Background Deregulation of the Wnt/ β-catenin signal transduction pathway has been implicated in the pathogenesis of tumours in the mammary gland, colon and other tissues. Mutations in components of this pathway result in β-catenin stabilization and accumulation, and the aberrant modulation of β-catenin/TCF target genes. Such alterations in the cellular transcriptional profile are believed to underlie the pathogenesis of these cancers. We have sought to identify novel target genes of this pathway in mouse mammary epithelial cells.
Background
Deregulation of the Wnt/ β-catenin signal transduction pathway has been implicated in the pathogenesis of tumours in the mammary gland, colon and other tissues. Mutations in components of this pathway result in β-catenin stabilization and accumulation, and the aberrant modulation of β-catenin/TCF target genes. Such alterations in the cellular transcriptional profile are believed to underlie the pathogenesis of these cancers. We have sought to identify novel target genes of this pathway in mouse mammary epithelial cells.Methods Gene expression microarray analysis of mouse mammary epithelial cells inducibly expressing a constitutively active mutant of β-catenin was used to identify target genes of this pathway.
Methods
Gene expression microarray analysis of mouse mammary epithelial cells inducibly expressing a constitutively active mutant of β-catenin was used to identify target genes of this pathway.Results The differential expression in response to ΔNβ-catenin for five putative target genes, Autotaxin, Extracellular Matrix Protein 1 (Ecm1), CD14, Hypoxia-inducible gene 2 (Hig2) and Receptor Activity Modifying Protein 3 (RAMP3), was independently validated by northern blotting. Each of these genes encodes either a receptor or a secreted protein, modulation of which may underlie the interactions between Wnt/β-catenin tumour cells and between the tumour and its microenvironment. One of these genes,  Hig2 , previously shown to be induced by both hypoxia and glucose deprivation in human cervical carcinoma cells, was strongly repressed upon ΔNβ-catenin induction. The predicted N-terminus of Hig2 contains a putative signal peptide suggesting it might be secreted. Consistent with this, a Hig2-EGFP fusion protein was able to enter the secretory pathway and was detected in conditioned medium. Mutation of critical residues in the putative signal sequence abolished its secretion. The expression of human  HIG2  was examined in a panel of human tumours and was found to be significantly downregulated in kidney tumours compared to normal adjacent tissue.
Results
The differential expression in response to ΔNβ-catenin for five putative target genes, Autotaxin, Extracellular Matrix Protein 1 (Ecm1), CD14, Hypoxia-inducible gene 2 (Hig2) and Receptor Activity Modifying Protein 3 (RAMP3), was independently validated by northern blotting. Each of these genes encodes either a receptor or a secreted protein, modulation of which may underlie the interactions between Wnt/β-catenin tumour cells and between the tumour and its microenvironment. One of these genes,  Hig2 , previously shown to be induced by both hypoxia and glucose deprivation in human cervical carcinoma cells, was strongly repressed upon ΔNβ-catenin induction. The predicted N-terminus of Hig2 contains a putative signal peptide suggesting it might be secreted. Consistent with this, a Hig2-EGFP fusion protein was able to enter the secretory pathway and was detected in conditioned medium. Mutation of critical residues in the putative signal sequence abolished its secretion. The expression of human  HIG2  was examined in a panel of human tumours and was found to be significantly downregulated in kidney tumours compared to normal adjacent tissue.Hig2HIG2Conclusions HIG2 represents a novel non-cell autonomous target of the Wnt pathway which is potentially involved in human cancer.
Conclusions
HIG2 represents a novel non-cell autonomous target of the Wnt pathway which is potentially involved in human cancer.

Background The Wnt/β-catenin signal transduction pathway plays a central role in metazoan development, controlling such diverse processes as cell growth, proliferation and organogenesis [ 1 ]. Wnt-1 is the prototypic member of this large family of secreted glycoproteins and was originally identified as a gene insertionally activated by mouse mammary tumour virus [ 2 ]. Wnt-1 is one of a number of Wnt family members which act to control the cellular level of β-catenin. Wnt proteins bind seven-pass transmembrane receptors of the Frizzled family, and a signal is transduced via Dishevelled to a complex which contains the Adenomatous Polyposis Coli (APC), Axin and Glycogen Synthase Kinase-3β (GSK-3β) proteins [ 3 , 4 ]. This signal antagonizes the phosphorylation of β-catenin by GSK-3β. There are four phosphorylation sites in the N-terminus of β-catenin which, in the absence of Wnt signal, are phosphorylated by Casein Kinase I alpha and GSK-3β [ 5 , 6 ]. This phosphorylation leads to the ubiquitination and subsequent proteasomal degradation of β-catenin [ 7 ]. Inhibition of β-catenin phosphorylation by Wnt signalling leads to the accumulation of β-catenin which forms a bipartite complex with members of the TCF/LEF transcription factor family and activates the transcription of target genes, a process which is regulated by multiple interacting factors [ 8 ]. Overexpression of Wnt-1 in the mammary glands of transgenic mice leads to extensive hyperplasia and tumorigenesis [ 9 ].  APC  was identified as the tumour suppressor gene mutated in the hereditary colorectal cancer syndrome, Familial Adenomatous Polyposis [ 10 , 11 ]. Mutations in  Axin  and β- catenin  have also been detected in tumours of the colon and other tissues [ 12 ]. Deregulation of this pathway appears to be play a contributory role in a significant proportion of human tumours of epithelial origin and hence, the identification of effector genes of this pathway is an important step towards the elucidation of the mechanisms involved. Many of the Wnt targets thus far identified are cell-cycle regulators [ 13 , 14 ] and transcription factors [ 15 - 20 ], and function in a cell-autonomous manner, providing insight into the mechanisms by which tumour cells deregulate proliferation and inhibit apoptosis. Tumours are complex organs composed of tumour cells, stromal fibroblasts, endothelial cells and cells of the immune system; and reciprocal interactions between these cell types in the tumour microenvironment are necessary for tumour growth [ 21 , 22 ]. Here we postulate that proteins secreted by Wnt/β-catenin tumour cells and receptors expressed by these cells may play roles in mediating interactions between neighbouring tumour cells or between tumour cells and their microenvironment. Consequently, in this study we have focussed our attention on identifying novel genes encoding receptors and secreted proteins.
Background
The Wnt/β-catenin signal transduction pathway plays a central role in metazoan development, controlling such diverse processes as cell growth, proliferation and organogenesis [ 1 ]. Wnt-1 is the prototypic member of this large family of secreted glycoproteins and was originally identified as a gene insertionally activated by mouse mammary tumour virus [ 2 ]. Wnt-1 is one of a number of Wnt family members which act to control the cellular level of β-catenin. Wnt proteins bind seven-pass transmembrane receptors of the Frizzled family, and a signal is transduced via Dishevelled to a complex which contains the Adenomatous Polyposis Coli (APC), Axin and Glycogen Synthase Kinase-3β (GSK-3β) proteins [ 3 , 4 ]. This signal antagonizes the phosphorylation of β-catenin by GSK-3β. There are four phosphorylation sites in the N-terminus of β-catenin which, in the absence of Wnt signal, are phosphorylated by Casein Kinase I alpha and GSK-3β [ 5 , 6 ]. This phosphorylation leads to the ubiquitination and subsequent proteasomal degradation of β-catenin [ 7 ]. Inhibition of β-catenin phosphorylation by Wnt signalling leads to the accumulation of β-catenin which forms a bipartite complex with members of the TCF/LEF transcription factor family and activates the transcription of target genes, a process which is regulated by multiple interacting factors [ 8 ].Overexpression of Wnt-1 in the mammary glands of transgenic mice leads to extensive hyperplasia and tumorigenesis [ 9 ].  