Oncogene (2002) 21, 1309 ± 1315 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc ORIGINAL PAPERS The LIM-domain Lmo2 is a key regulator of tumour angiogenesis: a new anti-angiogenesis drug target

Yoshihiro Yamada1,2, Richard Pannell1, Alan Forster1 and Terence H Rabbitts*,1

1Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

The growth of solid tumours requires a blood supply where this remodelling process of angiogenesis is provided by re-modeling of existing blood vessel required such as wound healing or menstruation and endothelium (angiogenesis). Little is known about in pathological conditions, such as tumour angiogen- transcription regulators which are speci®c for the control esis, ischaemia, in¯ammation and diabetic retinopathy of tumour angiogenesis. The proto-oncogene LMO2 (Folkman, 1995). In cancer, angiogenesis is a key part encodes a LIM domain transcription regulator which of solid tumour growth and invasion (metastasis). Like controls angiogenesis during mouse embryogenesis where all cells, cancer cells require oxygen and carbon dioxide it regulates remodelling of the capillary network into exchange, and therefore solid tumours must develop a mature vessels. We now show that Lmo2 expression is blood supply to facilitate their growth (Folkman, 1971, augmented in tumour endothelium such as mouse 1972). This is achieved by subverting local vessel thymomas and human lung tumours. The functional endothelium in the process of tumour angiogenesis signi®cance of this Lmo2 expression was assessed in (Hanahan and Folkman, 1996; Weidner et al., 1991) teratocarcinomas induced in nude mice by subcutaneous which allows for sprouting of existing endothelium into implantation of Lmo2-lacZ targeted ES cells. CD31- the tumour deposits (Carmeliet and Jain, 2000). This positive, sprouting endothelium of ES-cell origin oc- angiogenic switch occurs when there is a relative curred in teratocarcinomas from heterozygous Lmo2- increase in angiogenesis stimulating factors and in lacZ ES cells but none occurred from null Lmo2-lacZ tumour growth, the cancer cells become angiogenic to ES cells. Therefore, in this model Lmo2 is an obligatory stimulate the necessary remodelling state. Furthermore, regulator of neo-vascularization of tumours. These data the concept of mosaic blood vessels is strongly suggest that LMO2 function may be a drug target in supported by recent data which shows that a signi®cant cancer and other conditions characterized by neo- percentage of the vessel wall can be made from tumour vascularization. cells (Chang et al., 2000) and these cells are continually Oncogene (2002) 21, 1309 ± 1315. DOI: 10.1038/sj/ shed into the vessel lumen, and therefore the blood onc/1205285 circulation contributing to the metastatic population. In addition, so-called angioblast-like endothelial pre- Keywords: Transcriptional regulation; tumour angio- cursors may be recruited from the bone marrow (Ra®i, genesis; chromosomal translocation; endothelium; can- 2000) providing another source of vessel wall cells. cer; neo-vascularization These features of angiogenesis have been proposed as a major target for patient treatment in cancer (Folkman, 1971, 1972). Several key molecules have been identi®ed which Introduction control angiogenesis, such as vascular endothelial growth factor (Ferrara and Alitalo, 1999) which The formation of the vascular system during embry- stimulates the process of vessel sprouting, but little is ogenesis occurs in two stages. In the ®rst stage or known of transcription regulators of vessel remodeling. vasculogenesis, de novo endothelial cells di€erentiate Some candidates have come from the study of from mesoderm to create the primary capillary net- chromosomal translocations in human leukaemias work. In the subsequent stage of angiogenesis, the which has led to the identi®cation of novel , existing endothelial layers are remodelled to form many of which are transcription factors (reviewed in mature blood vessels and hence the ®nal vasculature Rabbitts (1994)). The LMO2 is an example, being of the adult. There are a number of situations in adults a gene discovered from the chromosomal translocation (t(11;14)(p13;q11) in T-cell acute leukaemia (Boehm et al., 1991; Royer-Pokora et al., 1991). The gene encodes a small protein comprising two zinc-binding LIM- *Correspondence: Dr TH Rabbitts; E-mail: [email protected] domains and this structural element appears to be a 2 Current address: Department of Pathology and Biology of Diseases, protein interaction domain capable of binding various Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan partners depending on the cellular environment in Received 26 October 2001; accepted 3 January 2002 which it is expressed (Rabbitts, 1998). For instance, Lmo2 in tumour neo-vascularization Y Yamada et al 1310 LMO2 can interact with the T-cell oncogenic protein anti-LMO2 antibody (Figure 2b). Sections were made TAL1/SCL in haematopoietic cells and in leukaemias from these specimens and either H&E stained or IHC (see (Rabbitts, 1998)). As with many of the genes performed with antibodies detecting the endothelial cell activated by chromosomal translocations, the LMO2 marker CD31 (PECAM) or with antibodies detecting function adopted in tumour cells belies its normal LMO2. The normal and adenocarcinoma blood vessel function. In the case of Lmo2, gene targeting endothelial cells had equivalent