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[Cancer Research 64, 6402-6409, September 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

{alpha}2HS-glycoprotein, an Antagonist of Transforming Growth Factor ß In vivo, Inhibits Intestinal Tumor Progression

Carol J. Swallow1,2, Emily A. Partridge1, Jennifer C. Macmillan1, Tania Tajirian1, Gianni M. DiGuglielmo1, Kazy Hay1, Melanie Szweras1, Willi Jahnen-Dechent3, Jeff L. Wrana1,2, Mark Redston1, Steven Gallinger1,2 and James W. Dennis1,2

1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; 2 Departments of Molecular and Medical Genetics and Surgery, University of Toronto, Toronto, Ontario, Canada; and 3 Interdisziplinären Zentrums für Klinische Forschung BIOMAT, Klinikum der Rheinisch-Westfälischen Technischen Hoschschule, Aachen, Aachen, Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor (TGF)-ß1 is associated with tumor progression and resistance to chemotherapy in established cancers, as well as host immune suppression. Here, we show that the serum glycoprotein {alpha}2-HS-glycoprotein (AHSG) blocks TGF-ß1 binding to cell surface receptors, suppresses TGF-ß signal transduction, and inhibits TGF-ß-induced epithelial-mesenchymal transition, suggesting that AHSG may play a role in tumor progression. In 66 consecutive sporadic human colorectal cancer specimens, we observed a 3-fold depletion of ASHG in tumor compared with normal tissue, whereas levels of other abundant plasma proteins, albumin and transferrin, were equivalent. Using the Multiple intestinal neoplasia/+ (Min/+) mouse model of intestinal tumorigenesis, we found twice as many intestinal polyps overall, twice as many large polyps (>3 mm diameter), and more progression to invasive adenocarcinoma in Min/+ Ahsg–/– mice than in littermates expressing Ahsg. Phosphorylated Smad2 was more abundant in the intestinal mucosa and tumors of Min/+ mice lacking Ahsg, demonstrating increased TGF-ß signaling in vivo. Furthermore, TGF-ß-mediated suppression of immune cell function was exaggerated in Ahsg–/– animals, as shown by inhibition of macrophage activation and reduction in 12-O-tetradecanoylphorbol 13-acetate–induced cutaneous inflammation. Reconstitution of Ahsg–/– mice with bovine Ahsg suppressed endogenous TGF-ß-dependent signaling to wild-type levels, suggesting that therapeutic enhancement of AHSG levels may benefit patients whose tumors are driven by TGF-ß.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor (TGF)-ß cytokines are conserved in metazoans and function as morphogens that regulate cell proliferation and differentiation during embryogenesis. In postnatal life, TGF-ß cytokines regulate tissue remodeling, wound repair, and immune cell functions. TGF-ß1 inhibits normal epithelial cell proliferation, and in a subset of epithelial malignancies, inactivating mutations in genes of the signaling pathway occur early, presumably allowing expansion of premalignant cell populations (1, 2, 3, 4) . However, in established human cancers, elevated TGF-ß1 expression correlates with progression and adverse outcome (5, 6, 7) . Transgenic mice that express TGF-ß1 in keratinocytes exhibit both reduced incidence of skin tumor initiation and enhanced malignant progression, exemplifying the disparate effects of TGF-ß1 at early and late stages in tumorigenesis (8) . Similarly, loss of TGF-ß responsiveness promotes carcinogenesis in nonmalignant human breast-derived cell lines but inhibits metastatic behavior in genetically related high-grade malignant cells (9) .

TGF-ß signaling promotes the epithelial-mesenchymal transition, a morphogenic event governing cell fate during embryogenesis. TGF-ß1-induced epithelial-mesenchymal transition is characterized by down-regulation of E-cadherin expression, loss of tight junctions, and increased cell motility (1 , 10) and is dependent on phosphatidylinositol 3'-kinase/Akt activation (11) , a key pathway promoting tumor cell invasiveness (12) . Epithelial-mesenchymal transition appears to be a close in vitro correlate of metastatic capacity, and both require cooperative signaling of TGF-ß and Ras/mitogen-activated protein kinase pathways (13 , 14) . Tumor-derived TGF-ß1 also has paracrine effects on host cells that promote angiogenesis, alter matrix turnover, and suppress immune cell functions (1 , 7 , 15 , 16) . Furthermore, mice treated with neutralizing anti-TGF-ß antibody or with the competitive inhibitor protein Fc:TßRII and mice expressing dominant negative TGF-ß type II receptor (TßRII) display suppression of tumor growth, invasion, and/or metastasis (17, 18, 19, 20, 21) .

TGF-ß/bone morphogenetic protein cytokines are present in most tissues but their availability to signaling receptors is regulated by soluble and matrix-associated binding proteins. During embryogenesis, chordin, noggin, twisted, and follistatin regulate local cytokine concentrations and thereby morphogenic activity (22 , 23) . In adult tissues, TGF-ß-binding proteins limit cytokine activity, thereby controlling tissue remodeling and inflammation. For example, TGF-ß latency-associated peptide and {alpha}2-macroglobulin regulate inflammation and response to infection (24 , 25) . {alpha}2-HS-glycoprotein (Ahsg)/fetuin is a glycoprotein present in serum and extracellular matrix that binds to TGF-ß cytokines (bone morphogenetic proteins 2, 4, and 6 and TGF-ß1 and TGF-ß2) via a 19 amino acid cytokine-binding domain homologous to an extracellular sequence in the TßRII (26) . Ahsg antagonized binding of TGF-ß1 to TßRII in surface plasmon resonance assays and inhibited the effect of TGF-ß1 on mink lung epithelial cell proliferation, rat bone marrow cell osteogenic differentiation (27) , and human monocyte matrix metalloproteinase 9 release (28) . Ahsg and TGF-ß1 are also concentrated in mineralized bone, and Ahsg–/– mice display a bone phenotype characterized by increased trabecular bone remodeling, osteoblast density, femur thickness, and bone mineral density (29) .

