A Role for Glial Cell–Derived Neurotrophic Factor–Induced Expression by Inflammatory Cytokines and RET/GFRA1 Receptor Up-Regulation in Breast Cancer

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

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(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 microarray and hybridization. The tissue microarray breast cancers. Using a signal-trap screen to identify transcripts contained 0.6-mm cores of 245 invasive breast carcinomas. Full details of differentially expressed in primary invasive breast tumor cells the tissue microarray characterization and the cohort of patients are described elsewhere (14, 15) and in the Supplementary Methods. Tumors compared with normal breast tissue, we have previously reported GFRA1 were classified according to the criteria of Nielsen et al. (16) into HER2 that mRNA was overexpressed in tumor cells. This up- [HER2+, estrogen receptor (ER) any, Ck 5/6, or epidermal growth factor regulation was validated by both reverse Northern blot and À receptor (EGFR) any], luminal (HER2 ,ER+, Ck5/6 or epidermal growth Northern blot analyses (11). To confirm that the increased levels À À factor receptor any), or basal-like (HER2 ,ER , Ck 5/6, or EGFR+) groups. of GFRA1 mRNA corresponds to an increase in GFRa1 protein, In situ hybridization probes for RET and GFRA1 were generated by PCR normal and breast tumor cryosections were stained with a GFRa1 amplification and cloned into the pGEM3Z vector (Promega). Both GFRA1 antibody and examined by confocal microscopy. As illustrated in RET ACTB and probes will detect all known receptor splice variants. An Fig. 1A, GFRa1 is expressed at very low levels in normal human h ( -actin) probe was used as a positive control. Generation and labeling of breast. In contrast, GFRa1 expression was readily detectable in a riboprobes, hybridization to the tissue microarray, scoring of the tissue series of ER-positive (ER+) breast cancer examined with expression microarray, and the statistical analysis are described in Supplementary Methods. levels ranging from low (as exemplified by tumor 42T) to high (as Small interference RNA transfection. Two different small interference exemplified by tumor 67T). RNA (siRNA) oligonucleotide pairs directed against human RET and human To extend this analysis, riboprobes for GFRA1 and RET were GFRa1 and GFRa2 different nontargeting oligonucleotide pairs (C1 and C2) generated and used to screen a tissue microarray of 245 invasive were purchased from Dharmacon (Thermo Fisher Scientific). Cells were breast cancers. Figure 1B illustrates representative in situ hybrid- transfected with 40 nmol/L siRNA oligonucleotides with DharmaFECT 4 in ization results obtained. A summary of the results for the univariate Opti-MEM (Invitrogen) and incubated at 37jC for 4 h before washing and analysis for each probe is shown in Table 1. In keeping with the culturing for 48 or 72 h with DMEM plus 10% FBS. immunofluorescence analysis (Fig. 1A), GFRA1 mRNA expression Cell proliferation assays. MCF7 cells were seeded onto glass coverslips was detected in 126 of 212 (59.4%) of tumors and associated with in DMEM plus 10% FCS. Where indicated, cells had been pretreated with lymphovascular invasion (P = 0.0051), lymph node metastasis at siRNAs for 72 h. The following day, cells were washed and incubated in time of diagnosis (P = 0.0278), and ER and progesterone receptor serum-free DMEM for 24 h before incubation in DMEM plus 0.5% FCS with P or without 10 ng/mL GDNF for 28 h. BrdUrd (10 Amol/L) was added for (PR; both < 0.0001). There was an inverse correlation between GFRA1 P 1 h before cells were fixed in 4% paraformaldehyde for 30 min, treated with mRNA expression and the expression EGFR ( < 0.0001), 2 mol/L HCl in 0.5% Triton X-100 for 30 min, neutralized in PBS (pH 8.5), basal markers (Ck 5/6. Ck 14, Ck 17, and EGFR, P < 0.0001), p53 and incubated with FITC-conjugated anti-BrdUrd antibody for 1 h. Cells (P = 0.0129), and MIB1 labeling index (Ki67; P < 0.0001). Fur- were washed, nuclei were counterstained with TO-PRO-3, and coverslips thermore, there was a positive correlation with luminal-type tumors were mounted in Vectashield. Images were collected from multiple fields for and an inverse correlation with basal-like tumors as defined by each treatment. For each treatment, >200 cells were scored for BrdUrd Nielsen et al. (ref. 16; P < 0.0001). RET mRNA expression was F staining. Data are shown as mean percentage of BrdUrd-positive cells SE. detected in 63 of 212 (29.7%) of tumors and, like GFRA1, this cor- P values between GDNF-treated and untreated samples were obtained related with the expression of ER (P = 0.0031) and PR (P = 0.0034). using a two-tailed unpaired t test with 95% confidence interval. Importantly, because this expression analysis was performed by in Cell survival assays. MCF7 cells were transfected with siRNAs. After situ RET GFRA1 24 h, the medium was replaced with serum-free DMEM and cells were hybridization, we were able to confirm that and cultured for a further 48 h before being trypsinized and plated at 1 Â 104 per mRNA up-regulation resulted from expression by the tumor cells well in 96-well plates in serum-free DMEM with or without 10 ng/mL GDNF. rather than the stromal cells. GFRA1 and RET mRNAs were coex- Cell survival was assayed the following day (day 1) and at days 3 and 8 using pressed in 18.1% of the tumors, whereas 41.9% of tumors were À À Cell Titre Blue (Promega) and measuring fluorescence in Wallac Victor 2 V GFRA1+/RET and 0.9% of tumors were GFRA1 /RET+ (Table 1). www.aacrjournals.org 11733 Cancer Res 2007; 67: (24). December 15, 2007

