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Cancer Research 67, 7124, August 1, 2007. doi: 10.1158/0008-5472.CAN-07-0686
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

IFN-ß Signaling Positively Regulates Tumorigenesis in Aggressive Fibromatosis, Potentially by Modulating Mesenchymal Progenitors

Sean S. Tjandra1, Claire Hsu1, Ingrid Goh1, Ananta Gurung1, Raymond Poon1, Puviindran Nadesan1 and Benjamin A. Alman1,2

1 Program in Developmental and Stem Cell Biology, The Hospital for Sick Children and 2 Department of Surgery, University of Toronto, Toronto, Ontario, Canada

Requests for reprints: Benjamin A. Alman, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G1X8. Phone: 416-813-2178; Fax: 416-813-6414; E-mail: Benjamin.alman{at}sickkids.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aggressive fibromatosis (also called desmoid tumor) is a benign, locally invasive, soft tissue tumor composed of cells with mesenchymal characteristics. These tumors are characterized by increased levels of ß-catenin–mediated T-cell factor (TCF)–dependent transcriptional activation. We found that type 1 IFN signaling is activated in human and murine aggressive fibromatosis tumors and that the expression of associated response genes is regulated by ß-catenin. When mice deficient for the type 1 IFN receptor (Ifnar1–/–) were crossed with mice predisposed to developing aggressive fibromatosis tumors (Apc/Apc1638N), a significant decrease in aggressive fibromatosis tumor formation was observed compared with littermate controls, showing a novel role for type 1 IFN signaling in promoting tumor formation. Type 1 IFN activation inhibits cell proliferation but does not alter cell apoptosis or the level of ß-catenin–mediated TCF-dependent transcriptional activation in aggressive fibromatosis cell cultures. Thus, these changes cannot explain our in vivo results. Intriguingly, Ifnar1–/– mice have smaller numbers of mesenchymal progenitor cells compared with littermate controls, and treatment of aggressive fibromatosis cell cultures with IFN increases the proportion of cells that exclude Hoechst dye and sort to the side population, raising the possibility that type 1 IFN signaling regulates the number of precursor cells present that drive aggressive fibromatosis tumor formation and maintenance. This study identified a novel role for IFN type 1 signaling as a positive regulator of neoplasia and suggests that IFN treatment is a less than optimal therapy for this tumor type. [Cancer Res 2007;67(15):7124–31]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Aggressive fibromatosis, also called desmoid tumor, is a benign locally invasive soft tissue tumor of mesenchymal origin composed of a clonal population of fibroblast-like cells (1). This tumor is characterized by mutations resulting in ß-catenin protein stabilization and activation of ß-catenin–mediated T-cell factor (TCF)-dependent transcription. Germ-line mutations in the adenomatous polyposis gene (APC), which are associated with the premalignant cancer syndrome familial adenomatous polyposis, also predispose to aggressive fibromatosis (2). Mutations residing near the 3' end of the APC gene predispose to a more severe aggressive fibromatosis phenotype (3, 4). The Apc/Apc1638N mouse harbors a targeted mutation in exon 15 of the Apc gene and develops large numbers of aggressive fibromatosis tumors with an average of 45 tumors in male mice at 6 months of age (4).

ß-Catenin is a crucial mediator of the canonical Wnt pathway. In the absence of Wnt ligand, ß-catenin is phosphorylated on serine residues near the NH2 terminus by a multiprotein regulatory complex including APC, which targets the protein for ubiquitin-mediated proteasomal degradation (5). When an appropriate Wnt ligand is present, this multiprotein complex is inhibited, allowing ß-catenin levels to accumulate. ß-Catenin can translocate to the nucleus and bind to members of the TCF/lymphoid enhancer factor family of transcription factors to regulate transcription (5). Mutations in components of the regulatory complex or in the phosphorylation sites of ß-catenin itself result in elevated levels of ß-catenin protein and aberrant TCF-dependent transcriptional activation (6). This dysregulated transcriptional activity alters gene expression. Many of the dysregulated genes regulate cell processes implicated in neoplasia, such as cell proliferation (6, 7).

IFNs are grouped into two major classes with distinct and overlapping functions based on the receptor they activate. Type 1 IFNs, which include IFN-{alpha} and IFN ß, activate the type 1 IFN receptor IFNAR and induce antiviral and antiproliferative activity. Type 2 IFNs, including IFN-{gamma}, activate the type 2 receptor IFNGR and have potent immunomodulatory activities such as activation and recruitment of immune mediators (8). IFNs are secreted by leukocytes (IFN-{alpha}), fibroblasts (IFN-ß), and cells involved in the immune response (IFN-{gamma}), often during insults such as viral infection. Once they bind their receptor, they transduce signals through several pathways such as through the Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) cascade (8). Activation of JAK-STAT signaling by type 1 IFNs up-regulates a series of IFN response genes that possess antiviral and antiproliferative functions such as MxA/Mx1, MxB/Mx2, and IFIT1 (9, 10).

