Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium Tumor Immunology: New Perspectives
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Peduto, L.
Right arrow Articles by Blobel, C. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Peduto, L.
Right arrow Articles by Blobel, C. P.
[Cancer Research 65, 9312-9319, October 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Critical Function for ADAM9 in Mouse Prostate Cancer

Lucie Peduto1, Victor E. Reuter2, David R. Shaffer1,3, Howard I. Scher1 and Carl P. Blobel4

1 Genitourinary Oncology Service; Departments of 2 Pathology and 3 Molecular Biology, Memorial Sloan-Kettering Cancer Center; and 4 Arthritis and Tissue Degeneration Program and Departments of Medicine and Physiology & Biophysics, Hospital for Special Surgery at Weill Medical College of Cornell University, New York, New York

Requests for reprints: Carl P. Blobel, Arthritis and Tissue Degeneration Program and Departments of Medicine and Physiology & Biophysics, Hospital for Special Surgery, Weill Medical College of Cornell University, Room 426, Caspary Research Building, 535 East 70th Street, New York, NY 10021. Phone: 212-606-1429; Fax: 212-774-2560; E-mail: blobelc{at}hss.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ADAM9 is a membrane-anchored metalloprotease that is markedly up-regulated in several human carcinomas. Here, we show that ADAM9 is similarly up-regulated in mouse models for prostate, breast, and intestinal carcinoma. To assess whether ADAM9 is critical for the pathogenesis of prostate carcinoma, one of the most common cancers in men, we evaluated how loss of ADAM9 affects tumorigenesis in W10 mice, a mouse model for this disease. In the absence of ADAM9, most tumors in 50-week-old W10 mice were well differentiated, whereas littermate controls expressing wild-type ADAM9 had predominantly poorly differentiated, and in some cases significantly larger, tumors. Moreover, gain-of-function experiments in which ADAM9 was overexpressed in mouse prostate epithelium resulted in significant abnormalities, including epithelial hyperplasia at 4 to 6 months of age, and prostatic intraepithelial neoplasia after 1 year. A potential underlying mechanism for the role of ADAM9 in prostate cancer emerged from cell-based assays: ADAM9 can cleave and release epidermal growth factor and FGFR2iiib from cells, both of which have pivotal functions in the pathogenesis of this disease. Taken together, these results suggest that ADAM9 contributes to the pathogenesis of prostate cancer and potentially also other carcinomas, raising the possibility that ADAM9 might be a good target for antitumor drugs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cell-to-cell communication is vital for development and adult tissue homeostasis. However, dysregulated or inappropriate cell-to-cell communication can contribute to the pathogenesis of diseases such as cancer. Several key components of the cell-to-cell signaling machinery, including all ligands of the epidermal growth factor receptor (EGFR) as well as other growth factors, cytokines, and their receptors, are synthesized as membrane-anchored proteins whose function can be modulated by proteolytic cleavage and release from the cell (1). The enzymes that are responsible for this process, which is called protein ectodomain shedding, are frequently members of the ADAM (a disintegrin and metalloprotease) family of membrane-anchored metalloproteases (1, 2). Protein ectodomain shedding functions as a molecular signaling switch that can activate or inactivate the released substrate protein and has emerged as a particularly important regulator of the function of EGFR ligands (1).

The ability of ADAMs to affect the function of growth factors and receptors raises questions about whether dysregulation of ADAMs might contribute to tumorigenesis. Recently, overexpression of ADAM9 has been reported in several human carcinomas, including breast (3), liver (4, 5), pancreas (6), and gastric cancer (7). The goal of this study was to address whether ADAM9 might play a causative role in tumor development. In situ mRNA analysis showed that ADAM9 is highly expressed in epithelial cells of mouse models for prostate, breast, and gastrointestinal carcinoma. More detailed analysis of prostate tumors revealed high levels of ADAM9 mRNA in well-differentiated carcinomas, but only low or undetectable levels in poorly differentiated carcinomas. This prompted an evaluation of how loss of ADAM9 function affects tumorigenesis in the W10 mouse model for prostate cancer (8), and how gain of function through transgenic overexpression of ADAM9 affects epithelial cells in the mouse prostate. The loss-of-function experiments suggest that ADAM9 is critical for tumor progression past the well-differentiated state, whereas gain-of-function experiments provided additional evidence for a causal role of ADAM9 in tumorigenesis. In cell-based assays, ADAM9 cleaves and releases membrane proteins with known roles in prostate epithelial proliferation and transformation, FGFR2iiib and EGF, establishing potential mechanisms for its role in carcinogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. PCR reagents were from Promega (Madison, WI), restriction enzymes from NEB (Beverly, MA), goat anti-mouse ADAM9 ectodomain antibodies from R&D Systems (Minneapolis, MN), and rabbit antisynaptophysin antibodies from Zymed Laboratories (San Francisco, CA).

In situ hybridization. Tumor sections from three mouse carcinoma models were used for ADAM9 expression analysis by mRNA in situ hybridization as previously described (9, 10). In the prostate cancer model (called W10 hereafter), the SV40 large T antigen is expressed in glandular epithelial cells of the prostate under control of the probasin promoter (8). In the breast cancer model, expression of the polyoma virus middle T antigen in mammary epithelium is driven by the murine mammary tumor virus promoter (MMTV-PyMT; ref. 11), and in the intestinal cancer model a germ line mutation in the adenomatosis polyposis coli gene (Apc) leads to multiple intestinal neoplasias (Min; ref. 12).

