Introduction

Pdcd4, a 64 kDa protein, is a novel tumor suppressor inhibiting TPA-induced neoplastic transformation (Cmarik et al., 1999), tumor promotion and progression (Jansen et al., 2005). Pdcd4 interacts with translation initiation factors eIF4A and eIF4G and inhibits translation (Yang et al., 2004; Zakowicz et al., 2005). Pdcd4 is upregulated in apoptosis in response to different inducers (Shibahara et al., 1995; Zhang and DuBois, 2001). Molecules regulated by Pdcd4 include p21 (Göke et al., 2004), Cdk4, ornithine decarboxylase (Jansen et al., 2005), carbonic anhydrase II (Lankat-Buttgereit et al., 2004), and JNK/c-Jun/AP-1 (Bitomsky et al., 2004; Yang et al., 2006). Recently, we showed that Pdcd4 suppresses expression of the invasion-related urokinase-receptor-(u-PAR)-gene, invasion and intra-vasation, via Sp1/Sp3 promoter motifs in cancer (Leupold et al., 2007) and initial studies suggest a downregulation of Pdcd4 especially in lung and colorectal cancers, which is associated with a poor patient prognosis (Chen et al., 2003; Mudduluru et al., 2007).

Relatively little is known about mechanisms regulating Pdcd4 expression in cancer. Initial studies suggested that Pdcd4 is regulated by topoisomerase-inhibitors (Onishi et al., 1998), COX-2-inhibitors (Zhang and DuBois, 2001), Myb (Schlichter et al., 2001), and Akt (Palamarchuk et al., 2005) and in a recent study, Pdcd4 was shown to be, at least in part, regulated by proteasomal degradation in response to mitogens (Dorrello et al., 2006). Further mechanisms leading to downregulation of this important tumor suppressor in cancer need to be elucidated.

MicroRNAs (miRNAs) have been discovered as naturally occuring non-coding RNAs, controlling gene expression via specific sites at the 3'-UTR of target-mRNAs, causing translational repression or degradation (Berezikov et al., 2005; Pillai et al., 2005; Zamore and Haley, 2005). Primary transcripts are cleaved by Drosha, leading to pre-miRNAs of 70 nt and processed to 17–24 nt-miRNAs via Dicer (Bartel, 2004). miRNAs have important regulatory functions in processes such as differentiation, proliferation and inhibiting apoptosis (Chen et al., 2004; Croce and Calin, 2005). Increasing numbers of reports implicate an aberrant expression of certain miRNAs, in particular miR-21, -17-92, -15, -16, -141, let-7, miR-103, miR-107 and others, in tumor growth, carcinogenesis, or response to chemotherapy in different malignancies (Calin et al., 2002; Chan et al., 2005; Lu et al., 2005; Esquela-Kerscher and Slack, 2006; Hammond, 2006; Meng et al., 2006; Roldo et al., 2006; Si et al., 2007). First cancer-related target genes of miR-21 have been described, among them being PTEN and TPM1 (Meng et al., 2006; Zhu et al., 2007).

So far, it has not been investigated whether Pdcd4 is being regulated by miRNAs. To our knowledge there has been no study investigating whether important miRNAs such as miR-21 contribute to tumor cell invasion or intravasation. The present study was undertaken (1) to elucidate the regulation of Pdcd4 by miRNAs and identify target motifs and mechanisms of this regulation, (2) to determine a role of miR-21 in invasion/intravasation. This is the first report demonstrating that miR-21, via a specific target motif at nt228–249 of the 3'-UTR, is negatively regulating Pdcd4 and induces invasion-related processes in colorectal cancer.

Results

An evolutionary conserved target sequence for miR-21 is found in the 3'-UTR of Pdcd4

The 652 nt-3'-UTR of Pdcd4 was screened for complementarity to seed sequences of known miRNAs via a bioinformatic search. A 100%-match target sequence for miR-21 at nt228–249 was found. As shown in Figure 1a, the minimum free energy predicted for hybridization with the Pdcd4-3'-UTR and miR-21 at this site is G-10.0 kcal mol^-1, determined by mFold analysis, this being consistent with authentic miRNA targeting (Doench and Sharp, 2004). Comparing the human sequence for interspecies homology, we found that the miR-21 target sequence at nt228–249 of the Pdcd4-3'-UTR is highly conserved among seven species (Figure 1b). We also found a conserved target site for miR-141, however, in initial reporter experiments; we found that it was not functional (see below). Therefore, we focused our studies on miR-21.

