(2012) 31, 3796–3806 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE The immediate early Ier2 promotes tumor cell motility and metastasis, and predicts poor survival of colorectal cancer patients

A Neeb1,5, S Wallbaum1,5, N Novac1,5, S Dukovic-Schulze1,2, I Scholl1, C Schreiber1,2, P Schlag3, J Moll1, U Stein4 and JP Sleeman1,2

1Karlsruhe Institute of Technology, Institut fu¨r Toxikologie und Genetik, Karlsruhe, Germany; 2Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany; 3Charite´ Comprehensive Cancer Center, Invalidenstrae 80, Berlin, Germany and 4Experimental and Clinical Research Center, Charite´ University Medicine Berlin at the Max-Delbru¨ck-Center for Molecular Medicine, Berlin, Germany

Here, we report unbiased screens for expressed in penetrate vessels of the circulatory system and are metastatic tumor cells that are associated with cell transported to distant sites where they need additional motility. These screens identified Ier2, an immediate early properties to form metastatic lesions. Much remains to gene of unknown function, as potentially having a role in be discovered about the changes in tumor cell motility and metastasis. Knockdown of Ier2 in required for metastasis. The identification of genes 3T3 fibroblasts inhibited their motility upon relief of specifically expressed or suppressed in metastatic tumor contact inhibition in monolayer wounding assays. Further- cells compared with their non-metastatic counterparts more, ectopic Ier2 expression promoted the motility and will not only aid in the understanding of the molecular invasiveness of poorly metastatic 1AS pancreatic tumor regulation of metastasis but will also have potential cells in vitro. Relief of contact inhibition was associated applications in the development of novel diagnostic and with translocation of the Ier2 protein from the cytoplasm therapeutic tools for use in the care of cancer patients. to the nucleus in both 3T3 fibroblasts and 1AS tumor cells. We have previously used suppression subtractive Importantly, ectopic Ier2 expression in 1AS cells stimu- hybridization to identify a library of genes the expres- lated metastasis formation when cells were implanted into sion of which correlates with the metastatic potential of experimental animals. Furthermore, we found elevated tumor cells (Nestl et al., 2001). Here, we describe the Ier2 expression in a wide variety of human tumor types. further screening of this library to identify genes that This correlated with poor metastasis-free and overall might have a role in the migration of metastasizing cells. survival in patients with colorectal adenocarcinomas. This was achieved by looking for the differential Together, these data reveal Ier2 as a new player in the expression of the library genes in activated and non- regulation of tumor progression and metastasis, and activated T lymphocytes. Antigen-activated T cells share suggest that Ier2 may be useful prognostically and many properties with metastatic tumor cells (Herrlich therapeutically in the management of cancer. et al., 1993). We reasoned that genes commonly Oncogene (2012) 31, 3796–3806; doi:10.1038/onc.2011.535; expressed by both activated T cells and metastatic published online 28 November 2011 tumor cells may be functionally relevant for tumor metastasis. One gene identified by the secondary screen- Keywords: Ier2; Pip92; ETR101; metastasis; motility; ing of the subtractive library was Ier2, the expression of invasion which was found to be elevated in both activated lymphocytes and in various metastatic tumor cells. Ier2 (also called ETR101, Pip92, 3CH92 and CHX1, hereafter referred to as Ier2, regardless of species) is an Introduction immediate early gene ( Rollins and Stiles, 1989) that was originally found to be upregulated in fibroblasts upon Tumor metastasis is a highly complex molecular and their activation (Lau and Nathans, 1985). Immediate cellular process (reviewed in Sleeman et al. (2011)). early genes are a group of genes that are rapidly induced Local invasion of tumor cells involves changes in in quiescent cells by proliferation- and migration- adhesive properties, activation of proteolysis and inducing stimuli. Mutation or inappropriate expression acquisition of cell motility. Thereafter, tumor cells of several of these genes (for example, members of the fos, jun and nuclear factor-kB gene families) has been Correspondence: Professor JP Sleeman, Universita¨tsmedizin shown to cause them to function as and/or to Mannheim, University of Heidelberg, Centre for Biomedicine and contribute to tumor progression (reviewed in Aggarwal Medical Technology Mannheim (CBTM), TRIDOMUS-Geba¨ude (2004) and Milde-Langosch (2005). Haus C, Ludolf-Krehl-Str. 13–17, Mannheim D-68167, Germany. Here, we show that expression of Ier2 correlates with E-mail: [email protected] 5These authors contributed equally to this work. the metastatic phenotype in a wide range of rodent and Received 28 July 2011; revised 7 October 2011; accepted 17 October human tumor types. Ectopic expression of Ier2 in poorly 2011; published online 28 November 2011 metastatic pancreatic tumor cells promotes their metas- Ier2 and metastasis A Neeb et al 3797 tasis in vivo and enhances their motility and invasiveness non-metastatic cells in a broad panel of tumor cell lines. in culture. Furthermore, high expression of Ier2 in To this end, we