PB-ADAM9 transgenic mice. The complete mouse ADAM9 cDNA coding sequence (bp 14-2,551; ref. 10) was amplified by PCR with primers that introduced a 5' EcoRV site and a 3' NotI site, inserted into the SK-PB plasmid, which contains a rat probasin (PB) minimal promoter (kindly provided by Dr. N.M. Greenberg, Baylor College of Medicine, Houston, TX) and sequenced to rule out mutations. Transgenic mice were generated by pronuclear microinjection of QiaexII (Qiagen, Valencia, CA) purified PB-ADAM9 cDNA into fertilized C57BL/6J x CBA-f2 eggs. Founder animals were identified by PCR on genomic DNA from tail biopsies with ADAM9 primers that only amplify transgenic ADAM9 cDNA, but not genomic DNA (forward primer, bp 734-757: 5'-CGCTTAGCAAACTACCTGGATAGC-3'; reverse primer, bp 1,489-1,513: 5'-CTGGCAATCTTTACAACAGTCGCC-3'). All histopathologic comparisons were between pairs of heterozygous transgenics and nontransgenic littermate controls. All animal experiments were approved by the Institutional Animal Care and Use Committees of the Memorial Sloan-Kettering Cancer Center and the Hospital for Special Surgery.

Generation of W10/Adam9–/– mice. Transgenic W10 females (8) were crossed with Adam9–/– males (mixed SV129-C57BL/6 background) to generate F1 W10/Adam9+/– mice and nontransgenic Adam9+/– littermates. For tumor studies, W10/Adam9+/– mice were bred with Adam9+/– mice to obtain the following genotypes: W10/Adam9+/+, W10/Adam9+/– and W10/Adam9–/–. All offsprings were genotyped by PCR on genomic DNA to detect the W10 transgene as described (8), and by Southern blot to detect the targeted ADAM9 allele (13). Male littermates containing the W10 transgene that were either Adam9+/+, Adam9+/–, Adam9–/– were saved and analyzed at 50 weeks of age.

Histopathologic analysis and immunostaining. Transgenic PB-ADAM9 mice and littermate controls from three different lines were sacrificed at 4, 6, and 12 months of age, and W10/Adam9+/+, W10/Adam9+/–, and W10/Adam9–/– were sacrificed at 50 weeks of age. The urogenital tract was removed en bloc and fat and connective tissue were trimmed off. Individual prostate lobes were isolated and fixed overnight with 4% paraformaldehyde for histologic analysis. A portion of the urogenital tracts of 4-month-old PB-ADAM9 mice were embedded in OCT Tissue-Tex (Sakura Miles, Elkhart, IN), frozen, cryosectioned, and subjected to immunofluorescent staining for ADAM9. Antisynaptophysin immunostaining was done on paraformaldehyde-fixed sections using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA).

Cell-based protein ectodomain shedding assays. Expression plasmids for alkaline phosphatase–tagged EGFR ligands and the ectodomain shedding assay have been described previously (14, 15). The FGFR2iiib cDNA was amplified by PCR using full-length human FGFR2iiib cDNA as template (kindly provided by D.M. Ornitz, Washington University, St. Louis, MO) and ligated into the pAPtag5 expression plasmid (Genehunter, Nashville, TN). The AP-FGFR2iiib deletion mutant (A363-Y376) was generated by overlapping PCR. All expression constructs were sequenced to rule out undesired mutations. Expression constructs for ADAM9 and the catalytically inactive E>A mutant were described previously (10, 16).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Up-regulation of ADAM9 in mouse models for prostate, breast, and intestinal cancer. To examine whether ADAM9 is up-regulated in mouse tumor models, we determined its expression by mRNA in situ hybridization at different stages of the W10 mouse model for prostate cancer, in which the SV40 large T antigen is expressed in prostate epithelium under control of the probasin promoter (8). Several histologically well defined states of prostate cancer are observed in mouse models for prostate cancer, including high-grade prostatic intraepithelial neoplasia, well, moderately, and poorly differentiated carcinoma (17). Whereas ADAM9 was only weakly expressed in prostates from wild-type (WT) mice (Fig. 1A-C) or in W10 mice with prostatic intraepithelial neoplasia (data not shown), we observed high expression of ADAM9 in epithelial cells of well-differentiated tumors (Fig. 1D-F), characterized by densely packed, well-formed glands. This pronounced expression of ADAM9 was no longer seen at more advanced states of prostate cancer, such as in poorly differentiated tumors (Fig. 1G-I) or in metastatic carcinoma (Fig. 1J-L). The expression pattern of mouse ADAM9 at different states of the W10 prostate cancer model is consistent with a gene chip analysis of ADAM9 expression at different states of human prostate cancer, which revealed a significantly increased expression of ADAM9 in nontreated primary prostate tumors compared with nonneoplastic prostate, whereas no significant increase was seen in metastatic prostate cancer compared with nonneoplastic prostate.5



View larger version (106K):
[in this window]
[in a new window]
  		Figure 1. ADAM9 is overexpressed in mouse models for prostate, breast, and intestinal carcinoma. In situ mRNA hybridization to detect expression of ADAM9 in prostate tumors from W10 mice (8), mammary tumors from MMTV-PyMT mice (11), and intestinal tumors from Apc/Min/+ mice (12). A to I, prostate sections. Whereas ADAM9 is weakly expressed in normal WT prostate (A-C), high expression is observed in epithelial cells of a well-differentiated prostate tumor (WDCA, arrows in D-F), relative to the adjacent negative stroma. Little ADAM9 is detected in a poorly differentiated carcinoma (PDCA, G-I) and a small lung metastasis (MET, J-L). M to O, MMTV-PyMT mammary tumor. High levels of ADAM9 mRNA are seen in tumor cells filling an enlarged duct (lobular carcinoma in situ, arrow). P to R, Apc/Min/+ model. High expression of ADAM9 is visible throughout the adenoma (arrow), whereas ADAM9 expression is low in the apparently normal adjacent villi (indicated by an asterisk in Q). Each panel shows result that is representative for at least five different tumors. Magnifications were x100 in the left and middle panel of each row and x600 in the right panel of each row.