Type 1 IFNs seem to have an anticancer effect because mice harboring mutations that abolish or reduce type 1 IFN activity show increased tumor formation, progression, and invasiveness (11, 12). Whereas IFN-{gamma} also has an antitumor effect, in some tumor types, notably medulloblastoma, IFN-{gamma} activity promotes tumorigenesis (13). In our previous work using gene profiling to identify genes differentially regulated in aggressive fibromatosis, we found an up-regulation of type 1 IFN response genes (14). In this study, we investigated the role of type 1 IFN signaling in aggressive fibromatosis in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human tumor samples. Samples of sporadic aggressive fibromatosis tumors were obtained at the time of surgery. Mutational analysis for ß-catenin on these samples has previously been reported (15). Tumor tissue and surrounding normal fibrous fascial tissue were harvested and processed as soon as possible after surgical excision. Tissues were cryopreserved and stored in liquid nitrogen vapor for future use. Additional material was prepared as primary cell cultures from five of the cases, as previously reported (6, 15).

Mice. Apc/Apc1638N mice harbor a targeted mutation in the Apc gene. Male mice develop an average of 6 gastrointestinal lesions and 45 fibromatoses by the age of 6 months. Female mice develop significantly fewer numbers of fibromatoses than males. These mice develop gastrointestinal tumors at a much lower rate than Min mice (4). Ifnar1–/– mice harbor a targeted deletion of the Ifnar1 gene, which completely abolished type 1 IFN–mediated signaling. These mice also exhibit abnormalities in the proportions of hematopoietic progenitors but are not reported to show anomalies in other organ systems. These mice were crossed using a previously described strategy (16) to generate Apc/Apc1638N;Ifnar1–/– and Apc/Apc1638N;Ifnar1+/+ littermates. In this way, the phenotype of mice expressing Ifnar1 was compared with that of littermates in which Ifnar1 was knocked out. Twelve male and 12 female mice of each genotype were investigated. Mice were sacrificed at 6 months of age, and the number and volume of tumors formed were scored as previously reported (17). Tcf reporter mice harbor a transgene containing three consensus TCF-binding motifs driving lacZ expression under the control of a c-fos minimal promoter (16). Primary cell cultures from normal fibrous tissues and aggressive fibromatosis tumors from these mice were generated as previously reported (18–20) and used to investigate the effects of type 1 IFN signaling on levels of Tcf-dependent transcription.

Cell culture studies. Primary cell cultures from the human and murine aggressive fibromatosis tumors and from fibrous tissue samples as described above were used. To activate ß-catenin–mediated signaling, Wnt3A-conditioned medium or lithium treatment was used. Wnt3A-conditioned medium was obtained from a cell line transfected with a Wnt3A expression vector, the CRL-2647 cell line from the American Type Culture Collection (ATCC). Control medium was obtained from the CRL-2648 control cell line, which is the same cell line but does not express Wnt3A. Conditioned medium was prepared according to the ATCC recommendations on their website and used at a 50% concentration in experiments. Lithium chloride (LiCl; Sigma) was used at a concentration of 50 mmol/L, with NaCl as a control. The ability of Wnt3A and Li to regulate ß-catenin levels was verified by Western blot analysis as previously reported (18), showing a >50% increase in level. To modulate IFN signaling, human and murine IFN-ß monoclonal antibodies (mAb; PBL InterferonSource) were used. These were used at concentrations previously reported to activate and inhibit IFN ß signaling (21, 22), 1,000 units/mL and 0.5 µg/mL, respectively. Changes in levels of phospho-STAT were detected by Western blot analysis (antibody from Abcam) to verify regulation of IFN signaling as previously reported (22).

Proliferation was measured by bromodeoxyuridine (BrdUrd) incorporation over a 12-h period. Cells that incorporated BrdUrd were identified by immunohistochemistry, and positively stained nuclei were counted over 10 high-powered fields running from the periphery to the center of the cell culture plate. Apoptosis was measured 12, 24, and 36 h posttreatment by flow cytometric analysis and staining for the cell death marker Annexin V (Molecular Probes) as previously reported (19). TCF-dependent transcriptional activation was analyzed using cells derived from the Tcf-reporter mice by measuring the level of ß-galactosidase activity as previously described (18).