performed northern blots using RNA human colorectal carcinoma patients was found to be derived from rat pancreatic, mammary and prostate significantly associated with short metastasis-free survi- tumor cell lines with verified metastatic potential in vivo. val and poor overall survival. Therefore, these data Only one of these nine clones was consistently upregu- identify Ier2 as a metastasis-relevant immediate early lated in a metastasis-relevant manner in all cell lines gene the expression of which represents a prognostic tested (Figure 1a). Sequence analysis of this clone marker and a potential target for cancer therapy. revealed a 128-bp insert with complete homology to the 50 coding sequence of Ier2. To confirm the RNA expression data at the protein Results level, we generated and affinity purified polyclonal anti- Ier2 antibodies that cross-reacted with the rat, mouse Ier2 is expressed in both activated T cells and metastatic and human Ier2 protein. Western blots of lysates from tumor cells tumor cell lines with defined metastatic potential in vivo 1AS pancreatic carcinoma cells form primary tumors were probed with these antibodies. These data con- but rarely metastasize in vivo, whereas ASML cells firmed that constitutive Ier2 protein expression in tumor derived from the same primary tumor as 1AS cells are cells correlates with metastatic potential (Figure 1b). highly metastatic and disseminate from subcutaneous sites through the lymphatics to colonize the lungs (Nestl Ier2 expression can promote cell motility et al., 2001). We have previously described a subtractive Activation of resting lymphocytes and stimulation of library that was created through suppression subtractive quiescent fibroblasts increase their migratory activity hybridization comparison of these two cell lines (Nestl (Herrlich et al., 1993; Bradley, 2003; Li et al., 2004). As et al., 2001). To identify genes in this library that may previously reported, we found that concanavilin A- have a role in invasive migration, we screened the library activated splenocytes and serum-stimulated quiescent to identify transcripts that are upregulated in activated murine 3T3 fibroblasts exhibit immediate early expres- T cells in comparison with non-activated T cells. For sion of Ier2 (Figure 1, Supplementary Figure 1a). this purpose, inserts of the cDNA library were PCR Therefore, we investigated whether Ier2 expression amplified and differentially hybridized with radiolabeled might influence cell motility, as enhanced motility is probes derived from concanavilin A-activated and non- associated with metastasis. activated splenocytes. Nine clones from the library were Using the well-established 3T3 fibroblast model system found to be upregulated in concanavilin A-treated cells for investigating cell motility, we first knocked down Ier2 compared with non-treated cells (data not shown). expression (Supplementary Figure 1b) and examined the We further screened the nine clones to identify those effect on motility after relief of contact inhibition in dense that are upregulated in metastatic cells compared with monolayers. To this end, we included a silicon insert in cell cultures during establishment of the monolayer that was subsequently removed to create a cell-free area, thereby relieving contact inhibition. Serum-starved fibroblasts filled the cell-free area more slowly than did serum- stimulated cells. In both cases, knockdown of Ier2 retarded closure of the cell-free area. This retardation was most pronounced in serum-stimulated cells (Figure 2). Knock- down of Ier2 had no effect on cell proliferation or apoptosis (Supplementary Figure 2), supporting the notion that Ier2 supports the motility of fibroblasts. To examine the consequences of constitutively aug- mented Ier2 expression in non-transformed cells, we used the ecdysone-based Rheoswitch-inducible expres- sion system to ectopically express Ier2 conditionally in 3T3 fibroblasts (Wallbaum et al., 2009). In these cells, Ier2 expression is upregulated after 3 h of treatment with the Rheoswitch ligand RSL1, and can be maintained in cycling cells over several days at a level that is above the Figure 1 (a) Northern blot and (b) western blot showing expression peak induction levels achieved in serum-stimulated post- of Ier2 in a panel of pancreatic (panc), prostate and mammary quiescent fibroblasts (Supplementary Figure 3). Using carcinoma cell lines with different metastatic potential. The northern blot was probed sequentially with Ier2 and GAPDH probes. The this system, we observed no significant increase in cell western blot was probed with anti-Ier2 antibodies and with anti- motility upon induction of Ier2 over and above that vinculin antibodies as a loading control. It must be noted that ASML supported by endogenous Ier2 (Figure 2c). Therefore, cells are deficient for vinculin expression. The metastatic potential of these data suggest that Ier2 expression has a role in the cell lines in vivo is indicated, with * indicating poorly metastatic fibroblast motility, as evidenced by the knockdown data, cells and *** indicating strongly metastatic cells. Induction of Ier2 expression in ConA-activated T cells, as well as in serum-starved 3T3 but that above a certain threshold of Ier2 expression, no fibroblasts treated for 45 min with 20% serum is also shown. further increase in motility can be induced.