Figure 1. Expression of GFRa1 and RET in human breast tumors. A, cryosections of one normal human breast (sample 34N) and two ER+ invasive ductal carcinomas (42T and 67T) were labeled with anti-GFRa1 antibody followed by Alexa 488 antirabbit immunoglobulin (green). Nuclei were counterstained with TO-PRO-3 (blue). Scale bar, 50 Am. B, riboprobes for RET, GFRA1, and ACTB (h-actin) were hybridized to sections of a human breast cancer tissue microarray. Representative images of one normal breast section (sample 228N) and four different tumors (samples 298T, 243T, 253T, and 287T) are shown with the score given to each sample indicated. The intensity of signals (white reflective silver grains) was scored from 0 to 4 for GFRA1 mRNA transcripts and from 0 to 3 for RET mRNA transcripts (see Materials and Methods for details of the scoring). For each probe, top panels show Giemsa counterstain; bottom panels show autoradiographic silver. Cores were 0.6 mm in diameter.

Coexpression of both receptors was directly correlated with the reverse transcription-PCR for RET and GFRA1 expression. Of the expression of the steroid hormone receptors (ER and PR) and eight cell lines tested, only MCF7 cells showed robust expression of inversely correlated with the expression of basal markers, including both GFRA1 and RET mRNA (data not shown). This is consistent EGFR, Ck 14, and Ck 17 (Table 1). The GFRA1+/RET+ phenotype with recent data showing up-regulation of RET expression in MCF7 À showed an inverse correlation with basal-like phenotype as defined cells compared with T47D cells and three ER cell lines (17) and in by Nielsen et al. (ref. 16; see Materials and Methods), being found in an expression profiling study of 51 breast cancer cells lines where it 28 of 137 (20%) luminal tumors, in 7 of 29 (24%) HER2 breast was shown that MCF7 cells showed the highest expression of RET cancers, and in none of 26 (0%) basal-like breast cancers. No and the second highest expression of GFRA1 mRNA (18). To verify correlation between coexpression of GFRA1 and RET and other protein expression, MCF7 cells were subjected to either flow clinicopathologic features or immunohistochemical markers was cytometry or immunoblotting (Fig. 2). By flow cytometry, GFRa1 found (Table 1). No correlations between GFRA1 and RET mRNA was readily detectable at the cell surface and this expression was expression or their coexpression and disease-free or overall survival unaffected by mock transfection of cells or transfection with a in the present cohort of patients were observed (data not shown). scrambled siRNA oligonucleotide. In contrast, transfection with GDNF signals via RET and GFRA1to promote cell either of two GFRa1 siRNAs resulted in a consistent reduction in proliferation and survival in breast cancer cells. To investigate GFRa1expression(Fig.2A). Immunoblotting revealed RET the role of RET and the coreceptor GFRa1inanin vitro model, ER+ expression in MCF7 cells as a characteristic protein doublet of À (MCF7, BT474, MDA-MB-361, T47D, and ZR75.1) and ER (BT20, 170 and 150 kDa (19). Again, expression was not affected by mock Cal51, MDA-MB-468) breast cancer cell lines were screened by transfection or transfection with scrambled siRNA. In contrast,