These observations suggest that Ahsg might inhibit cancer progression in vivo both by blocking TGF-ß1 activity in tumor cells and by opposing TGF-ß-dependent immunosuppression. Here, we demonstrate that AHSG inhibits TGF-ß1 binding to cell surface TGF-ß receptors and blocks phosphorylation and nuclear translocation of Smad2/3. Ahsg inhibited signaling by endogenous TGF-ß in colon cancer cells and blocked induction of epithelial-mesenchymal transition by TGF-ß1, suggesting that its depletion in vivo could promote cancer cell autonomous invasiveness. Indeed, in compound mutant Min/+ mice of three Ahsg genotypes, we found that the presence of Ahsg inhibited intestinal tumor progression, reducing polyp number, size, and invasion. Compared with Ahsg wild-type Min/+ mice, littermates lacking Ahsg displayed increased phosphorylated Smad2 in intestinal epithelium and tumors, indicating enhanced TGF-ß signaling in vivo. Treatment of Ahsg–/– mice with exogenous Ahsg restored normal TGF-ß signaling, as demonstrated in freshly harvested peritoneal macrophages. We additionally show that ASHG levels are significantly and selectively reduced in tumor tissue compared with adjacent normal mucosa in human colorectal cancer. These results indicate that AHSG repletion holds promise as a treatment strategy to combat epithelial cancer progression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human Tissue Specimens.
Sixty-six sequential sporadic colorectal cancer specimens from our tumor bank were analyzed (Table 1)Citation . Median patient age was 70 years (range, 22 to 96 years). None of the patients had received preoperative radiation or chemotherapy. Samples of colorectal tumor tissue and paired adjacent normal colonic mucosa were obtained fresh at the time of resection and snap frozen in liquid nitrogen or fixed in 10% formalin. Normal liver samples were handled similarly.


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  		Table 1 Clinical characteristics and AHSG N/T levels in 66 colorectal cancer patients

 
Frozen tissue samples were homogenized, and protein samples (10, 1, and 20 µg for detection of AHSG, albumin, and transferrin, respectively) were separated by SDS-PAGE. Proteins were identified with mouse or goat antihuman primary antibody followed by application of secondary sheep antimouse antibody or rabbit antigoat antibody. All antibodies were purchased from Sigma (St. Louis, MO), except mouse antihuman AHSG, which was prepared as described previously (30) . The enhanced chemiluminescence photodetection system was used and densitometric analysis (Imagequant Iq) performed. For each protein, three separate blots of tumor and normal mucosa protein extracts were analyzed, and the final normal/tumor (N/T) ratio calculated was the mean of these three. Use of human tissue specimens was approved by the Mount Sinai Hospital Institutional Review Board.

Mice.
Ahsg-deficient mice were generated by targeted gene mutation in ES cells that removed the entire coding sequence of the gene, as described previously (30) . Ahsg–/– mice of 129/Sv x C57BL6 background were crossed with C57BL/6 mice; female Ahsg+/– offspring were bred with male C57BL/6 Min/+ mice from a colony established at our institution. Min/+Ahsg+/– offspring were then crossed to produce a F2 generation of Min/+ mice of three Ahsg genotypes, which were obtained at the expected Mendelian frequency. Littermates were maintained on standard chow, weighed weekly, aged to the indicated time points, and sacrificed by CO2 inhalation. The small and large intestines were harvested, washed with PBS, split longitudinally, preserved in formalin, and examined for lesions as previously described (31) . No mice required sacrifice before 180 days. One Min/+ Ahsg+/+ mouse, one Min/+ Ahsg+/– mouse, and two Min/+ Ahsg–/– mice developed wasting symptoms requiring sacrifice before 340 days; these mice were eliminated from subsequent analysis. An observer blind to the genotype of the mice examined the intestinal segments. Tumor number and location and tumor diameter to a precision of 0.1 mm in 340-day-old mice were scored using a dissecting microscope as described previously (31) . A second observer verified tumor counts in a subset of samples. For histologic analysis, sections of the proximal small intestine were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with H&E. Polyp histology was analyzed by a pathologist blinded to the genotype of the samples. Phospho-Smad2 (Ser465/467) antibody (1:1500; Cell Signaling Technology, Inc., Beverly, MA) was used for immunohistochemical analysis of deparaffinized sections of proximal small intestine, according to the manufacturer’s suggested protocol. Photographs of three crypts, three villi, and two to four polyps per mouse were taken under oil immersion using identical charge-coupled device settings by an observer blinded to the genotype, and luminosity of nuclei was determined using Adobe Photoshop. In total, three random groups of five nuclei were assessed for each crypt, each villus, and each polyp to yield a mean nuclear luminosity score for each. This was subtracted from the mean background luminosity for each slide, which was measured and calculated similarly, to yield a nuclear intensity score. A mean crypt, villus, and polyp nuclear intensity score was then calculated for each mouse.