Figure 1.

A miR-21 target site resides at nt228–249 of the Pdcd4-3'-UTR, and is highly conserved in seven species. (a) The location of the putative miR-21 target site is shown. First eight nucleotide of miR-21 and its target region: bold, unpaired bases: above and below the duplex. (b) Comparison of nucleotides between the miR-21 seed-sequence and its target in seven species.

Full figure and legend (103K)

Pdcd4 protein correlates inversely with miR-21 amounts in colorectal cancer cell lines

We first determined expression levels of miR-21 and Pdcd4 (mRNA and protein) in 10 different colorectal cancer cell lines (Figure 2). In cell lines with high endogenous miR-21 (for example, RKO, Hct116, Widr, Figure 2c, lanes 1, 3 and 10) as measured by miRNA-qRT–PCR, a low amount of Pdcd4 protein at 64 kDa was observed (Figures 2a and b lanes 1, 3 and 10), whereas cell lines with low miR-21 (for example, Geo, Colo206f, CaCo2, Figure 2c, lanes 7, 9 and 2) showed high amounts of Pdcd4 protein (Figures 2a and b, lanes 7, 9 and 2). Across all 10 cell lines tested, we found a significant inverse correlation between miR-21 and Pdcd4 protein levels (P=0.01). For miR-21 and Pdcd4-mRNA; however, there was no significant association (P=0.10), and the fold differences in Pdcd4-mRNA were less than for Pdcd4-protein (Figure 2d). These initial experiments suggest that miR-21 might negatively regulate Pdcd4 at the post-transcriptional level.

Figure 2.

miR-21 and Pdcd4 correlate inversely in 10 colorectal cancer cell lines. (a) Pdcd4-western (b) densitometric western analysis (NIH-ImageJ). Density ratio Pdcd4/-Actin: bar diagram. (c) miR-21-expression, (d) Pdcd4 mRNA-expression (TaqMan-qRT–PCR). Fold differences in Pdcd4 protein are higher than for Pdcd4-mRNA, and inversely correlate with miR-21 (P=0.01), in contrast to Pdcd4-mRNA (P=0.10).

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The Pdcd4-3'-UTR is a target for miR-21

Given the hypothesis that Pdcd4 might be a target of miR-21, we asked whether the 3'-UTR of Pdcd4 is a functional target of miR-21. We cloned a reporter plasmid driven by the SV40 basal promoter, harboring the 652 nt-wild-type-3'-UTR of Pdcd4 at the 3'-position of the luciferase reporter gene (wt-UTR, Figure 3a). In parallel, we cloned a second reporter construct in which the conserved targeting region AUAAGCUA of miR-21 within nt228–249 was specifically mutated which is predicted to abolish binding (Figure 3a). Transient transfection of Colo206f cells, with a low endogenous miR-21 expression, with the wild-type-3'-UTR-reporter construct and pre-miR-21, led to a significant decrease of reporter activity as compared to the control (Figure 3b). However, the activity of the reporter construct mutated at the specific miR-21 target site at nt228–249 was unaffected by a simultaneous transfection with pre-miR-21 (Figure 3b). The same observation was made with HeLa cells (data not shown). Further experiments were carried out using anti-miR-21, which binds to endogenous miR-21 and thereby antagonizes its activity (Meister et al., 2004). When RKO or HCT116 cells with a high endogenous miR-21 expression (Figure 2) were transfected with anti-miR-21 and the wild-type-3'-UTR-reporter construct, a significant increase in activity of the wild-type reporter was observed (Figures 3c and d). This was abolished when the same cell lines were transfected with the reporter construct mutated at the miR-21 target-site instead (Figures 3c and d). Similar results were not observed when HeLa and RKO cells were transfected with reporter construct and pre-miR-141, moreover, there was no additional effect in the presence of pre-miR-21 (see Supplementary Figure 1).

Figure 3.

The Pdcd4-3'-UTR is a target for miR-21. (a) Diagram of Pdcd4-3'-UTR-containing reporter constructs. Mut: contains 7-base-mutation at the miR-21-target region, abolishing its binding. (b) Reporter assay, Colo206f, with cotransfection of 500 ng Wt-or mut-reporter and 50 nM control-miR, or pre-miR-21 as indicated. Each bar represents values from three independent experiments performed in quadruplicates. P<0.05. (c and d) Reporter assay as in (b), with control anti-miR and anti-miR-21.