Oncogene Ier2 and metastasis A Neeb et al 3798

Oncogene Ier2 and metastasis A Neeb et al 3799 Next, we examined the subcellular distribution of Ier2 had similar effect on these parameters as in transiently before and after relief of contact inhibition subsequent transfected cells. The clones were then injected subcuta- to removal of the silicon insert. In the densely growing neously into syngeneic rats, and tumors were allowed to fibroblast monolayer before relief of contact inhibition, grow to 5 cm in one dimension. Animals were then killed Ier2 was cytoplasmic and largely excluded from the and autopsied for evidence of metastases. nucleus. Lack of incorporation of 5-bromo-20-deoxyur- Ier2 expression had no effect on the growth of primary idine into cells confirmed that contact inhibition induced tumors (Figure 4a). However, the size of the axillary quiescence. Three hours after relief of contact inhibition, lymph nodes ipsilateral and regional to the primary tumor the Ier2 protein translocated to the nucleus in cells on was significantly increased in comparison with the the edge of the monolayer. Nuclear translocation was contralateral axillary lymph nodes with all Ier2-expressing even more pronounced after 6 h as cells at the edge of independent 1AS clones tested. In contrast, no significant the monolayer delaminated and began to migrate into increase in the size of the ipsilateral axillary lymph nodes the cell-free area (Figure 2d). was observed with any of the independent vector- transfected 1AS clones tested (Figure 4b). Examination Ier2 expression promotes invasiveness of 1AS tumor cells of the enlarged lymph nodes draining Ier2-expressing To determine whether Ier2 expression in tumor cells can tumors revealed them to be full of metastatic tumor cells also increase motility and influence invasiveness, we (Figure 4c). Together, these data suggest that ectopic Ier2 created an expression construct for Ier2 and used it to expression is sufficient to stimulate the metastasis of 1AS transiently transfect poorly metastatic 1AS tumor cells. tumor cells to regional lymph nodes. Transient expression of Ier2 had no effect on the proliferation, apoptosis or adhesion of 1AS cells (Supple- Ier2 expression is upregulated in many human tumor types mentary Figure 4). We then determined whether the speed and correlates with poor metastasis-free and overall of closure of scratch wounds made in monolayers of Ier2- survival transfected 1AS cells was different compared with vector- The data above suggest that Ier2 expression can be transfected control cells. As can be seen in Figure 3a, Ier2 sufficient to promote metastasis in experimental tumors. expression substantially and consistently increased the To determine whether Ier2 expression is of relevance to speed of closure of wounded monolayers. Similar to the human tumors, we used affinity-purified polyclonal anti- case with non-transformed fibroblasts, relief from contact Ier2 antibodies to perform immunohistochemistry on inhibition resulted in a nuclear localization of Ier2 at the human tumor sections. We found that Ier2 is robustly edge of the monolayer (Figure 3b). overexpressed in a number of different types of primary In transwell motility/invasion assays, 1AS cells tumors compared with the corresponding non-trans- transfected with either Ier2 or empty vector were seeded formed tissue (Table 1), including cancers of the onto Matrigel-coated filters of transwell migration pancreas, breast, endometrium, cervix, liver and intes- chambers, and migration across the filter in response tine (Figure 5a). to a chemotactic gradient was assessed. As shown in To determine whether Ier2 expression is associated Figure 3c, ectopic expression of Ier2 dramatically with tumor metastasis in human tumors, we performed increased cell invasion and migration across the filter. quantitative reverse transcriptase–PCR analysis using Together, these data demonstrate that Ier2 expression microdissected tumor material obtained from 39 pa- can promote tumor cell motility and invasiveness. tients with colorectal adenocarcinomas. None of them had metastases at surgery, but 17 developed distant metastases metachronously. Analysis of Ier2 expression Ectopic Ier2 expression promotes metastasis of in these samples revealed that expression of Ier2 in experimental tumors to regional lymph nodes primary tumors of these patients is a strong predictor of To determine whether Ier2 expression influences tumor poor prognosis (Figure 5b). Importantly, Ier2 expres- metastasis in vivo, we stably transfected 1AS cells with the sion significantly correlates with a short metastasis-free Ier2 expression construct (Supplementary Figure 5). survival (P ¼ 0.0338) and with a poor overall survival Clones of 1AS cells either ectopically expressing Ier2 or (P ¼ 0.0029). transfected with the empty vector were checked in the Together, these data indicate that tumor cells can motility, proliferation apoptosis, adhesion and invasion constitutively express Ier2 in a manner that correlates assays outlined above to verify that stable Ier2 expression with tumor progression, metastasis and poor prognosis.