To minimize platelet degranulation, whole blood obtained by cardiac puncture was allowed to clot at room temperature for 1 hour, centrifuged at 3000 rpm, and serum samples collected. Total serum TGF-ß1 was determined by ELISA (R&D Systems, Minneapolis, MN), using acid activation. For Western blot analysis of serum Ahsg, 10 µL of serum (diluted 1:10 with PBS) were loaded per well, separated by SDS-PAGE, and Ahsg identified using mouse antihuman AHSG monoclonal antibody (30) , which reacts with both mouse and bovine Ahsg. For some experiments, mice received i.p. injections of bovine Ahsg (Sigma), 3 mg in 1 mL of PBS, 72 and 24 hours before sacrifice.

All animal protocols were reviewed and approved by the Samuel Lunenfeld Research Institute Institutional Review Board.

Peritoneal Macrophage Preparation and Analysis.
Except where indicated, mice were injected intraperitoneally with 25 µg lipopolysaccharide (from Escherichia coli serotype 026:B6; Sigma) in 1 mL of PBS 5 days before sacrifice. The peritoneal cavity was lavaged with 10 mL of sterile ice-cold Ca2+- and Mg2+-free PBS, lavage fluid centrifuged at 4°C at 1100 rpm, and pellets resuspended in serum-free macrophage specific medium (Invitrogen Life Technologies, Burlington, Ontario, Canada) with penicillin and streptomycin. The cell suspension was plated onto wells or coverslips, incubated for 2 h at 37°C in 5% CO2, washed vigorously with warm PBS, and fresh serum-free macrophage-specific medium added. For measurement of nitric oxide release and tumor necrosis factor {alpha}, macrophages in 12-well plates were incubated for an additional 48 hours in serum-free macrophage-specific medium ± human AHSG or ± TGF-ß1 (R&D Systems). Supernatants were collected, nitrites quantitated using the Griess method (Sigma), tumor necrosis factor {alpha} by ELISA assay (R&D Systems), and both normalized to protein content in each well (BCA assay). For immunofluorescence assays, macrophages on coverslips were fixed with 4% formaldehyde x 15 minutes, washed with PBS, permeabilized with 100% methanol x 2 minutes, washed, and incubated with mouse antihuman AHSG monoclonal antibody (30) at 1:100 x 2 hours, followed by Cy3-labeled sheep antimouse antibody (1:50) plus Hoechst (1:1000) x 1 hour.

Skin Inflammation Assay.
12-O-Tetradecanoylphorbol 13-acetate (2 µg in 25 µL of ethanol) was applied to the right ear and ethanol alone to the left ear of each mouse. Ear swelling was measured with a micrometer at 24-hour intervals and reported as the difference in thickness between the right and left ears. Five mice of each genotype (Ahsg+/+ and Ahsg–/–) were tested.

TGF-ß Receptor Binding, Smad2/3 Nuclear Translocation, and Phospho/Total Smad2 Analysis.
TGF-ß1 cross-linking to cell surface receptor was performed as described previously (32) . Briefly, MvLu1 cells were incubated for 30 minutes at 37°C in Krebs-Ringer-HEPES (KRH) plus 0.5% BSA and placed on ice. 125Iodine-labeled TGF-ß1 at 250 pmol/L was preincubated for 30 minutes at 20°C with Ahsg or transferrin, then added to cells and incubated for 2 hours at 4°C with agitation. The cells were then incubated with DSS (60 µg/mL) for 15 minutes at 4°C. Cells lysates were centrifuged, and the supernatants subjected to SDS-PAGE. 125I-TGF-ß1 bound to proteins corresponding in size to TßRIII, TßRII, and TßRI, and immunoprecipitation of 125I-TGF-ß1 receptor complexes with anti-TßR antibodies confirmed their identity.

Smad2/3 localization was measured in MvLu1, SW620 cells and peritoneal macrophages plated in 96-well plates at a density of 5000 cells/well, and serum starved for 24 h. TGF-ß1 was preincubated with human AHSG (Calbiochem), bovine Ahsg or human transferrin (Sigma) for 30 minutes at 37°C, then added to cells for 30 min. For experiments assessing the effect of endogenous TGF-ß, Ahsg alone was added. The cells were washed with warm PBS, fixed for 10 minutes with 3.7% formaldehyde at 37°C, then washed with PBS plus 1% FBS, and permeabilized with methanol for 2 minutes. PBS plus 10% FBS was added and left overnight at 4°C. Mouse anti-Smad2/3 antibody (BD Transduction Laboratories, Mississauga, Ontario, Canada) was added at 1:200 in PBS plus 10% FBS for 1 h at 20°C. After washing three times with PBS plus 1% FBS, Alexa Fluor 488 goat antimouse immunoglobulin (Molecular Probes) was added at 1:200 with Hoechst x 1 hour at 20°C. The plates were scanned using the Scan Array automated fluorescence microscope and cytoplasmic-nuclear software (Cellomics, Inc., Pittsburgh, PA). The difference in nuclear-cytoplasmic staining intensity was determined for 100 cells per well, generating a mean ± SE for each condition. Experiments were performed in triplicate.