APC  was identified as the tumour suppressor gene mutated in the hereditary colorectal cancer syndrome, Familial Adenomatous Polyposis [ 10 , 11 ]. Mutations in  Axin  and β- catenin  have also been detected in tumours of the colon and other tissues [ 12 ]. Deregulation of this pathway appears to be play a contributory role in a significant proportion of human tumours of epithelial origin and hence, the identification of effector genes of this pathway is an important step towards the elucidation of the mechanisms involved.APCAxincateninMany of the Wnt targets thus far identified are cell-cycle regulators [ 13 , 14 ] and transcription factors [ 15 - 20 ], and function in a cell-autonomous manner, providing insight into the mechanisms by which tumour cells deregulate proliferation and inhibit apoptosis. Tumours are complex organs composed of tumour cells, stromal fibroblasts, endothelial cells and cells of the immune system; and reciprocal interactions between these cell types in the tumour microenvironment are necessary for tumour growth [ 21 , 22 ]. Here we postulate that proteins secreted by Wnt/β-catenin tumour cells and receptors expressed by these cells may play roles in mediating interactions between neighbouring tumour cells or between tumour cells and their microenvironment. Consequently, in this study we have focussed our attention on identifying novel genes encoding receptors and secreted proteins.Methods Cell culture All reagents were purchased from Sigma unless otherwise noted. HC11 mouse mammary epithelial cells were cultured in 5% CO 2  at 37°C in RPMI 1640, supplemented with 10% Foetal Bovine Serum, 2 mM L-glutamine, 2.5 μg/ml insulin, 5 ng/ml epidermal growth factor and 50 μg/ml gentamycin [ 23 ]. HC11- lacZ  and HC11-Δ N β- catenin  cells were routinely cultured in 2 μg/ml tetracycline to repress transcription of the tetracycline-regulated transgene. HEK293 and MDCK cells were grown in DMEM supplemented with 10% Foetal Bovine Serum. The HC11- lacZ  and HC11-Δ N β- catenin  cell lines were generated by infecting the cells with an ecotropic retrovirus (TRE-tTA) in which the  tTA  cDNA is under the control of a tetracycline responsive promoter. Consequently, tTA expression is minimal in the presence of tetracycline and, upon tetracycline withdrawal, tTA activates its own transcription in an autoregulatory manner [ 24 ]. HC11 cells expressing tTA were subsequently infected with ecotropic retroviruses derived from RevTRE (Clontech) which directed the expression of either β-galactosidase or ΔNβ-catenin in a tetracycline dependent manner. Bosc23 cells were used to produce ecotropic retroviruses [ 25 ]. Cells were transiently transfected with the appropriate retroviral construct and the supernatant was collected 48 hours post-transfection. Polybrene was added to a final concentration of 5 μg/ml and the supernatant was added to HC11 cells for 24 hours. HC11 cells were then subjected to antibiotic selection using either 250 μg/ml G418 or 200 μg/ml hygromycin B as appropriate. RNA isolation Cell monolayers were washed twice in ice-cold Phosphate Buffered Saline and lysed by addition of Trizol (Invitrogen). Total RNA was isolated according to the manufacturer's instructions. PolyA+ RNA was purified from total RNA using Oligotex (Qiagen) according to the manufacturer's instructions. Northern blotting 10 μg of total RNA from each cell line was fractionated on a denaturing formaldehyde agarose gel and transferred to a positively charged nylon membrane (Hybond N+, Amersham Pharmacia Biotech) in 10x SSC. Membranes were prehybridised for four hours in 50% (v/v) formamide, 5X SSPE, 2X Denhardt's reagent, 0.1% (w/v) SDS and 100 μg/ml denatured herring sperm DNA. Radiolabelled probes were prepared from PCR-amplified cDNA clones using the Rediprime II kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. EST sequences corresponding to the coding sequence of the genes-of-interest were identified by BLAST [ 26 ] and obtained from the I.M.A.G.E. consortium through the UK Human Genome Mapping Project Resource Centre (Hinxton, UK). ESTs bearing the following I.M.A.G.E. cloneIDs were used:  Autotaxin  – 533819;  CD14  – 2936787;  Ecm1  717050;  Hig2  – 367488;  Ramp3  – 615797,  HIG2  4366895). Following overnight hybridisation with the labelled probe, the membranes were washed twice in 1X SSC, 0.1% (w/v) SDS at room temperature for 20 mins, and twice in 0.2X SSC, 0.1% (w/v) SDS at 68°C for 10 mins and exposed to film at -80°C for 48 hours. Bound probe was quantitated using a phosphorimager (Molecular Dynamics). Western blotting Cell monolayers were rinsed twice with ice-cold Phosphate Buffered Saline and total cell lysates were prepared by scraping cells into a minimal volume of 50 mM Tris. HCl pH 7.5, 150 mM NaCl, 0.5% NP40 and Complete protease inhibitor cocktail (Roche). Aliquots containing 80 μg protein from each sample were analysed by SDS-PAGE [ 27 ], and transferred electrophoretically to a PVDF membrane. Mouse monoclonal antibodies were used to detect tTA (Clontech), β-catenin (Transduction Laboratories) and EGFP (Santa Cruz Biotechnology). Samples of conditioned medium were concentrated 12-fold using Microcon YM-10 centrifugal filter units (Millipore) prior to analysis. Construction of plasmids A BgIII fragment containing the  lacZ  cDNA was excised from the CMV- lacZ  construct (a gift of Trevor Dale) and sub-cloned into BamHI digested RevTRE to make RevTRE- lacZ . A plasmid containing a myc-tagged ΔNβ-catenin was obtained from Hans Clevers. The myc-tagged ΔNβ-catenin was excised with KpnI and NotI and the ends were blunted, and subcloned into HpaI digested RevTRE to make RevTRE-Δ N β- catenin . The mouse  Hig2  open reading frame was amplified by PCR from I.M.A.G.E. cDNA clone 367488 using the primers TTTACTAGTAGGAGCTGGGCACCGTCGCC and TTTTACCGGTGCCTGCACTCCTCGGGATGGATGG. The PCR product was digested with AgeI and SpeI and subcloned into the AgeI and NheI sites in pEGFP-C1 (Clontech) to make the Hig2-EGFP fusion gene. Site directed mutagenesis was carried out by the method of Sawano and Miyawaki (2000) [ 28 ]. The primer TGCTGAACCTCGAGGAGCTGGGCATCATG was used to make the Hig2-EGFP(Y8V9/D8D9) mutant. Transient transfections Transient transfections were performed using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Briefly, 1.5 × 10 5  cells were plated in 3.5 cm wells on the day prior to transfection. Each well was transfected with a total of 0.9 μg DNA under serum-free conditions for six hours, after which the cells were washed and incubated for a further 48 hours before assaying expression. β-galactosidase activity assay For the tetracycline dose response curve, 5000 HC11- lacZ  cells for each condition, were cultured in triplicate in 96 well plates for 72 hours, and beta-galactosidase activity was determined as previously described [ 24 ].
Methods
Cell culture All reagents were purchased from Sigma unless otherwise noted. HC11 mouse mammary epithelial cells were cultured in 5% CO 2  at 37°C in RPMI 1640, supplemented with 10% Foetal Bovine Serum, 2 mM L-glutamine, 2.5 μg/ml insulin, 5 ng/ml epidermal growth factor and 50 μg/ml gentamycin [ 23 ]. HC11- lacZ  and HC11-Δ N β- catenin  cells were routinely cultured in 2 μg/ml tetracycline to repress transcription of the tetracycline-regulated transgene. HEK293 and MDCK cells were grown in DMEM supplemented with 10% Foetal Bovine Serum. The HC11- lacZ  and HC11-Δ N β- catenin  cell lines were generated by infecting the cells with an ecotropic retrovirus (TRE-tTA) in which the  tTA  cDNA is under the control of a tetracycline responsive promoter. Consequently, tTA expression is minimal in the presence of tetracycline and, upon tetracycline withdrawal, tTA activates its own transcription in an autoregulatory manner [ 24 ]. HC11 cells expressing tTA were subsequently infected with ecotropic retroviruses derived from RevTRE (Clontech) which directed the expression of either β-galactosidase or ΔNβ-catenin in a tetracycline dependent manner. Bosc23 cells were used to produce ecotropic retroviruses [ 25 ]. Cells were transiently transfected with the appropriate retroviral construct and the supernatant was collected 48 hours post-transfection. Polybrene was added to a final concentration of 5 μg/ml and the supernatant was added to HC11 cells for 24 hours. HC11 cells were then subjected to antibiotic selection using either 250 μg/ml G418 or 200 μg/ml hygromycin B as appropriate.
Cell culture
All reagents were purchased from Sigma unless otherwise noted. HC11 mouse mammary epithelial cells were cultured in 5% CO 2  at 37°C in RPMI 1640, supplemented with 10% Foetal Bovine Serum, 2 mM L-glutamine, 2.5 μg/ml insulin, 5 ng/ml epidermal growth factor and 50 μg/ml gentamycin [ 23 ]. HC11- lacZ  and HC11-Δ N β- catenin  cells were routinely cultured in 2 μg/ml tetracycline to repress transcription of the tetracycline-regulated transgene. HEK293 and MDCK cells were grown in DMEM supplemented with 10% Foetal Bovine Serum.lacZNcateninThe HC11- lacZ  and HC11-Δ N β- catenin  cell lines were generated by infecting the cells with an ecotropic retrovirus (TRE-tTA) in which the  tTA  cDNA is under the control of a tetracycline responsive promoter. Consequently, tTA expression is minimal in the presence of tetracycline and, upon tetracycline withdrawal, tTA activates its own transcription in an autoregulatory manner [ 24 ]. HC11 cells expressing tTA were subsequently infected with ecotropic retroviruses derived from RevTRE (Clontech) which directed the expression of either β-galactosidase or ΔNβ-catenin in a tetracycline dependent manner.lacZNcatenintTABosc23 cells were used to produce ecotropic retroviruses [ 25 ]. Cells were transiently transfected with the appropriate retroviral construct and the supernatant was collected 48 hours post-transfection. Polybrene was added to a final concentration of 5 μg/ml and the supernatant was added to HC11 cells for 24 hours. HC11 cells were then subjected to antibiotic selection using either 250 μg/ml G418 or 200 μg/ml hygromycin B as appropriate.RNA isolation Cell monolayers were washed twice in ice-cold Phosphate Buffered Saline and lysed by addition of Trizol (Invitrogen). Total RNA was isolated according to the manufacturer's instructions. PolyA+ RNA was purified from total RNA using Oligotex (Qiagen) according to the manufacturer's instructions.