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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 treatment with either of two RET siRNA oligonucleotides resulted In neuronal systems, GDNF has been shown to promote both cell in loss of RET expression (Fig. 2B). These data show that MCF7 proliferation and cell survival (20–22). To monitor whether cells express both RET and GFRa1 protein and that their signaling through GFRa1 and RET promoted MCF7 cells prolifer- expression can be effectively down-regulated by siRNA treatment. ation, it was first shown that GDNF stimulation of MCF7 cells

Table 1. Correlation between expression and coexpression of GFRa1 and RET, clinicopathologic features, and immunohistochemical markers in a cohort of 245 invasive breast cancers

Variable n GFRa1+ PnRET+ PnGFRa1À/ GFRa1+/ GFRa1À/ GFRa1+/ P* (%) (%) RetÀ (%) RetÀ (%) Ret+ (%) Ret+ (%)

c c c Grade 208 0.0540 209 0.7335 198 0.2468 1 11 (57.9) 4 (22.2) 3 (1.5) 10 (5.1) 3 (1.5) 1 (0.5) 2 40 (72.7) 18 (30) 9 (4.5) 31 (15.7) 5 (2.5) 8 (4.0) 3 72 (53.7) 41 (31.3) 47 (23.7) 42 (21.2) 12 (6.1) 27 (13.6) b b b LVI 211 0.0051 211 0.9762 200 0.1796 + 92 (66.2) 41 (29.9) 32 (16) 61 (30.5) 9 (4.5) 27 (13.5) À 33 (45.8) 22 (29.7) 27 (13.5) 24 (12.0) 11 (5.5) 9 (4.5) b b b LN mets 207 0.0278 206 0.5965 196 0.5654 + 86 (64.7) 39 (29.3) 33 (16.8) 59 (30.1) 9 (4.6) 25 (12.8) À 36 (48.6) 24 (32.9) 26 (13.3) 23 (11.7) 10 (5.1) 11 (5.6) b b b ER 209 0.0001 208 0.0031 198 0.0005 + 118 (70.7) 57 (33.7) 30 (15.2) 80 (40.4) 15 (7.6) 35 (17.7) À 6 (14.3) 4 (10.3) 29 (14.6) 5 (2.5) 4 (2.0) 0 (0) b b b PR 209 0.0001 208 0.0034 198 0.0210 + 105 (69.5) 53 (35.1) 26 (13.1) 71 (35.9) 15 (7.6) 31 (15.7) À 19 (32.8) 8 (14) 33 (16.7) 14 (7.1) 4 (2.0) 4 (2.0) b b b HER2 206 0.2435 205 0.4831 195 0.2823 + 16 (50) 9 (36) 10 (10.5) 7 (7.4) 4 (4.2) 7 (7.4) À 107 (61.5) 51 (28.3) 47 (49.5) 78 (82.1) 15 (15.8) 27 (28.4) b b b EGFR 212 0.0001 212 0.0661 201 0.0278 + 2 (10.5) 2 (10.5) 15 (7.5) 2 (1.0) 2 (1.0) 0 (0) À 124 (64.2) 61 (31.6) 44 (21.9) 84 (41.8) 18 (9.0) 36 (17.9) b b b Ck 14 211 0.0001 211 0.0425 200 0.0278 + 3 (15) 2 (10) 15 (7.5) 3 (1.5) 2 (1.0) 0 (0) À 122 (63.9) 61 (31.9) 44 (22.0) 82 (41.0) 18 (9.0) 36 (18) b b b Ck 5/6 204 0.0004 204 0.3220 194 0.0842 + 5 (22.7) 4 (18.2) 14 (7.2) 4 (2.1) 3 (1.5) 1 (0.5) À 118 (64.8) 56 (30.8) 42 (21.6) 80 (41.2) 15 (7.7) 35 (18) b b b Ck 17 210 0.0001 210 0.1618 199 0.0096 + 4 (15.4) 4 (16.7) 16 (8) 4 (2.0) 4 (2.0) 0 (0) À 120 (65.2) 58 (31.2) 43 (21.6) 81 (40.7) 16 (8) 35 (17.6) b b b Basal markers 211 0.0001 211 0.0273 200 0.0072 + 7 (18.9) 5 (14.3) 24 (12.0) 6 (3.0) 4 (2.0) 1 (0.5) À 118 (67.8) 58 (33) 35 (17.5) 79 (39.5) 16 (8) 35 (17.5) c c c Nielsen groups 201 0.0001 200 0.0478 191 0.0314 Basal 2 (7.4) 3 (11.5) 21 (11) 2 (1.0) 3 (1.6) 0 (0) Luminal 103 (72.5) 45 (31) 24 (12.6) 74 (38.7) 11 (5.8) 28 (14.7) HER2 16 (50) 12 (41.4) 11 (5.8) 7 (3.7) 4 (2.1) 7 (3.7) b b b p53 198 0.0129 200 0.3923 189 0.2968 + 28 (45.9) 14 (24.1) 24 (12.7) 20 (10.6) 6 (3.2) 7 (3.7) À 89 (65) 44 (31) 33 (17.5) 61 (32.3) 12 (6.3) 26 (13.8) c c c MIB-1 198 0.0001 199 0.0742 188 0.1016 <10% 51 (65.4) 26 (31.7) 16 (8.5) 36 (19.1) 8 (4.3) 13 (6.9) 10–30% 61 (67.8) 30 (33.3) 20 (10.6) 40 (21.3) 9 (4.8) 19 (10.1) >30% 6 (20) 3 (11.1) 19 (10.1) 5 (2.7) 2 (1.1) 1 (0.5)

