in a 6-well plate and, after a 48 hour incubation with PG-J2 and/or NRGs, directly stained with 1 ml of 0.1 µg/ml final concentration of the fluoresccent stain Nile red (Sigma Chemical Co.) in TD (prepared by dilution of a stock solution which was 0.1 mg/ml in acetone) for 5 minutes. Sample observation was carried out immediately after its preparation. Nile red-stained cells were then examined with a Zeiss Axiophot microscope.
Apoptosis assay
To calculate the extent of cell death, 0.5x106 cells were cultured in 10-cm diameter tissue culture plates and grown in RPMI for 24 hours before switching to serum-free medium containing the factors indicated in the figure legends for an additional 72 hours. For analyses, both the floating cells in the supernatant and in the PBS wash were collected from each plate. Apoptotic cells were assayed by analyzing annexin V conjugated to fluoresceinisothiocyanate (annexin-V-FITC) (Bender MedSystems, Vienna, Austria) to determine the translocation of phosphatidylserine from the inside to the outside of the plasma membrane. Cell staining was performed according to the manufacturers instructions.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To ascertain the possible involvement of PTPases in this reduction of receptor phosphorylation by PG-J2, MCF-7 cells were treated with 1 mM pervanadate prior to PG-J2 stimulation. We determined that the dephosphorylation of the ErbB2 and ErbB3 receptors was almost completely blocked by the addition of pervanadate to the culture medium (Fig. 1B). These data suggest that the reduction in phosphotyrosine content of the ErbB receptors induced by PG-J2 is mediated by PTPases.
To determine whether PG-J2 effects were restricted to ErbB2 and ErbB3 phosphorylation, the effect of this prostaglandin on phosphorylation of two other receptor tyrosine kinases: epidermal growth factor receptor (EGF-R) and insulin-like growth factor I receptor (IGF-IR) was examined. As shown in Fig. 2, the level of tyrosine phosphorylation of EGF-R and IGF-IR in MCF-7 cells was significantly increased by treatment with EGF and IGF-I, respectively. The addition of PG-J2 markedly decreased the observed phosphorylation of IGF-IR without affecting the phosphotyrosine content of EGF-R.
|
|
To identify intracellular signal transduction pathways linked to NRG1 and NRG2-induced cell cycle progression, we treated MCF-7 cells with specific kinase inhibitors. Treatment of MCF-7 cells with LY294002, an inhibitor of PI 3-K, prevented NRG1 and NRG2-induced G1 progression (Fig. 3B). However, PD98509, an inhibitor of the mitogen-activated protein kinase pathway that selectively inhibits the MAPK activating enzyme, MAP kinase kinase (MEK), did not prevent NRG1 and NRG2-induced cell cycle progression. These data suggest that it is the PI 3-K pathway and not the MAPK pathway that is required for neuregulin-induced DNA synthesis.
To better understand the regulation of the PI 3-K signaling pathway by neuregulins, and the possible involvement of PG-J2, MCF-7 cells were preincubated with PG-J2 and then treated with either NRG1 or NRG2. Phosphorylation of Akt was analyzed by using a phospho-specific antibody (which recognizes Akt only when phosphorylated at the Ser-473 residue). As shown in Fig. 4A, Akt phosphorylation was not present in unstimulated serum-starved MCF-7 cells. Stimulation with both NRG1 and NRG2 dramatically increased phosphorylation of Akt within 10 minutes of treatment. When cells were preincubated with prostaglandin, a significant suppression of Akt phosphorylation was detected.