For analysis of total and phosphorylated Smad2, SW620 cells at 75% confluence were washed three times with PBS, preincubated without or with Ahsg (20 µmol/L) in serum-free RPMI medium at 37°C for 30 minutes, after which, 200 pmol/L TGF-ß1 was added for the indicated time, cells washed three times with PBS, and lysed with Tris-NaCL-Triton X 100-EDTA (TNTE) buffer with protease inhibitor mixture tablets (Roche Diagnostics, Mannheim, Germany) at 4°C x 20 minutes. Protein lysates were diluted with TNT buffer, immunoprecipitated with anti-Smad2/3 antibody (N-19; Santa Cruz Biotechnology, Santa Cruz, CA) on ice x 3 hours, shaken with a 1:5 slurry of protein G-Sepharose beads (Pharmacia, Uppsala, Sweden) in Tris-NaCL-Tween (TNT) buffer at 4°C x 1 hour, centrifuged, washed, resuspended in loading buffer with ß-mercaptoethanol, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Blots were incubated at 4°C overnight with anti-Smad2/3 antibody at a 1:2000 dilution (BD Transduction Laboratories) or anti-phospho-Smad2 antibody at a 1:1000 dilution (Upstate Biotechnology, Inc., Lake Placid, NY), followed by antimouse IgG conjugated to horseradish peroxidase (Amersham Biosciences, Inc., Piscataway, NJ) or antirabbit IgG conjugated to horseradish peroxidase (Amersham Biosciences, Inc.), and blots developed using the Renaissance chemiluminescence kit (NEN-PerkinElmer, Boston, MA).

E-Cadherin Staining.
NMuMG cells were plated at 30% confluence and cultured in DMEM for 48 hours with 10 µg/mL insulin ± TGF-ß1 (200 pmol/L) and/or Ahsg (20 µmol/L). E-Cadherin was detected with mouse monoclonal antibody (500 ng/mL x 2 hours; Transduction Laboratories), followed by Alexa Fluor 488 goat antimouse immunoglobulin (2 µg/mL x 1 hour; Molecular Probes) and imaged using a deconvolution microscope.

Statistical Analysis.
All values are reported as mean ± SE. Statistical analysis to compare means was by ANOVA or Mann-Whitney when data were or were not distributed normally, respectively, and proportions were compared by {chi}2 test. Statistical significance was established at P = 0.05 (two-sided).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ahsg Competes for TGF-ß1 Binding to Cell Surface Receptors and Blocks Signaling.
The disulfide-looped sequence in Ahsg from Cys114 to Cys130 (bovine sequence) shares homology with the extracellular domain of TßRII (Cys84 to Cys101). In surface plasmon resonance assays, these peptides bind to TGF-ß and bone morphogenetic protein cytokines with specificity characteristic of native Ahsg and TßRII, suggesting these are the major cytokine-binding domains in both glycoproteins (26) . Because Ahsg binds to TGF-ß1 with a dissociation constant (KD) of ~2.0 µmol/L (26) and serum AHSG concentration is ~12 µmol/L, the laws of mass action indicate that Ahsg should influence cytokine availability in vivo. To determine whether Ahsg competes for TGF-ß1 binding to cell surface signaling receptors, MvLu1 cells were incubated with 125I-labeled TGF-ß1 and a chemical cross-linking agent to reveal the ligand-receptor complexes (Fig. 1A)Citation . Preincubation of 125I-TGF-ß1 with increasing concentrations of Ahsg resulted in dose-dependent inhibition of cytokine binding to TßRIII, TßRII, and TßRI, whereas the control glycoprotein transferrin had no effect (Fig. 1, A and B)Citation . Ahsg reduced 125I-TGF-ß1 cross-linking to receptors with an estimated IC50 for the individual receptors of 20 to 30 µmol/L (Fig. 1B)Citation .



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  		Fig. 1. Ahsg inhibits TGF-ß1 binding to cell surface receptors and blocks Smad2/3 nuclear translocation. A, inhibition of TGF-ß1 binding to cell surface receptors by Ahsg. 125I-labeled TGF-ß1 was preincubated with either Ahsg or transferrin, added to MvLu1 cells, chemically cross-linked, and protein extracts separated by SDS-PAGE. The position of 125I-TGF-ß1 in complex with TßRIII, TßRII, and TßRI is indicated by the arrows. The four lanes on the right represent transferrin controls. B, competition by Ahsg ({bullet}, {blacksquare}, {blacktriangleup}), or transferrin ({circ}, {square}, {triangleup}), for 125I-TGF-ß1 binding to TßRIII ({blacktriangleup}, {triangleup}), TßRII ({blacksquare}, {square}), and TßRI ({bullet}, {circ}) quantified by densitometry. C, localization of Smad2/3 in MvLu1 cells treated with AHSG (20 µmol/L), TGF-ß1 (200 pmol/L), or both for 30 minutes, imaged by Scan Array automated fluorescence microscopy. D, nuclear-cytoplasmic Smad2/3 intensity difference in MvLu1 cells treated with 200 pmol/L TGF-ß1 plus varying concentrations of AHSG ({blacksquare}) or transferrin ({blacktriangleup}) or treated with AHSG alone ({circ}), measured by Scan Array. Data are means ± SE for 100 cells. E, nuclear-cytoplasmic Smad2/3 intensity difference in MvLu1 cells treated with varying doses of TGF-ß1 in the presence of increasing concentrations of AHSG arrayed in two dimensions in a 96-well plate. Data are means ± SE for 100 cells per well.