NOTE: Significant P values are shown in boldface. Abbreviations: Ck, cytokeratin; LN mets, lymph node metastasis; LVI, lymphovascular invasion; n, number. À À À À *P values derived from a comparison between GFRa1+/RET+ cases versus the remaining three groups (GFRa1 /Ret+, GFRa1+/RET , and GFRa1 /RET ). cm2 test. bFisher’s exact test.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Cancer Research resulted in stimulation of RET autophosphorylation and activation of the ERK1/2 and AKT pathways (Fig. 3A). Cells were then treated with 10 ng/mL GDNF for 28 h followed by 1 h BrdUrd labeling, and BrdUrd incorporation was monitored by immunostaining. GDNF treatment resulted in a 1.4-fold increase in BrdUrd-positive cells (P = 0.0049; Fig. 3B). In titration experiments, no further increase in cell proliferation was observed with 15 or 45 ng/mL GDNF (data not shown). To show that this effect of GDNF was mediated via RET/GFRa1 signaling, cells were pretreated with siRNAs or were mock transfected. GDNF treatment of mock-transfected cells (P = 0.021) or cells transfected with either of two different control siRNAs (both P < 0.001) resulted in a statistically significant increase in BrdUrd incorporation, whereas pretreatment with either RET siRNA or GFRa1 siRNA oligonucleotides completely blocked this response (Fig. 3C). To investigate the effects of GDNF on MCF7 cell survival, serum- starved cells were replated into 96-well plates in serum-free medium. Under these conditions, the cells did not attach to the tissue culture plastic. After 3 days in culture, there was a decrease in the number of live cells present in all of the untreated (no GDNF) samples, and this was further reduced by 8 days in culture (Fig. 3D). In the nontransfected cells, the presence of GDNF resulted in a statistically significant increase in the number of live cells at day 3 (P = 0.0025) and at day 8 (P = 0.0042). This increase was retained in mock-transfected cells (day 3, P = 0.0003; day 8, P = 0.0026) and in cells transfected with either of two different control siRNA oligonucleotides, siRNA-C1 (days 3 and 8, both P < 0.0001) and siRNA-C2 (days 3 and 8, both P < 0.0001). In contrast, treatment with RET or GFRa1 siRNA blocked the effects of GDNF (Fig. 3D). In these assays, it was consistently noted that GFRa1 siRNA treatment was not as effective as RET siRNA treatment in abrogating the effects of GDNF on cell survival. The most likely reason for this is that GFRa1 down-regulation by siRNA treatment was not maintained for the length of the assay (data not shown). GDNF induces MCF7 cell scattering. In vivo and ex vivo experiments have shown that expression of GDNF by the nephrogenic mesenchyme is required for development and branching of Figure 2. MCF7 cells express GFRa1 and RET. MCF7 cells were either the ureteric bud during kidney morphogenesis (6, 7). Similarly, untreated, mock transfected, or transfected with siRNA and cultured for 72 h. A, flow cytometry: Cells were detached and stained with isotype-matched control Madin-Darby canine kidney epithelial cells transfected with RET antibody (solid profiles) or anti-GFRa1 antibody (open profiles) followed by and either cotransfected with GFRa1 or treated with soluble Alexa 488–conjugated antigoat immunoglobulin. Data shown are from untreated a cells, mock-transfected cells, or cells transfected with scrambled control siRNA GFR 1 displayed enhanced migration and chemotaxis in response (C1) or two independent GFRa1 siRNAs (siRNA-2 and siRNA-3). Values given to GDNF (23, 24). As GFRA1 mRNA expression in breast tumors are for the relative fluorescence index. Two additional independent GFRa1 was associated with lymphovascular invasion and lymph node siRNAs gave an identical reduction in GFRa1 expression with RFI values of 3.07 and 2.65 (data not shown). B, Western blotting: cells were left untreated (lane 1), metastasis, we examined the ability of GDNF to promote the mock transfected (lane 2), or transfected with scrambled siRNA C1 (lane 3), scattering of MCF7 cell colonies. Further, as TGF-h has been RET siRNA-5 (lane 4), or RET siRNA-6 (lane 5) for 72 h before lysates were reported to contribute to the recruitment of GFRa1 to the subject to immunoblotting with anti-RET antibody (top) and anti–a-tubulin antibody (bottom) followed by HRP-conjugated antirabbit and antimouse multisubunit signaling complex (25), cells were also treated with immunoglobulin. *, nonspecific band. or without TGF-h1. In untreated colonies, phase contrast imaging and staining of cells with phalloidin to visualize the actin cytoskeleton revealed closely packed, tightly adherent cells with GDNF and TGF-h1 and associated with this phenotype, was the strong cortical actin filaments (Fig. 4). GDNF treatment resulted presence of larger and more abundant actin stress fibers and pro- in scattering of the cells, particularly at the edge of the colonies, minent focal adhesions (Fig. 4; Supplementary Fig. S1). Together with the concomitant loss of cortical actin organization and the with the data shown in Fig. 3, these results show that GDNF pro- formation of actin stress fibers. Costaining with an antivinculin motes cell proliferation, survival, and cell motility in RET+/GFRa1+ antibody showed that many of these actin stress fibers terminated breast cancer cells. in focal adhesions (Supplementary Fig. S1). TGF-h is well recog- GDNF expression is modulated by inflammatory signals. nized to promote the transition of epithelial cells to a more During development, GDNF is expressed by support cells adjacent mesenchymal phenotype (26) and, as expected, treatment of MCF7 to target cell types, for example by the nephrogenic mesenchyme in cells with TGF-h1 resulted in a decrease in cell-to-cell adhesion and the developing kidney, Sertoli cells in the testis, and nonneuronal in the acquisition of a migratory phenotype. This scattered phe- cells in the nervous system (7). In the healthy adult, the expression notype was further accentuated by the cotreatment of cells with of GDNF is generally low in all tissues examined. Here, we have