Full figure and legend (34K)

Taken together, these data suggest that the 3'-UTR of Pdcd4 is a functional target site for miR-21 in cultured colorectal cancer cells.

miR-21 downregulates Pdcd4 protein and upregulates tumor cell invasion in cultured colon cancer cells

Next, we determined whether transfection of cell lines with pre-miR-21, or anti-miR-21, affects Pdcd4 protein expression and invasion. In RKO colon cancer cells characterized by a high miR-21 expression, downregulation of endogenous miR-21 with anti-miR-21 (Figure 4a) led to a significant increase in Pdcd4 protein (Figure 4c) without any change in Pdcd4-mRNA (Figure 4b). As a control, PTEN, a known target of miR-21, was also induced (Figure 4c). In parallel, we observed a significant (50%) downregulation of invasion into Matrigel in anti-miR-21-transfected RKO cells (Figure 4d). This was not due to a downregulation of proliferation since incubation of RKO with similar amounts of anti-miR-21 and even longer time periods as compared to the Matrigel assay led to only 10% reduction in proliferation (Supplementary Figure 2).

Figure 4.

miR-21 regulates Pdcd4 expression at the post-transcriptional level and influences cell invasion. (a–c) RKO cells were transfected with 50 nM control-anti-miR, or anti-miR-21. After 3 days, total RNA was isolated and analysed for miR-21 and Pdcd4 mRNA as described in Materials and methods. Proteins from the same experiment were used to detect Pdcd4 and PTEN by western blotting. (d) Matrigel invasion was performed in triplicates as described in Materials and methods. Invaded cells: expressed as relative light units. (e–g) Colo206f were transfected with 50 nM control-miR or pre-miR-21, and analysed for miR-21-expression, Pdcd4-mRNA and protein. (h) Matrigel invasion assay was performed as in (d). P<0.05.

Full figure and legend (93K)

Furthermore, to determine whether overexpression of miR-21 in low-miR-21 expressing cells will have vice versa effects on Pdcd4 protein and invasion, Colo206f cells were transiently transfected with pre-miR-21, or control-miRNA (Figure 4). The transfection was efficient with an almost 250-fold expression of miR-21 as compared to the control (Figure 4e). Whereas Pdcd4 mRNA was almost unaltered in pre-miR-21-transfected cells (Figure 4f), there was a significant reduction of Pdcd4 protein amounts in pre-miR-21-transfected cells (Figure 4g), this again being paralleled by a downregulation of PTEN as a known miR-21 target. In Matrigel assays, we found a significant increase in invasive capacity in pre-miR-21-transfected cells (Figure 4h). These data suggest that miR-21 specifically downregulates Pdcd4 at the post-transcriptional level, and that miR-21 positively regulates invasion of cultured colon cancer cells.

Anti-miR-21 transfection leads to reduced intravasation and distal metastasis

To study the effect of anti-miR-21 treatment on intra-vasation and metastasis, we employed the chicken–embryo–metastasis assay. RKO cells transfected with either control-anti-miR, or anti-miR-21, were inoculated on the upper chorioallantoic membrane (CAM) of 10-day-old chicken embryos. Two days post-inoculation, the lower CAM was isolated and the number of intravasated cells were measured by real-time Alu-PCR. Significantly less intravasation was observed for the anti-miR-21 treated group (Figure 5a).

Figure 5.

Anti-miR-21 treatment attenuates intravasation and metastatic capacity of RKO colon cancer cells. 1 10⁶ of control anti-miR-, or anti-miR-21-transfected RKO cells were inoculated on the upper chorioallantoic membrane (CAM) of chicken embryos. The DNAs of the lower CAM and lungs were extracted 2 days and 7 days post inoculation, respectively. The genomic DNA were analysed by real-time Alu-PCR to determine the number of (a) intravasated and (b) metastasized RKO cells, respectively. Bar graphs represent fold difference in cell numbers withs.d. of four embryos per group (individual cell number is given in the adjacent box). P<0.05.