 
When we examined ADAM9 expression in a second mouse carcinoma model—breast tumors from 4-month-old MMTV-PyMT mice—we found very high ADAM9 expression in tumor cells (an acinus distended by tumor cells is shown in Fig. 1M-O), whereas little expression of ADAM9 was evident in the surrounding mammary tissue, or in high grade, poorly differentiated sections of solid tumors (data not shown). ADAM9 is expressed in a region that is similar to one that was previously described as early carcinoma resembling human ductal carcinoma in situ with early invasion in tumor sections from MMTV-PyMT mice (18). In a third mouse carcinoma model—intestinal tumors from Apc/Min/+ mice—ADAM9 was highly expressed in adenomas with strong epithelial proliferation, whereas little ADAM9 was expressed in adjacent apparently normal villi (Fig. 1P-R). Thus, ADAM9 expression was strongly up-regulated in epithelial cells of three different mouse carcinoma models.

More well-differentiated tumors are seen in W10 mice lacking ADAM9 compared with W10 littermate controls. To determine whether loss of ADAM9 affects development of prostate cancer or its progression, we crossed Adam9–/– mice with W10 mice, a model for prostate cancer (8). Through appropriate matings, we generated W10 mice lacking ADAM9 as well as littermate controls carrying one or both WT alleles of ADAM9 (see Materials and Methods). At 50 weeks of age, prostates of all mice were harvested and weighed in a double-blinded manner. We found that 5 of 22 W10 mice with one or both WT alleles of ADAM9 (W10/Adam9+/+ or W10/Adam9+/–) had tumors weighing >200 mg at 50 weeks, whereas all tumors in the 18 W10/Adam9–/– mice analyzed weighed <200 mg (P < 0.05, t test; Fig. 2A).



View larger version (59K):
[in this window]
[in a new window]
  		Figure 2. Targeted deletion of ADAM9 results in more well-differentiated carcinoma in the W10 mouse model for prostate cancer at 50 weeks of age. A, weights of prostate tumors from W10/Adam9–/–, W10/Adam9+/–, and W10/Adam9+/+ mice at 50 weeks of age (determined in a double-blinded manner). No large tumors (>200 mg) were observed in W10/Adam9–/– mice (n = 18) compared with W10/Adam9+/– and W10/Adam9+/+ mice (n = 22; P < 0.05, t test). B, results of histologic scoring of tumors. The top three panels show representative examples of tumors that were scored as well-differentiated carcinoma, moderately differentiated carcinoma (MDCA; arrow, well-formed epithelia surrounding a solid nest of cells), and poorly differentiated carcinoma. Magnifications were x100 (top) and x400 (middle). In W10/Adam9–/– mice, 58% of the tumors were well-differentiated carcinoma and 42% had at least a some moderately differentiated carcinoma, but none of the tumors examined had progressed to poorly differentiated carcinoma (n = 12; P < 0.005, t test). In W10/Adam9+/– and W10/Adam9+/+ mice, 50% of the tumors contained areas of poorly differentiated carcinoma. *, a poorly differentiated carcinoma region (B, top); only 16% contained well-differentiated carcinoma (n = 12; P < 0.005, t test).

 
To determine whether ADAM9, which is highly expressed at the well-differentiated carcinoma state, might affect the progression of prostate cancer to poorly differentiated carcinoma, we did a double-blinded analysis of the tumor histology of representative sections of tumors from 50-week-old W10/Adam9–/–, W10/Adam9–/+, and W10/Adam9+/+ mice. The lesions were classified as well-differentiated carcinoma when gland formations lined with epithelial cells were present throughout the tumor sections, as moderately differentiated carcinoma when focal to extensive areas of solid cell nests and fused glands were mixed with well formed glands, and as poorly differentiated carcinoma when large areas of the tumor were composed of more solid sheets or nests, with rare gland formation (Fig. 2B, top, shows examples of each tumor state). This analysis revealed that 58% of tumors from W10/Adam9–/– mice contained only well-differentiated carcinoma, 42% of tumors contained some moderately differentiated carcinoma, whereas none had areas of poorly differentiated carcinoma (n = 12; Fig. 2B, bottom). On the other hand, 50% of tumors in littermate controls (W10/Adam9+/+ or W10/Adam9+/– mice) contained poorly differentiated carcinoma, 33% contained moderately differentiated carcinoma whereas 17% contained only well-differentiated carcinoma (n = 12; Fig. 2B, bottom). Thus, there was significantly more well-differentiated carcinoma and less poorly differentiated carcinoma in W10 mice in the absence of ADAM9, compared with controls with one or both copies of WT ADAM9.

Transgenic overexpression of ADAM9 in mouse prostate results in abnormal acinar epithelium and high-grade prostatic intraepithelial neoplasia. The high expression of ADAM9 in epithelial cells of three mouse carcinoma models coupled with the results of the loss-of-function studies prompted us to assess how transgenic overexpression of ADAM9 affects prostate epithelial cells. For this purpose, we generated three independent mouse lines that overexpress ADAM9 under control of the minimal probasin promoter (PB-ADAM9). PCR and Western blot analysis confirmed that overexpression of ADAM9 was restricted to the dorsolateral prostate lobes in all three lines (data not shown). Urogenital tracts from 4-, 6-, and 12-month-old male PB-ADAM9 mice and nontransgenic littermates were collected for further analysis. In 4- and 6-month-old PB-ADAM9 mice, histopathologic analysis of dorsolateral prostate lobes uncovered disorganized prostate ducts with areas that seemed to lack a proper epithelial lining and lumens filled with rounded cells (Fig. 3A). These abnormalities were not seen in any sections of prostate glands of WT littermates (Fig. 3D). Immunofluorescent analysis of adjacent cryosections stained with anti-ADAM9 antibodies confirmed high ADAM9 expression in transgenic glands and in cells filling the lumen of these glands (Fig. 3B).