RNA isolation
Cell monolayers were washed twice in ice-cold Phosphate Buffered Saline and lysed by addition of Trizol (Invitrogen). Total RNA was isolated according to the manufacturer's instructions. PolyA+ RNA was purified from total RNA using Oligotex (Qiagen) according to the manufacturer's instructions.Northern blotting 10 μg of total RNA from each cell line was fractionated on a denaturing formaldehyde agarose gel and transferred to a positively charged nylon membrane (Hybond N+, Amersham Pharmacia Biotech) in 10x SSC. Membranes were prehybridised for four hours in 50% (v/v) formamide, 5X SSPE, 2X Denhardt's reagent, 0.1% (w/v) SDS and 100 μg/ml denatured herring sperm DNA. Radiolabelled probes were prepared from PCR-amplified cDNA clones using the Rediprime II kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. EST sequences corresponding to the coding sequence of the genes-of-interest were identified by BLAST [ 26 ] and obtained from the I.M.A.G.E. consortium through the UK Human Genome Mapping Project Resource Centre (Hinxton, UK). ESTs bearing the following I.M.A.G.E. cloneIDs were used:  Autotaxin  – 533819;  CD14  – 2936787;  Ecm1  717050;  Hig2  – 367488;  Ramp3  – 615797,  HIG2  4366895). Following overnight hybridisation with the labelled probe, the membranes were washed twice in 1X SSC, 0.1% (w/v) SDS at room temperature for 20 mins, and twice in 0.2X SSC, 0.1% (w/v) SDS at 68°C for 10 mins and exposed to film at -80°C for 48 hours. Bound probe was quantitated using a phosphorimager (Molecular Dynamics).
Northern blotting
10 μg of total RNA from each cell line was fractionated on a denaturing formaldehyde agarose gel and transferred to a positively charged nylon membrane (Hybond N+, Amersham Pharmacia Biotech) in 10x SSC. Membranes were prehybridised for four hours in 50% (v/v) formamide, 5X SSPE, 2X Denhardt's reagent, 0.1% (w/v) SDS and 100 μg/ml denatured herring sperm DNA. Radiolabelled probes were prepared from PCR-amplified cDNA clones using the Rediprime II kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. EST sequences corresponding to the coding sequence of the genes-of-interest were identified by BLAST [ 26 ] and obtained from the I.M.A.G.E. consortium through the UK Human Genome Mapping Project Resource Centre (Hinxton, UK). ESTs bearing the following I.M.A.G.E. cloneIDs were used:  Autotaxin  – 533819;  CD14  – 2936787;  Ecm1  717050;  Hig2  – 367488;  Ramp3  – 615797,  HIG2  4366895). Following overnight hybridisation with the labelled probe, the membranes were washed twice in 1X SSC, 0.1% (w/v) SDS at room temperature for 20 mins, and twice in 0.2X SSC, 0.1% (w/v) SDS at 68°C for 10 mins and exposed to film at -80°C for 48 hours. Bound probe was quantitated using a phosphorimager (Molecular Dynamics).AutotaxinCD14Ecm1Hig2Ramp3HIG2Western blotting Cell monolayers were rinsed twice with ice-cold Phosphate Buffered Saline and total cell lysates were prepared by scraping cells into a minimal volume of 50 mM Tris. HCl pH 7.5, 150 mM NaCl, 0.5% NP40 and Complete protease inhibitor cocktail (Roche). Aliquots containing 80 μg protein from each sample were analysed by SDS-PAGE [ 27 ], and transferred electrophoretically to a PVDF membrane. Mouse monoclonal antibodies were used to detect tTA (Clontech), β-catenin (Transduction Laboratories) and EGFP (Santa Cruz Biotechnology). Samples of conditioned medium were concentrated 12-fold using Microcon YM-10 centrifugal filter units (Millipore) prior to analysis.
Western blotting
Cell monolayers were rinsed twice with ice-cold Phosphate Buffered Saline and total cell lysates were prepared by scraping cells into a minimal volume of 50 mM Tris. HCl pH 7.5, 150 mM NaCl, 0.5% NP40 and Complete protease inhibitor cocktail (Roche). Aliquots containing 80 μg protein from each sample were analysed by SDS-PAGE [ 27 ], and transferred electrophoretically to a PVDF membrane. Mouse monoclonal antibodies were used to detect tTA (Clontech), β-catenin (Transduction Laboratories) and EGFP (Santa Cruz Biotechnology). Samples of conditioned medium were concentrated 12-fold using Microcon YM-10 centrifugal filter units (Millipore) prior to analysis.Construction of plasmids A BgIII fragment containing the  lacZ  cDNA was excised from the CMV- lacZ  construct (a gift of Trevor Dale) and sub-cloned into BamHI digested RevTRE to make RevTRE- lacZ . A plasmid containing a myc-tagged ΔNβ-catenin was obtained from Hans Clevers. The myc-tagged ΔNβ-catenin was excised with KpnI and NotI and the ends were blunted, and subcloned into HpaI digested RevTRE to make RevTRE-Δ N β- catenin . The mouse  Hig2  open reading frame was amplified by PCR from I.M.A.G.E. cDNA clone 367488 using the primers TTTACTAGTAGGAGCTGGGCACCGTCGCC and TTTTACCGGTGCCTGCACTCCTCGGGATGGATGG. The PCR product was digested with AgeI and SpeI and subcloned into the AgeI and NheI sites in pEGFP-C1 (Clontech) to make the Hig2-EGFP fusion gene. Site directed mutagenesis was carried out by the method of Sawano and Miyawaki (2000) [ 28 ]. The primer TGCTGAACCTCGAGGAGCTGGGCATCATG was used to make the Hig2-EGFP(Y8V9/D8D9) mutant.
Construction of plasmids
A BgIII fragment containing the  lacZ  cDNA was excised from the CMV- lacZ  construct (a gift of Trevor Dale) and sub-cloned into BamHI digested RevTRE to make RevTRE- lacZ . A plasmid containing a myc-tagged ΔNβ-catenin was obtained from Hans Clevers. The myc-tagged ΔNβ-catenin was excised with KpnI and NotI and the ends were blunted, and subcloned into HpaI digested RevTRE to make RevTRE-Δ N β- catenin . The mouse  Hig2  open reading frame was amplified by PCR from I.M.A.G.E. cDNA clone 367488 using the primers TTTACTAGTAGGAGCTGGGCACCGTCGCC and TTTTACCGGTGCCTGCACTCCTCGGGATGGATGG. The PCR product was digested with AgeI and SpeI and subcloned into the AgeI and NheI sites in pEGFP-C1 (Clontech) to make the Hig2-EGFP fusion gene. Site directed mutagenesis was carried out by the method of Sawano and Miyawaki (2000) [ 28 ]. The primer TGCTGAACCTCGAGGAGCTGGGCATCATG was used to make the Hig2-EGFP(Y8V9/D8D9) mutant.lacZlacZlacZNcateninHig2Transient transfections Transient transfections were performed using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Briefly, 1.5 × 10 5  cells were plated in 3.5 cm wells on the day prior to transfection. Each well was transfected with a total of 0.9 μg DNA under serum-free conditions for six hours, after which the cells were washed and incubated for a further 48 hours before assaying expression.
Transient transfections
Transient transfections were performed using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Briefly, 1.5 × 10 5  cells were plated in 3.5 cm wells on the day prior to transfection. Each well was transfected with a total of 0.9 μg DNA under serum-free conditions for six hours, after which the cells were washed and incubated for a further 48 hours before assaying expression.β-galactosidase activity assay For the tetracycline dose response curve, 5000 HC11- lacZ  cells for each condition, were cultured in triplicate in 96 well plates for 72 hours, and beta-galactosidase activity was determined as previously described [ 24 ].