Figure 2 Ier2 promotes motility and translocates from the cytoplasm to the nucleus upon relief of contact inhibition in 3T3 fibroblasts. Confluent monolayers of 3T3 fibroblasts that had been transfected with either scrambled (Control) or Ier2 siRNA (Ier2 knockdown) were grown in the presence of a silicon insert, and either (a) serum-starved or (b) stimulated with 20% serum. Upon removal of the silicon insert, movement of cells into the resulting cell-free area was monitored microscopically. The reduction in cell-free area was calculated at the indicated time points. The data represent mean and s.e. of three independent experiments. (c) Monolayers of Ier2-inducible 3T3 fibroblasts grown in the presence of a silicon insert were treated with either RSL1 (RSL1) or solvent control (DMSO). Upon removal of the silicon insert, movement of cells into the resulting cell-free area was monitored microscopically and reduction in cell-free area was calculated at the indicated time points. The data represent mean and s.e. of three independent experiments. (d) Confluent monolayers of 3T3 fibroblasts were grown densely under normal culture conditions for 64 h in the presence of a silicon insert. Cells were BrdU pulsed (10 mM)3hbeforefixation. Upon removal of the insert to relieve contact inhibition, cells were immunostained at the indicated time points with the anti-Ier2 antibody 1A2 (green) and anti-BrdU antibodies (red). DAPI staining (blue) was used to visualize nuclei.

Oncogene Ier2 and metastasis A Neeb et al 3800

Figure 3 (a) Ier2 expression promotes the closure of monolayer wounds. 1AS cells were transiently transfected with Ier2 expression vector (P) or empty vector (V) 24 h before the experiments. The expression of Ier2 was ensured on western blots probed with the V5 antibody. Subsequent probing of the blot with anti-b-actin antibodies served as a loading control. Monolayers were wounded and the position of the wound marked. Migration of cells into the monolayer wound was assessed 24 h later (see photographs; lines represent the original edges of the wound). The number of cells migrating into the wounded area was quantified. The mean and s.e. of four representative independent 800 mm2 fields of view is presented. (b) 1AS cells were grown as confluent monolayers, as wounded monolayers and as single cells. Immunostaining was performed with the anti-Ier2 antibody 1A2 (green) and anti-FAK (red) antibodies. Similar to the situation with 3T3 fibroblasts, Ier2 is located in the cytoplasm in densely growing cells, but translocates to the nucleus upon relief from contact inhibition and in sparsely growing single cells. (c) Ier2 expression promotes migration and invasion. Two independent 1AS cell cultures were stably transfected with the Ier2 expression construct or with empty vector, and cultivated as total pooled selected cells. Cells were plated onto the surface of Matrigel-coated transwell chambers. After 6 h, the number of cells that had invaded through the Matrigel was determined.

Oncogene Ier2 and metastasis A Neeb et al 3801 Discussion constitutively upregulated in various tumor types in comparison with non-transformed cellular counterparts Here, we report a functional role for Ier2 in tumor cell (Gutmann et al., 2002; Hippo et al., 2002; Yu et al., motility and metastasis. A number of reports have 2004; French et al., 2005; Skotheim et al., 2005). Ier2 has previously associated Ier2 expression with neoplasia. In also been reported to be induced in gastric epithelial contrast to the transient increased expression typical of cells in response to Helicobacter infection (Chiou et al., immediate early genes, a number of gene expression 2001). Moreover, the Tax protein of the human T-cell profiling studies indicate that Ier2 expression can be leukemia virus type-1 upregulates the expression of Ier2 at the transcriptional level (Chen et al., 2003). The results we present here extend these observations and demonstrate that Ier2 expression can promote tumor cell motility and metastasis in animal models, is strongly upregulated in a wide variety of human tumors in comparison with the corresponding non-transformed normal tissue and is a predictor of poor metastasis-free and overall survival of patients with colorectal adeno- carcinomas. Although little is known about the function of Ier2, its expression has been shown to be induced in a wide range of biological contexts. It is activated upon stimulation of quiescent fibroblasts, T cells and other cell types with serum, 12-O-tetradecanoylphorbol-13-acetate (TPA) or growth factors (Charles et al., 1990; Coleclough et al., 1990; Scott et al., 1994; Fambrough et al., 1999; Keeton et al., 2002). Ier2 is also associated with apoptosis, as its expression is induced in after N-methyl-D- aspartic acid (NMDA) administration in vivo, and in fibroblasts after anisomycin treatment (Chung et al., 2000a, 2000b). Furthermore, Ier2 is expressed in the ischemic , and its ectopic expression induces apoptosis in this context (Schneider et al., 2004). Ier2 expression also correlates with cell differentiation, for example, during neuronal differentiation of PC12 and H19-7 cells (Charles et al., 1990; Eschelbach et al., 1998; Chung et al., 1998), and after TPA-induced differentia- tion of HL-60 cells (Shimizu et al., 1991). During embryonic development, Ier2 has been reported to determine left–right asymmetry patterning in an fibro- blast growth factor (FGF)-dependent manner through interacting with fibp1 (Hong and Dawid, 2009).