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To determine the effect on metastatic capacity of these transfected cells, chicken embryos were grown for 5 more days, and on day 17 the lungs were harvested and the number of metastasized cells detected as described above. Anti-miR-21 treated cells showed reduced metastasis compared to control-anti-miR transfected cells (Figure 5b). These two observations clearly emphasize the role of miR-21 in the invasion/intra-vasation and metastatic potential of RKO colon cancer cells.

miR-21 and Pdcd4 are inversely expressed in resected patient tumors in vivo

Finally, we sought to corroborate a negative regulation of endogenous Pdcd4 protein by endogenous miR-21 in vivo. Resected tumor and corresponding normal tissues of 22 patients with colorectal cancer were analysed for Pdcd4-protein (western), Pdcd4-mRNA and miR-21 (qRT–PCR). Representative examples are shown in Figure 6a. A comprehensive analysis of miR-21, Pdcd4-mRNA and -protein for all patients is shown in Figures 6b–d. Tumor characteristics of the samples used in the study are given in Supplementary Table 1. As reported from other tumor entities (for example, Meng et al., 2006; Roldo et al., 2006), we observed that all of the tumors investigated showed an expression of miR-21 higher than in the corresponding normal tissues (Figures 6a and b). This increase of miR-21 expression in tumor tissues was highly significant with P=3.89E-06 (Figure 6b). Within all 22 patients and all tissues, we observed a highly significant negative correlation between Pdcd4 protein and miR-21 (P=0.0001), high expression of miR-21 being associated with low amounts of Pdcd4 protein in many cases (Figure 6a). Even though we observed a weak negative correlation (P=0.03) between miR-21 and Pdcd4-mRNA in resected tumors, as reported in another tumor entity (Roldo et al., 2006), there was no significant difference between Pdcd4-mRNA levels in tumor and corresponding normal tissues (Figure 6c, P=0.36).

Figure 6.

miR-21 expression is negatively associated with Pdcd4 protein, and is significantly upregulated in 22 resected colorectal tumors. (a) Representative examples for 8 of 22 matched normal/tumor tissues (N/T) investigated. Numbers below the western represent ratios Pdcd4/-Actin (densitometry). Below: normalized Pdcd4-mRNA and miR-21. Bold: tumor tissue. (b) Relative miR-21 amounts were determined in 22 cases (TaqMan-miRNA-assay) and expressed as fold change after normalization to U6-snRNA. (c) Relative Pdcd4-mRNA was determined by TaqMan-qRT–PCR and expressed as fold change after normalization (-Actin-mRNA). (d) Pdcd4-protein was quantified as the ratio Pdcd4/-actin. Significant differences: P=3.89E-06; P<0.005; NS not significant.

Full figure and legend (88K)

This in vivo data support the notion that Pdcd4 is negatively regulated by miR-21 at the post-transcriptional level.

Discussion

This is the first study to show that tumor suppressor Pdcd4 is negatively regulated by miR-21 at the post-transcriptional level, via a specific target site (nt228–249) within the 3'-UTR. It is also the first study to demonstrate that miR-21 induces invasion/intra-vasation/metastasis in colorectal cancer cells. The in vivo relevance of these findings is supported by correlational studies in resected tissues of colorectal cancer patients, where high miR-21 correlates significantly with low Pdcd4 protein.

Besides being a suppressor of malignant transformation (Cmarik et al., 1999; Yang et al., 2003), tumorigenesis, tumor progression (Jansen et al., 2005), Pdcd4 has been shown to be upregulated in apoptosis (Shibahara et al., 1995; Zhang and DuBois, 2001) and cellular senescence (Kang et al., 2002). This is interesting since miR-21 has been shown to act as an antiapoptotic factor in glioblastoma cells (Chan et al., 2005). Previous studies reported that miR-21 can inhibit apoptosis by regulating Bcl-2 in a breast cancer mouse model (Si et al., 2007), and that gemcitabine-induced apoptosis is specifically inhibited by miR-21 via PTEN and the PI-3-kinase pathway (Meng et al., 2006). Based on our present study showing that miR-21 is a negative regulator of Pdcd4, it is interesting to additionally speculate that miR-21 might achieve antiapoptosis, at least in part, via negatively regulating Pdcd4. Since in our transfection experiments (Figure 4) Pdcd4-mRNA was unaltered as opposed to a significant change in Pdcd4-protein, we propose that the main mechanism of miR-21-induced Pdcd4 suppression is post-transcriptional. However, since there was also a trend for correlation between Pdcd4-mRNA and miR-21 in cell lines and tissues, we cannot exclude the possibility that additional means of Pdcd4-regulation such as affecting mRNA-stability (Esquela-Kerscher and Slack, 2006), add to miR-21-induced Pdcd4-suppression.