|
|
|
|
Finally, to address the question of whether the dephosphorylation/deactivation effect of PG-J2 was restricted to MCF-7 cells, we next examined the effects of PG-J2 in two other human mammary epithelial cell lines: T47D and SKBR3, the latter having a high basal ErbB2 phosphotyrosine content. We first analyzed the phosphorylation state of ErbB2 and ErbB3 in cells stimulated with NRG1 or NRG2 and preincubated or not with PG-J2. As shown in Fig. 8A, NRG1-induced phosphorylation of ErbB2 and ErbB3 in T47D was completely abolished by PG-J2 preincubation. The same results were obtained with NRG2 (data not shown). In SKBR3 cells, as expected, ErbB2 was already highly phosphorylated in basal conditions and no further effect of the NRGs was observed. By contrast, ErbB3 phosphorylation was clearly induced by NRGs (only NRG1 data are shown). Preincubation with PG-J2 dramatically blocked the basal phosphorylation levels of ErbB2. NRG1-induced phosphorylation of ErbB3 was also significantly inhibited by this prostaglandin (Fig. 8A). Next, we examined the effect of ligand activation of PPAR on cell growth of T47D and SKBR3 by analyzing thymidine incorporation (Fig. 8B). Consistent with its effects on ErbB2 and ErbB3 phosphorylation, PG-J2 also caused a dramatic decrease in basal and neuregulin-induced T47D and SKBR3 cell proliferation.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although PPAR was initially described in adipocytes and hepatocytes, recently it was shown that this receptor is expressed in macrophages and in human mammary epithelial cell lines. Elstner et al. have recently found that breast cancer cells express high levels of this receptor, whereas normal human breast epithelial cells lining the mammary ducts express only a low level of PPAR
protein (Elstner et al., 1998). These authors have also shown that some ligands of PPAR
, such as troglitazone (TGZ), inhibited the clonal growth of several breast cancer cell lines and slightly increased the levels of the apoptotic population. Also, an anti-tumor effect of PPAR
ligands was observed in mice injected with prostate tumor cells and it has been shown that troglitazone promotes terminal differentiation of human liposarcoma cells in vitro and in patients suffering from advanced liposarcoma (Demetri, 1999). These data, along with the findings of others (Kilgore et al., 1997; Gimble, 1998; Mueller et al., 1998) suggest a possible role for ligand-activated PPAR
as an anti-tumor agent in breast cancer. The evidence presented here not only supports this hypothesis but also indicates a possible mechanism by which prostaglandins could affect the proliferation, differentiation and death of breast cancer cells.
Our studies provide a link between PPAR and ErbB signaling, with PPAR
being able to suppress ErbB activation dramatically. The negative effects of PPAR
on ErbB signaling appear to block the cells ability to mount proliferative and anti-apoptotic responses, thereby ensuring a non-proliferative outcome regardless of the presence of activating ErbB ligands. Based on the relatively long period of time (10 hours) required to achieve its effect, and its sensitivity to actinomycin D, it appears that the effects of PG-J2 are directly mediated through PPAR
activation. The NRGs have been implicated in a wide variety of physiological and developmental processes including cardiac development, the proliferation and differentiation of oligodendroglial and Schwann cell precursors, the formation of the neuromuscular synapse, epithelial morphogenesis, as well as in pathological states (Burden and Yarden, 1997). The discovery of additional NRG genes has increased the potential signaling complexity of the NRG/ErbB network. NRGs have been shown to be potent mitogens for Schwann cells and astrocytes in cell culture as well as inhibitors of apoptosis in cardiac myocytes (Lemke, 1996; Zhao et al., 1998). However, several reports show that, in addition to promoting proliferation, NRGs can induce apoptosis (Daly et al., 1997; Kirchhoff and Hauser, 1999; Daly et al., 1999). Our results further support a role for the NRGs in promoting the proliferation of breast cancer cells, but differ from the pro-apoptotic effects reported by other authors, as we have observed that NRG1 and NRG2 have a significant anti-apoptotic effect in MCF-7 cells.