 
TGF-ß1 binding to TßRII stimulates transphosphorylation of TßRI, which in turn phosphorylates the RSmads Smad2/Smad3. The latter heterodimerize with Smad4 and translocate into the nucleus where in complexes with other proteins, they regulate gene transcription. Anti-Smad2/3 antibody was used to probe the translocation of protein from the cytoplasm to the nucleus after TGF-ß1 stimulation in MvLu1 cells (Fig. 1C)Citation . Preincubation of TGF-ß1 with either bovine Ahsg or human AHSG blocked the TGF-ß1-induced nuclear translocation of Smad2/3 in MvLu1 cells, whereas transferrin protein had no effect (Fig. 1, C and D)Citation . The IC50 for AHSG was 10 µmol/L when TGF-ß1 was added at 200 pmol/L, a concentration 10-fold greater than the D50 for maximal signaling (Fig. 1, D and E)Citation . Furthermore, the competitive interaction between TGF-ß1 and AHSG was observed over a range of concentrations for both proteins (Fig. 1E)Citation .

To determine whether Ahsg can regulate TGF-ß signaling in malignant cells, we examined the SW620 human colorectal cancer line, which releases ~4 ng of TGF-ß1 per 106 cells in 24 hours when cultured in serum-free medium. Ahsg suppressed Smad2 phosphorylation induced by both endogenous and exogenous TGF-ß (Fig. 2A)Citation . At concentrations in the physiologic range, Ahsg suppressed the basal level of Smad2/3 nuclear localization in SW620 cancer cells (Fig. 2B)Citation . Ahsg also blocked TGF-ß1-induced loss of E-cadherin at cell tight junctions, a hallmark of the epithelial-mesenchymal transition phenotype associated with progression of epithelial tumors to an invasive phenotype (Fig. 2C)Citation . These results suggest that Ahsg has the potential to block TGF-ß1-driven tumor progression in vivo.



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  		Fig. 2. AHSG inhibits signaling by endogenous TGF-ß and blocks TGF-ß1-induced epithelial-mesenchymal transition. A, total Smad2/3 and phosphorylated Smad2, assessed by Western blot, in protein lysates prepared from SW620 colorectal cancer cells incubated without (control) or with Ahsg (20 µmol/L) plus 200 pmol/L TGF-ß1 for the indicated time. B, nuclear-cytoplasmic Smad2/3 intensity difference in SW620 colorectal cancer cells incubated with Ahsg at the indicated concentration for 48 hours. C, E-cadherin localization in NMuMG cells incubated for 48 hours ± TGF-ß1 (200 pmol/L) ± Ahsg (20 µmol/L), examined by confocal fluorescence microscopy.

 
Depletion of AHSG in Human Colorectal Cancer.
Tumor promotion by autocrine TGF-ß might be enhanced by selective depletion of TGF-ß antagonist proteins in the tumor microenvironment. To explore this possibility, we measured the relative abundance of AHSG in tumor versus paired normal mucosa in 66 consecutive sporadic colorectal cancer specimens from our tumor bank (Fig. 3, A and B)Citation . The AHSG transcript was below the level of detection in tumor and normal mucosa but readily detected in liver by Northern blotting and by in situ RNA hybridization, confirming that tissue AHSG was derived largely from serum. As a control for serum-derived proteins in normal and tumor tissues, albumin and transferrin content were assessed by the same method (Fig. 3, A and B)Citation . The mean normal mucosa:tumor ratio (N/T) for AHSG was significantly greater than that for transferrin or albumin (2.73 ± 0.42 versus 1.11 ± 0.05 and 1.13 ± 0.05, respectively, P = 0.001; Fig. 3BCitation ). In addition, the mean compound N/T ratios for AHSG:transferrin and AHSG:albumin were both significantly >1 (2.71 and 2.43, respectively, P = 0.001). These results indicate that AHSG is selectively depleted in tumor tissue when compared with adjacent normal colonic mucosa. Furthermore, the AHSG:transferrin N/T and the AHSG:albumin N/T ratios were >1 in 69 and 68% of specimens, respectively, with congruence between the two ratios in 85% of patients, demonstrating selective depletion of AHSG in the majority of colorectal tumors. The AHSG N/T ratio did not correlate with patient gender, age, or tumor location (Table 1)Citation but did correlate with tumor stage: the AHSG N/T ratio was >1 in 65.6% of stage I/II versus 91.1% of stage III/IV cancers (n = 32 and 34, respectively, P = 0.03 by {chi}2), suggesting an association of AHSG depletion with tumor progression. Immunohistochemical analysis of AHSG in tissue sections revealed staining in the extracellular matrix of the mucosal and submucosal layers of the bowel wall in normal colon, but tumor tissue stained only weakly (Fig. 3C)Citation .



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  		Fig. 3. AHSG is depleted in human colorectal cancer. A, three representative cases of colorectal cancer tumor (T) and paired normal colonic mucosa (N) protein extracts probed for AHSG, transferrin, and albumin by Western blotting. B, AHSG, transferrin, and albumin content expressed as a ratio of normal mucosa to tumor tissue, mean ± SE, n = 66, *, P = 0.001, AHSG versus transferrin and albumin. C, serial sections of invasive colon cancer (T, bottom panels) and adjacent normal colon (N, top panels) stained with H&E or mouse monoclonal antihuman AHSG antibody, showing staining of blood vessels in normal and tumor, and of extracellular stroma in normal only.