Furthermore, Pdcd4 has been shown to inhibit invasion and intravasation, this being achieved in part by inhibiting matrix-metalloproteinase activation, and u-PAR-gene expression (Yang et al., 2006; Leupold et al., 2007). We now demonstrate that miR-21 is a potent stimulator of the invasive/intravasative capacity of colorectal cancer cells. Thus, the stimulation of invasion by miR-21 might in part be mediated via a negative regulation of Pdcd4. The observation of others (Roldo et al., 2006; Si et al., 2007) that miR-21 also acts as an activator of tumor cell proliferation was also observed in our present study (see Supplementary Figure 2), nevertheless, this effect on proliferation (observed after 6-days) was not as prominent in our study as the stimulating effect of miR-21 on invasion (observed after 12 h), which excludes a bias of the invasion assays by an additional effect on proliferation. Certainly, additional mechanisms and targets of miR-21 besides Pdcd4 are likely to contribute to miR-21-induced tumor cell invasion. However, these additional mechanisms still need to be elucidated. In general, our present work on miR-21 does not exclude other important mechanisms of Pdcd4-regulation in tumors, such as the regulation by phosphorylation-induced degradation of Pdcd4 by ubiquitin ligase -TRCP and the proteasome, following mitogen stimulation (Dorrello et al., 2006).

miR-21 is one of the most prominent miRNAs implicated in the genesis and progression of human cancer. Not only has it been implicated in the promotion of tumor growth (Si et al., 2007), proliferation (Roldo et al., 2006), antiapoptosis (Chan et al., 2005) and response to gemcitabine-based chemotherapy (Meng et al., 2006), but diverse studies have shown that miR-21 is being overexpressed in different tumor types. Thus, Si et al. (2007), investigated 157 miRNAs in five human breast cancers and matched normal tissues, and found an overexpression of miR-21 in tumors, confirming an earlier study on human breast cancer samples of Chan et al. (2005), Iorio et al. (2005) found an overexpression of miR-21 in glioblastoma tissues, and Meng et al. (2006) reported that malignant cholangiocytes overexpress miR-21, besides other miRNAs such as let7a-1/miR-16-1. Roldo et al. (2006) compared expression patterns of miRNAs, applying a global screening method, between normal pancreas and pancreatic cancers of different histological types and identified a miRNA expression pattern able to distinguish any pancreatic tumor type from normal pancreatic tissue. In their study, high miR-21 was associated with the presence of liver metastasis, supporting our observation that miR-21 induces invasion-related processes. One of the largest and most systematic studies on miRNA expression was published by Lu et al. (2005) and determined several patterns of differentially expressed miRNAs, some of them again including miR-21, in diverse human tumors, considering potential biases by factors such as tumor differentiation. Taken together, a number of studies including ours support the hypothesis that miR-21 might be one of the most relevant oncogene-like factors (Esquela-Kerscher and Slack, 2006) among the class of miRNAs. Our additional observation that Pdcd4 is not regulated by miR-141, although a putative binding site for this miRNA was found within the 3'-UTR of Pdcd4 mRNA, supports the specificity of our findings for miR-21 and emphasizes that the presence of a putative miRNA-target sequence does not necessarily imply functionality.

In conclusion, our present study suggests that tumor suppressor Pdcd4 is negatively regulated at the post-transcriptional level by miR-21 via a specific target motif at nt228–249 of the Pdcd4-3'-UTR. Furthermore, miR-21 induces invasion/intra-vasation/metastasis in colorectal cancer cells. This together with our correlational data in resected patient colorectal tumors, and previous translational clinical studies on miR-21 and Pdcd4, implicates that inhibitory strategies against miR-21, strategies interfering with the miR-21/Pdcd4-interaction, or rescuing Pdcd4-expression, will have a strong rationale for therapeutic applications in cancer in the future.

Materials and methods

Materials, antibodies, cell lines and patient tissues

Media/FBS were purchased from Invitrogen/Gibco (Karlsruhe, Germany) and Sigma (Taufkirchen, Germany), transwell chambers (1 cm², 12 m pores) from Machery-Nagel (Düren, Germany), Matrigel from BDBiosciences (Bedfird, MA, USA), control-miR, pre-miR21, control-anti-miR and anti-miR-21 from Ambion (Austin, TX, USA). Taqman primer-probe for quantification of miR-21 and Pdcd4 were from Applied Biosystems (Foster City, CA, USA), oligonucleotides from Metabion (Martinsried, Germany), PTEN-antibody from Cell Signalling and -actin-antibody from Sigma. An affinity-purified antibody against carboxy-terminal Pdcd4 (Jansen et al., 2005) was used as described. Human colorectal cancer cell lines RKO, Hct116, Hct15, HT29, CaCo2, Colo206F, Colo320, Sw480 and Widr were from ATCC, Geo was a gift from D Boyd (MD Anderson Cancer Center, Houston, TX, USA). Tissue specimens (tumor, adjacent normal mucosa) of 22 patients with colorectal cancer were collected after informed consent and verification by a pathologist, and immediately frozen in liquid nitrogen.