In this work we have also examined the signaling pathways involved in neuregulin-induced cell proliferation and survival. Our results suggest that, in MCF-7 cells, G1 progression is associated with the phosphatidylinositol 3-kinase pathway, whereas the anti-apoptotic effects were dependent on ERK1/2 activation. The inhibition of ERK1 and ERK2 by the MEK inhibitor PD98059 largely blocks the survival effects of NRG1 and NRG2 but has little effect on cell cycle progression. These results are noteworthy as, in most systems, activation of the MAPK signaling pathway has been associated with cell proliferation (Pages et al., 1993; Dhnasekaran and Reddy, 1998). The PI 3-K inhibitor LY294002 blocks Akt phosphorylation in MCF-7 cells, a finding that correlates with the abolishment of NRG1 and NRG-2-induced proliferation. This inhibitor has little effect on the reduction of apoptosis. These results are in agreement with previous data showing an induction of G1 progression in the breast cancer cell line SKBR3 through the PI 3-K pathway (Daly et al., 1999). However, studies with cells of the oligodendrocyte lineage show that activation of Akt by heregulin plays a major role in cell survival but has no effect upon proliferation (Flores et al., 2000). The role of Akt in the regulation of the cell cycle has received significantly less attention than its role in the regulation of apoptosis. Cell cycle regulation by Akt was first observed by Ahmed et al. (Ahmed et al., 1997) and was later confirmed by Brennan et al. who showed that Akt transduces PI-3K-dependent IL-2 signals leading to the phosphorylation of Rb and promoting the activation of E2F (Brennan et al., 1997).
The present data demonstrate that PG-J2, a specific ligand of PPAR, is a robust inhibitor of ErbB signaling pathways in the MCF-7 breast cancer cell line and is capable of completely blocking the transforming capacity of ErbB2 and ErbB3. These findings indicate that prostaglandins inhibit mammary epithelial proliferation and induce apoptosis, at least in part by antagonizing the actions of the neuregulins via receptor dephosphorylation. The strong blocking effect of PG-J2 on cell cycle progression induced by serum (Fig. 3B) suggests that other growth factor signaling pathways may be the target of the action of this prostaglandin. In fact, we have also demonstrated that, in addition to blocking the ErbB signaling pathway, PG-J2 caused a significant decrease in the phosphotyrosine content of IGF-IR, without modifying the phosphorylation levels of EGF-R. These results suggest that, although not all signaling pathways are affected, PG-J2 blocks other tyrosine kinase systems in breast cancer cells. Interestingly, the effects of PG-J2 seem to be quite general, since the ErbB tyrosine phosphorylation is also completely abolished in other breast cancer cell lines, such as T47D and SKBR3.
In conclusion, these observations suggest that PPAR and locally produced prostaglandin D2 metabolites may be involved in the regulation of cancer cell growth and development. These findings raise the possibility that synthetic PPAR
ligands may be of therapeutic value in human diseases, such as breast cancer, in which activated ErbB receptors play prominent pathogenic roles.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahmed, N. N., Grimes, H. L., Bellacosa, A., Chan, T. O. and Tsichlis, P. N. (1997). Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase. Proc. Natl. Acad. Sci. 94, 3627-3632.
Alroy, I. and Yarden, Y. (1997). The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand-receptor interactions. FEBS Lett. 410, 83-86.[Medline]
Andrulis, I. L. (1998). Neu/erbB2 amplification identifies a poor prognosis group of women with node-negative breast cancer. J. Clin. Oncol. 16, 1340-1349.[Abstract]
Brennan, P., Babbage, J. W., Burgering, B. M., Groner, B., Reif, K. and Cantrell, D. A. (1997). Phosphatidylinositol 3-kinase couples interleukin-2 receptor to the cell cycle regulator E2F. Immunity 7, 679-689.[Medline]
Burden, S. and Yarden, Y. (1997). Neuregulins and their receptors: a versatile signaling module in organogenesis and oncogenesis. Neuron 18, 847-855.[Medline]
Carraway, K. L., III, Weber, J. L., Unger, M. J., Ledesma, J., Yu, N., Gassmann, M. and Lai, C. (1997). Neuregulin-2, a new ligand for ErbB3/ErbB4-receptor tyrosine kinases. Nature 387, 512-516.[Medline]
Daly, J. M., Jannot, C. B., Beerli, R. R., Graus-Porta, D., Maurer, F. G. and Hynes, N. E. (1997). Neu differentiation factor induces ErbB2 down-regulation and apoptosis of ErbB2-overexpressing breast tumor cells. Cancer Res. 57, 3804-3811.[Abstract]
Daly, J. M., Olayioye, M. A., Wong, A. M., Neve, R., Lane, H. A., Maurer, F. G. and Hynes, N. E. (1999). NDF/heregulin-induced cell cycle changes and apoptosis in breast tumor cells: role of PI3 kinase and p38 MAP kinase pathways. Oncogene 18, 3440-3451.[Medline]
Demetri, G. D. (1999). Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor-gamma ligand troglitazone in patients with liposarcoma. Proc. Natl. Acad. Sci. USA 96, 3951-3956.