 
Ahsg Inhibits Intestinal Tumor Progression.
The progressive loss of AHSG in late-stage tumors suggests that this TGF-ß antagonist may oppose intestinal tumor progression in vivo. To test this hypothesis, adenomatous intestinal tumors were studied in Min/+Ahsg+/+, Min/+Ahsg+/–, and Min/+Ahsg–/– mice. Min/+ mice have a loss-of-function point mutation in one allele of the Apc gene, and with stochastic loss of the wild-type allele, they develop multiple small bowel adenomas (33) . This is an apt model because sporadic human colorectal cancers commonly have loss-of-function mutations in APC and loss of the wild-type allele (34) . At 180 days of age, there was no statistically significant difference between groups in the number of small bowel adenomatous polyps (Fig. 4A)Citation , although there was a trend toward increased polyp multiplicity in Min/+Ahsg–/– mice compared with Min/+Ahsg+/+ and Min/+Ahsg+/– littermates. By 340 days, however, Ahsg-deficient Min/+ mice harbored an average of 21.2 ± 2.4 small bowel tumors per mouse, significantly more than either wild-type (10.4 ± 1.6, P = 0.005) or heterozygous (13.8 ± 2.7, P = 0.047) littermates (Fig. 4A)Citation .



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  		Fig. 4. Ahsg inhibits intestinal tumor progression in Min/+ mice. A, mean number ± SE of small bowel polyps per mouse at 180 and 340 days of age. At 340 days, polyp number was significantly greater in Ahsg–/– compared with Ahsg+/+ and Ahsg+/– Min/+ mice; *, P = 0.005 and 0.047, respectively. Number of mice per group is indicated. B, mean number of polyps per segment of three equal small bowel segments at 340 days of age. *, P = 0.048 for Ahsg–/– versus Ahsg+/+ (proximal segment) and P = 0.018 for Ahsg–/– versus Ahsg+/+ and Ahsg+/– (distal segment). C, incidence of small bowel polyps < 1, 1 to 3, or >3 mm diameter at 340 days of age; *, P = 0.008 for Ahsg–/– versus Ahsg+/+ and Ahsg+/–. D, histologic sections of small bowel tumors from Min/+Ahsg+/+ (adenoma, top panel) and Min/+Ahsg–/– (invasive carcinoma, bottom panel; arrow shows penetration through the muscle layer of the bowel wall toward the adjacent fat) mice. Scale bar represents 1 mm.

 
The polyp counts were corrected for intestinal surface area at 340 days of age. Although the mean small bowel and colonic surface areas displayed a trend to increased dimensions in mice lacking Ahsg compared with wild-type and heterozygous littermates, the polyp incidence per small bowel surface area was still significantly elevated (~2-fold) in Ahsg-deficient Min/+ mice (Table 2)Citation . For each segment of the small bowel (proximal, middle, and distal), the number of polyps per centimeter of bowel length, measured at 340 days, was greater in mice lacking Ahsg than in wild-type or heterozygous littermates, and the difference reached statistical significance for the proximal and distal segments. The greatest difference between genotypes was observed in the distal small bowel (Fig. 4B)Citation , where Ahsg-deficient Min/+ mice harbored almost four times the number of polyps as Min/+Ahsg+/+ mice and twice that found in Min/+Ahsg+/– mice.


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  		Table 2 Intestinal surface area and polyp multiplicity at 340 days of age

 
The differences between genotypes for polyp incidence increased in the late stages of disease (340 days), suggesting that Ahsg opposes tumor progression rather than initiation. In support of this interpretation, an increased frequency of large tumors (>3 mm in diameter) was observed in the small bowel of Ahsg-deficient Min/+ mice compared with their wild-type and heterozygous littermates at 340 days (Fig. 4C)Citation . Invasion into the muscularis propria layer of the bowel wall (Fig. 4DCitation , bottom panel) was observed in 6 of 73 tumors > 1 mm diameter isolated from the proximal small bowel segment in Ahsg-deficient mice and in none of 18 similarly sized tumors from Min/+Ahsg+/+ mice (P = 0.01, {chi}2).

Ahsg Regulates TGF-ß-Dependent Signaling and Immunosuppression In vivo.
TGF-ß1-deficient mice display an overwhelming inflammatory response shortly after weaning (35) , which is mediated by hyperactivation of lymphocytes and monocytes. To determine whether TGF-ß1 signaling in immune cells was sensitive to changes in Ahsg levels in vivo, we compared freshly harvested peritoneal macrophages from Ahsg+/+ and Ahsg–/– mice. Monocytes, including Kupffer cells (Fig. 5A)Citation and peritoneal macrophages from wild-type mice (Fig. 5B)Citation , accumulate Ahsg protein in vivo, presumably via serum, because the cells do not express Ahsg mRNA (Fig. 5C)Citation . In the basal state, macrophages from Ahsg–/– mice displayed {approx}3-fold more Smad2/3 protein localized in the nucleus compared with Ahsg+/+ mice (Fig. 5, D and E)Citation . Smad2/3 nuclear localization in response to exogenous TGF-ß1 added for 30 or 60 minutes was also significantly higher in mutant than wild-type macrophages (Fig. 5, D and E)Citation . These results demonstrate hyper-TGF-ß signaling in Ahsg–/– cells. TGF-ß1 acts on monocytes and macrophages to suppress the release of cytokines and nitric oxide, thereby reducing tissue inflammation (1) . Macrophages from Ahsg–/– mice released 53% less nitric oxide in the basal state and were ~10-fold more sensitive to suppression by exogenous TGF-ß1 than wild-type cells (Fig. 5F)Citation . Addition of Ahsg protein to the serum-free culture medium enhanced nitric oxide production by both Ahsg–/– and Ahsg+/+ macrophages (Fig. 5G)Citation . Tumor necrosis factor {alpha} release by macrophages was affected by endogenous and exogenous Ahsg in an analogous manner (data not shown). Serum TGF-ß1 levels were not significantly different in Min/+Ahsg+/+ and Min/+Ahsg–/– animals.