β-galactosidase activity assay
For the tetracycline dose response curve, 5000 HC11- lacZ  cells for each condition, were cultured in triplicate in 96 well plates for 72 hours, and beta-galactosidase activity was determined as previously described [ 24 ].lacZResults Generation of HC11- lacZ  and HC11-Δ N β- catenin  cell lines Stable cell lines were generated in which either ΔNβ-catenin or β-galactosidase was expressed in a tetracycline dependent manner. These cell lines were established using a novel autoregulatory system in which the expression level of the tetracycline transactivator (tTA) protein is minimised during routine culture and is induced upon withdrawal of tetracycline with concomitant upregulation of the transgene-of-interest [ 24 ]. This strategy helps to minimise deleterious effects due to tTA toxicity. A dose-response analysis for the HC11- lacZ  cell line is shown in Figure  1A . β-galactosidase expression is effectively repressed at tetracycline concentrations in excess of 20 ng/ml and is strongly induced in the absence of tetracycline. The N-terminal truncation mutant of β-catenin can be detected by western blotting by both its myc-epitope tag and an anti-β-catenin antibody (Figure  1B ). tTA expression is detectable only in the absence of tetracycline demonstrating the autoregulatory nature of this system. Microarray analysis Transgene expression was induced in HC11- lacZ  and HC11-Δ N β- catenin  cells by withdrawal of tetracycline for 72 hours. Total RNA was isolated, from which mRNA was purified. cDNAs were labelled and hybridized to an 8962 element Incyte mouse GEM1 cDNA microarray (Incyte Genomics, Palo Alto, CA). These data are provided as supplementary material (See  Additional file 1 ). Among those genes upregulated were two genes shown by other workers to be transcriptional targets of this pathway –  Fibronectin  [ 29 ] and  Autotaxin  [ 30 ] (data not shown) – suggesting that our model of Wnt/β-catenin signalling deregulation results in the activation of a set of target genes which overlaps, at least partially, with pathway targets in other cell lines. The microarray experiment described here was performed only once but differential expression was repeatedly validated by northern blotting from independent samples for the genes discussed here. Validation of targets Five genes were selected for further study –  Extracellular Matrix Protein 1  ( Ecm1 ),  Autotaxin ,  Receptor Activity Modifying Protein 3  ( Ramp3 ),  Cd14  and  Hypoxia Inducible Gene 2  ( Hig2 ). Each putative target gene was initially subjected to a secondary screen by Northern blotting to confirm the differential expression in response to ΔNβ-catenin (Figure  2 ). RNA samples used for Northern blotting were from independent induction experiments to those used for microarray analysis, thus demonstrating repeatedly by two distinct methods that the transcript levels of these genes are altered in cells overexpressing ΔNβ-catenin. The expression level of each of the transcripts was quantitated using a phosphorimager and normalised to the expression of  Gapdh  mRNA in the samples. The data in Figure  2  represent film exposure times ranging between 24 and 72 hours. Quantitations were performed using short (one hour or less) exposures to a phosphorimager screen, such that the signal intensity was not saturating. Molecular cloning of mouse  Hig2 Hypoxia Inducible Gene 2  encodes a 63 amino acid polypeptide and was one of several genes identified in a screen for genes regulated by hypoxia in a human cervical epithelial cell line [ 31 ]. HIG2 shares no sequence similarity with other known proteins. In order to facilitate the functional analysis of this gene, ESTs were identified which encoded mouse and rat Hig2, and the sequences of chimpanzee and baboon were inferred from genomic sequence data. A multiple alignment of the inferred amino acid sequences shows that these polypeptides are highly similar (Fig  3A ). Analysis of these sequences using a Kyte-Doolittle hydrophobicity plot showed that the N-termini of these proteins contain a series of hydrophobic amino acids (Fig  3B ). This region of hydrophobicity was reminiscent of a signal peptide and sequence analysis using the signal peptide prediction program, SignalP [ 32 , 33 ] supported this possibility. Hig2 has an N-terminal signal peptide and is secreted To investigate the subcellular localisation of Hig2, a  Hig2 - EGFP  fusion gene was constructed and expressed in both HC11 and Madin-Darby Canine Kidney (MDCK) cells by transient transfection (Figure  4a  and  4c ). In both cell lines, Hig2-EGFP is localised to large round vesicle-like structures in the cytoplasm. Similar observations were made in HEK-293 cells (data not shown). The fluorescence was detected predominantly around the periphery of these structures suggesting that they do not consist of solid masses of aggregated protein. When two aspartate residues were introduced to the putative signal peptide by site-directed mutagenesis, Hig2(Y8V9/D8D9)EGFP, this distinctive subcellular localization was abolished (Fig  4B  and  4D ). These large structures did not colocalize with either markers of mitochondria (pDsRed2-mito, Clontech), nor lipid droplets (Nile Red, Molecular Probes) nor with markers of endosomes or lysosomes (pulse-chase analysis with TRITC-dextran); data not shown. However, in live HC11 cells transfected with Hig2-EGFP, observations at high magnification revealed that the cytoplasm of these cells contained many very small solid green vesicles moving along the cytoskeleton. These vesicles were approx 1/100 the size of the large vesicles shown in Fig  4A  and  4C , and were not observed in cells transfected with either Hig2(Y8V9/D8D9)EGFP or EGFP alone. The rapidity of this motion in live cells, even at room temperature, precluded capture of these images but suggested the possibility that measurable amounts of secreted Hig2-EGFP might be found in the culture medium. HEK-293 cells were chosen as they could be transfected at high efficiency (approx 80%), the presence of the green transport vesicles was confirmed, and 48 hours after transfection samples of total cell lysate and conditioned medium were analysed by western blotting. Secreted Hig2-EGFP was detected in the conditioned medium of Hig2-EGFP cells, but not Hig2(Y8V9/D8D9)EGFP cells (Fig  5 ). Multiple bands were detected in cell lysates for both Hig2-EGFP fusion proteins: whether these represent artifactual degradation products or physiologically relevant biological entities is as yet unknown. Such multiple banding has also been observed with other EGFP-fusion proteins targeted to the secretory pathway (Amphiregulin-EGFP, PK unpublished observations). At least one of the bands may result from internal translation initiation at the consensus Kozak initiation sequence of pEGFP-C1 which is located between the Hig2 and EGFP open reading frames. EGFP was also detected in the conditioned medium. This is consistent with previous reports of GFP secretion via a non-classical Brefeldin A-insensitive pathway [ 34 ]. In this study, several cell lines are described (including HEK293) in which wild-type GFP is released from the cell without passing through the golgi apparatus. Thus, it is formally possible that, instead of its secretion being directed by the putative signal peptide, HIG2-EGFP might be released from the cell via this pathway in a manner specifically dependent on the EGFP moiety. The presence of post-translational modifications acquired during endoplasmic reticulum/golgi apparatus mediated secretion would exclude the latter hypothesis. The altered mobility of HIG-2-EGFP in the medium suggested that it might be glycosylated, however the mobility was not changed by treatment with the glycosidase PNGaseF, suggesting that this secreted protein is not glycosylated (data not shown). Previous studies using GFP fused to a signal peptide directing entry into the ER demonstrated that, in this redox environment, the cysteine residues of GFP form intermolecular disulphide bridges which result in oligomerization of GFP molecules [ 35 ]. Oligomers of Hig2-EGFP were detected (Fig  5 , black arrowheads) but no oligomerization of EGFP was observed. Hig2 itself does not contain cysteine residues, thus the oligomerization is mediated by the EGFP domains. These data are consistent with HIG2-EGFP entry into the classical secretory pathway. Collectively, these data demonstrate that Hig2 contains a functional N-terminal signal peptide and is likely a secreted protein. Expression of  HIG2  in human tumours To investigate the relevance of  HIG2  in human tumours, the expression level of this gene was examined in 68 tumour cDNA samples compared to normal adjacent tissue from the same patients using a Matched Tumour/Normal cDNA blot (Clontech) (Figure  6A ). The levels of  HIG2  were approximately similar in most of the tumour types examined but were strongly and consistently downregulated in most of the cases of kidney and stomach tumours analysed. These data suggest that the downregulation of  HIG2  observed upon deregulated β-catenin signalling  in vitro  may be of clinical relevance in human tumours.
Results
Generation of HC11- lacZ  and HC11-Δ N β- catenin  cell lines Stable cell lines were generated in which either ΔNβ-catenin or β-galactosidase was expressed in a tetracycline dependent manner. These cell lines were established using a novel autoregulatory system in which the expression level of the tetracycline transactivator (tTA) protein is minimised during routine culture and is induced upon withdrawal of tetracycline with concomitant upregulation of the transgene-of-interest [ 24 ]. This strategy helps to minimise deleterious effects due to tTA toxicity. A dose-response analysis for the HC11- lacZ  cell line is shown in Figure  1A . β-galactosidase expression is effectively repressed at tetracycline concentrations in excess of 20 ng/ml and is strongly induced in the absence of tetracycline. The N-terminal truncation mutant of β-catenin can be detected by western blotting by both its myc-epitope tag and an anti-β-catenin antibody (Figure  1B ). tTA expression is detectable only in the absence of tetracycline demonstrating the autoregulatory nature of this system.
Generation of HC11- lacZ  and HC11-Δ N β- catenin  cell lines
lacZNcateninStable cell lines were generated in which either ΔNβ-catenin or β-galactosidase was expressed in a tetracycline dependent manner. These cell lines were established using a novel autoregulatory system in which the expression level of the tetracycline transactivator (tTA) protein is minimised during routine culture and is induced upon withdrawal of tetracycline with concomitant upregulation of the transgene-of-interest [ 24 ]. This strategy helps to minimise deleterious effects due to tTA toxicity.A dose-response analysis for the HC11- lacZ  cell line is shown in Figure  1A . β-galactosidase expression is effectively repressed at tetracycline concentrations in excess of 20 ng/ml and is strongly induced in the absence of tetracycline. The N-terminal truncation mutant of β-catenin can be detected by western blotting by both its myc-epitope tag and an anti-β-catenin antibody (Figure  1B ). tTA expression is detectable only in the absence of tetracycline demonstrating the autoregulatory nature of this system.lacZMicroarray analysis Transgene expression was induced in HC11- lacZ  and HC11-Δ N β- catenin  cells by withdrawal of tetracycline for 72 hours. Total RNA was isolated, from which mRNA was purified. cDNAs were labelled and hybridized to an 8962 element Incyte mouse GEM1 cDNA microarray (Incyte Genomics, Palo Alto, CA). These data are provided as supplementary material (See  Additional file 1 ). Among those genes upregulated were two genes shown by other workers to be transcriptional targets of this pathway –  Fibronectin  [ 29 ] and  Autotaxin  [ 30 ] (data not shown) – suggesting that our model of Wnt/β-catenin signalling deregulation results in the activation of a set of target genes which overlaps, at least partially, with pathway targets in other cell lines. The microarray experiment described here was performed only once but differential expression was repeatedly validated by northern blotting from independent samples for the genes discussed here.
