BMC Cancer 2005, 5:3 http://www.biomedcentral.com/1471-2407/5/3 both pyrophosphatase and phosphodiesterase activity [37]. Transplantation experiments in athymic mice showed that ras-transformed NIH-3T3 fibroblasts became significantly more tumorigenic, invasive and metastatic when transfected with Autotaxin [38], and purified recom- binant Autotaxin has potent angiogenic activity in vivo [39]. Autotaxin has been shown to be regulated by both Wnt-1 and retinoic acid [30]. Autotaxin has been shown to have lysophospholipase activity and the effects of Auto- taxin on tumour cell motility are mediated by its conversion of lysophosphatidylcholine to lysophospha- tidic acid (LPA), a potent signalling molecule [40,41]. Extracellular Matrix Protein 1 was first identified as a novel 85 KDa protein secreted by a mouse osteogenic stromal cell line [42]. In situ hybridisation showed that Ecm1 was strongly expressed in most newly formed blood vessels and experiments using purified recombinant Ecm1 showed that it could increase the proliferation rate of vas- cular endothelial cells in vitro and also stimulate angio- genesis in vivo. The ability to induce de novo angiogenesis is an absolute requirement for tumours to grow beyond a size which can be readily perfused by oxygen and nutri- ents from the interstitial fluid. ECM1 is overexpressed in many epithelial tumours including 73% of breast tumours analyzed [43]. Homozygous loss-of-function mutations in the human ECM1 gene were recently identi- fied by linkage analysis as the causative mutations behind Lipoid Proteinosis, a rare autosomal recessive disorder characterized by hyaline deposition in the skin, mucosae and viscera [44]. The identification of Autotaxin and Ecm1 as genes upregulated by activation of this pathway, together with VEGF [45] suggests that deregulation of Wnt/β-catenin signalling during tumour initiation and progression may be one of the factors which promotes tumour angiogenesis. CD14, which can function as both a receptor and a secreted protein, was downregulated upon ∆Nβ-catenin expression. CD14 is a glycosyl-phosphatidylinositol- linked cell surface protein, preferentially expressed in monocytes, where it acts as a receptor for Lipopolysaccha- ride Binding Protein:Lipopolysaccharide complexes [46]. Soluble CD14 (sCD14) is also expressed in mammary epi- thelial cells in vitro and has been detected in human milk where it is postulated to play a role in neonatal immunity [47], and is strongly upregulated in mammary luminal epithelial cells in vivo at the onset of involution [48]. Receptor Activity Modifying Protein 3 (RAMP3) was down- regulated upon ∆Nβ-catenin induction and is one of three members of the RAMP family. These proteins are involved in mediating the cellular response to the neuropeptides calcitonin, calcitonin gene related peptide, amylin and adrenomedullin. The RAMP family members function as chaperones for the seven transmembrane domain G-pro- tein coupled receptors for these neuropeptides, shuttling the receptor to the cell surface and altering receptor glycosylation. The ligand binding phenotype of the receptor is dependent on the RAMP family member with which it is associated [49]. RAMP3-Calcitonin Receptor (CR) heterodimers form a functional receptor for amylin [50], and RAMP3-Calcitonin-Receptor-Like-Receptor (CRLR) heterodimers act as an adrenomedullin receptor [51]. Expression of both CR and CRLR was detected in HC11 by RT-PCR (data not shown) suggesting that functional receptor-RAMP complexes are present in this cell line. Adrenomedullin, the ligand for the CRLR/RAMP3 recep- tor dimer, functions as a growth factor in several human tumour cell lines [52], in addition to promoting angio- genesis in vivo [53] via CRLR/RAMP3 and CRLR/RAMP2 receptor dimers [54,55]. Hypoxia-inducible gene 2 (Hig2) was one of several genes identified in a representational difference analysis screen for genes regulated by hypoxia in a human cervical epithe- lial cell line. The human gene encodes a 63 amino acid polypeptide of unknown function [31]. Expression of mouse Hig2 was downregulated in HC11 cells overex- pressing ∆Nβ-catenin. The identification of a group of mammalian orthologues revealed a well conserved hydro- phobic region in the N-terminus, reminiscent of a signal peptide. A Hig2-EGFP fusion protein entered the secretory pathway and was detected in conditioned medium of transfected cells. The introduction of a pair of charged amino acids into the hydrophobic region abolished secre- tion, lending support to the hypothesis that this region contains a functional signal peptide. The nature of the large vesicular structures observed in Hig2-EGFP overex- pressing cells is as yet unclear. Mammary epithelial cells are known to contain membrane-enclosed lipid droplets, as well as a variety of vesicular compartments involved in the secretion of casein, citrate, lactose and calcium [56], however the presence of these vesicles in MDCK and HEK293 cells argues that they are not mammary specific. Indeed, co-localization experiments suggest that these structures are neither mitochondria, lysosomes, endo- somes nor lipid droplets. Given the demonstration that Hig2 is secreted, these structure most likely correspond to overexpressed Hig2-EGFP in transit through the endoplas- mic reticulum and golgi apparatus. As no antibody is available against Hig2, it was not possible to investigate the localisation of the endogenous protein, but these data represent a useful initial step in the functional characteri- sation of this gene. Analysis of the expression of HIG2 using a matched Tumour/Normal tissue cDNA array showed that HIG2 is Page 7 of 9 (page number not for citation purposes)