Figure 4 Ectopic expression of Ier2 promotes metastasis in vivo. Groups of eight rats were injected subcutaneously with clones of 1AS cells ectopically expressing Ier2 (P35, P38, P40 and P43) or with control clones transfected with empty vector (V14, V15, V17). Tumor volume was regularly monitored. (a) Animals were killed once their tumors exceeded 50 mm in one dimension. Ier2 expression does not affect the growth of tumors in vivo. The volume of all tumors was compared at the time the first animal needed to be killed. The mean and s.e. for all eight animals in each group is depicted. (b) Development of metastases in the axillary lymph nodes draining the primary tumors. At autopsy, the volume of the axillary lymph nodes ipsilateral (ip) and contralateral (co) to the primary tumor was measured. The contralateral lymph nodes served as a negative control for metastasis formation. The volume of each axillary lymph node is plotted for all eight rats injected with the different 1AS clones. The volume of the ipsilateral and contralateral lymph nodes for each clone was statistically compared using the Mann–Whitney rank test. (c) Representative H and E staining of a section of a paraffin-embedded enlarged ipsilateral lymph node taken from a rat bearing an Ier2-expressing tumor. The staining demonstrates that most of the tissue within the lymph node is composed of metastatic tumor cells.

Oncogene Ier2 and metastasis A Neeb et al 3802 Table 1 Expression of Ier2 in human tumors and their corresponding non-neoplastic tissue Tumor type Tumor Matched non-neoplastic tissue

Gastric carcinoma 3/10 þ Strong staining of parietal cells 7/10 À Esophagus SCC 6/8 þþ/ þþþ Strong epithelial staining 1/8 ( þ )/ þ 1/8 À Lung cancer 3/10 þþ À 5/10 ( þ )/ þ 2/10 À Colorectal carcinoma 5/10 þþ/ þþþ À 5/10 ( þ )/ þ Thyroid papillary carcinoma 9/10 þþ/ þþþ Variable epithelial staining, weak to strong 1/10 þ Kidney RCC 7/9 þþ/ þþþ Renal corpuscles negative 2/9 ( þ ) Proximal convoluted tubules weakly positive Distal convoluted tubules strongly positive Mammary carcinoma 7/8 þþ/ þþþ 3/8 À 1/8 þ 5/8 Heterogeneous staining of ductal epithelium Heterogeneous staining Hepatic carcinoma 6/10 þþ/ þþþ À 2/10 ( þ )/ þ ) 2/10 À Urinary bladder carcinoma 5/8 þþ Epithelial layer luminally positive 2/8 ( þ )/ þ 1/8 À Ovarian carcinoma 3/10 þþ/ þþþ ( þ )/À 5/10 ( þ )/ þ 2/10 À Heterogeneous staining Pacreatic carcinoma 5/10 þþ/ þþþ Strong staining of intracalated and intercalated ducts 5/10 ( þ )/ þ Islets of Langerhans weakly positive Prostate carcinoma 4/9 þþ þ/À weak heterogeneous epithelial staining 4/9 ( þ )/ þ 1/9 À Endometrial carcinoma 3/9 þþ/ þþþ À/ þ 3/9 þ /( þ ) 3/9 À Gall bladder carcinoma 1/6 þþþ À 4/6 þ 1/6 À Head and neck SCC 3/8 þ / þþ Weak staining of salivary glands 2/8 ( þ )/ þ 3/8 À heterogeneous staining Cervical carcinoma 7/10 þþ/ þþ À/ þ 3/10 ( þ )/ þ Lymphoma 10/10 ÀÀ/ þ Melanoma 4/9 ( þ )/ þÀ/ þ 6/9 À