Microarray analysis
Transgene expression was induced in HC11- lacZ  and HC11-Δ N β- catenin  cells by withdrawal of tetracycline for 72 hours. Total RNA was isolated, from which mRNA was purified. cDNAs were labelled and hybridized to an 8962 element Incyte mouse GEM1 cDNA microarray (Incyte Genomics, Palo Alto, CA). These data are provided as supplementary material (See  Additional file 1 ). Among those genes upregulated were two genes shown by other workers to be transcriptional targets of this pathway –  Fibronectin  [ 29 ] and  Autotaxin  [ 30 ] (data not shown) – suggesting that our model of Wnt/β-catenin signalling deregulation results in the activation of a set of target genes which overlaps, at least partially, with pathway targets in other cell lines. The microarray experiment described here was performed only once but differential expression was repeatedly validated by northern blotting from independent samples for the genes discussed here.lacZNcateninFibronectinAutotaxinValidation of targets Five genes were selected for further study –  Extracellular Matrix Protein 1  ( Ecm1 ),  Autotaxin ,  Receptor Activity Modifying Protein 3  ( Ramp3 ),  Cd14  and  Hypoxia Inducible Gene 2  ( Hig2 ). Each putative target gene was initially subjected to a secondary screen by Northern blotting to confirm the differential expression in response to ΔNβ-catenin (Figure  2 ). RNA samples used for Northern blotting were from independent induction experiments to those used for microarray analysis, thus demonstrating repeatedly by two distinct methods that the transcript levels of these genes are altered in cells overexpressing ΔNβ-catenin. The expression level of each of the transcripts was quantitated using a phosphorimager and normalised to the expression of  Gapdh  mRNA in the samples. The data in Figure  2  represent film exposure times ranging between 24 and 72 hours. Quantitations were performed using short (one hour or less) exposures to a phosphorimager screen, such that the signal intensity was not saturating.
Validation of targets
Five genes were selected for further study –  Extracellular Matrix Protein 1  ( Ecm1 ),  Autotaxin ,  Receptor Activity Modifying Protein 3  ( Ramp3 ),  Cd14  and  Hypoxia Inducible Gene 2  ( Hig2 ). Each putative target gene was initially subjected to a secondary screen by Northern blotting to confirm the differential expression in response to ΔNβ-catenin (Figure  2 ). RNA samples used for Northern blotting were from independent induction experiments to those used for microarray analysis, thus demonstrating repeatedly by two distinct methods that the transcript levels of these genes are altered in cells overexpressing ΔNβ-catenin. The expression level of each of the transcripts was quantitated using a phosphorimager and normalised to the expression of  Gapdh  mRNA in the samples. The data in Figure  2  represent film exposure times ranging between 24 and 72 hours. Quantitations were performed using short (one hour or less) exposures to a phosphorimager screen, such that the signal intensity was not saturating.Extracellular Matrix Protein 1Ecm1AutotaxinReceptor Activity Modifying Protein 3Ramp3Cd14Hypoxia Inducible Gene 2Hig2GapdhMolecular cloning of mouse  Hig2 Hypoxia Inducible Gene 2  encodes a 63 amino acid polypeptide and was one of several genes identified in a screen for genes regulated by hypoxia in a human cervical epithelial cell line [ 31 ]. HIG2 shares no sequence similarity with other known proteins. In order to facilitate the functional analysis of this gene, ESTs were identified which encoded mouse and rat Hig2, and the sequences of chimpanzee and baboon were inferred from genomic sequence data. A multiple alignment of the inferred amino acid sequences shows that these polypeptides are highly similar (Fig  3A ). Analysis of these sequences using a Kyte-Doolittle hydrophobicity plot showed that the N-termini of these proteins contain a series of hydrophobic amino acids (Fig  3B ). This region of hydrophobicity was reminiscent of a signal peptide and sequence analysis using the signal peptide prediction program, SignalP [ 32 , 33 ] supported this possibility.
Molecular cloning of mouse  Hig2
Hig2Hypoxia Inducible Gene 2  encodes a 63 amino acid polypeptide and was one of several genes identified in a screen for genes regulated by hypoxia in a human cervical epithelial cell line [ 31 ]. HIG2 shares no sequence similarity with other known proteins. In order to facilitate the functional analysis of this gene, ESTs were identified which encoded mouse and rat Hig2, and the sequences of chimpanzee and baboon were inferred from genomic sequence data. A multiple alignment of the inferred amino acid sequences shows that these polypeptides are highly similar (Fig  3A ). Analysis of these sequences using a Kyte-Doolittle hydrophobicity plot showed that the N-termini of these proteins contain a series of hydrophobic amino acids (Fig  3B ). This region of hydrophobicity was reminiscent of a signal peptide and sequence analysis using the signal peptide prediction program, SignalP [ 32 , 33 ] supported this possibility.Hypoxia Inducible Gene 2Hig2 has an N-terminal signal peptide and is secreted To investigate the subcellular localisation of Hig2, a  Hig2 - EGFP  fusion gene was constructed and expressed in both HC11 and Madin-Darby Canine Kidney (MDCK) cells by transient transfection (Figure  4a  and  4c ). In both cell lines, Hig2-EGFP is localised to large round vesicle-like structures in the cytoplasm. Similar observations were made in HEK-293 cells (data not shown). The fluorescence was detected predominantly around the periphery of these structures suggesting that they do not consist of solid masses of aggregated protein. When two aspartate residues were introduced to the putative signal peptide by site-directed mutagenesis, Hig2(Y8V9/D8D9)EGFP, this distinctive subcellular localization was abolished (Fig  4B  and  4D ). These large structures did not colocalize with either markers of mitochondria (pDsRed2-mito, Clontech), nor lipid droplets (Nile Red, Molecular Probes) nor with markers of endosomes or lysosomes (pulse-chase analysis with TRITC-dextran); data not shown. However, in live HC11 cells transfected with Hig2-EGFP, observations at high magnification revealed that the cytoplasm of these cells contained many very small solid green vesicles moving along the cytoskeleton. These vesicles were approx 1/100 the size of the large vesicles shown in Fig  4A  and  4C , and were not observed in cells transfected with either Hig2(Y8V9/D8D9)EGFP or EGFP alone. The rapidity of this motion in live cells, even at room temperature, precluded capture of these images but suggested the possibility that measurable amounts of secreted Hig2-EGFP might be found in the culture medium. HEK-293 cells were chosen as they could be transfected at high efficiency (approx 80%), the presence of the green transport vesicles was confirmed, and 48 hours after transfection samples of total cell lysate and conditioned medium were analysed by western blotting. Secreted Hig2-EGFP was detected in the conditioned medium of Hig2-EGFP cells, but not Hig2(Y8V9/D8D9)EGFP cells (Fig  5 ). Multiple bands were detected in cell lysates for both Hig2-EGFP fusion proteins: whether these represent artifactual degradation products or physiologically relevant biological entities is as yet unknown. Such multiple banding has also been observed with other EGFP-fusion proteins targeted to the secretory pathway (Amphiregulin-EGFP, PK unpublished observations). At least one of the bands may result from internal translation initiation at the consensus Kozak initiation sequence of pEGFP-C1 which is located between the Hig2 and EGFP open reading frames. EGFP was also detected in the conditioned medium. This is consistent with previous reports of GFP secretion via a non-classical Brefeldin A-insensitive pathway [ 34 ]. In this study, several cell lines are described (including HEK293) in which wild-type GFP is released from the cell without passing through the golgi apparatus. Thus, it is formally possible that, instead of its secretion being directed by the putative signal peptide, HIG2-EGFP might be released from the cell via this pathway in a manner specifically dependent on the EGFP moiety. The presence of post-translational modifications acquired during endoplasmic reticulum/golgi apparatus mediated secretion would exclude the latter hypothesis. The altered mobility of HIG-2-EGFP in the medium suggested that it might be glycosylated, however the mobility was not changed by treatment with the glycosidase PNGaseF, suggesting that this secreted protein is not glycosylated (data not shown). Previous studies using GFP fused to a signal peptide directing entry into the ER demonstrated that, in this redox environment, the cysteine residues of GFP form intermolecular disulphide bridges which result in oligomerization of GFP molecules [ 35 ]. Oligomers of Hig2-EGFP were detected (Fig  5 , black arrowheads) but no oligomerization of EGFP was observed. Hig2 itself does not contain cysteine residues, thus the oligomerization is mediated by the EGFP domains. These data are consistent with HIG2-EGFP entry into the classical secretory pathway. Collectively, these data demonstrate that Hig2 contains a functional N-terminal signal peptide and is likely a secreted protein.
