Research Article A Role for Glial Cell–Derived Neurotrophic Factor–Induced Expression by Inflammatory Cytokines and RET/GFRA1 Receptor Up-regulation in Breast Cancer Selma Esseghir,1 S. Katrina Todd,1 Toby Hunt,2 Richard Poulsom,2 Ivan Plaza-Menacho,1 Jorge S. Reis-Filho,1 and Clare M. Isacke1 1Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research and 2In Situ Hybridisation Service and Histopathology Unit, Cancer Research UK-London Research Institute, London, United Kingdom Abstract system homeostasis and the inflammatory response (3, 4) and in By screening a tissue microarray of invasive breast tumors, we tumor progression (5). The neurotrophic factor glial cell–derived have shown that the receptor tyrosine kinase RET (REar- neurotrophic factor (GDNF) was first identified for its trophic ranged during Transfection) and its coreceptor GFRA1(GDNF activity on midbrain dopaminergic neurons; however, GDNF has receptor family A-1) are overexpressed in a subset of estrogen subsequently been shown to have broader effects in regulating receptor–positive tumors. Germ line–activating oncogenic growth, survival, and migration of neurons in the brain, spinal cord, mutations in RET allow this receptor to signal independently and periphery as well as having an essential role in the growth and A branching of the ureteric buds of the developing kidney (6, 7). Mice of GFR 1and its ligand glial cell–derived neurotrophic factor Gdnf (GDNF) to promote a spectrum of endocrine neoplasias. with a homozygous deletion of die shortly after birth due to However, it is not known whether tumor progression can also severe defects in renal differentiation and the absence of an enteric nervous system (8, 9). GDNF exerts its effect on target cells by be driven by receptor overexpression and whether expression binding to a glycosyl phosphatidylinositol–linked GDNF family of GDNF, as has been suggested for other neurotrophic factors, a a is regulated in response to the inflammatory microenviron- receptor- (GFR ), which, in turn, recruits the receptor tyrosine ment surrounding many epithelial cancers. Here, we show kinase RET (REarranged during Transfection) to form a multi- that GDNF stimulation of RET+/GFRA1+ MCF7 breast cancer subunit signaling complex. Formation of this complex results in cells in vitro enhanced cell proliferation and survival, and RET autophosphorylation and a cascade of intracellular signaling (7, 9, 10). Of the four GFRa receptors, GDNF preferentially binds promoted cell scattering. Moreover, in tumor xenografts, a GDNF expression was found to be up-regulated on the GFR 1 and it is notable that mice harboring a homozygous deletion in either Ret or Gfra1 have a phenotype similar to that infiltrating endogenous fibroblasts and to a lesser extent by GdnfÀ/À the tumor cells themselves. Finally, the inflammatory cyto- seen in mice and die shortly after birth due to kidney kines tumor necrosis factor-A and interleukin-1B, which are defects and an absence of enteric innervation (8, 9), thus providing involved in tumor promotion and development, were found to strong evidence for the requirement of all three components for act synergistically to up-regulate GDNF expression in both effective downstream signaling. In a screen setup specifically to identify mRNA transcripts of cell fibroblasts and tumor cells. These data indicate that GDNF can act as an important component of the inflammatory surface and secreted proteins that are differentially expressed in invasive breast tumor cells compared with normal breast tissue, we response in breast cancers and that its effects are mediated by GFRA1 both paracrine and autocrine stimulation of tumor cells via identified transcripts as being overexpressed in invasive signaling through the RET and GFRA1receptors. [Cancer Res breast carcinomas (11). As GDNF has been shown to stimulate the 2007;67(24):11732–41] migration and activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways of a RET+/GFRa1+ pancreatic carcinoma cell line (12), this led us to Introduction hypothesize that the neurotrophic factor GDNF may play a role in There is now a substantial body of evidence for a causative link promoting breast cancer growth by signaling via the RET and between cancer promotion and the presence of a chronic GFRa1 complex expressed on tumor cells. Here, we have tested this inflammatory response in the surrounding microenvironment hypothesis and provided evidence to support a role for inflamma- and for the benefit of treating patients with premalignant disease tory cytokines in regulating GDNF expression and for GDNF with inflammatory inhibitors (1, 2). To date, the majority of studies signaling in promoting