Construction of 3'-UTR-luciferase plasmid and reporter assays

The full-length 3' UTR of Pdcd4-isoform1 (632 nt) was amplified using cDNA from MCF7 (primers Fwd-5'-GAATCTAGAATATAAGAACTCTTGCAGTC-3', Rev-5'-CTTCTAGAACCAGGTTCATTTTTCC-3'), cloned into the Xba1-site of pGL3 (Promega, Madison, WI, USA), checked for orientation, sequenced and named Luc-Pdcd4Wt. Site-directed mutagenesis of the miR-21 target-site in the Pdcd4-3'-UTR was carried out using Quik change-mutagenesis kit (Stratagene, Heidelberg, Germany), with Luc-Pdcd4Wt as a template. For reporter assays, Colo206f, RKO and Hct116 were transiently transfected with wt or mutant reporter plasmid and microRNA (as indicated in Figure 3) using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Reporter assays were performed 36 h post-transfection using the Dual-luciferase-assay-system (Promega), normalized for transfection efficiency by cotransfected Renilla-luciferase.

Preparation of cell/tissue lysates, western blots

Cells were washed with phosphate-buffered saline and lysed in extraction-buffer (Biosource, Camarillo, CA, USA). 50–100 mg tissues were homogenized using a dismembrator, and proteins were extracted. Protein concentration was determined by BCA (Pierce, Rockford, IL, USA). Aliquots (25 g) were separated on a 10% SDS–PAGE and transferred to nitrocellulose membrane. The membrane was incubated with the specific antibody followed by horseradish–peroxidase-linked immunoglobulin G, and visualized by chemiluminescence (ECL, Amersham, Freiburg, Germany).

Real-time PCR-based detection of miR-21 and Pdcd4-mRNA

Total RNA from cells or human normal tissue/matched tumor samples was extracted using Trizol (Invitrogen). Expression of mature miRNAs was determined by the TaqMan miRNA-assay (Applied Biosystems, Foster City, CA, USA), and normalized using the 2^-DDC_T-method (Pfaffl, 2001) relative to U6-snRNA. Pdcd4-mRNA was quantified by TaqMan-qRT–PCR and normalized to -Actin (AppliedBiosystems). All TaqMan-PCRs were performed in triplicates.

Matrigel invasion

Cells were transfected with 50 nM control-anti-miR, anti-miR-21, control-miR, or pre-miR-21 respectively. Post-transfection cells (48 h) were trypsinized, and 0.1 10⁶ cells plated on transwell chambers precoated with 20 g Matrigel. Medium-containing 10% FBS in the lower chamber served as chemoattractant. After 12/24 h, non-invading cells were removed with cotton swabs. Invading cells were trypsinized and counted using the ATP-luminescence-based motility-invasion assay as described (Leupold et al., 2007). Remaining cells were plated into a 6-well plate for protein- and RNA-isolation 24 h later.

CAM assay

The CAM assay was performed as described previously (Zijlstra et al., 2002). Genomic DNA from lower CAM and lungs were prepared using Puregene DNA purification system. Quantification of human cells in the extracted DNA was done as described (Leupold et al., 2007). Fluorogenic TaqManQPCR probes were applied as above, and DNA copy numbers were quantified.

Statistical analysis

This was performed using StatView5.0. (Macintosh/Windows). Values were expressed as means.e.m. Differences/correlations between groups were calculated with Student's t-, Pearson, or Wilcoxon-test. A P-value<0.05 was defined as significant, a P-value<0.1 as a trend.

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Acknowledgements

HA was supported by Alfried Krupp von Bohlen und Halbach Foundation, Essen, Wilhelm-Sander-Stiftung, Munich, Auguste-Schaedel-Dantscher-Stiftung, Garmisch, Germany, Dr Hella-Buehler-Foundation, Heidelberg, B Braun Foundation, Malsung, The Hector Foundation, Weinheim and Dr Ingrid-zu-Solms Foundation, Frankfurt, Germany. We thank Dr Laura Nelson for critical reading of the article. This publication contains parts of the dissertation of Irfan A Asangani performed in partial fulfilment of the requirements for the PhD at DKFZ/University-Heidelberg.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).