Hig2 has an N-terminal signal peptide and is secreted
To investigate the subcellular localisation of Hig2, a  Hig2 - EGFP  fusion gene was constructed and expressed in both HC11 and Madin-Darby Canine Kidney (MDCK) cells by transient transfection (Figure  4a  and  4c ). In both cell lines, Hig2-EGFP is localised to large round vesicle-like structures in the cytoplasm. Similar observations were made in HEK-293 cells (data not shown). The fluorescence was detected predominantly around the periphery of these structures suggesting that they do not consist of solid masses of aggregated protein. When two aspartate residues were introduced to the putative signal peptide by site-directed mutagenesis, Hig2(Y8V9/D8D9)EGFP, this distinctive subcellular localization was abolished (Fig  4B  and  4D ). These large structures did not colocalize with either markers of mitochondria (pDsRed2-mito, Clontech), nor lipid droplets (Nile Red, Molecular Probes) nor with markers of endosomes or lysosomes (pulse-chase analysis with TRITC-dextran); data not shown. However, in live HC11 cells transfected with Hig2-EGFP, observations at high magnification revealed that the cytoplasm of these cells contained many very small solid green vesicles moving along the cytoskeleton. These vesicles were approx 1/100 the size of the large vesicles shown in Fig  4A  and  4C , and were not observed in cells transfected with either Hig2(Y8V9/D8D9)EGFP or EGFP alone. The rapidity of this motion in live cells, even at room temperature, precluded capture of these images but suggested the possibility that measurable amounts of secreted Hig2-EGFP might be found in the culture medium. HEK-293 cells were chosen as they could be transfected at high efficiency (approx 80%), the presence of the green transport vesicles was confirmed, and 48 hours after transfection samples of total cell lysate and conditioned medium were analysed by western blotting. Secreted Hig2-EGFP was detected in the conditioned medium of Hig2-EGFP cells, but not Hig2(Y8V9/D8D9)EGFP cells (Fig  5 ). Multiple bands were detected in cell lysates for both Hig2-EGFP fusion proteins: whether these represent artifactual degradation products or physiologically relevant biological entities is as yet unknown. Such multiple banding has also been observed with other EGFP-fusion proteins targeted to the secretory pathway (Amphiregulin-EGFP, PK unpublished observations). At least one of the bands may result from internal translation initiation at the consensus Kozak initiation sequence of pEGFP-C1 which is located between the Hig2 and EGFP open reading frames.Hig2EGFPEGFP was also detected in the conditioned medium. This is consistent with previous reports of GFP secretion via a non-classical Brefeldin A-insensitive pathway [ 34 ]. In this study, several cell lines are described (including HEK293) in which wild-type GFP is released from the cell without passing through the golgi apparatus. Thus, it is formally possible that, instead of its secretion being directed by the putative signal peptide, HIG2-EGFP might be released from the cell via this pathway in a manner specifically dependent on the EGFP moiety. The presence of post-translational modifications acquired during endoplasmic reticulum/golgi apparatus mediated secretion would exclude the latter hypothesis. The altered mobility of HIG-2-EGFP in the medium suggested that it might be glycosylated, however the mobility was not changed by treatment with the glycosidase PNGaseF, suggesting that this secreted protein is not glycosylated (data not shown). Previous studies using GFP fused to a signal peptide directing entry into the ER demonstrated that, in this redox environment, the cysteine residues of GFP form intermolecular disulphide bridges which result in oligomerization of GFP molecules [ 35 ]. Oligomers of Hig2-EGFP were detected (Fig  5 , black arrowheads) but no oligomerization of EGFP was observed. Hig2 itself does not contain cysteine residues, thus the oligomerization is mediated by the EGFP domains. These data are consistent with HIG2-EGFP entry into the classical secretory pathway. Collectively, these data demonstrate that Hig2 contains a functional N-terminal signal peptide and is likely a secreted protein.Expression of  HIG2  in human tumours To investigate the relevance of  HIG2  in human tumours, the expression level of this gene was examined in 68 tumour cDNA samples compared to normal adjacent tissue from the same patients using a Matched Tumour/Normal cDNA blot (Clontech) (Figure  6A ). The levels of  HIG2  were approximately similar in most of the tumour types examined but were strongly and consistently downregulated in most of the cases of kidney and stomach tumours analysed. These data suggest that the downregulation of  HIG2  observed upon deregulated β-catenin signalling  in vitro  may be of clinical relevance in human tumours.
Expression of  HIG2  in human tumours
HIG2To investigate the relevance of  HIG2  in human tumours, the expression level of this gene was examined in 68 tumour cDNA samples compared to normal adjacent tissue from the same patients using a Matched Tumour/Normal cDNA blot (Clontech) (Figure  6A ). The levels of  HIG2  were approximately similar in most of the tumour types examined but were strongly and consistently downregulated in most of the cases of kidney and stomach tumours analysed. These data suggest that the downregulation of  HIG2  observed upon deregulated β-catenin signalling  in vitro  may be of clinical relevance in human tumours.HIG2HIG2HIG2in vitroDiscussion cDNA microarray analysis of the transcriptional changes resulting from overexpression of a constitutively active β-catenin revealed a panel of putative target genes of the Wnt/β-catenin pathway in mouse mammary epithelial cells. This differential expression was confirmed by Northern blotting in five cases. Autotaxin was originally identified as a secreted enzyme with potent motility stimulating activity [ 36 ] and has 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 recombinant 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 Autotaxin on tumour cell motility are mediated by its conversion of lysophosphatidylcholine to lysophosphatidic 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 vascular endothelial cells  in vitro  and also stimulate angiogenesis  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 nutrients 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 identified 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 ΔNβ-catenin expression. CD14 is a glycosyl-phosphatidylinositol-linked cell surface protein, preferentially expressed in monocytes, where it acts as a receptor for Lipopolysaccharide Binding Protein:Lipopolysaccharide complexes [ 46 ]. Soluble CD14 (sCD14) is also expressed in mammary epithelial 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 downregulated upon ΔNβ-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-protein 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 receptor dimer, functions as a growth factor in several human tumour cell lines [ 52 ], in addition to promoting angiogenesis  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 epithelial cell line. The human gene encodes a 63 amino acid polypeptide of unknown function [ 31 ]. Expression of mouse  Hig2  was downregulated in HC11 cells overexpressing ΔNβ-catenin. The identification of a group of mammalian orthologues revealed a well conserved hydrophobic 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 secretion, lending support to the hypothesis that this region contains a functional signal peptide. The nature of the large vesicular structures observed in Hig2-EGFP overexpressing 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, endosomes nor lipid droplets. Given the demonstration that Hig2 is secreted, these structure most likely correspond to overexpressed Hig2-EGFP in transit through the endoplasmic 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 characterisation of this gene. Analysis of the expression of  HIG2  using a matched Tumour/Normal tissue cDNA array showed that  HIG2  is widely expressed. In most cases, the levels of  HIG2  in the tumours and the associated normal tissue controls were similar.  HIG2  was, however, strongly and consistently downregulated in the majority of the kidney and stomach tumours analysed. This represents an significant validation of our  in vitro  findings in human tumours, and suggests that HIG2 may exert a tumour suppressive effect  in vivo . Human HIG2 is located on 7q32.2, a commonly deleted region in several tumour types, most prominently leukaemias and lymphomas [ 57 ]. Deletion analysis of 7q in a panel of patients with Splenic Lymphoma with Villous Lymphocytes by Catovsky and colleagues suggests that a critical tumour suppressor is located on 7q32 [ 58 ].
Discussion
cDNA microarray analysis of the transcriptional changes resulting from overexpression of a constitutively active β-catenin revealed a panel of putative target genes of the Wnt/β-catenin pathway in mouse mammary epithelial cells. This differential expression was confirmed by Northern blotting in five cases.Autotaxin was originally identified as a secreted enzyme with potent motility stimulating activity [ 36 ] and has 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 recombinant 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 Autotaxin on tumour cell motility are mediated by its conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA), a potent signalling molecule [ 40 , 41 ].Autotaxinin vivoExtracellular 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 vascular endothelial cells  in vitro  and also stimulate angiogenesis  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 nutrients 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 identified 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.In situEcm1in vitroin vivode novoAutotaxinEcm1CD14 , which can function as both a receptor and a secreted protein, was downregulated upon ΔNβ-catenin expression. CD14 is a glycosyl-phosphatidylinositol-linked cell surface protein, preferentially expressed in monocytes, where it acts as a receptor for Lipopolysaccharide Binding Protein:Lipopolysaccharide complexes [ 46 ]. Soluble CD14 (sCD14) is also expressed in mammary epithelial 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 ].CD14in vitroin vivoReceptor Activity Modifying Protein 3  ( RAMP3 ) was downregulated upon ΔNβ-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-protein 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 ].Receptor Activity Modifying Protein 3RAMP3Expression 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 receptor dimer, functions as a growth factor in several human tumour cell lines [ 52 ], in addition to promoting angiogenesis  in vivo  [ 53 ] via CRLR/RAMP3 and CRLR/RAMP2 receptor dimers [ 54 , 55 ].in vivoHypoxia - 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 epithelial cell line. The human gene encodes a 63 amino acid polypeptide of unknown function [ 31 ]. Expression of mouse  Hig2  was downregulated in HC11 cells overexpressing ΔNβ-catenin. The identification of a group of mammalian orthologues revealed a well conserved hydrophobic 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 secretion, lending support to the hypothesis that this region contains a functional signal peptide. The nature of the large vesicular structures observed in Hig2-EGFP overexpressing 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, endosomes nor lipid droplets. Given the demonstration that Hig2 is secreted, these structure most likely correspond to overexpressed Hig2-EGFP in transit through the endoplasmic 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 characterisation of this gene.Hypoxiainducible gene 2Hig2Hig2Analysis of the expression of  HIG2  using a matched Tumour/Normal tissue cDNA array showed that  HIG2  is widely expressed. In most cases, the levels of  HIG2  in the tumours and the associated normal tissue controls were similar.  HIG2  was, however, strongly and consistently downregulated in the majority of the kidney and stomach tumours analysed. This represents an significant validation of our  in vitro  findings in human tumours, and suggests that HIG2 may exert a tumour suppressive effect  in vivo . Human HIG2 is located on 7q32.2, a commonly deleted region in several tumour types, most prominently leukaemias and lymphomas [ 57 ]. Deletion analysis of 7q in a panel of patients with Splenic Lymphoma with Villous Lymphocytes by Catovsky and colleagues suggests that a critical tumour suppressor is located on 7q32 [ 58 ].HIG2HIG2HIG2HIG2in vitroin vivoConclusions The identification of this panel of candidate target genes for this clinically important signal transduction pathway adds to those identified by other workers in a variety of model systems and suggests that, as well as promoting tumour cell proliferation and survival in a cell autonomous manner, this activation of this pathway is likely to have a series of non-cell autonomous effects. Here we have focussed on the identification of Wnt/β-catenin target genes that are either secreted signalling molecules or receptors. It is likely that such targets are involved in mediating autocrine proliferation, promotion of angiogenesis and the mediation of reciprocal communication between Wnt/β-catenin tumours and their microenvironmental milieu.