Immunofluorescence for ß-catenin localization in human and murine cells treated with IFN-ß was done to detect nuclear localization by confocal microscopy. After treatment, cells were fixed in 4% paraformaldehydye and stained for ß-catenin with mouse monoclonal anti–ß-catenin immunoglobulin M (IgM; BD Biosciences) and anti-mouse-fluorescein IgM (Vector Laboratories). Coverslips were mounted onto slides and stained with 4',6-diamidino-2-phenylindole mounting medium (Vector Laboratories). Fluorescence microscopy was done using a Zeiss LSM 510 confocal microscope to assess nuclear and cytoplasmic staining.

RNA analysis and Western blot analysis. Total RNA was isolated using TriZol reagents. Semiquantitative reverse transcription-PCR (RT-PCR) was done as previously described. Expression was compared with ß-actin as a housekeeping control. Although our previous work found a close correlation between semiquantitative PCR and real-time PCR data (23), we used real-time PCR to verify the observed changes. Real-time PCR primers for the genes of interest were obtained from Bioscience Corporation and 28S rRNA was used as a control gene. Validation curves were carried out for the primer sets using RNA from tumor tissue diluted to 1:5, 1:10, 1:50, 1:100, and 1:1,000. The {Delta}{Delta}Ct method (24) was used for the analysis of the data. The threshold cycle, Ct, was determined using the analysis software SDS 2.1 (Applied Biosystems). The expression levels were represented as the fold difference from normal control tissues or cell cultures. Expression profiling for genes implicated in maintaining stem cells in an undifferentiated or differentiated state was done using the human stem cell RT2 profiler PCR array (SuperArray Bioscience) according the manufacturer's instructions, comparing RNA from the side population and non–side population cells. All expression studies were done in triplicate. Northern blot analysis was done as previously reported using oligoprobes generated from the RT-PCR products. Western blot analysis was done to determine protein expression. Nitrocellulose membranes (Amersham) were probed with an antibody against gene of interest. Target protein bands were detected using horseradish peroxidase–conjugated secondary antibody and the enhanced chemiluminescence detection system (Amersham).

Fibroblastic colony-forming unit assay. The femurs of 8- and 12-week-old male Ifnar1–/– and Ifnar1+/+ (littermates) mice were harvested for bone marrow to measure colony-forming units as previously reported (25). Cells (7.5 x 106) were seeded in triplicate in 100-mm cell culture dishes and grown for 8 days in fibroblast-conditioned medium ({alpha}MEM supplemented with 9% horse serum, 9% fetal bovine serum, and 1x antibiotic/antimycotic). Cells were stained with 0.5% crystal violet solution and the number of fibroblastic colony-forming units (CFU-F) formed was counted by microscopy. A CFU-F was defined as a colony containing ≥50 cells. Five mice from each genotype at each age were assayed.

Side population assay. Cells were trypsinized, resuspended in PBS supplemented with 2% fetal bovine serum at a concentration of 1 x 106/mL, and treated with 2.5 µg/mL Hoechst 33342 dye (Sigma) for 90 min at 37°C, alone or in combination with 50 µmol/L verapamil (Sigma). Counterstaining with 1 µg/mL propidium iodide (Molecular Probes) was done and positive (nonviable) cells were excluded from the analysis. To detect the side population, cells were analyzed by using a dual-wavelength analysis (blue, 424–444 nm; red, 675 nm) after excitation with 350-nm UV light (MoFlow, Cytomation). The percent of cells sorting to the side population was determined from the total number of propidium iodide–negative cells (26). Cultures from five tumors were analyzed.

Statistical analysis. Means, SDs, and 95% confidence intervals (95% CI) were calculated for each experiment. The Student t test was used to compare means between different experimental conditions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increased expression of type 1 IFN response genes in human and murine aggressive fibromatosis. The level of expression of various type 1 IFN response genes was compared between human aggressive fibromatosis tumors and adjacent normal fibrous tissues from the same patients in 10 cases. The IFN response genes identified as up-regulated in aggressive fibromatosis in our gene profiling analysis, MxA, MxB, IFIT1, and IFNAR1, were verified to be up-regulated in all of the human tumors analyzed (Fig. 1A ). Using real-time PCR, there was a substantial increase in expression of each gene (all P < 0.01; Fig. 1B). Although Northern blot analysis was not possible in the human samples because of limited quantities of RNA available, it was possible in the murine tumors. Mx1, Mx2, and Ifit1 mRNA expression was also increased significantly in murine aggressive fibromatosis tumors compared with normal fibrous tissues as detected by Northern blot analysis (Fig. 1C). No significant differences were observed in the expression of the IFN ligands IFN-{alpha} and IFN-ß between the tumors and normal tissues.