tumor cell growth, survival, and scattering. have focused on the role of inflammatory chemokines and cytokines in these events. However, it is clear that neurotrophic factors, originally characterized for their role in the development Materials and Methods and maintenance of the nervous system, also play a role in immune Studies using human breast cancer samples and normal breast tissue collected from reduction mammoplasties were approved by the Research Ethics Committee of the Royal Marsden NHS Trust. All animal procedures Note: Supplementary data for this article are available at Cancer Research Online were done in accordance with United Kingdom Home Office legislation. (http://cancerres.aacrjournals.org/). Antibodies, reagents, and cells. Antibodies were as follows: anti-RET Requests for reprints: Clare M. Isacke, Breakthrough Breast Cancer Research polyclonal antibody (C-19), anti-GDNF polyclonal antibody (D-20-G), anti- Centre, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, United GFRa1 (H-70), and anti–phosphoTyr1062-RET were obtained from Santa Kingdom. Phone: 44-20-7153-5510; Fax: 44-20-7153-5340; E-mail: [email protected]. I2007 American Association for Cancer Research. Cruz Biotechnology, Inc. FITC-conjugated anti-bromodeoxyuridine doi:10.1158/0008-5472.CAN-07-2343 (BrdUrd) antibody (BD Biosciences); anti-vinculin monoclonal antibody Cancer Res 2007; 67: (24). December 15, 2007 11732 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Up-regulation of GDNF, GFRa1, and RET in Breast Cancer (V9131) and FITC-conjugated anti–a-smooth muscle actin (aSMA; Sigma- microplate reader (Perkin-Elmer). For each treatment, cells were plated in Aldrich); extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2, quadruplicate, and cell survival at days 3 and 8 is shown relative to the day 1 AKT, and phospho-AKT (Cell Signalling Technology, New England Biolabs); values for that treatment (set at 100%). No decrease in cell survival was Alexa Fluor–conjugated antibodies and Alexa 555–conjugated phalloidin observed between day 0 and day 1 (not shown). Data from independent (Molecular Probes, Invitrogen); and horseradish peroxidase (HRP)–conjugated experiments are shown as mean percentage of live cells F SE. P values antibodies (Jackson Immunoresearch). Transforming growth factor-h1 between GDNF-treated and untreated samples were obtained using a two- (TGF-h1), recombinant mouse interleukin-1h (IL-1h), recombinant human tailed unpaired t test with 95% confidence interval. IL-1h, recombinant mouse tumor necrosis factor-a (TNF-a), recombinant Real-time quantitative PCR. NIH-3T3 cells were cultured overnight, human TNF-a, and recombinant human GDNF were purchased from washed, and incubated for a further 48 h in serum-free DMEM. Cells were R&D Systems Europe Ltd.; BrdUrd was from Sigma-Aldrich. MCF7 cells then stimulated in serum-free DMEM with 5 ng/mL mouse IL-1h and/or were cultured in DMEM plus 10% fetal bovine serum (FBS, Invitrogen), 10 ng/mL mouse TNF-a for 24 h before being lysed in TRIzol (Invitrogen), whereas NIH-3T3 cells were cultured in DMEM plus 10% donor calf serum and RNA was extracted using chloroform-phase separation. MCF7 cells (DCS, Invitrogen). Western blot analysis was performed as previously were treated similarly except that they were stimulated for 48 h with human described (13). IL-1h and human TNF-a. Full details of the quantitative PCR (qPCR) Staining tumor sections. Representative 7-Am cryosections were cut analysis are provided in Supplementary Methods. from fresh-frozen human breast cancer material and breast reduction Tumor xenografts. Sustained release estradiol pellets (0.36 mg, 90 days; mammoplasties and stored at À80jC. For immunohistochemical analysis, Innovative Research of America) were implanted s.c. on the back of the neck sections were thawed, fixed in ice-cold methanol for 5 min, incubated for of 6-week-old athymic female mice (Ncr-nude). Five days later, 3 Â 104 MCF7 1 h at room temperature with anti-GFRa1 antibody (0.4 Ag/mL) or over- cells mixed with Matrigel in a ratio of 1:1 were injected into the mammary night at 4jC with anti-GDNF antibody (10 Ag/mL), followed by Alexa 488 fat pad. Tumors were removed after 6 weeks and snap frozen. Seven- anti-rabbit immunoglobulin or Alexa 555 anti-goat immunoglobulin, micrometer cryosections were stained as described for human tumors. respectively. Nuclei were counterstained with TO-PRO-3 (Molecular Probes) and sections were mounted in Vectashield H-1000 (Vector Laboratories, Inc.). Images were collected sequentially in three channels on a Leica TCS Results SP2 confocal microscope. Expression of GFRA1and RET is up-regulated in human in situ Tissue