Conclusions
The identification of this panel of candidate target genes for this clinically important signal transduction pathway adds to those identified by other workers in a variety of model systems and suggests that, as well as promoting tumour cell proliferation and survival in a cell autonomous manner, this activation of this pathway is likely to have a series of non-cell autonomous effects. Here we have focussed on the identification of Wnt/β-catenin target genes that are either secreted signalling molecules or receptors. It is likely that such targets are involved in mediating autocrine proliferation, promotion of angiogenesis and the mediation of reciprocal communication between Wnt/β-catenin tumours and their microenvironmental milieu.Competing interests The authors declare that they have no competing interests.
Competing interests
The authors declare that they have no competing interests.Authors' contributions PK carried out all of the experimental procedures and drafted the manuscript. PK, TE and AA contributed to the design of the study. All authors read and approved the final version of this manuscript.
Authors' contributions
PK carried out all of the experimental procedures and drafted the manuscript. PK, TE and AA contributed to the design of the study. All authors read and approved the final version of this manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:Supplementary Material Additional File 1 cDNA microarray dataset: HC11 ΔNβ-catenin v. HC11 lacZ. This file contains the dataset from the Incyte GEM1 cDNA microarray comparison between HC11 cells overexpressing ΔNβ-catenin (Probe 1) and β-galactosidase (Probe 2). This file may be easily imported into MS Excel, Genespring or other microarray analysis software to facilitate further analysis. Click here for file
Supplementary Material
Additional File 1
cDNA microarray dataset: HC11 ΔNβ-catenin v. HC11 lacZ. This file contains the dataset from the Incyte GEM1 cDNA microarray comparison between HC11 cells overexpressing ΔNβ-catenin (Probe 1) and β-galactosidase (Probe 2). This file may be easily imported into MS Excel, Genespring or other microarray analysis software to facilitate further analysis.Click here for file


Background Lymphoma-associated hemophagocytic syndrome (LAHS) occurs in mostly extra nodal non-Hodgkin's lymphoma. LAHS arising from gastrointestinal lymphoma has never been reported. Here we report a case of gastric T-cell lymphoma-associated hemophagocytic syndrome.
Background
Lymphoma-associated hemophagocytic syndrome (LAHS) occurs in mostly extra nodal non-Hodgkin's lymphoma. LAHS arising from gastrointestinal lymphoma has never been reported. Here we report a case of gastric T-cell lymphoma-associated hemophagocytic syndrome.Case presentation A 51-year-old woman presented with pain, redness of breasts, fever and hematemesis. Hematological examination revealed anemia. Gastroscopy revealed small bleeding ulcers in the stomach and the computed tomography scan showed liver tumor. She underwent total gastrectomy for gastrointestinal bleeding and the histopathology revealed gastric T-cell lymphoma. She continued to bleed from the anastomosis and died on the 8th postoperative day. Autopsy revealed it to be a LAHS.
Case presentation
A 51-year-old woman presented with pain, redness of breasts, fever and hematemesis. Hematological examination revealed anemia. Gastroscopy revealed small bleeding ulcers in the stomach and the computed tomography scan showed liver tumor. She underwent total gastrectomy for gastrointestinal bleeding and the histopathology revealed gastric T-cell lymphoma. She continued to bleed from the anastomosis and died on the 8th postoperative day. Autopsy revealed it to be a LAHS.Conclusions If Hemophagocytic syndrome (HPS) occurs in lymphoma of the gastrointestinal tract, bleeding from the primary lesion might be uncontrollable. Early diagnosis and appropriate treatment are needed for long-term survival.
Conclusions
If Hemophagocytic syndrome (HPS) occurs in lymphoma of the gastrointestinal tract, bleeding from the primary lesion might be uncontrollable. Early diagnosis and appropriate treatment are needed for long-term survival.

Background Hemophagocytic syndrome (HPS) in adults is characterized by reactive and systemic proliferation of benign histiocytes that phagocytose blood cells [ 1 ]. It is often associated with infections, malignant neoplasms, autoimmune diseases and various immunodeficiencies. Lymphoma-associated hemophagocytic syndrome (LAHS) mostly occurs from extra nodal lymphoma and is known to have a poor prognosis. Here we report a case of LAHS arising from gastric lymphoma with a fulminant clinical course and difficult diagnosis until the time of autopsy.
Background
Hemophagocytic syndrome (HPS) in adults is characterized by reactive and systemic proliferation of benign histiocytes that phagocytose blood cells [ 1 ]. It is often associated with infections, malignant neoplasms, autoimmune diseases and various immunodeficiencies. Lymphoma-associated hemophagocytic syndrome (LAHS) mostly occurs from extra nodal lymphoma and is known to have a poor prognosis. Here we report a case of LAHS arising from gastric lymphoma with a fulminant clinical course and difficult diagnosis until the time of autopsy.Case presentation A 51-year-old female was admitted on May 9, 1995, because of severe hematemesis. The patient had been treated elsewhere for one month for pain and redness of both breasts and fever (≥ 38°C). There was no generalized lymphadenopathy. On gastroscopic examination multiple small ulcers were observed in the stomach. An abdominal computed tomographic (CT) scan showed liver tumor and a normal spleen. Hematological and biochemical examination at admission showed the following results: RBC 352 × 10 4 /mm 3 , hemoglobin 10.3 g/dl (post transfusion), WBC 4,900/mm 3 , Platelets 51,000/mm 3 , serum albumin 1.5 g/dl, total bilirubin 0.6 mg/dl, AST 691 IU/l, ALT 187 IU/l, LDH 2976 IU/l, fibrinogen 134 mg/dl, FDP 10 μg/ml, and AT-III 40%. Bleeding from the stomach continued and did not stop with conservative treatment; therefore, two days later the patient underwent total gastrectomy and a partial liver resection. Histopathology of the resected specimen showed it to be a gastric lymphoma (pleomorphic medium-large cell type, non-Hodgkin's T-cell lymphoma) with liver metastasis (Fig.  1 ). From first postoperative day (POD), bleeding from the esophagojejunostomy continued; the patient developed disseminated intravascular coagulopathy and died on 8 th  postoperative day. Figure 1 Photomicrograph showing medium-large sized atypical lymphoid cells with pleomorphic features in the stomach suggesting a gastric lymphoma (Hematoxylin and Eosin, ×170). On autopsy, malignant lymphoid cell infiltration and hemophagocytosis were observed in the liver, spleen, heart, small bowel, lung, both breasts, kidney, pancreas, uterus, and gastroduodenal lymph nodes (Fig.  2 ). The bone marrow presented hyperplasia and hemophagocytic macrophages but no infiltration by lymphoma cells. Immunohistochemically the neoplastic cells were positive for T-cell marker UCHL1 (CD45RO) and EBV by EBER  in situ  hybridization. The final diagnosis was EBV-related T-cell LAHS. Figure 2 Photomicrograph of the lymph node at autopsy illustrating histiocytes that show hemophagocytosis of normoblast in a lymph node (Hematoxylin and Eosin, ×200).