View larger version (91K):
[in this window]
[in a new window]
  		Figure 3. Histologic analysis of PB-ADAM9 mice shows structurally abnormal prostate acini at 4 months and high-grade prostatic intraepithelial neoplasia lesions by 12 months of age. A to F, sections of prostates from 4-month-old PB-ADAM9 mice (A-C) or WT littermates (D-F) were stained with H&E (A and D), anti-ADAM9 (B and E), or antisynaptophysin (C and F). Ducts filled with ADAM9-expressing cells are seen in PB-ADAM9 mice, but not in WT littermates (B and E). Arrows, synaptophysin-positive cells; arrowhead, neuronal bundle (C and F). G to J, H&E-stained sections of prostate epithelia from a 1-year-old PB-ADAM9 mouse (G and H) and WT littermate (I and J). Normal nuclei of similar size and uniform staining without proliferation into the lumen are seen in sections of a WT prostate (J), whereas similar sections of PB-ADAM9 mice display abnormally enlarged nuclei and increased nuclear/cytoplasmic ratio together with stratification of the epithelia (H), characteristic features of high-grade prostatic intraepithelial neoplasia. The changes shown here were seen in all three PB-ADAM9 mouse lines and in all transgenic animals that were analyzed. Magnifications were x100 in (A), (B), (G), and (I); x200 in (C) to (F); and x400 in (H) and (J).

 
Prostate ducts with a similar appearance as those seen in 4- and 6-month-old PB-ADAM9 mice (disorganized epithelia, lumen filled with cells) have also been described in transgenic mice overexpressing a dominant-negative form of the FGFR2iiib under the probasin promoter (19). In prostates from these animals, increased expression of the neuroendocrine marker synaptophysin was observed in the smooth muscle area adjacent to the basement membrane. When prostates from 4-month-old PB-ADAM9 mice and WT littermate controls were stained with antibodies against synaptophysin, we observed a clear increase of stained cells within the smooth muscle areas adjacent to the basement membrane in PB-ADAM9 mice compared with nontransgenic littermates (compare Fig. 3C and F).

When we inspected prostates of 12-month-old PB-ADAM9 mice, we observed considerable epithelial cell hyperplasia with stratification of the nuclei (Fig. 3G and H), whereas prostates from nontransgenic littermates did not have any evident abnormalities (Fig. 3I and J). In some sections of PB-ADAM9 prostates, more severe abnormalities could be seen, including extensive layering of the acinar epithelial cells, cells with increased nuclear to cytoplasmic ratio, variability in nuclear shape, condensed chromatin, prominent nucleoli, and appearance of mitotic figures. These abnormalities are consistent with the changes associated with prostatic intraepithelial neoplasia, considered to be a precursor of invasive prostate carcinoma in humans.

ADAM9 cleaves FGFR2iiib and epidermal growth factor in cell-based assays. The similarities between the histopathologic changes seen in 4-month-old PB-ADAM9 animals (Fig. 3A-C) and in mice expressing a dominant-negative FGFR2iiib under control of the probasin promotor (19) raised the question whether overexpressed ADAM9 could cleave and potentially down-regulate FGFR2iiib. If this were the case, ADAM9 might affect signaling via FGFR2iiib in a similar manner as overexpression of a dominant-negative receptor. To address this possibility, we cotransfected WT ADAM9 or a catalytically inactive E>A mutant together with FGFR2iiib in Cos-7 cells. Overexpression of ADAM9 resulted in a strong increase in ectodomain shedding of FGFR2iiib into the supernatant, whereas overexpression of the inactive ADAM9E>A control did not (Fig. 4B). ADAM9-dependent shedding of FGFR2iiib was only weakly enhanced by phorbol 12-myristate 13-acetate (PMA), a phorbol ester that is commonly used to stimulate ectodomain shedding. No shedding was observed when a mutant FGFR2iiib lacking 14 membrane-proximal amino acid residues (del 363-376aa) was cotransfected with ADAM9 WT, strongly suggesting that FGFR2iiib is cleaved in its juxtamembrane region (Fig. 4B).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 4. Overexpression of ADAM9 results in ectodomain shedding of FGFR2iiib and EGF. A, schematic representation of FGFR2iiib indicates the position of the transmembrane domain (TM), juxtamembrane domain (JM), and the carboxy-terminal alkaline phosphatase tag (AP). B, FGFR2iiib-AP shedding. Cotransfection experiments in Cos-7 cells show increased release of AP-tagged FGFR2iiib into the cell supernatant (SN) of cells expressing WT ADAM9, compared with cells expressing its catalytically inactive E>A mutant. No shedding of FGFR2iiib lacking its juxtamembrane domain (del 363-376aa) is observed. C, EGFR ligand shedding. Cos-7 cells were cotransfected with AP-tagged EGF, HB-EGF, TGF-{alpha}, amphiregulin, epiregulin, or ß-cellulin and ADAM9 or ADAM9E/A, and the alkaline phosphatase–labeled ectodomains released into the culture supernatant were separated by SDS-PAGE and visualized as described in Materials and Methods. In all experiments, the expression level of alkaline phosphatase–tagged substrates in the cell lysate (CL) was comparable in ADAM9 and ADAM9E/A-transfected cells, and the expression level of ADAM9 and ADAM9E/A was also similar as confirmed by Western blot analysis (data not shown).