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  		Fig. 5. Ahsg localizes to macrophages in vivo and regulates TGF-ß-dependent signaling and function. A, serial sections of normal human liver stained with H&E, the monocyte-specific monoclonal antibody KP-1, and mouse monoclonal antihuman AHSG antibody, showing colocalization of staining in Kupffer cells (arrows). B, peritoneal macrophages from 10-week-old Ahsg+/+ or Ahsg–/– mice stained with anti-AHSG antibody. Some Ahsg–/– mice were injected intraperitoneally with bovine Ahsg (3 mg in 1 mL of PBS) 72 and 24 hours before lavage (bottom panel). Nuclei are stained with Hoechst. C, Northern analysis of Ahsg mRNA performed on total RNA isolated from peritoneal macrophages or liver homogenates from Ahsg+/+ or Ahsg–/– mice that had (+) or had not (–) been treated with 25 µg of lipopolysaccharide (LPS) i.p. 5 days previously. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was measured as a loading control. D, localization of Smad2/3 in peritoneal macrophages from Ahsg+/+ or Ahsg–/– mice, at baseline (control) or treated with 40 pmol/L TGF-ß1 for 60 minutes, imaged by Scan Array automated fluorescence microscopy. E, nuclear-cytoplasmic Smad2/3 intensity difference in peritoneal macrophages from Ahsg+/+ or Ahsg–/– mice treated with 40 pmol/L TGF-ß1 for the indicated times; n = 6 mice per genotype. Data are means ± SE for 100 cells. E and F, nitric oxide release from peritoneal macrophages incubated with the indicated concentration of TGF-ß1 (E) or Ahsg (F) for 48 hours.

 
To assess the effect of Ahsg on TGF-ß signaling in normal and neoplastic intestinal epithelium, we compared phospho-Smad2 staining in Min/+Ahsg+/+ and Min/+Ahsg–/– littermates (Fig. 6A)Citation . For epithelial cells located in crypts, villi, or polyps, the intensity of phospho-Smad2 nuclear staining was significantly greater in mice lacking Ahsg (Fig. 6B)Citation , demonstrating enhanced endogenous TGF-ß signaling. Consistent with this, we have also found that Ahsg-deficient mice have a greater capacity to suppress the inflammatory response. Cutaneous inflammation induced by topical application of 12-O-tetradecanoylphorbol 13-acetate was reduced at its peak and resolved more rapidly in Ahsg–/– mice (Fig. 6C)Citation . To determine whether extrinsic manipulation of Ahsg levels in vivo could modulate TGF-ß1 responses, Ahsg–/– mice received i.p. injections of bovine Ahsg protein. This partially restored Ahsg in serum (Fig. 6D)Citation and in peritoneal macrophages (Fig. 5B)Citation . Nuclear localization of Smad2/3 in macrophages from the Ahsg–/– mice that received injections of Ahsg protein was normalized, i.e., rendered comparable with that in cells from Ahsg+/+ mice (Fig. 6E)Citation . This proves that Ahsg protein can be administered to mice to suppress the TGF-ß response in macrophages and quite possibly other tissues, including tumors.



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  		Fig. 6. Enhanced endogenous TGF-ß signaling in mice lacking Ahsg. A and B, immunostaining for phosphorylated Smad2 on sections of intestinal epithelium from 340-day-old mice. Intensity of nuclear staining in crypts, villi (A), and polyps (B) was greater in Min/+Ahsg–/– than Min/+Ahsg+/+ littermates; *, P < 0.05, **, P < 0.01. C, mean difference in ear thickness between the right (treated) and left (control) ears for each individual mouse at the indicated time after topical application of 12-O-tetradecanoylphorbol-13-acetate to the right ear. P < 0.05 in Ahsg–/– versus Ahsg+/+ mice; n = 5 per group. D, serum Ahsg in Ahsg+/+, Ahsg+/–, and Ahsg–/– mice or Ahsg–/– mice that received i.p. injections of bovine Ahsg 72 and 24 hours before lavage, detected by Western blot. E, Smad2/3 nuclear localization in peritoneal macrophages from Ahsg+/+, Ahsg–/– or Ahsg–/– mice that received i.p. injections of bovine Ahsg; n = 6 mice per group. Data are means ± SE for 100 cells.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this article, we demonstrate that Ahsg competes with cell surface receptors for TGF-ß1 binding, blocks TGF-ß1-induced Smad2/3 activation in vivo, and suppresses tumor progression in the Min/+ model of intestinal neoplasia. Furthermore, AHSG levels were reduced in the majority of human colorectal cancer tumors compared with adjacent normal mucosa, a reduction that was not dependent on vascularity or plasma content. Postnatally, Ahsg is expressed almost exclusively by hepatocytes, and Ahsg protein localized to other tissues is plasma derived. In normal colonic mucosa, AHSG appeared to be localized to the extracellular matrix, and it is likely that altered matrix structure and composition in tumor tissue underlies the loss of AHSG.