Case presentation
A 51-year-old female was admitted on May 9, 1995, because of severe hematemesis. The patient had been treated elsewhere for one month for pain and redness of both breasts and fever (≥ 38°C). There was no generalized lymphadenopathy. On gastroscopic examination multiple small ulcers were observed in the stomach. An abdominal computed tomographic (CT) scan showed liver tumor and a normal spleen. Hematological and biochemical examination at admission showed the following results: RBC 352 × 10 4 /mm 3 , hemoglobin 10.3 g/dl (post transfusion), WBC 4,900/mm 3 , Platelets 51,000/mm 3 , serum albumin 1.5 g/dl, total bilirubin 0.6 mg/dl, AST 691 IU/l, ALT 187 IU/l, LDH 2976 IU/l, fibrinogen 134 mg/dl, FDP 10 μg/ml, and AT-III 40%. Bleeding from the stomach continued and did not stop with conservative treatment; therefore, two days later the patient underwent total gastrectomy and a partial liver resection. Histopathology of the resected specimen showed it to be a gastric lymphoma (pleomorphic medium-large cell type, non-Hodgkin's T-cell lymphoma) with liver metastasis (Fig.  1 ). From first postoperative day (POD), bleeding from the esophagojejunostomy continued; the patient developed disseminated intravascular coagulopathy and died on 8 th  postoperative day.On autopsy, malignant lymphoid cell infiltration and hemophagocytosis were observed in the liver, spleen, heart, small bowel, lung, both breasts, kidney, pancreas, uterus, and gastroduodenal lymph nodes (Fig.  2 ). The bone marrow presented hyperplasia and hemophagocytic macrophages but no infiltration by lymphoma cells. Immunohistochemically the neoplastic cells were positive for T-cell marker UCHL1 (CD45RO) and EBV by EBER  in situ  hybridization. The final diagnosis was EBV-related T-cell LAHS.in situDiscussion HPS is a clinicopathological entity characterized by systemic proliferation of benign hemophagocytic histiocytes, fever, cytopenia, liver dysfunction, hepatosplenomegaly, and coagulopathy [ 1 ]. This syndrome has been observed during the clinical course of a wide variety of disorders, including viral infections and malignant neoplasms. Diagnostic guidelines of Henter  et al , [ 2 ] are widely used for the diagnosis of HPS. However, these guidelines are not satisfactory in diagnosing HPS in adults; therefore, a number of studies on adult HPS have used their own criteria [ 1 , 3 , 4 ]. On the other hand for the diagnosis of LAHS, in addition to the clinical features, it is also important to confirm the presence of malignant lymphoid cells histopathologically. Takahashi  et al , [ 5 ] has proposed a set of new diagnostic criteria for adult LAHS that has been detailed in Table  1 . Table 1 Diagnostic criteria for adult lymphoma associates hemophagocytic syndrome (LAHS) 1 High fever for more than a week (peak 38.5°C) 2 Anemia (Hb < 9 g/dl) or thrombocytopenia (platelet < 100,000 μ/l) 3 a) LDH ≥ 2 × upper limit  b) Hyperferritinemia (≥ 1,000 ng/dl)  c) Hepatosplenomegaly on CT, US or MRI  d) FDP ≥ 10 μg/ml 4 Hemophagocytosis in bone marrow, spleen or liver 5 No evidence of infection 6 Histopathologically confirmed malignant lymphoma     A diagnosis of LAHS requires that all of the above conditions are fulfilled.     Of the item 3, at least two of the four sub-items (a~d) should be fulfilled.     When item 1 to item 5 are present for 2 weeks and glucocorticoid or γ-globulin therapy is not effective, a diagnosis of probable LAHS can be made and chemotherapy against malignant lymphoma can be started. In Japan, T-cell LAHS accounts for 48.5% of all adult LAHS [ 5 ]. T-cell LAHS mostly occurs in extra nodal, especially nasal, cutaneous, or malignant lymphoma involving liver and spleen. There have been no reports on LAHS from gastric lymphoma. As the diagnosis in the present case was made at autopsy it is not clear as to when the HPS occurred initially. One possibility is the setting of disseminated T-cell lymphoma. This is supported by the patient's fever, which continued for one month, liver dysfunction, and coagulopathy, which existed from the initial stage of the disease, however the bone marrow did not show any lymphoma infiltration. It could also be considered that the hemophagocytic syndrome occurred as a result of the surgery as pancytopenia and hepatosplenomegaly were not observed before the operation and hemophagocytosis was not recognized on histopathological examination in the resected stomach. In T-cell lymphoma, the hemophagocytic syndrome is assumed to be caused by cytokines, especially, tumor necrosis factor-α, and interferon-γ released from neoplastic T-cells [ 4 , 6 ]. Uncontrolled secretion of cytokines may stimulate the proliferation and phagocytic activity of macrophages. It seems likely that hypercytokinemia due to surgical resection might have contribute to the development of HPS in the present case. In our opinion the former is more likely however based on the findings of this case the second hypothesis too cannot be rejected. The poor prognosis of LAHS, especially T-LAHS, is well known. The median survival time from the diagnosis is reported to be 143 and 69 days respectively in Japan [ 5 ]. For LAHS prompt initiation of treatment with multi agent chemotherapy is required to improve the symptoms and survival [ 7 ]. Bone marrow transplantation is considered to be a treatment for chemotherapy-resistant LAHS [ 8 ]. The median survival time of LAHS patients without chemotherapy is only 11 days [ 5 ]. In this case, the initial presentation was mastalgia and hence it took a considerable amount of time to reach a diagnosis. Furthermore, bleeding from the anastomosis continued leading to a rapidly progressive fatal clinical course. In HPS occurring in lymphoma of the gastrointestinal tract uncontrollable bleeding from the primary lesion might occur. Therefore, an earlier diagnosis of HPS should be made by bone marrow aspirates, and appropriate treatments should be started as soon as possible. Surgery if performed, must be performed with utmost caution.
Discussion
HPS is a clinicopathological entity characterized by systemic proliferation of benign hemophagocytic histiocytes, fever, cytopenia, liver dysfunction, hepatosplenomegaly, and coagulopathy [ 1 ]. This syndrome has been observed during the clinical course of a wide variety of disorders, including viral infections and malignant neoplasms. Diagnostic guidelines of Henter  et al , [ 2 ] are widely used for the diagnosis of HPS. However, these guidelines are not satisfactory in diagnosing HPS in adults; therefore, a number of studies on adult HPS have used their own criteria [ 1 , 3 , 4 ]. On the other hand for the diagnosis of LAHS, in addition to the clinical features, it is also important to confirm the presence of malignant lymphoid cells histopathologically. Takahashi  et al , [ 5 ] has proposed a set of new diagnostic criteria for adult LAHS that has been detailed in Table  1 .et alet alDiagnostic criteria for adult lymphoma associates hemophagocytic syndrome (LAHS)In Japan, T-cell LAHS accounts for 48.5% of all adult LAHS [ 5 ]. T-cell LAHS mostly occurs in extra nodal, especially nasal, cutaneous, or malignant lymphoma involving liver and spleen. There have been no reports on LAHS from gastric lymphoma. As the diagnosis in the present case was made at autopsy it is not clear as to when the HPS occurred initially. One possibility is the setting of disseminated T-cell lymphoma. This is supported by the patient's fever, which continued for one month, liver dysfunction, and coagulopathy, which existed from the initial stage of the disease, however the bone marrow did not show any lymphoma infiltration. It could also be considered that the hemophagocytic syndrome occurred as a result of the surgery as pancytopenia and hepatosplenomegaly were not observed before the operation and hemophagocytosis was not recognized on histopathological examination in the resected stomach. In T-cell lymphoma, the hemophagocytic syndrome is assumed to be caused by cytokines, especially, tumor necrosis factor-α, and interferon-γ released from neoplastic T-cells [ 4 , 6 ]. Uncontrolled secretion of cytokines may stimulate the proliferation and phagocytic activity of macrophages. It seems likely that hypercytokinemia due to surgical resection might have contribute to the development of HPS in the present case. In our opinion the former is more likely however based on the findings of this case the second hypothesis too cannot be rejected.The poor prognosis of LAHS, especially T-LAHS, is well known. The median survival time from the diagnosis is reported to be 143 and 69 days respectively in Japan [ 5 ]. For LAHS prompt initiation of treatment with multi agent chemotherapy is required to improve the symptoms and survival [ 7 ]. Bone marrow transplantation is considered to be a treatment for chemotherapy-resistant LAHS [ 8 ]. The median survival time of LAHS patients without chemotherapy is only 11 days [ 5 ].In this case, the initial presentation was mastalgia and hence it took a considerable amount of time to reach a diagnosis. Furthermore, bleeding from the anastomosis continued leading to a rapidly progressive fatal clinical course.In HPS occurring in lymphoma of the gastrointestinal tract uncontrollable bleeding from the primary lesion might occur. Therefore, an earlier diagnosis of HPS should be made by bone marrow aspirates, and appropriate treatments should be started as soon as possible. Surgery if performed, must be performed with utmost caution.Conclusions LAHS could also occur from lymphoma of the gastrointestinal tract. For long-term survival; early diagnosis and appropriate treatment are needed. Surgery if performed without a proper diagnosis could prove fatal.
Conclusions
LAHS could also occur from lymphoma of the gastrointestinal tract. For long-term survival; early diagnosis and appropriate treatment are needed. Surgery if performed without a proper diagnosis could prove fatal.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions RF, FH, TY, RD, KO and KH  were gastrointestinal surgeons. MO  referred this patient to us. KK and MS  performed pathological examination and the autopsy. KS  was a member of the intensive care team. TH, YY, HN  gave us helpful comments about the manuscript
Authors' contributions
RF, FH, TY, RD, KO and KH  were gastrointestinal surgeons.RF, FH, TY, RD, KO and KHMO  referred this patient to us.MOKK and MS  performed pathological examination and the autopsy.KK and MSKS  was a member of the intensive care team.KSTH, YY, HN  gave us helpful comments about the manuscriptTH, YY, HN