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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 shown that in a subset of breast tumors, the GDNF receptors RET endogenous mouse fibroblasts recruited to a tumor site up-regulate and GFRa1 are up-regulated. This raises the question as to whether GDNF expression. MCF7 cells were grown as xenografts in the GDNF expression is coordinately up-regulated and if so, by which mouse mammary fat pad; the tumors were excised and sectioned; cell types. It has previously been reported that the inflammatory and expression of GDNF and aSMA was monitored by confocal cytokines TNF-a and IL-1h induce GDNF expression in astrocytes microscopy. In the tumors, infiltrating fibroblasts identified by (27, 28). Consequently, we initially examined the effects of these aSMA positivity were clearly detected, and a subset of these factors on GDNF expression in mouse fibroblasts by real-time showed strong GDNF expression (Fig. 5B). In contrast, no GDNF qPCR. TNF-a treatment of NIH-3T3 cells resulted in a 2.2-fold expression was detected in fibroblasts located in skin tissue increase in GDNF mRNA levels (P = 0.011), whereas there was no adjacent to the xenograft tumors (Fig. 5B) or in the kidney (data significant increase in expression levels following IL-1h treatment not shown). These data show that GDNF expression in mesenchy- (Fig. 5A). Addition of TNF-a and IL-1h together resulted in a 3.5- mal cells can be induced by factors that are released in the tumor fold increase in GDNF expression (P = 0.032), indicating that there microenvironment. However, in these studies, it was also noted may be a small synergistic effect of both cytokines. that the MCF7 cells in the tumor xenograft showed a low level of As tumor and inflammatory cells secrete cytokines such as TNF- GDNF staining (Fig. 5B). As GDNF is a secreted factor, it was not a and IL-1h at the site of tumor growth, we next examined whether known whether this staining reflected GDNF that had been