Figure 1
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  		Figure 1. Elevated expression of IFN response genes MxA/Mx1, MxB/Mx2, IFIT1, and the type 1 IFN receptor IFNAR1 in aggressive fibromatosis. A, representative RT-PCR data from three cases of human aggressive fibromatosis showing that MxA, MxB, IFIT1, and IFNAR1 are expressed at higher levels in tumors compared with normal fibrous tissue from the same patients. B, real-time PCR data from 10 cases of aggressive fibromatosis, showing an increased expression of these genes in tumor tissue compared with normal control tissue in the cases. Data are given as relative expression compared with the normal fibroblast tissues. Bars, 95% CIs. There is a statistically significant difference for instances in which the 95% CIs do not cross the mean of the comparison. C, Northern blot analysis shows that Mx1, Mx2, and Ifit1 are up-regulated in murine aggressive fibromatosis tumors from Apc/Apc1638N mice compared with littermate normal fibrous tissue.

 
ß-Catenin signaling induces expression of type 1 IFN response genes independent of the type 1 IFN receptor. To determine if ß-catenin–mediated signaling could regulate the expression of type 1 IFN signaling targets, the expression of IFN target genes was examined in primary fibroblast cell cultures derived from normal human and murine tissues treated with Wnt3A-conditioned medium or LiCl. Treatment with either Wnt3A or lithium resulted in a 2-fold increase in ß-catenin protein level. Phospho-STAT1 levels showed a 3-fold increase with IFN treatment. Treatment with IFN also caused an expected increase in expression levels of MxA/Mx1 and MxB/Mx2. Using semiquantitative and real-time RT-PCR, we found that both Wnt3A and lithium treatment increased MxA/Mx1 and MxB/Mx2 mRNA expression at least 4-fold (P < 0.01) compared with nontreated controls, whereas there was no difference in expression of IFN-{alpha}, IFN-ß, the type 1 IFN receptor, or Ifnar1, or in phospho-STAT1 levels (Fig. 2A and B ). Primary fibroblast cultures from Ifnar1–/– mice were treated with Wnt3A-conditioned medium or LiCl and the expression of Mx1 and Mx2 was compared with control cultures. Mx1 and Mx2 mRNA levels were significantly up-regulated in Ifnar1–/– cultures treated with Wnt3A-conditioned medium or LiCl by at least 6-fold (P < 0.01) compared with nontreated control (Fig. 2C). The regulation of IFN response genes was also analyzed in cell cultures derived from human aggressive fibromatosis tumors treated with a mAb to IFN-ß ({alpha}-IFN-ß) or immunoglobulin G (IgG). Treatment with the antibody abolished the ability of IFN to regulate phospho-STAT1 levels or expression of IFN response genes. The cultures were treated with either a mAb to IFN-ß ({alpha}-IFN-ß) or IgG. There was no difference in MxA/Mx1 or MxB/Mx2 mRNA levels between treated and control samples. Taken together, these data suggest that ß-catenin regulates IFN response gene expression in a type 1 IFN ligand–independent manner.


Figure 2
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  		Figure 2. ß-Catenin signaling up-regulates expression of MxA/Mx1 and MxB/Mx2 in human and murine mesenchymal cells independent of IFNAR1 signaling. A, treatment of human normal fibrous cultures with IFN, LiCl, or Wnt3A results in increased expression of MxA and MxB compared with untreated controls. Whereas the increased level of expression was not as high as with IFN treatment, activation of ß-catenin–mediated signaling substantially increased expression of the IFN response genes. B, murine normal fibroblast cultures treated with LiCl or Wnt3A increased Mx1 and Mx2 expression compared with untreated controls. Ifnar1 expression was unchanged with LiCl and Wnt3A treatments in human and murine cultures. C, fibroblasts from Ifnar1–/– mice do not show up-regulation of Mx1 and Mx2 expression with IFN treatment but show up-regulation of expression of these genes with LiCl and Wnt3A treatment. As such, ß-catenin signaling is able to regulate expression of type 1 IFN response genes independent of the presence of the type 1 IFN receptor.