binding with anti- experiments in mice have shown that it is crucial for CD31 antibody (Figure 2b, middle sections). However, (both primitive and de®nitive) (Warren LMO2 protein was only detected in the lung et al., 1994; Yamada et al., 1998) and there is a adenocarcinoma vessels, while it was almost undetect- complete failure of adult haematopoiesis in the absence able on normal vessels of lung (Figure 2b, right hand of Lmo2 (Yamada et al., 1998). In addition, while the panel). We have examined two separate adenocarcino- yolk sac endothelium appears to develop normally in mas and two separate squamous cell carcinomas of the Lmo2 homozygous null mice (Warren et al., 1994), null lung. Therefore there seems to be augmentation of embryos do not sustain development of a mature blood LMO2 protein in these human lung tumours like that vascular system because of a speci®c failure of the in mouse T-cell lymphomas. remodelling of the capillary endothelium into mature These observations show that LMO2 is expressed in vessels (angiogenesis) (Yamada et al., 2000). LMO2 is tumour endothelium and that the level appears to be therefore a candidate factor for controlling tumour augmented in tumour angiogenic situations. This in angiogenesis in view of these embryonic roles in blood turn implies a functional role for LMO2 in tumour vessel endothelium. We have now evaluated a possible angiogenesis. Accordingly, a teratocarcinoma assay function of LMO2 in angiogenesis of primary tumours was established to determine if the Lmo2 protein is and in tumours implanted in mice. Our data suggest functional in endothelial cells during tumour angiogen- that LMO2 is required for tumour angiogenesis. esis. An Lmo27/7 ES line has been described in which one null Lmo2 allele carries the lacZ knock-in and the second allele Lmo2 has been targeted by Results and Discussion hygromycin insertion (illustrated in Figure 1b) (Yama- da et al., 2000). Teratocarcinomas were developed by The expression of Lmo2 has been followed using a gene subcutaneous implantation of either Lmo2+/7 or targeting knock-in strategy in which lacZ was cloned in Lmo27/7 ES cells in nude mice. ES cells were injected frame with Lmo2 exon 2 allowing Lmo2 gene into both ¯anks of a cohort of nude mice and tumours expression to be monitored in vivo in mice via b- were excised at 2, 3, 3.5 and 4 weeks after implantation galactosidase (b-gal) enzyme activity (Yamada et al., for analysis of ES-derived vasculature using b-gal 2000). ES cells were generated with one Lmo2 allele staining for Lmo2-lacZ gene transcription and counter disrupted by this homologous recombination (Lmo2+/7 staining with the endothelial cell surface marker CD31. where the null allele is the lacZ knock-in) (Figure 1a) Two issues are pertinent to the interpretation. Firstly, and heterozygous mice carrying this allele have been both ES-derived and host (nude mouse) derived described (Yamada et al., 2000). These mice were vasculature will contribute in this model and secondly crossed with tumour-prone p53 null mice (Harvey et b-gal expression will still occur in any Lmo27/7 ES al., 1993) to generate p537/7 ; Lmo2+/7 mice, cell derivatives in which the Lmo2-lacZ knock-in gene which frequently develop thymomas. When signs of ill- is transcribed. health indicated lymphomatous deposits of sucient In all we examined in detail 19 tumours derived from size, the mice were sacri®ced and thymomas subjected either Lmo2+/7 or Lmo27/7 targeted ES cells to whole mount staining. Histological sections of the (Table 1). There appeared to be little signi®cant tumour specimens were obtained and b-gal expression di€erence in the rate of growth of the teratocarcinomas in blood vessel lining was compared with that of consistent with a blood supply being contributed by normal thymus tissue from Lmo2+/7 mice. Figure 2a angiogenesis from the host endothelium. However, shows signi®cant b-gal staining in two examples of T- major di€erences were observed in the pattern of b-gal cell lymphoma blood vessel endothelium (one is an expression within the developing tumours. Macro- abdominal lymph node T-cell lymphoma associated scopic examination of resected tumours using a with thymoma, central panel, and the other is a T-cell dissecting microscope, after b-gal staining, showed that thymoma). No obvious b-gal expression was present in after about three weeks, an intrinsic Lmo2+/7 ES the normal Lmo2+/7 thymus samples (Figure 2a, left cell-derived vascular system was visible and could be hand panel). Note that the malignant T-cells in the distinguished from host (nude mice) vessels because of lymphomatous masses are not expressing b-gal (Figure Lmo2-lacZ fusion protein in endothelial cells of ES cell 2a). These data indicate an augmentation of Lmo2 origin. Two weeks (2W) after implantation, there was expression in tumour vessel endothelium. no sign of obvious ES cell-derived vascular tree This conclusion was con®rmed by examining development in either Lmo2+/7 and Lmo27/7 whether human tumours also exhibit augmented tumours (Figure 3). However, clusters of lacZ-positive LMO2 expression in endothelium. Lung tumour cells, possibly angioblasts or precursors of sprouting biopsies and matched peripheral parts of normal lung endothelium, were present in both sets of tumours. were used for immunohistochemistry (IHC) with the From 2 weeks onwards, vascular trees, displaying