Figure 3. GDNF stimulates MCF7 proliferation and survival. A, MCF7 cells cultured in serum-free DMEM overnight were stimulated for 20 min with or without 10 ng/mL GDNF and subject to Western blotting using the indicated antibodies. B, MCF7 cells cultured in serum-free DMEM for 24 h were then incubated for 28 h with DMEM plus 0.5% FCS with or without 10 ng/mL GDNF. BrdUrd (10 Amol/L) was added for 1 h before fixation and staining with FITC-conjugated anti-BrdUrd antibody. Nuclei were counterstained with TO-PRO-3. Representative monochrome images. Values given are percentage of BrdUrd-positive nuclei F SD from z3 independent experiments (P = 0.0049). Scale bar, 100 Am. C, MCF7 transfected with siRNA for 72 h and then treated as described in A to monitor BrdUrd incorporation following stimulation with or without 10 ng/mL GDNF. Columns, % BrdUrd-positive nuclei from three independent experiments; bars, SD. *, P = 0.05; ***, P < 0.001. D, 1 Â 104 siRNA transfected MCF7 cells were seeded in anchorage-independent conditions and treated with or without 10 ng/mL GDNF for 3 or 8 d and cell survival was monitored with the Cell Titre Blue assay as described in Materials and Methods. Columns, mean % live cells from quadruplicate samples in two independent experiments; bars, SE. *, P < 0.005.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Cancer Research secreted by the infiltrating fibroblasts and bound to the tumor cells 67T, which has high-level GFRa1 expression, GDNF staining was or that the tumor cells themselves were capable of expressing observed both in the associated stromal fibroblasts and in a subset GDNF. To address this, the expression of GDNF by MCF7 cells was of the tumor cells (Fig. 5D). assessed by qPCR. In untreated MCF7 cells, GDNF mRNA levels approached the limit of detection in the TaqMan assay with a mean Discussion CT value of (36.04 F 1.46) compared with a mean CT value for glyceraldehyde-3-phosphate dehydrogenase of 23.79 F 1.65. This A subset of luminal breast cancers show up-regulation of large difference in cycle number indicates that untreated MCF7 GDNF receptors. Activating germ line mutations in RET are cells have very little endogenous GDNF mRNA. Treatment with oncogenic and give rise to the spectrum of cancer phenotypes seen TNF-a or IL-1h resulted in a 4- to 5-fold increase in the levels in multiple endocrine neoplasia type 2 and familial medullary of GDNF mRNA (P = 0.041 and P = 0.022, respectively), whereas thyroid carcinoma. In addition, somatic gene rearrangements in TNF-a and IL-1h together synergized to produce a 17-fold increase which the RET tyrosine kinase domain is fused with the 5¶ region in GDNF transcripts (P < 0.0001; Fig. 5C). Moreover, this prolonged of heterologous genes (RET/PTC) give rise to papillary thyroid treatment with cytokines resulted in down-regulation of RET carcinoma (31, 32). These mutations/rearrangements result in the expression in MCF7 cells (Supplementary Fig. S2) consistent with constitutive activation of RET independent of its association with previous reports that prolonged GDNF treatment results in RET GDNF and GFRa1 and show that aberrant RET signaling is tumo- degradation (19, 29, 30). The up-regulation of GDNF by MCF7 cells rigenic. In contrast to these well-studied oncogenic RET mutants, indicates that tumor cells can be induced to express GDNF and to date few studies have addressed whether up-regulation of wild- suggests that in vivo, tumor cells expressing RET and GFRa1 may type RET and/or its GFRa coreceptor may also play a role in tumor exploit paracrine and/or autocrine GDNF signaling. To investigate progression. Using relatively small sample sets, it has been reported whether these xenograft models accurately reflect the expression of that RET is overexpressed in high-grade prostatic intraepithelial GDNF in human cancers, normal breast and tumor samples shown neoplasia and prostate cancer (33), in pancreatic cancer (where in Fig. 1A were stained with anti-GDNF antibodies. In normal expression correlates with poor survival; ref. 34), and in some breast and in tumors shown to have low GFRa1 expression, only neural crest–derived tumors (35), and that RET is amplified in a low-level GDNF staining was detected. In contrast, in tumor sample subset of radiation-induced and anaplastic thyroid cancers (36).