Dhnasekaran, N. and Reddy, E. P. (1998). Signaling by dual specificity kinases. Oncogene 17, 1447-1455.[Medline]
Drebin, J. A., Link, V. C., Weinberg, R. A. and Greene, M. I. (1986). Inhibition of tumor growth by a monoclonal antibody reactive with an oncogen-encoded tumor antigen. Proc. Natl. Acad. Sci. USA 83, 9129-9133.[Abstract]
Dreyer, C., Krey, G., Keller, H., Givel, J., Helftenbein, G. and Wahli, W. (1992). Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear receptors. Cell 68, 879-887.[Medline]
Elstner, E., Muller, C., Koshizuka, K., Williamson, E. A., Park, D., Asou, H., Shintaku, P., Said, J. W., Heber, D. and Koeffler, H. P. (1998). Ligands for peroxisome proliferator-activated receptorg and retinoic acid inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice. Proc. Natl. Acad. Sci. USA 95, 8806-8811.
Flores, A. I., Mallon, B. S., Matsui, T., Agawa, W., Rosenzweig, A., Okamoto, T. and Macklin, W. B. (2000). Akt-mediated survival of oligodendrocytes induced by neuregulins. J. Neurosci. 20, 7622-7630.
Gimble, J. M. (1998). Expression of peroxisome proliferator activated receptor mRNA in normal and tumorigenic rodent mammary glands. Biochem. Biophys. Res. Comm. 253, 813-817.[Medline]
Greenspan, P., Mayer, E. P. and Fowler, S. D. (1985). Nile red: a selective fluorescent stain for intracellular lipoid droplets. J. Cell. Biol. 100, 965-973.[Abstract]
Harari, D., Tzahar, E., Romano, J., Shelly, M., Pierce, J. H., Andrews, G. C. and Yarden Y. (1999). Neuregulin-4: a novel growth factor that acts through the ErbB-4 receptor tyrosine kinase. Oncogene 18, 2681-2689.[Medline]
Johnson, M. D., Campbell, L. K. and Campbell, R. K. (1998). Troglitazone: review and assessment of its role in the treatment of patients with impaired glucose tolerance and diabetes mellitus. Ann. Pharmacother. 32, 337-348.
Kilgore, M. W., Tate, P. L., Rai, S., Sengoku, E. and Price, T. M. (1997). MCF-7 and T47D human breast cancer cells contain a functional peroxisomal response. Mol. Cell. Endocrinol. 129, 229-235.[Medline]
Kirchhoff, S. and Hauser, H. (1999). Cooperative activity between HER oncogenes and the tumor suppressor IRF-1 results in apoptosis. Oncogene 18, 3725-3736.[Medline]
Kliewer, S. A., Forman, B. M., Blumberg, B., Ong, E. S., Borgmeyer, U., Mangelsdorf, D. J., Umesono, K. and Evans, R. M. (1994). Differential expression and activation of a family of murine peroxisome-proliferator-activated receptors. Proc. Natl. Acad. Sci. 91, 7355-7359.[Abstract]
Kraus, M. H., Popescu, N. C., Amsbaugh, S. C. and King, C. R. (1987). Overexpression of the EGF receptor-related protooncogene erbB-2 in human mammary tumor cell lines by different molecular mechanisms. EMBO J. 6, 605-610.[Abstract]
Krisst, D. A. and Yarden, Y. (1996). Differences between phosphotyroisne accumulation and Neu/ErbB2 receptor expression in astrocytic proliferative processes. Implications for glial oncogenesis. Cancer 78, 1272-1283.[Medline]
Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T. M. and Kliewer, S. A. (1995). An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J. Biol. Chem. 270, 12953-12956.
