[CANCER RESEARCH 64, 6402–6409, September 15, 2004] ␣2HS-glycoprotein, an Antagonist of Transforming Growth Factor ␤ In vivo, Inhibits Intestinal Tumor Progression Carol J. Swallow,1,2 Emily A. Partridge,1 Jennifer C. Macmillan,1 Tania Tajirian,1 Gianni M. DiGuglielmo,1 Kazy Hay,1 Melanie Szweras,1 Willi Jahnen-Dechent,3 Jeff L. Wrana,1,2 Mark Redston,1 Steven Gallinger,1,2 and James W. Dennis1,2 1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; 2Departments of Molecular and Medical Genetics and Surgery, University of Toronto, Toronto, Ontario, Canada; and 3Interdisziplina¨ren Zentrums fu¨r Klinische Forschung BIOMAT, Klinikum der Rheinisch-Westfa¨lischen Technischen Hoschschule, Aachen, Aachen, Germany ABSTRACT ␤1-induced epithelial-mesenchymal transition is characterized by down-regulation of E-cadherin expression, loss of tight junctions, and ␤ Transforming growth factor (TGF)- 1 is associated with tumor pro- increased cell motility (1, 10) and is dependent on phosphatidylinosi- gression and resistance to chemotherapy in established cancers, as well as tol 3Ј-kinase/Akt activation (11), a key pathway promoting tumor cell host immune suppression. Here, we show that the serum glycoprotein ␣2-HS-glycoprotein (AHSG) blocks TGF-␤1 binding to cell surface re- invasiveness (12). Epithelial-mesenchymal transition appears to be a ceptors, suppresses TGF-␤ signal transduction, and inhibits TGF-␤- close in vitro correlate of metastatic capacity, and both require coop- induced epithelial-mesenchymal transition, suggesting that AHSG may erative signaling of TGF-␤ and Ras/mitogen-activated protein kinase play a role in tumor progression. In 66 consecutive sporadic human pathways (13, 14). Tumor-derived TGF-␤1 also has paracrine effects colorectal cancer specimens, we observed a 3-fold depletion of ASHG in on host cells that promote angiogenesis, alter matrix turnover, and tumor compared with normal tissue, whereas levels of other abundant suppress immune cell functions (1, 7, 15, 16). Furthermore, mice plasma proteins, albumin and transferrin, were equivalent. Using the treated with neutralizing anti-TGF-␤ antibody or with the competitive -Multiple intestinal neoplasia/؉ (Min/؉) mouse model of intestinal tumor inhibitor protein Fc:T␤RII and mice expressing dominant negative igenesis, we found twice as many intestinal polyps overall, twice as many TGF-␤ type II receptor (T␤RII) display suppression of tumor growth, large polyps (>3 mm diameter), and more progression to invasive adeno- .(carcinoma in Min/؉ AhsgϪ/Ϫ mice than in littermates expressing Ahsg. invasion, and/or metastasis (17–21 Phosphorylated Smad2 was more abundant in the intestinal mucosa and TGF-␤/bone morphogenetic protein cytokines are present in most tumors of Min/؉ mice lacking Ahsg, demonstrating increased TGF-␤ tissues but their availability to signaling receptors is regulated by signaling in vivo. Furthermore, TGF-␤-mediated suppression of immune soluble and matrix-associated binding proteins. During embryogene- Ϫ Ϫ cell function was exaggerated in Ahsg / animals, as shown by inhibition sis, chordin, noggin, twisted, and follistatin regulate local cytokine of macrophage activation and reduction in 12-O-tetradecanoylphorbol concentrations and thereby morphogenic activity (22, 23). In adult Ϫ/Ϫ 13-acetate–induced cutaneous inflammation. Reconstitution of Ahsg tissues, TGF-␤-binding proteins limit cytokine activity, thereby con- mice with bovine Ahsg suppressed endogenous TGF-␤-dependent signal- trolling tissue remodeling and inflammation. For example, TGF-␤ ing to wild-type levels, suggesting that therapeutic enhancement of AHSG ␣ levels may benefit patients whose tumors are driven by TGF-␤. latency-associated peptide and 2-macroglobulin regulate inflamma- tion and response to infection (24, 25). ␣2-HS-glycoprotein (Ahsg)/ fetuin is a glycoprotein present in serum and extracellular matrix that INTRODUCTION binds to TGF-␤ cytokines (bone morphogenetic proteins 2, 4, and 6 and TGF-␤1 and TGF-␤2) via a 19 amino acid cytokine-binding Transforming growth factor (TGF)-␤ cytokines are conserved in domain homologous to an extracellular sequence in the T␤RII (26). metazoans and function as morphogens that regulate cell proliferation Ahsg antagonized binding of TGF-␤1toT␤RII in surface plasmon and differentiation during embryogenesis. In postnatal life, TGF-␤ resonance assays and inhibited the effect of TGF-␤1 on mink lung cytokines regulate tissue remodeling, wound repair, and immune cell epithelial cell proliferation, rat bone marrow cell osteogenic differen- functions. TGF-␤1 inhibits