Figure 4. MCF7 cells display scattering in response to GDNF. MCF7 cells plated onto glass coverslips were incubated in DMEM plus 0.5% FCS overnight and then stimulated with 10 ng/mL GDNF, 5 ng/mL TGF-h1, or 10 ng/mL GDNF plus 5 ng/mL TGF-h1 for 48 h. Cells were fixed, permeabilized, and stained with Alexa 555–conjugated phalloidin. Scale bars, 100 Am (left and middle) and 25 Am(right). Images of cells costained with phalloidin and antivinculin antibody are shown in Supplementary Fig. S1.

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Figure 5. GDNF expression is up-regulated in response to proinflammatory signals. A, qPCR was performed to determined the fold change in Gdnf mRNA expression in NIH-3T3 cells following treatment with 5 ng/mL IL-1h and/or 10 ng/mL TNF-a. Relative expression was determined from a fold difference of 1, which was the value assigned to untreated control (comparator). Columns, mean fold changes in Gdnf mRNA levels from at least seven independent experiments; bars, SE. *, P < 0.05. B, cryosections of two MCF7 tumor xenografts and adjacent mouse skin stained with FITC-conjugated anti-aSMA antibody (green) and anti-GDNF antibody followed by Alexa-555 antigoat immunoglobulin (red). Nuclei were counterstained with TO-PRO-3 (blue). Scale bar, 50 Am. Arrowheads in tumor samples, GDNF expression in aSMA-positive infiltrating fibroblasts; arrows, MCF7 tumor cells. Arrowhead in skin sample, aSMA-positive blood vessel; arrow, GDNF-negative fibroblasts. C, qPCR analysis of MCF7 cells was performed as described in A. Columns, mean fold changes in GDNF mRNA levels from eight independent experiments; bars, SE. *, P < 0.05; ***, P < 0.0001. D, cryosections of normal human breast (34N) and two breast tumor samples (42T and 67T) shown in Fig. 1A were labeled with anti-GDNF antibody followed by Alexa 555 antigoat immunoglobulin (red). Nuclei were counterstained with TO-PRO-3 (blue). Arrowheads in sample 34N, normal myoepithelial and luminal epithelial cells. Arrowheads in samples 42T and 67T, tumor cells. Arrows, stromal cells. Scale bar, 50 Am.

In breast cancer where few kinase-activating mutations have been set of 36 breast cancers that RET mRNA expression significantly identified, it is notable that overexpression of wild-type receptor correlates with expression of ER (17). However, these studies were tyrosine kinases such as HER2, EGFR, and FGFR1 can drive tumor performed on homogenates of tumor samples. In contrast, the growth (37–39). Using a breast cancer tissue microarray, we have in situ hybridization approach we have used confirms that shown here that both RET and GFRA1 mRNA are preferentially expression of RET and GFRa1 is localized to the tumor cells expressed in hormone receptor–positive (ER+ and PR+) tumors and rather than to the stromal compartment. These expression studies À coexpressed in 20.4% and 24.1% of invasive breast cancers with a also revealed that 41.9% of tumors were GFRA1+/RET ,an luminal and HER2 phenotype (16), respectively. Conversely, no expression pattern that is observed in many developing tissues basal-like carcinomas displayed concurrent mRNA expression of (41), and has led to the suggestion that soluble shed GFRa1may these genes. Data mining also indicates that RET and GFRA1 signal in trans to nearby RET+ cells and/or that GDNF and GFRa1 mRNAs are preferentially expressed in ER+ and in nonbasal-like may be able to signal independently of RET. In neuronal systems, tumors3 (40) and, similarly, it has been reported previously using a GFRa1 has been shown to form a functional complex with NCAM (42) and LICAM (43) and, consequently, it will be of interest to determine whether GFRa1 expressed in the absence of RET in 3 http://www.oncomine.org tumor cells signals via cis interactions with alternative transmem- www.aacrjournals.org 11739 Cancer Res 2007; 67: (24). December 15, 2007