Abbreviations: RCC, renal cell carcinoma; SCC, squamous cell carcinoma. Staining of multiple tissue arrays of human tumors with matched normal tissues with anti-Ier2 antibodies. Biocat common cancer arrays MA, MB and MC and matched normal tissue arrays MAN, MBN and MCN were used. Full details of the histopathology of the sections on these multiple tissue arrays can be found on http://www.biocat.de. Staining was graded: À: no staining; þ : weak staining; þþ: moderate staining; þþþ: strong staining.

The Ier2 protein contains two putative nuclear influences subcellular localization of the Ier2 protein. localization signals (Shimizu et al., 1991) and there is In particular, release of cells from contact inhibition some limited homology to the nuclear proteins JunB and resulted in a translocation of Ier2 from the cytoplasm to JunD (Coleclough et al., 1990; Scott et al., 1994). the nucleus. Given recent evidence suggesting a direct Published evidence to date using nuclear/cytoplasmic role in transcriptional regulation for Ier2 (Takaya et al., fractionation techniques suggests that Ier2 localizes 2009), the regulated compartmentalization of Ier2 is exclusively in the cytoplasm of 3T3 and PC12 cells presumably an important means by which the cell (Charles et al., 1990), although at least a partial nuclear regulates the activity of this protein. localization has also been reported (Chung et al., 2000a; Our data indicate that Ier2 expression in 3T3 Takaya et al., 2009). Furthermore, Ier2 can transcrip- fibroblasts and 1AS pancreatic tumor cells promotes tionally regulate the ISYNA1 (Takaya et al., cell motility. The enhanced motility and invasiveness of 2009). Here, we show that cell density strongly 1AS cells we observed upon ectopic Ier2 expression is

Oncogene Ier2 and metastasis A Neeb et al 3803

Figure 5 (a) Immunohistochemical staining of Ier2 in various human tumor types (large panels) and the corresponding non- transformed tissues (small panels). Positive staining is indicated by brown coloration, the blue coloration is the hematoxylin counterstaining. (b, c) Ier2 expression correlates with poor metastasis-free (panel b) and overall survival (panel c) of patients with colorectal adenocarcinomas. Ier2 expression was analyzed by qRT–PCR, and compared with time to metastasis development and survival of the patients. The Kaplan–Meier plots show the proportion of patients with and without metastasis against time (panel b) and the proportion of patients surviving against time (panel c). likely to contribute to the induction of metastasis that in serum-induced post-quiescent fibroblasts did not occurred when cells were injected into experimental further enhance motility, suggesting that there may be animals. However, we also observed that enhanced Ier2 an upper threshold to Ier2-induced motility. Further- expression does not invariably promote motility. In the more, using RSL1-inducible Ier2 expression in case of 3T3 cells, although knockdown of Ier2 reduced non-transformed NMuMG mammary epithelial cells motility after relief of contact inhibition, increased Ier2 (Wallbaum et al., 2009), we also did not observe expression over and above endogenous levels observed increased motility in response to induction of Ier2