 
The EGFR signaling pathway has also been implicated in the pathogenesis of prostate cancer (20–22), and proteolytic processing of EGFR ligands is important for regulating the function of these growth factors (1, 23–25). Because ADAMs participate in the shedding of six of seven EGFR ligands (15), we tested whether overexpression of ADAM9 triggers the release of one or more of these proteins. When ADAM9 WT was overexpressed in Cos-7 cells together with different EGFR ligands, we observed a significant increase in the release of EGF, but not of HB-EGF, transforming growth factor-{alpha} (TGF-{alpha}), betacellulin, amphiregulin, or epiregulin (Fig. 4C). ADAM9-dependent shedding of EGF was only weakly enhanced by treatment with PMA, and was not seen in the presence of the overexpressed catalytically inactive ADAM9E>A mutant (Fig. 4C). The apparent mass of soluble EGF released by ADAM9 differed from that of endogenously released EGF, indicating that ADAM9 has a different cleavage site than the endogenous EGF sheddase (most likely ADAM10; ref. 15).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Membrane-anchored peptidases can function as critical regulators of cell-to-cell signaling. This study was initiated because the membrane anchored metalloprotease-disintegrin ADAM9 is highly expressed in several human carcinomas (3–6). We found that ADAM9 is also prominently expressed in epithelial cells undergoing neoplastic changes in mouse models for prostate, breast, and intestinal cancer. In addition, the more detailed analysis of different tumor states in the W10 mouse model for prostate cancer uncovered highest expression of ADAM9 in well-differentiated tumors of the prostate. The observed up-regulation of ADAM9, a cell surface peptidase with a potential role in cell-to-cell signaling, raised questions about whether the enhanced expression is merely correlative, or whether it might also have a causative role in tumorigenesis. To address this, we did loss-of-function experiments with W10 mice crossed with mice lacking ADAM9, and gain-of-function experiments by transgenic overexpression of ADAM9 under the prostate specific probasin promoter (PB-ADAM9 mice).

The loss-of-function experiments revealed a critical function of ADAM9 in prostate cancer growth and progression in W10 mice. Remarkably, most of the tumors in W10/Adam9–/– mice were well-differentiated carcinomas, whereas most of the tumors in littermates with one or both copies of WT ADAM9 contained significant areas of poorly differentiated carcinoma. Moreover, 5 of 22 W10 mice with WT ADAM9 had large or very large tumors. No poorly differentiated or very large tumors were seen in W10 mice lacking ADAM9. These results suggest that prostate tumor initiation or progression is delayed in W10/Adam9–/– mice, or that tumor progression is possibly even arrested at the well-differentiated tumor state, which is the state where ADAM9 shows the highest expression in the W10 tumor model. These findings raise the possibility that ADAM9 has a critical role in the transition to more poorly differentiated carcinomas.

Gain-of-function experiments provided additional evidence for a causal role of ADAM9 in tumorigenesis. We found that PB-ADAM9 mice at different ages had significant abnormalities compared with age matched littermate controls. Specifically, at 4 and 6 months of age, transgenic animals of three different lines invariably displayed areas of structurally abnormal and disorganized acinar ducts that were filled with round epithelial cells, with a concomitant increase of synaptophysin-positive cells in the adjacent smooth muscle surrounding the ducts. After 1 year, the dorsolateral prostate lobes of mice from all three lines displayed high-grade prostatic intraepithelial neoplasia lesions, with crowded epithelial cells showing nuclear and nucleolar enlargement.

Taken together, the gain- and loss-of-function studies provide independent lines of evidence for a role of ADAM9 in carcinogenesis. Whereas the loss-of-function studies establish a critical role for ADAM9 in an in vivo tumor model, the gain-of-function experiments show that overexpression of ADAM9 alone is sufficient to trigger preneoplastic changes, including high-grade prostatic intraepithelial neoplasia. Further studies will be necessary to determine whether transgenic overexpression of ADAM9 at the well-differentiated carcinoma state in W10 mice accelerates tumor growth and progression. Nevertheless, it is tempting to speculate that ADAM9, which is highly expressed at the well-differentiated carcinoma state in W10 mice (see Fig. 1D-F), might contribute to cell proliferation and promote neoplastic changes in W10 mice in a similar manner as when it is overexpressed in normal prostate epithelia in PB-ADAM9 mice.

The early lesions observed in PB-ADAM9 mice share striking similarities with the disorganized prostatic ducts in mice expressing a dominant-negative FGFR2iiib in the prostate to disrupt FGF-7 signaling (19). Because changes in the FGF signaling pathway are believed to be critical for the development of prostate cancer (26), we tested whether ADAM9 can cleave FGFR2iiib in cell-based assays. We found that overexpressed ADAM9 enhances shedding of FGFR2iiib from cells, which is consistent with a model in which ADAM9 overexpressed in prostate epithelium could release and thus down-regulate FGFR2iiib. In addition, the soluble ectodomain of FGFR2iiib has been reported to function in a dominant-negative fashion and disrupt FGF signaling in vivo (27). In PB-ADAM9 transgenics, cleavage of FGFR2iiib by ADAM9 could, therefore, conceivably contribute to the early changes seen at 4 and 6 months by disrupting FGFR2iiib signaling. This notion is also supported by the observed up-regulation of the neuroendocrine marker synaptophysin in PB-ADAM9 mice, which was also seen in transgenic mice expressing the dominant-negative FGFR2iiib.

