Esseghir et al. (2007)

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Supplementary Methods and Supplementary Figures 1 &2

Supplementary Methods

Tissue microarray (TMA)

The tissue microarray (TMA) contained 0.6 mm cores of 245 invasive breast

carcinomas. Full details of the TMA characterization and the cohort of patients are

described elsewhere (1, 2). All patients were treated primarily with curative surgery

(63 mastectomy and 155 wide local excision) followed by anthracycline-based

adjuvant chemotherapy, in combination with hormone therapy for patients with

estrogen receptor (ER)-positive tumors. Follow-up was available for 244 patients,

ranging from 0.5 to 125 months (median – 67 months, mean – 67 months). Tumors

were graded according to a modified Bloom-Richardson scoring system (3) and size

was categorized according to the TNM staging criteria (4). Tumors were classified

according to the criteria of Nielsen et al. (5) into HER2 (HER2+ve, ER any, Ck 5/6 or

EGFR any), luminal (HER2-ve, ER+ve, Ck5/6 or EGFR any) or basal-like (HER2-ve, ER-

ve, Ck 5/6 or EGFR+ve) groups. Results of immunohistochemical analysis to detect

ER, progesterone receptor (PgR), HER2, EGFR, cytokeratin (Ck) 14, Ck 5/6, Ck 17,

Ki67 and p53 have been described previously (1, 6).

In situ hybridization

In situ hybridization probes for RET (RT-PCR amplification from MCF7 mRNA, nt

781-1440) and GFRA1 (IMAGE clone 4874042, nt 1081-1620, Geneservice Ltd.,

Cambridge, UK; (7)) were generated by PCR amplification and cloned into the

pGEM3Z vector (Promega). Both GFRA1 and RET probes will detect all known

receptor splice variants. Generation and labeling of riboprobes and hybridization to

the tissue microarray was as previously described (8). An ACTB (β-actin) probe was

used as a positive control. Levels of mRNA in the tumor cells were scored

concurrently by two observers (RP, JSR-F) on a dark-field microscope coupled with a

digital camera. Expression was scored blinded to clinicopathological data, patients’

outcome and results of immunohistochemical analysis, and classified semi-

quantitatively into five categories: 0, negative, 1, weak, 2 moderate expression, 3

strong expression 4, strong and widespread expression. Tumors were scored

positive if morphologically unequivocal tumor cells showed >1 expression. For the

correlation between GFRA1 and RET mRNA expression on the TMA and

clinicopathologic and immunohistochemical data, Statview 5.0 (SAS Institute Inc)

software package was used. Correlations between categorical variables were done

Esseghir et al. (2007)

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using the χ2 test and Fisher’s exact test. Disease-free and overall survival were

expressed as the number of months from diagnosis to the occurrence of an event

(local/distant relapse or disease-related death, respectively). Cumulative survival

probabilities were calculated using the Kaplan-Meier method. Difference between

survival rates were tested with the log-rank test. All tests were two-tailed with a

confidence interval of 95%.

Real-time quantitative PCR (qPCR)

NIH-3T3 cells were cultured overnight, washed and incubated for a further 48 h in

serum-free DMEM. Cells were then stimulated in serum-free DMEM with 5 ng/ml

mouse IL-1β and/or 10 ng/ml mouse TNF-α for 24 h before being lysed in Trizol

(Invitrogen) and RNA extracted using chloroform-phase separation. MCF7 cells were

treated similarly except they were stimulated for 48 h with human IL-1β and human

TNF-α. RNA clean up and DNase digestion were performed using the RNeasy Micro

kit (Qiagen, Crawley, UK) and 150 ng of total RNA used in the reverse transcription

reactions (Omniscript, Qiagen, Crawley, UK). qPCR was performed on the ABI Prism

7900HT sequence detection system (Applied Biosystems, Foster City, California)

using Taqman® Gene Expression assays (Applied Biosystems). The reference

numbers for the assays employed were: 4310884E (human GAPDH), 4352339E

(mouse GAPDH), Hs00181185_m1 (human GDNF), Mm00599849_m1 (mouse

GDNF). Independent experiments were performed on 7 (MCF7 cells) or 8 (NIH-3T3

cells) occasions. Each experiment was represented on a single qPCR plate, where

GAPDH and GDNF were amplified in triplicate, over the course of 40 cycles for NIH-

3T3 cells and 45 cycles for MCF7 cells. The baseline level of expression was set as

untreated cells and GAPDH as the endogenous control. Applied Biosystems SDS

v2.2 software was used to analyze results generating expression levels (RQ values),

relative to untreated cells (comparator). Cycle threshold (CT) was set to 0.1. The CT

was positioned in the exponential phase of all the amplification plots, where the

fluorescence generated by the TaqMan reaction was deemed to be significantly

greater than background levels. Data from all of the plates were imported into the

software to generate a mean value for the untreated controls, over replicate

experiments. RQ values for each experiment were generated for the remaining

treatment groups, relative to this untreated baseline value (comparator). Significant

deviation of the mean value of the data points from a fold difference of 1 (which

indicates no changed compared to the comparator sample) was tested using a two-

tailed t test on Log10 transformed data.

Esseghir et al. (2007)

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Supplementary Figure 1 (Esseghir et al., 2007)

PBS

GDNF

TGF-β1

GDNF &TGF-β1

phalloidin vinculin merge

MCF7 cells display scattering and formation of focal adhesions 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- 1 or 10 ng/ml GDNF plus 5 ng/ml TGF- 1 for 48 h. Cells were then fixed, permeabilized and stained with anti-vinculin mAb followed by Alexa488 anti-mouse Ig ( green) and Alexa555-conjugated phalloidin (red). Nuclei were counterstained with TO-PRO-3 (blue). Scale bar, 50 m.

Treatment of MCF7 cells for 48 hours with TNF-α and IL-1β results indownregulation of RET. MCF7 cells were cultured for 48 hours in serum-free DMEMin the presence or absence of TNF-α (10 ng/ml) and IL-1β (5 ng/ml) and then cells wereincubated with or without GDNF (10 ng/ml) for 20 minutes prior to lysis. Cell lysateswere subject to Western blotting to assess the level of RET and activation ofdownstream signaling pathways as monitored by phosphorylation of ERK1/2 and AKT.All samples were run on the same gel.

TNF-α and IL-1β treatment results in downregulation of the RET receptor andattenuation of downstream signalling. This is consistent with a model in which TNF-αand IL-1β treatment result in enhanced GDNF expression by MCF7 cells (see Figure5C) and that, as previously reported (1-3), long term GDNF treatment results in RETdegradation.

1. Pierchala BA, Milbrandt J, Johnson EM, Jr. Glial cell line-derived neurotrophic factor-dependent recruitment of Ret into lipid rafts enhances signaling by partitioning Ret fromproteasome-dependent degradation. J Neurosci 2006;26:2777-87.2. Richardson DS, Lai AZ, Mulligan LM. RET ligand-induced internalization and itsconsequences for downstream signaling. Oncogene 2006;25:3206-11.3. Scott RP, Eketjall S, Aineskog H, Ibanez CF. Distinct turnover of alternatively splicedisoforms of the RET kinase receptor mediated by differential recruitment of the Cblubiquitin ligase. J Biol Chem 2005;280:13442-9.

Supplementary Figure 2 (Esseghir et al., 2007)