Oncogene Ier2 and metastasis A Neeb et al 3804 expression (data not shown). In 1AS tumor cells, acids 25–142 of GenBank accession number M59821) was endogenous levels of Ier2 may be lower than the cloned downstream of and in-frame with the GST gene in the putative upper threshold, explaining why ectopic Ier2 pGEX-1 expression vector, and soluble GST–Ier2 fusion expression was able to increase motility. Consistent with protein was produced as described previously (Smith and this notion, we observed that expression levels of Ier2 in Johnson, 1988). Rabbits were immunized with the fusion serum-induced post-quiescent fibroblasts were higher protein and bleeds were assessed for anti-Ier2 activity in ELISA (enzyme-linked immunosorbent assay) assays. Positive than the constitutive expression in tumor cells serum was purified using GST-sepharose to remove anti-GST (Figure 1b). Alternatively, an upper threshold may not antibodies, followed by affinity purification over GST-Ier2 exist in tumor cells. Either way, these data indicate that sepharose. The same fusion protein was used to produce the ability of Ier2 to promote motility is likely to be cell monoclonal antibodies. Immunization of Balb/C mice with the context dependent. fusion protein and production of hybridomas were performed As outlined above, some studies have shown en- as described previously (Harlow and Lane, 1998; Niebuhr hanced Ier2 expression during the induction of apopto- et al., 1998). Hybridoma supernatants were differentially sis, and there is some evidence that Ier2 expression may screened in ELISA assays for their ability to bind to GST- be able to promote apoptosis (Schneider et al., 2004). Ier2 but not to the GST protein. One of these monoclonal However, in the experiments presented here, we found antibodies (1A2) was found to bind to endogenous mouse and rat Ier2 in a number of immunoassays (Wallbaum et al., 2009). no evidence for apoptosis induction in response to Ier2 expression. Therefore, any role for Ier2 in the induction of apoptosis may be dependent on the levels and kinetics Cloning of a Ier2 expression construct of Ier2 protein expression, as well as on the cellular The entire coding sequence of Ier2 was amplified by reverse transcriptase–PCR and cloned in-frame with the V5 tag in the context. pcDNA3.1. vector. The inclusion of the C-terminal V5 In summary, the data we present here provide tag permitted identification of ectopically expressed Ier2 evidence of a role for Ier2 in regulating cell motility. in transfected cells. The following primers were used: In the context of tumor cells this translates into 50-GCGGATCCTATGGAAGTACAGAAAGAAGCGCAGC increased invasiveness and an enhanced ability to form GCATCATGACTGTC-30 and 50-CGCAAGCTTGAAGGC metastases. Accordingly, expression of Ier2 in the CACCACAGCTCGCACC-30. The underlined bases corre- primary tumors of colorectal cancer patients correlates spond to the initiating methionine codon and the penultimate with poor prognosis. Future work will focus on under- codon of Ier2. standing the molecular function of Ier2, with a view towards the development of therapeutic strategies that Ier2 siRNA inhibit the positive effects of Ier2 on tumor progression. For Ier2 knockdown analyses in 3T3 cells, mouse Ier2-specific stealth siRNA (Invitrogen, Darmstadt, Germany, Cat. No. MSS236850; Stealth RNAi siRNA duplexes; 20 nmol) was 0 Materials and methods used with the following sequence: 5 -CCAUGGAUACGCAA GAGGAAGUGCU-30 and 50-AGCACUUCCUCUUGCGU 0 Cells and cell lines AUCCAUGG-3 . Transient transfections were performed The culture conditions and properties of the tumor cell lines using siRNA (200 nM) and Lipofectamine 2000 (Invitrogen) used in this paper have been described previously (Nestl et al., according to the manufacturer’s instructions. For controls, 2001; Mengwasser et al., 2004). We have previously described scrambled stealth RNAi-negative control duplexes (Invitrogen, 3T3 cells, Ier2-inducible 3T3 cells, their cultivation under dense Cat. No: 12935-400) were transfected similarly. and sparse conditions, stimulation of Ier2 expression with RSL1 (New England Biolabs, Frankfurt a. M., Germany) and Transfections 5-bromo-20-deoxyuridine pulsing and staining used in this paper 1AS cells were stably or transiently transfected with the Ier2 (Wallbaum et al., 2009). For serum deprivation experiments, expression construct using Tfx50 (Promega, Mannheim, cells were cultivated in medium containing 0.3% serum for 48 h. Germany) or Geneporter 2 (Genlantis, San Diego, CA, Fluorescence-activated cell sorting cell-cycle profile analysis and USA) according to the manufacturer’s instructions. For fluorescence immunostaining were performed as described controls, cells were transfected with the corresponding empty previously (Wallbaum et al., 2009). The anti-FAK antibody vector as appropriate. Stably transfected cells were selected (C903) was purchased from Santa Cruz (Heidelberg, Germany). with 800 mg/ml G418. Selected cells were either pooled or were Freshly isolated splenocytes were incubated with and without picked as colonies as indicated in the text. Expression of the concanavilin A (5 mg/ml) for 72 h. transfected expression construct was confirmed by western blotting. RNA preparation and northern blots Preparation of polyadenylated RNA and northern blots was Western blots performed as described previously (Hofmann et al., 1998). Western blots were performed as described previously Blots were probed sequentially at high stringency using a full- (Baumann et al., 2005). Anti-V5 tag antibodies were purchased length Ier2 probe and a GAPDH (glyceraldehyde 3-phosphate from Invitrogen, and anti-b-actin and anti-vinculin antibodies dehydrogenase) probe. In between each probing, the blots were were purchased from Sigma-Aldrich (Taufkirchen, Germany). stripped in 0.1% SDS at 100 1C. Migration, adhesion, apoptosis and proliferation assays Generation of anti-Ier2 antibodies Monolayer wounding assays, adhesion, proliferation and To generate a glutathione-S-transferase (GST)–Ier2 fusion transwell migration and invasion assays were performed as protein, a fragment of the Ier2 cDNA (corresponding to amino described previously (Baumann et al., 2005), except for the