With respect to the possible role of the FGFR2iiib at later states of tumorigenesis, it is noteworthy that this receptor is frequently down-regulated during progression of human prostate cancer and animal tumor models to malignancy, such as in the TRAMP model (28). This down-regulation of FGFR2iiib is considered a feature of malignant stroma-independent epithelial cells (29, 30). On the other hand, transfection of WT FGFR2iiib into malignant prostate cancer cells is sufficient to inhibit growth and promote differentiation (31, 32). In the context of the loss-of-function experiments in W10 mice, it is tempting to speculate that less FGFR2iiib might be shed from Adam9–/– epithelial cells than from Adam9+/+ cells. Less FGFR2iiib shedding in Adam9–/– cells could conceivably lead to an accumulation of FGFRiiib, providing one possible explanation for the higher percentage of well-differentiated carcinomas in 50-week-old W10/Adam9–/– compared with littermate W10/Adam9+/+ controls.

Whereas the changes observed in 4- and 6-month-old transgenic PB-ADAM9 mice resemble those seen in animals expressing a dominant-negative FGFR2iiib, the development of prostatic intraepithelial neoplasia in 1-year-old transgenic PB-ADAM9 mice suggests that additional mechanisms might be affected by overexpression of ADAM9 in prostate epithelium (33). Because ADAMs have also been implicated as regulators of EGFR ligands, we asked whether ADAM9 can shed various EGFR ligands. We found that overexpression of ADAM9 leads to a clear enhancement of EGF shedding, but not of any of the other EGFR ligands tested here, including HB-EGF, which was previously reported as a substrate for ADAM9 (34). Whereas the reason for this discrepancy remains to be determined, the unequivocal ability of ADAM9 to cleave EGF has potential implications for its role in prostate cancer. EGF is present in large amounts in human prostatic fluid (35), is up-regulated in benign prostatic hyperplasia and tumors (36), has been implicated in the proliferation of epithelial and stromal prostate cells (37), and is expressed in epithelial cells in the TRAMP mouse model for prostate cancer (38), which is similar to the W10 model. Moreover, chronic systemic treatment with EGF induces prostate growth in young rats (39), and EGF has a role in androgen-independent transactivation of the androgen receptor, which in turn is thought to contribute to androgen-independent tumor growth (40, 41). Because ADAM9 can shed EGF in cell-based assays, we hypothesize that it might also increase EGF release and, thus, EGFR activation when it is overexpressed in prostate epithelial cells. In theory, the combination of EGFR activation and inactivation of FGFR2iiib could both promote epithelial cell proliferation, which in turn might raise the likelihood of acquiring additional deleterious mutations in the transgenic PB-ADAM9 mice. Likewise, ADAM9-dependent release of EGF at the well-differentiated state of prostate cancer in W10 mice could also contribute to progression of prostate cancer in this model, although ADAM9 might also have other mechanisms of action.

Taken together, these results provide the first evidence for a causal role of ADAM9 in the pathogenesis of cancer. ADAM9 is up-regulated, directly or indirectly, in three different mouse tumor models, in several human carcinomas, and its expression is also known to be induced by the oncogene c-myc (42). ADAM9 deficiency delays or prevents prostate tumor progression to advanced states in a mouse prostate cancer model, and dysregulated expression of ADAM9 initially disrupts epithelial-stromal homeostasis in the prostate epithelium, and later leads to prostatic intraepithelial neoplasia lesions. Cell-based studies show that ADAM9 has the ability to process and shed FGFR2iiib and EGF, both of which have well-established roles in prostate cancer. Moreover, ADAM9 could also have other yet to be identified substrates or other functions that contribute to its role in tumorigenesis, such as roles in cell-to-cell interactions or signaling via its cytoplasmic domain. These findings suggest that ADAM9 might, therefore, be a novel target for the treatment of carcinomas in which this enzyme is highly expressed.


   Acknowledgments
 
Grant support: Koch and Pepsico Funds (C.P. Blobel, H.I. Scher, and L. Peduto) and by NIH grant GM64750 (C.P. Blobel).

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.

We thank Dr. A. Koff (Memorial Sloan-Kettering Cancer Center, New York, NY) for providing W10 mice and for helpful comments while preparing the manuscript, Drs. B. Fingleton and L. Matrisian (Vanderbilt University, Nashville, TN) for providing tumors from MMTV-PyMT and Apc/Min/+ mice, Dr. S. Higashiyama (Ehime University, Ehime, Japan) for providing alkaline phosphatase–tagged EGFR ligands, Dr. Steve Swendeman for advice, and Brian Hutchinson and Katia Bozhilova for excellent technical assistance.