Polyp number was unchanged between 180 and 340 days in Min/+ Ahsg+/+ mice, whereas Min/+ Ahsg–/– mice showed a significant increase in multiplicity, size, and invasion at the later time point. Gene mutations that increase polyp multiplicity in Min/+ mice at an early age probably enhance tumor initiation because the histologic grade remains preinvasive. This has been observed for Min/+ mice lacking the mismatch repair gene Msh2 (31) . Similarly, Min/+ mice lacking the growth factor insulin-like growth factor II have fewer polyps, whereas an insulin-like growth factor II transgene increases polyp multiplicity in young mice (36) . Spontaneous and radiation-induced tumors in Min/+ mice commonly have loss of heterozygosity at the Smad4 locus (37) , and Smad4–/– Min/+ mice display increased polyp multiplicity (38) . Smad4 loss and insulin-like growth factor II overexpression may enhance intestinal epithelial cell proliferation at an early stage in tumorigenesis. By contrast, a delayed increase in polyp number in the Min/+ Ahsg–/– mice was associated with increased tumor size and invasive histology. This suggests that growth of individual polyps is enhanced with tumor progression and allows more foci to reach the size of macroscopic detection (39) . Indeed, TGF-ß-responsive tumor cells have a selective advantage in late-stage neoplasms, both in humans and in mouse models (5, 6, 7, 8, 9 , 18, 19, 20 , 40) . TGF-ß1 down-regulates the tumor suppressor E-cadherin and thus promotes epithelial-mesenchymal transition, signaling via phosphatidylinositol 3'-kinase/protein kinase B/glycogen synthase kinase-3 (12 , 13) , presumably unopposed by the tumor suppressor APC/ß-catenin in late-stage Min/+ Ahsg–/– tumors.

In addition to tumor cell autonomous effects, the selective depletion of AHSG in tumor tissue may enhance TGF-ß-dependent immune suppression and thereby promote tumor growth (15 , 41 , 42) . In this regard, cyclosporine promotes tumor progression by suppressing host immune function and by directly enhancing tumor cell invasiveness, and notably, both mechanisms are TGF-ß dependent (43) . Kupffer cells and peritoneal macrophages do not express the Ahsg gene, but the protein is taken up from extracellular sources, and we observed strong Ahsg staining in an intracellular vesicular compartment. Macrophages produce and activate TGF-ß1, which suppresses release of inflammation-associated nitric oxide and tumor necrosis factor {alpha}. The extent and duration of 12-O-tetradecanoylphorbol 13-acetate-induced skin inflammation was reduced in Ahsg-deficient mice compared with wild-type littermates, consistent with an increased availability of TGF-ß in Ahsg–/– mice. Mirroring this effect, reduced serum AHSG has been correlated with impaired delayed-type hypersensitivity response in malnourished patients with solid tumors (44) . Cultured splenic T cells from Ahsg–/– mice are less responsive to T-cell receptor agonists than cells from Ahsg+/+ mice.4 T-Cell–specific blockade of TGF-ß signaling is sufficient to restore an immune response capable of eradicating tumors in mice (21) . This indicates the potential for immune cell regulation by Ahsg in the control of tumor progression.

The efficacy of TGF-ß-neutralizing antibodies in short-term preclinical tumor models, and suppression of cancer progression in mice by long-term exposure to the TGF-ß antagonist Fc:TßRII highlight the potential therapeutic value of cytokine antagonists in cancer treatment (17, 18, 19 , 42) . In contrast to the exaggerated immune response observed in TGF-ß1–/– mice, mice transgenic for Fc:TßRII exhibited no chronic adverse effects and a normal life span (20) . Wakefield et al. (20) speculate that the incomplete blockade of endogenous TGF-ß activity effected by Fc:TßRII was sufficient to inhibit tumor progression in their model without uncontrolled immune cell activation. Our results indicate that Ahsg similarly effects an incomplete blockade of endogenous TGF-ß and could offer an attractive safety profile in vivo. Elevated tumor TGF-ß1 expression is associated with resistance to cytotoxic chemotherapy (45) , and colon cancer patients with microsatellite instability and TßRII mutations show a more favorable response to adjuvant chemotherapy (46) . This suggests that TGF-ß antagonists could complement the antineoplastic activity of conventional cytotoxic agents. On the basis of our results, Ahsg could also have therapeutic applications in other pathologies. Notably, excess TGF-ß1 contributes to the pathogenesis of asthma, persistent infections, chronic inflammation, and tissue fibrosis, and neutralization of TGF-ß in animal models has therapeutic benefit (25 , 47 , 48) .


   ACKNOWLEDGMENTS
 
We thank Aaron Pollett for helpful discussion, Patricia Wegrynowki, Naomi Dore, Pamela Cheung, Laura Daly, Carina Klemens, and Cindy Law for technical assistance, and Nancy Baxter, Michael A. Ko, and Erin Kennedy for assistance with statistical analyses.


   FOOTNOTES
 
Grant support: Canadian Institutes for Health Research (J. Dennis) and the Physicians Services Incorporated (C. Swallow).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Carol J. Swallow, Mount Sinai Hospital, 600 University Avenue, #1224, Toronto, Ontario, M5G 1X5 Canada. E-mail: cswallow{at}mtsinai.on.ca

4 M. Szweras, personal communication. Back

Received 3/29/04. Revised 5/20/04. Accepted 7/16/04.


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 RESULTS
 DISCUSSION
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