normal epithelial cell proliferation, and in tiation (27), and human monocyte matrix metalloproteinase 9 release a subset of epithelial malignancies, inactivating mutations in genes of (28). Ahsg and TGF-␤1 are also concentrated in mineralized bone, the signaling pathway occur early, presumably allowing expansion of Ϫ/Ϫ premalignant cell populations (1–4). However, in established human and Ahsg mice display a bone phenotype characterized by in- cancers, elevated TGF-␤1 expression correlates with progression and creased trabecular bone remodeling, osteoblast density, femur thick- adverse outcome (5–7). Transgenic mice that express TGF-␤1in ness, and bone mineral density (29). keratinocytes exhibit both reduced incidence of skin tumor initiation These observations suggest that Ahsg might inhibit cancer progres- ␤ and enhanced malignant progression, exemplifying the disparate ef- sion in vivo both by blocking TGF- 1 activity in tumor cells and by ␤ fects of TGF-␤1 at early and late stages in tumorigenesis (8). Simi- opposing TGF- -dependent immunosuppression. Here, we demon- ␤ ␤ larly, loss of TGF-␤ responsiveness promotes carcinogenesis in non- strate that AHSG inhibits TGF- 1 binding to cell surface TGF- malignant human breast-derived cell lines but inhibits metastatic receptors and blocks phosphorylation and nuclear translocation of ␤ behavior in genetically related high-grade malignant cells (9). Smad2/3. Ahsg inhibited signaling by endogenous TGF- in colon TGF-␤ signaling promotes the epithelial-mesenchymal transition, a cancer cells and blocked induction of epithelial-mesenchymal transi- morphogenic event governing cell fate during embryogenesis. TGF- tion by TGF-␤1, suggesting that its depletion in vivo could promote cancer cell autonomous invasiveness. Indeed, in compound mutant ϩ Received 3/29/04; revised 5/20/04; accepted 7/16/04. Min/ mice of three Ahsg genotypes, we found that the presence of Grant support: Canadian Institutes for Health Research (J. Dennis) and the Physi- Ahsg inhibited intestinal tumor progression, reducing polyp number, cians Services Incorporated (C. Swallow). size, and invasion. Compared with Ahsg wild-type Min/ϩ mice, 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 littermates lacking Ahsg displayed increased phosphorylated Smad2 18 U.S.C. Section 1734 solely to indicate this fact. in intestinal epithelium and tumors, indicating enhanced TGF-␤ sig- Requests for reprints: Carol J. Swallow, Mount Sinai Hospital, 600 University Ϫ/Ϫ Avenue, #1224, Toronto, Ontario, M5G 1X5 Canada. E-mail: [email protected]. naling in vivo. Treatment of Ahsg mice with exogenous Ahsg ©2004 American Association for Cancer Research. restored normal TGF-␤ signaling, as demonstrated in freshly har- 6402 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2004 American Association for Cancer Research. Ahsg AND INTESTINAL NEOPLASIA vested peritoneal macrophages. We additionally show that ASHG immunohistochemical analysis of deparaffinized sections of proximal small levels are significantly and selectively reduced in tumor tissue com- intestine, according to the manufacturer’s suggested protocol. Photographs of pared with adjacent normal mucosa in human colorectal cancer. These three crypts, three villi, and two to four polyps per mouse were taken under oil results indicate that AHSG repletion holds promise as a treatment immersion using identical charge-coupled device settings by an observer strategy to combat epithelial cancer progression. 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 MATERIALS AND METHODS each. This was subtracted from the mean background luminosity for each slide, which was measured and calculated similarly, to yield a nuclear intensity Human Tissue Specimens. Sixty-six sequential sporadic colorectal cancer score. A mean crypt, villus, and polyp nuclear intensity score was then specimens from our tumor bank were analyzed (Table 1). Median patient age calculated for each mouse. was 70 years (range, 22 to 96 years). None of the patients had received To minimize platelet degranulation, whole blood obtained by cardiac punc- preoperative radiation or chemotherapy. Samples of colorectal tumor tissue ture was allowed to clot at room temperature for 1 hour, centrifuged at 3000 and paired adjacent normal colonic mucosa were obtained fresh at the time of rpm, and serum samples collected. Total serum TGF-␤1 was determined by resection and snap frozen in liquid nitrogen or fixed in 10% formalin. Normal ELISA (R&D Systems, Minneapolis, MN), using acid activation. For Western liver samples were handled similarly. blot