   Footnotes
 
5 W. Gerald, Memorial Sloan-Kettering Cancer Center, personal communication. Back

Received 3/30/05. Revised 7/14/05. Accepted 8/ 9/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Blobel CP. ADAMs: key players in EGFR-signaling, development and disease. Nat Rev Mol Cell Biol 2005;6:32–43.[CrossRef][Medline]
  2. White JM. ADAMs: modulators of cell-cell and cell-matrix interactions. Curr Opin Cell Biol 2003;15:598–606.[CrossRef][Medline]
  3. O'Shea C, McKie N, Buggy Y, et al. Expression of ADAM-9 mRNA and protein in human breast cancer. Int J Cancer 2003;105:754–61.[CrossRef][Medline]
  4. Le Pabic H, Bonnier D, Wewer UM, et al. ADAM12 in human liver cancers: TGF-ß-regulated expression in stellate cells is associated with matrix remodeling. Hepatology 2003;37:1056–66.[CrossRef][Medline]
  5. Tannapfel A, Anhalt K, Hausermann P, et al. Identification of novel proteins associated with hepatocellular carcinomas using protein microarrays. J Pathol 2003;201:238–49.[CrossRef][Medline]
  6. Grutzmann R, Luttges J, Sipos B, et al. ADAM9 expression in pancreatic cancer is associated with tumour type and is a prognostic factor in ductal adenocarcinoma. Br J Cancer 2004;90:1053–8.[CrossRef][Medline]
  7. Carl-McGrath S, Lendeckel U, Ebert M, Roessner A, Rocken C. The disintegrin-metalloproteinases ADAM9, ADAM12, and ADAM15 are upregulated in gastric cancer. Int J Oncol 2005;26:17–24.[Medline]
  8. Shaffer DR, Viale A, Ishiwata R, et al. Evidence for a p27 tumor suppressive function independent of its role regulating cell proliferation in the prostate. Proc Natl Acad Sci U S A 2005;102:210–5.[Abstract/Free Full Text]
  9. Manova K, Nocka K, Besmer P, Bachvarova RF. Gonadal expression of c-kit encoded at the W locus of the mouse. Development 1990;110:1057–69.[Abstract/Free Full Text]
  10. Weskamp G, Krätzschmar JR, Reid M, Blobel CP. MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains. J Cell Biol 1996;132:717–26.[Abstract/Free Full Text]
  11. Guy CT, Cardiff RD, Muller WJ. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol Cell Biol 1992;12:954–61.[Abstract/Free Full Text]
  12. Moser AR, Pitot HC, Dove WF. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 1990;247:322–4.[Abstract/Free Full Text]
  13. Weskamp G, Cai H, Brodie TA, et al. Mice lacking the metalloprotease-disintegrin MDC9 (ADAM9) have no evident major abnormalities during development or adult life. Mol Cell Biol 2002;22:1537–44.[Abstract/Free Full Text]
  14. Zheng Y, Schlondorff J, Blobel CP. Evidence for regulation of the tumor necrosis factor {alpha}-convertase (TACE) by protein-tyrosine phosphatase PTPH1. J Biol Chem 2002;277:42463–70.[Abstract/Free Full Text]
  15. Sahin U, Weskamp G, Zhou HM, et al. Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR-ligands. J Cell Biol 2004;164:769–79.[Abstract/Free Full Text]
  16. Roghani M, Becherer JD, Moss ML, et al. Metalloprotease-disintegrin MDC9: intracellular maturation and catalytic activity. J Biol Chem 1999;274:3531–40.[Abstract/Free Full Text]
  17. Shappell SB, Thomas GV, Roberts RL, et al. Prostate pathology of genetically engineered mice: definitions and classification. The consensus report from the Bar Harbor meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee. Cancer Res 2004;64:2270–305.[Abstract/Free Full Text]
  18. Lin EY, Jones JG, Li P, et al. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. Am J Pathol 2003;163:2113–26.[Abstract/Free Full Text]
  19. Foster BA, Evangelou A, Gingrich JR, Kaplan PJ, DeMayo F, Greenberg NM. Enforced expression of FGF-7 promotes epithelial hyperplasia whereas a dominant negative FGFR2iiib promotes the emergence of neuroendocrine phenotype in prostate glands of transgenic mice. Differentiation 2002;70:624–32.[CrossRef][Medline]
  20. Di Fiore PP, Pierce JH, Fleming TP, et al. Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells. Cell 1987;51:1063–70.[CrossRef][Medline]
  21. Baselga J, Mendelsohn J. Receptor blockade with monoclonal antibodies as anti-cancer therapy. Pharmacol Ther 1994;64:127–54.[CrossRef][Medline]
  22. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001;2:127–37.[CrossRef][Medline]
  23. Peschon JJ, Slack JL, Reddy P, et al. An essential role for ectodomain shedding in mammalian development. Science 1998;282:1281–4.[Abstract/Free Full Text]
  24. Jackson LF, Qiu TH, Sunnarborg SW, et al. Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling. EMBO J 2003;22:2704–16.[CrossRef][Medline]
  25. Prenzel N, Zwick E, Daub H, et al. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature 1999;402:884–8.[Medline]
  26. Evangelou AI, Winter SF, Huss WJ, Bok RA, Greenberg NM. Steroid hormones, polypeptide growth factors, hormone refractory prostate cancer, and the neuroendocrine phenotype. J Cell Biochem 2004;91:671–83.[CrossRef][Medline]
  27. Celli G, LaRochelle WJ, Mackem S, Sharp R, Merlino G. Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning. EMBO J 1998;17:1642–55.[CrossRef][Medline]
  28. Foster BA, Kaplan PJ, Greenberg NM. Characterization of the FGF axis and identification of a novel FGFR1iiic isoform during prostate cancer progression in the TRAMP model. Prostate Cancer Prostatic Dis 1999;2:76–82.[CrossRef][Medline]
  29. Naimi B, Latil A, Fournier G, Mangin P, Cussenot O, Berthon P. Down-regulation of (IIIb) and (IIIc) isoforms of fibroblast growth factor receptor 2 (FGFR2) is associated with malignant progression in human prostate. Prostate 2002;52:245–52.[CrossRef][Medline]