Lemke, G. (1996). Neuregulins in development. Mol. Cell. Neurosci. 7, 247-262.[Medline]
Mangelsdorf, D. and Evans, R. M. (1995). The RXR heterodimers and orphan receptors. Cell 83, 841-850.[Medline]
Michalik, L. and Wahli, W. (1999). Peroxisome proliferator-activated receptors: three isotypes for a multitude of functions. Curr. Opin. Biotechnol. 10, 564-570.[Medline]
Mueller, E., Sarraf, P., Tontonoz, P., Evans, R. M., Martin, K. J., Zhang, M., Fletcher, C., Singer, S. and Spiegelman, B. M. (1998). Terminal differentiation of human breast cancer through PPAR. Mol. Cell 1, 465-470.[Medline]
Nguyen, B., Keane, M. M. and Johnston, P. J. (1995). The biology of growth regulation in normal and malignant breast epithelium: from bench to clinic. Crit. Rev. Oncol. Hematol. 20, 223-236.[Medline]
Olayioye, M. A., Neve, R. M., Lane, H. A. and Hynes, N. E. (2000). The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J. 19, 3159-3167.
Pages, G., Lenormand, P., LAllemain, G., Chambard, J. C., Meloche, S. and Pouyssegur, J. (1993). Mitogen-activated protein kinases p42mapk and p44mapk are required for fibrobalst proliferation.Proc. Natl. Acad. Sci. USA 90, 8319-8323.
Paterson, M. C. (1991). Correlation between c-erbB2 amplification and risk of recurrent disease in node-negative breast cancer. Cancer Res. 51, 556-567.[Abstract]
Riese, D. J., II and Stern, D. F. (1998). Specificity within the EGF family/ErbB receptor family signaling network. BioEssays 20, 41-48.[Medline]
Sarraf, P., Mueller, E., Jones, D., King, F. J., DeAngelo, D. J., Partridge, J. B., Holden, S. A., Chen, L. B., Singer, S., Fletcher, C. and Spiegelman, B. M. (1998). Differentiation and reversal of malignant changes in colon cancer through PPAR. Nat. Med. 4, 1046-1052.[Medline]
Schoonjans, K., Martin, G., Staels, B. and Auwerx, J. (1997). Peroxisome proliferator-activated receptors, orphans with ligands and junctions. Curr. Opin. Lipidol. 8, 159-166.[Medline]
Siegel, P. M., Ryan, E. D., Cardiff, R. D. and Muller, W. J. (1999). Elevated expression of activated forms of Neu/erbB2 and erbB3 are involved in the induction of mammary tumors in transgenic mice: implications for human breast cancer. EMBO J. 18, 2149-2164.
Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J., Ullrich, A. and Press, M. F. (1989). Studies of the Her2/neu proto-oncogene in human breast and ovarian cancer. Science 244, 707-712.[Medline]
Spiegelman, B. M. (1998). PPARg in monocytes: less pain, any gain? Cell 93, 153-155.[Medline]
Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. and Spiegelman, B. M. (1994a). mPPAR: tissue-specific regulator of an adipocyte enhancer. Genes. Dev. 8, 1224-1234.[Abstract]
Tontonoz, P., Hu, E. and Spiegelman, B. M. (1994b). Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell 79, 1147-1156.[Medline]
Walker, R. A. (1998). The ErbB/HER type 1 tyrosine kinase receptor family. J. Pathol. 185, 234-235.[Medline]
Zhang, D., Sliwkowski, M. X., Mark, M., Frantz, G., Akita, R., Sun, Y., Hillan, K., Crowley, C., Brush, J. and Godowski, P. J. (1997). Neuregulin-3 (NRG3): a novel neural tissu-enriched protein that binds and activates ErbB4. Proc. Natl. Acad. Sci. USA 94, 9562-9567.
Zhao, Y. Y., Sawyer, D. R., Baliga, D. R., Opel, D. J., Han, X., Marchionni, M. and Kelly, R. A. (1998). Neuregulins promote survival and growth of cardiac myocytes. Persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular myocytes. J. Biol. Chem. 273, 10261-10269.