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Cancer Research brane receptors. Although no correlation between patient outcome in stromal fibroblasts associated with GFRa1+ human breast and mRNA expression of GFRa1 or RET was identified in the cancers strongly supports our prediction that GDNF expression in present study, one could hypothesize that expression of these genes breast cancer can be up-regulated in response to inflammatory may be associated with the development rather than the cytokines. This is the first study to investigate the expression of aggressiveness of a subgroup of tumors with a luminal and HER2 GDNF and its receptors in breast cancer; however, it is unlikely that phenotype, in a way akin to the expression of caveolin 1 and EGFR GDNF is the only neurotrophic factor that can promote tumor in basal-like breast cancer (15, 16, 38, 44). progression. Expression of nerve growth factor (NGF) has similarly GDNF promotes proliferation, survival, and scattering of been shown to be up-regulated by inflammatory signals and to breast tumor cells via GFRA1and RET signaling. To date, the contribute to both survival and proliferation of breast cancer cells majority of in vitro studies on wild-type RET/GFRa1 signaling have (14, 50). Although signaling via RET and GFRa1 on tumor cells may focused either on neuronal lines that have endogenous expression be initiated by expression of GDNF in the stroma, it is commonly of these receptors or on epithelial and fibroblast lines in which the observed that tumor progression is accompanied by a switch from receptors are expressed ectopically. Studies in these systems have paracrine to autocrine signaling. For example, in breast cancer, the corroborated the phenotype of Ret-, Gfra1-, and Gdnf-deficient neurotrophic factor NGF is expressed by the stromal and tumor cells. mice in demonstrating a role for GDNF signaling in promoting cell Here, we noted that although GDNF expression was barely proliferation, neuroprotection, and migration. Less well studied is detectable in MCF7 cells under normal culture conditions, a low the role of GDNF signaling in cancer, although it has been shown level of GDNF staining was observed in MCF7 xenografts. Similarly, that GDNF can promote cell migration/chemotaxis and invasion of GDNF+ tumor cells were observed in breast cancers with high GFRa1 a RET+/GFRa1+ pancreatic cell line and that these activities are expression, and, interestingly, these GDNF+ cells were located at the dependent on the activation of the MAPKand PI3Kpathways (12). invasive margin of the tumor. In support of an autocrine role for Here, we have identified the MCF7 breast cancer cell line as GDNF signaling, expression in vitro was significantly increased expressing RET and GFRa1 and showed that both of these following either TNF-a or IL-1h treatment with a strong synergistic receptors are required for mediating GDNF-dependent cellular increase following stimulation with both TNF-a and IL-1h. Although responses. Further, the demonstration that GDNF treatment of these experiments show that GDNF expression can be up-regulated these cells promotes proliferation, survival in an anchorage- by TNF-a and IL-1h treatment of MCF7 cells in culture, further independent assay, and scattering supports the notion that GDNF studies will be required to assess the role and specificity of these signaling may have an important role in the development of ER+ cytokines in regulating GDNF expression in vivo. breast cancers. In conclusion, we provide evidence that the receptor tyrosine GDNF and inflammation. The observation that GDNF could act kinase RET and its coreceptor GFRa1 are expressed in a subset of as a trophic factor for RET+/GFRa1+ breast tumors raised the breast cancers and that the cytokine GDNF, which activates their question as to the source of GDNF in tumor tissue. GDNF was first signaling cascade, is induced by inflammatory signals in stromal identified as a neurotrophic factor expressed by glial cells in fibroblasts and tumor cells, thereby supporting both paracrine and response to neuronal cell injury and further studies have shown autocrine stimulation of tumor cells in response to their that GDNF expression is induced in vitro by inflammatory microenvironment. cytokines such as TNF-a and IL-1h in astrocytes (27, 28) as well as in lipopolysaccharide-induced spinal inflammation (45) and chronic inflammatory conditions such as inflammatory bowel Acknowledgments a disease (46). Here, we show that TNF- stimulation results in Received 6/22/2007; revised 10/9/2007; accepted 10/12/2007. increased GDNF expression by mouse fibroblasts in vitro and that Grant support: Breakthrough Breast Cancer (C.M. Isacke and J.S. Reis-Filho), h Cancer Research UK(R. Poulsom and T. Hunt), and Ministerio de Educacion y Ciencia this up-regulation is further enhanced by the addition of IL-1 . of Spain (I. Plaza-Menacho). TNF-a and IL-1h are major inflammatory cytokines secreted by The costs of publication of this article were defrayed in part by the payment of page tumor-associated macrophages in breast carcinomas (2, 47–49). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The demonstration here that GDNF expression is also up-regulated We thank David Robertson, Damian Johnson, Alan Kennedy, and the Breakthrough in infiltrating aSMA-positive fibroblasts in a xenograft model and Breast Cancer Histopathology Facility who provided invaluable help in this project.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. A Role for Glial Cell−Derived Neurotrophic Factor−Induced Expression by Inflammatory Cytokines and RET/GFR α1 Receptor Up-regulation in Breast Cancer

Selma Esseghir, S. Katrina Todd, Toby Hunt, et al.

Cancer Res 2007;67:11732-11741.

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