  30. Yan G, Fukabori Y, McBride G, Nikolaropolous S, McKeehan WL. Exon switching and activation of stromal and embryonic fibroblast growth factor (FGF)-FGF receptor genes in prostate epithelial cells accompany stromal independence and malignancy. Mol Cell Biol 1993;13:4513–22.[Abstract/Free Full Text]
  31. Feng S, Wang F, Matsubara A, Kan M, McKeehan WL. Fibroblast growth factor receptor 2 limits and receptor 1 accelerates tumorigenicity of prostate epithelial cells. Cancer Res 1997;57:5369–78.[Abstract/Free Full Text]
  32. Matsubara A, Kan M, Feng S, McKeehan WL. Inhibition of growth of malignant rat prostate tumor cells by restoration of fibroblast growth factor receptor 2. Cancer Res 1998;58:1509–14.[Abstract/Free Full Text]
  33. Jin C, McKeehan K, Guo W, et al. Cooperation between ectopic FGFR1 and depression of FGFR2 in induction of prostatic intraepithelial neoplasia in the mouse prostate. Cancer Res 2003;63:8784–90.[Abstract/Free Full Text]
  34. Izumi Y, Hirata M, Hasuwa H, et al. A metalloprotease-disintegrin, MDC9/meltrin-{gamma}/ADAM9 and PKC{delta} are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor. EMBO J 1998;17:7260–72.[CrossRef][Medline]
  35. Fuse H, Sakamoto M, Okumura M, Katayama T. Epidermal growth factor contents in seminal plasma as a marker of prostatic function. Arch Androl 1992;29:79–85.[Medline]
  36. De Miguel P, Royuela, Bethencourt R, Ruiz A, Fraile B, Paniagua R. Immunohistochemical comparative analysis of transforming growth factor {alpha}, epidermal growth factor, and epidermal growth factor receptor in normal, hyperplastic and neoplastic human prostates. Cytokine 1999;11:722–7.[CrossRef][Medline]
  37. Schuurmans AL, Bolt J, Mulder E. Androgens and transforming growth factor ß modulate the growth response to epidermal growth factor in human prostatic tumor cells (LNCaP). Mol Cell Endocrinol 1988;60:101–4.[CrossRef][Medline]
  38. Wang J, Eltoum IE, Lamartiniere CA. Genistein alters growth factor signaling in transgenic prostate model (TRAMP). Mol Cell Endocrinol 2004;219:171–80.[CrossRef][Medline]
  39. Torring N, Jensen LV, Wen JG, Sorensen FB, Djurhuus JC, Nexo E. Chronic treatment with epidermal growth factor induces growth of the rat ventral prostate. Scand J Urol Nephrol 2001;35:339–44.[CrossRef][Medline]
  40. Culig Z, Hobisch A, Cronauer MV, et al. Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte growth factor, and epidermal growth factor. Cancer Res 1994;54:5474–78.[Abstract/Free Full Text]
  41. Gregory CW, Fei X, Ponguta LA, et al. Epidermal growth factor increases coactivation of the androgen receptor in recurrent prostate cancer. J Biol Chem 2004;279:7119–30.[Abstract/Free Full Text]
  42. Schuhmacher M, Kohlhuber F, Holzel M, et al. The transcriptional program of a human B cell line in response to Myc. Nucleic Acids Res 2001;29:397–406.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
A. J. Najy, K. C. Day, and M. L. Day
ADAM15 Supports Prostate Cancer Metastasis by Modulating Tumor Cell-Endothelial Cell Interaction
Cancer Res., February 15, 2008; 68(4): 1092 - 1099.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Ali and V. Knauper
Phorbol Ester-induced Shedding of the Prostate Cancer Marker Transmembrane Protein with Epidermal Growth Factor and Two Follistatin Motifs 2 Is Mediated by the Disintegrin and Metalloproteinase-17
J. Biol. Chem., December 28, 2007; 282(52): 37378 - 37388.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. L. Moss, M. Bomar, Q. Liu, H. Sage, P. Dempsey, P. M. Lenhart, P. A. Gillispie, A. Stoeck, D. Wildeboer, J. W. Bartsch, et al.
The ADAM10 Prodomain Is a Specific Inhibitor of ADAM10 Proteolytic Activity and Inhibits Cellular Shedding Events
J. Biol. Chem., December 7, 2007; 282(49): 35712 - 35721.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
P. M. McGowan, B. M. Ryan, A. D.K. Hill, E. McDermott, N. O'Higgins, and M. J. Duffy
ADAM-17 Expression in Breast Cancer Correlates with Variables of Tumor Progression
Clin. Cancer Res., April 15, 2007; 13(8): 2335 - 2343.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
N. Kawaguchi, K. Horiuchi, J. D. Becherer, Y. Toyama, P. Besmer, and C. P. Blobel
Different ADAMs have distinct influences on Kit ligand processing: phorbol-ester-stimulated ectodomain shedding of Kitl1 by ADAM17 is reduced by ADAM19
J. Cell Sci., March 15, 2007; 120(6): 943 - 952.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
K. Horiuchi, S. Le Gall, M. Schulte, T. Yamaguchi, K. Reiss, G. Murphy, Y. Toyama, D. Hartmann, P. Saftig, and C. P. Blobel
Substrate Selectivity of Epidermal Growth Factor-Receptor Ligand Sheddases and their Regulation by Phorbol Esters and Calcium Influx
Mol. Biol. Cell, January 1, 2007; 18(1): 176 - 188.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
C. Frohlich, R. Albrechtsen, L. Dyrskjot, L. Rudkjaer, T. F. Orntoft, and U. M. Wewer
Molecular Profiling of ADAM12 in Human Bladder Cancer
Clin. Cancer Res., December 15, 2006; 12(24): 7359 - 7368.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S.-Y. Sung, H. Kubo, K. Shigemura, R. S. Arnold, S. Logani, R. Wang, H. Konaka, M. Nakagawa, S. Mousses, M. Amin, et al.
Oxidative Stress Induces ADAM9 Protein Expression in Human Prostate Cancer Cells
Cancer Res., October 1, 2006; 66(19): 9519 - 9526.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
H. Ohtsu, P. J. Dempsey, and S. Eguchi
ADAMs as mediators of EGF receptor transactivation by G protein-coupled receptors
Am J Physiol Cell Physiol, July 1, 2006; 291(1): C1 - C10.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Peduto, L.
Right arrow Articles by Blobel, C. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Peduto, L.
Right arrow Articles by Blobel, C. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online