Oncogene Ier2 and metastasis A Neeb et al 3805 silicon insert used to create a cell-free area in the monolayer performed with 25 ng of total RNA (MuLV Reverse Tran- wounding assays described here. Apoptosis assays were per- scriptase; Perkin-Elmer, Weiterstadt, Germany). Quantitative formed using the Cell Death Detection ELISAPLUS kit (Roche real-time reverse transcriptase–PCR was carried out as Diagnostics, Mannheim, Germany) according to the manu- described previously (Stein et al., 2002). Ier2 expression facturer’s instructions. analysis was performed based on the hybridization probe detection format using amplicon-specific hybridization probes Tumor experiments in vivo (Roche LightCycler format, Roche Diagnostics). The follow- Syngeneic rats were injected subcutaneously with 1 Â 106 cells ing primers (synthesized by BioTeZ, Berlin, Germany) and in phosphate-buffered saline. Animals were monitored until probes (synthesized by TIB MOLBIOL, Berlin, Germany) 0 0 their tumors grew to the German legal limit (50 mm in one were used: forward 5 -CAGACACACGGACACAATCAG-3 , 0 0 dimension), or until they became moribund, at which time they reverse 5 -CCGCACGAAAGGTAAACAAAG-3 , fluorescein 0 0 were killed and an autopsy was performed. At autopsy, the size isothiocyanate 5 -CCAATCTGGGGAGGGGAACATC-3 ; 0 0 of the primary tumor was measured and the number, size and LCRed640 5 -TTGCCAGGGAGTTTCTGAGGGTCT-3 . location of metastases in the draining lymph nodes and other PCR was carried out as follows: 30 s 95 1C, then 45 Â (10 s organs were assessed. The lymph nodes and lungs were also 95 1C, 10 s 60 1C, 10 s 72 1C), melting curve 40–95 1C. For analyzed histologically in parallel to determine the presence of calibration, we used total RNA from the Ier2-expressing metastases. Statistical significance of the results was deter- human prostate cancer cell line PC-3. The PC-3-derived mined using the Mann–Whitney rank test. calibrator cDNA was used in serial dilutions for generating a standard curve in each quantitative PCR run. The Ier2 mRNA expression of each tissue sample was calculated as percentage Immunostaining of the Ier2 mRNA expression of the defined calibrator sample, Sections of paraffin-embedded human tumor samples (Biocat, which was set at 100%. Each sample was run in duplicate, and Heidelberg, Germany) were deparaffinized and blocked for the mean was used for analysis of the correlation between Ier2 15 min with 10% goat serum in phosphate-buffered saline. The expression and patient survival. Statistical significance of samples were incubated with 5 mg/ml purified polyclonal anti- Kaplan–Meier curves was evaluated with the log-rank test. Ier2 antibodies and then for 30 min with biotinylated secondary antibody (Dako, Hamburg, Germany). Antibody staining was detected using StreptABComplex (Dako) accord- Conflict of interest ing to the manufacturer’s instructions and color development with 3-amino-9-ethylcarbazole (AEC). Sections were counter- The authors declare no conflict of interest. stained with hematoxylin. In other experiments, tissues derived from tumor-bearing rats were paraffin embedded and stained with H and E using standard procedures. Acknowledgements

Quantitative reverse transcriptase–PCR We are very grateful to Norma Howells, Selma Huber and Tissue specimens from 39 colon cancer patients were obtained Manuela Sauer for their assistance with the animal experi- with written consent. All patients were previously untreated, ments, to Anja Steffen for expert technical support and to did not have a history of familial colon cancer and underwent Wilko Thiele for critical reading of this manuscript. The surgical resection at the Robert-Ro¨ssle Cancer Hospital contribution of Franziska Arlt and Markus Niederstrasser is (Berlin, Germany). None of them had metastases at the time also gratefully acknowledged. This work was supported by of surgery, but 17 developed distant metastases metachro- grants to JPS and US from the BMBF NGFN2 CancerNet nously. Tumor specimens (all adenocarcinomas) were snap Programme and to JPS from the European Union (FP6 frozen in liquid nitrogen and blinded for analysis. Tumor cell STREP project BRECOSM, contract no. LSHC-CT-2004- populations were microdissected from serial cryosections and 503224 and FP7 Collaborative Project TuMIC, contract no. total RNA was isolated. Reverse transcriptase reactions were HEALTH-F2-2008-201662).

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