Oncogene Lmo2 in tumour neo-vascularization Y Yamada et al 1311

Figure 1 Structure of Lmo2 targeted alleles in ES cells. The Lmo2 gene targeting has been reported (Yamada et al., 2000). Heterozygous and homozygous null mutations were generated in ES cells to give Lmo2+/7 (called KZ26+/7 in Yamada et al. (2000)) and Lmo27/7 (called KZ267/7 in Yamada et al. (2000)). (a) The null allele in Lmo2+/7 cells was made using neomycin selection and caused a fusion of the bacterial lacZ gene with the second exon of Lmo2; this results in a b-galactosidase fusion protein comprising a short stretch of Lmo2 encoded amino acids and the whole b-galactosidase protein (Yamada et al., 2000). (b) The Lmo2+/7 ES cells (clone KZ26+/7 ) was used for targeting the second Lmo2 allele by insertion of the hygromycin resistance marker

sprouting neo-vasculature, developed from lacZ posi- histological sign of di€erentiation, other than b-gal- tive cell clusters in Lmo2+/7 tumours (Figure 3, top positive neuro-epithelial cells (Figure 4). The undi€er- panels), but there was no sign of any vascular tree entiated cells did not co-express CD31 while CD31- development in Lmo27/7 tumours (Figure 3, bottom positive endothelial cells in Lmo27/7 tumours were panels). In the set of 19 tumours from each ES cell b-gal negative, and thus of host nude mice origin type, we found b-gal expressing vascular trees in 84% (Figure 4, lower panels). of those from Lmo2+/7 cells but none from the Lmo2 was identi®ed as a T-cell oncogene activated Lmo27/7 cells (Table 1). Vascular trees which by speci®c chromosomal translocations (Rabbitts, appeared in Lmo2+/7 tumours kept growing until 1998). The gene is normally expressed in endothelial they formed the more mature vascular system in the cells of mouse embryos where it is required for tumour at 3.5 weeks and 4 weeks (Figure 3). However, angiogenesis after vascularization has occurred (Yama- there was no ES-derived vascular tree development in da et al., 2000). Our results show augmentation of Lmo27/7 tumours even at this stage (Figure 3). LMO2 expression in tumour neo-vasculature. Further- Histological analysis of Lmo2+/7 and Lmo27/7 more, in the absence of Lmo2, the development of a tumours at 3 ± 4 weeks showed the di€erence in the two mature vascular system in solid tumours is prevented in kinds of ES-derived tumour vasculature in more detail an ES-cell tumour model. Neo-vascularisation is a (Figure 4). In Lmo2+/7 tumours, the development of crucial step in development of several human diseases ES cell intrinsic vascular system was obvious, because and the regulation of this process could control these the vascular wall cells were mostly b-gal positive progressive diseases. Although growth factors and their (Figure 4) and the endothelial nature of these cells cell surface receptors are important for control of were clearly shown by b-gal and CD31 double staining. angiogenesis (e.g. vascular endothelial growth factor, Although b-gal positive cells were sometimes seen in angiopoietin, ®broblast growth factor and their cell immature neuroepithelial component, most of them surface receptors, reviewed in (Carmeliet and Jain, were endothelial cells. On the other hand, in Lmo27/7 2000)) and their clinical application has been exten- tumours, we found b-gal activity was only evident in sively studied, the molecular mechanism of transcrip- undi€erentiated cell clusters which showed little tional control of angiogenesis remains unclear. Our

Oncogene Lmo2 in tumour neo-vascularization Y Yamada et al 1312

Figure 2 Lmo2 expression in tumour vasculature. Tissues were dissected from normal thymus of an Lmo2+/7 mouse or T-cell tumour-bearing Lmo2+/7 ; p537/7 mice (9 ± 15 weeks old) followed by whole mount X-gal staining and histological sectioning (4 mM). These were counter-stained with nuclear fast red. Blue endothelial staining (b-gal) is evident in the T cell lymphoma sections but little in the normal thymus. Middle section is an abdominal lymph node tumour (T cell lymphoma) and right hand section is from a thymic T-cell lymphoma. en=endothelium. (b) Human lung adenocarcinoma (upper panels) and normal part of lung from the same surgical sample (lower panels) were studied for LMO2 expression. Haematoxylin and eosin (H&E) staining of lung adenocarcinoma showed that cancer cell nests (cells with large nucleus) are surrounded by a vascular network, which is stained with anti-CD31 (pan-endothelial marker, PECAM) (positive=brown colour). Large vessels of normal lung are CD31 positive in the same manner. LMO2 protein is detected in tumour vessels as shown in anti-LMO2 staining of adenocarcinoma vessels, while LMO2 protein is almost undetectable in normal vessels

Table 1 ES-derived sprouting angiogenesis during teratocarcinoma and furthermore highlights a potential therapeutic development application to cancer treatment, especially because Lmo2 +/7 Lmo2 7/7 LMO2 has a speci®c role in neo-vascularization whilst Time after injection Tumours vt Tumours vt existing endothelial cells do not seem to require the 2W 4140protein. 3W 5550The LIM domain provides an interface for protein 3.5W 3 3 5 0 interactions (Schmeichel and Beckerle, 1994; Valge- 4W 7750Archer et al., 1994; Wadman et al., 1994) and the LIM- Total 19 16 19 0 (84%) (0%) only protein LMO2 binds to several partners in haematopoietic cells and in T-cell tumours. By ES cell-derived teratocarcinomas were induced by subcutaneous modifying the LIM-domain protein ± protein interac- injection of Lmo2+/7 or Lmo27/7 ES cells into nude mice. In tions of LMO2, it might be possible to control the total 19 tumours from each implantation were examined. At each point, the number of tumours examined is indicated and the number critical speci®c step of tumour neo-vascularization. One in which vascular trees (vt) were detected interesting LMO2 interaction which could be a drug target is with the transcription factor TAL1/SCL as this protein also has a role in vascular formation (Visvader et al., 1998). Finally by inhibiting the ®nding that the LIM-only protein LMO2 is a nuclear transcriptional mechanism of neo-vascularization, it regulator of tumour neo-vascularization may help to should be possible to simultaneously control angiogen- understand transcription programmes in this process esis-related downstream genes, which may be a potent

Oncogene Lmo2 in tumour neo-vascularization Y Yamada et al 1313

Figure 3 The development of ES-cell derived vascular networks in subcutaneous teratocarcinomas. Subcutaneous teratocarcinomas were produced in nude mice by implantation of heterozygous Lmo2+/7 or homozygous null Lmo27/7 ES cells. 26106 of either Lmo2+/7 or Lmo27/7 ES cells were implanted into both ¯anks of nude mice. Subcutaneous tumours were dissected from the recipient mice at 2, 3, 3.5 and 4 weeks (W) after implantation, followed by whole mount X-gal staining and photography (sections and IHC is shown in Figure 4). Vessels of ES-cell origin are distinguishable from those of host (nude mice) origin by their blue colour after X-gal staining due to the knock-in fusion of lacZ into the Lmo2 gene (Yamada et al., 2000). ES-derived vascular development is apparent about 2 ± 3 weeks after implantation in Lmo2+/7 ES cell tumours (upper panels) but not in Lmo27/7 ES cell tumours (lower panels). At 3 ± 4 weeks, well developed vascular trees were seen in Lmo2+/7 tumours, whilst there was still no sign of vascular development from Lmo27/7 ES-derived endothelium at 4 weeks. vt=vascular tree

Figure 4 Immunohistochemistry of teratocarcinoma vasculature. Teratocarcinomas, described in Figure 3, where ®xed, whole mount stained with X-gal to detect b-gal expression and sections prepared (4 mM). Immunohistochemistry was performed with anti- mouse CD31 antibody and sections were countered stained with haematoxylin. The top panels show sections of tumours from Lmo2+/7 ES cells stained with X-gal (left hand panel) or with X-gal and anti-CD31 (right hand panel); b-gal-positive cells (blue) are vascular endothelial cells are also CD31-positive cells (brown). The lower panels show sections of tumours from Lmo27/7 ES cells stained with X-gal only (left hand panel) or X-gal and anti-CD31 (right hand and central lower panels). b-gal positive cell clusters comprise undi€erentiated cells and no double b-gal/CD31-double positive cells were observed with Lmo27/7 derived tumours

Oncogene Lmo2 in tumour neo-vascularization Y Yamada et al 1314 method to regulate neo-vascularization and eventually Xgal reagents. After ®xing in 4% paraformaldehyde solution to control tumour growth and metastasis. Alternatively in phosphate bu€ered saline (PBS) for 45 min at room the identity of these downstream gene products may temperature, samples were subjected to X-gal staining of b- themselves be drug targets. galactosidase activity (resulting from Lmo2-lacZ gene expres- sion) according to the procedure described (Yamada et al., 2000). For histological studies, samples were post-®xed in 10% (vol/vol) bu€ered formalin for at least 48 h after X-gal Materials and methods staining. The specimens were embedded in paran for sectioning (4 mM). Sections were mounted on microscope ES cells and gene targeted mice slides and counter-stained with nuclear fast red or haematox- The targeting of the Lmo2 locus in ES cells creating an in- ylin. CD31 immunohistochemistry was done with MEC 13.3 frame gene fusion of the lacZ gene with Lmo2 has been anti-mouse CD31 (Pharmingen) (12.5 mg/ml) by Avidin- described and the mouse strain called previously called KZ26 Biotin conjugated peroxidase method as described (TanieÁ re (Yamada et al., 2000). As the homozygous null mutation of et al., 2001). Lmo2 is embryonic lethal (Warren et al., 1994), we maintain the mouse line in the heterozygous state (designated Lmo2 is Immunohistochemical study of human lung carcinoma embryonic lethal (Warren et al., 1994), we maintain the mouse line in the heterozygous state (designated Lmo2+/7 ). Lung adenocarcinoma and squamous cell carcinomas and Both heterozygous Lmo2-targeted ES cells (Lmo2+/7 cells) matched peripheral part of normal lung were taken from and homozygous Lmo27/7 targeted ES cells have been surgical resection specimens and preserved in liquid nitrogen. described (Yamada et al., 2000). Frozen sections of 8 mm thickness were prepared from each sample for haematoxylin and eosin staining or immunohis- tochemistry with anti-CD31 antibody (clone JC/70A, DAKO, Tumour growth Copenhagen, Denmark) used at concentration 100 ng/ml. Lmo2+/7 mice were crossed with p537/7 (Harvey et al., Immunohistochemistry with anti-LMO2 rabbit polyclonal 1993) to generate Lmo2+/7 ; p53+/7 which were inter- antibody (Warren et al., 1994) was carried out at concentra- bred to generate Lmo2+/7 ; p537/7 . tion 5 mg/ml. Immunohistochemistry was performed as Teratocarcinomas were produced from ES cells implanted described by Avidin-Biotin conjugated peroxidase method in nude mice. ES cells were grown on neomycin-resistant as described (TanieÁ re et al., 2001). feeders in the presence of LIF prior to trypsinisation. The washed cells were resuspended in PBS at a concentration of 26106/100 ml. 100 ml of cell suspension was injected sub- cutaneously into both ¯anks of adult MF1 female nude mice. A cohort of mice was used for each of Lmo2+/7 and Lmo27/7 ES cells. Tumours were measured every 4 days Acknowledgments with a dial-caliper for decision points on dissection. We would like to thank Matt McCormack for discussions during this work and Gareth King, Theresa Langford, Angela Middleton and Tina Hamilton for expert help. Y Whole mount staining and histology of mouse tissues and tumour Yamada was generously supported by an award from the samples National Foundation for Cancer Research, USA. We also Organs and tumours were excised from mice and cut into two wish to thank Peter Goldstraw for the lung tumour pieces or into slices to allow maximum penetration of the biopsies used in this work.

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