 
Deficiency of the type 1 IFN receptor results in a reduction in the number of aggressive fibromatoses in vivo. Because type 1 IFN–regulated genes are expressed in both human and murine aggressive fibromatosis, we investigated the role of type 1 IFN signaling in aggressive fibromatosis in vivo. We crossed Ifnar1–/– (27) mice with Apc/Apc1638N mice. At 6 months of age, the mice were sacrificed and the number and volume of tumors that formed were scored as previously reported (17). Intriguingly, both male and female Apc/Apc1638N;Ifnar1–/– mice developed fewer aggressive fibromatosis tumors compared with their Apc/Apc1638N;Ifnar1+/+ littermate controls (males: P < 0.01, 7.58 versus 13.33; females: P < 0.05, 1.83 versus 3.08; Fig. 3 ). There was no significant difference in tumor size between male and female mice when compared with littermate controls. We also did not observe any differences in the number of gastrointestinal polyps present between Apc/Apc1638N;Ifnar1–/– and Apc/Apc1638N;Ifnar1+/+ mice. Thus, although a deficiency in type 1 IFN signaling does not have an effect on the development of the gastrointestinal tumors that develop in Apc mutant mice, it does result in the development of fewer aggressive fibromatosis tumors.


Figure 3
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  		Figure 3. Type 1 IFN receptor deficiency results in a reduction in the number of aggressive fibromatosis tumors formed in vivo. Columns, mean number of aggressive fibromatosis tumors that formed from male and female mice; bars, 95% CI. There is a statistically significant difference in cases in which the 95% CIs do not cross the mean of the comparison. Female mice are known to develop substantially fewer tumors than male mice.

 
IFN signaling negatively regulates cell proliferation. IFN activity can regulate cell proliferation and apoptosis. Because changes in these cell functions could cause the changes we observed in Apc/Apc1638N;Ifnar1–/– mice, we examined how IFN signaling regulated cell proliferation and apoptosis in normal fibroblasts and in aggressive fibromatosis cell cultures. BrdUrd incorporation increased in cultures from tumors derived from Ifnar1–/– mice compared with littermate controls (Fig. 4A ). In addition, human and murine (Apc/Apc1638N) aggressive fibromatosis tumor and normal fibrous tissue cultures showed a significant decrease in cell proliferation on treatment with IFN-ß compared with controls (Fig. 4B). Primary aggressive fibromatosis cultures from Apc/Apc1638N;Ifnar1–/– mice and littermate controls exhibited no difference in apoptosis rate as measured by Annexin V staining. IFN-ß treatment did not result in any difference in apoptosis between human and murine aggressive fibromatosis tumor and normal cultures at any treatment time points analyzed (Fig. 4C). These data show that type 1 IFN signaling regulates cell proliferation in the opposite manner that would be expected to give the phenotypic findings in Apc1638N;Ifnar1–/– mice, and as such cannot explain the decrease in tumor numbers observed in these mice.


Figure 4
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  		Figure 4. IFN type 1 signaling regulates cell proliferation but does not alter apoptosis rate. A, tumor cells from type 1 receptor–deficient mice (Apc/Apc1638N;Ifnar1–/–) exhibited increased cell proliferation compared with tumor cells from Apc/Apc1638N;Ifnar1+/+ mice. B, IFN-ß treatment of human aggressive fibromatosis tumor and normal fibroblast cultures decreased cell proliferation compared with untreated controls. C, there was no difference in apoptosis rate between tumor cells from type 1 receptor–deficient mice (Apc/Apc1638N;Ifnar1–/–) and Apc/Apc1638N;Ifnar1+/+ mice as measured by percent Annexin V staining. D, IFN-ß treatment of human aggressive fibromatosis tumor cultures did not alter apoptosis rates compared with untreated controls. Treatment with a mAb to IFN-ß did not affect cell proliferation or apoptosis in any group. Columns, mean; bars, 95% CI.

 
Type 1 IFN signaling does not modulate ß-catenin–mediated TCF-dependent transcriptional activation. We next examined the possibility that IFN signaling could positively regulate ß-catenin itself in a type of positive feedback loop. Human and murine primary aggressive fibromatosis tumor and normal fibrous tissue cultures were treated with IFN-ß or with an IFN antibody and observed for changes in total ß-catenin protein level, its localization within the cell, or TCF-dependent transcriptional activity. Treating the cultures with either IFN or a neutralizing antibody did not alter ß-catenin protein level or localization, whereas treatment with Wnt or lithium increased both the amount of ß-catenin present and the amount that localized to the nucleus as determined by confocal microscopy (Fig. 5A and B ). We did not observe any change in ß-catenin protein level of cellular location at any of the concentrations of IFN examined, or with any of the durations of treatment. Cell cultures from Tcf-reporter mice treated with IFN-ß also showed no change in TCF-dependent transcriptional activation, as measured by ß-galactosidase activity (Fig. 5C). Thus, type 1 IFN signaling has little effect on ß-catenin and TCF transcriptional activity in these mesenchymal cell cultures.


Figure 5
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  		Figure 5. Type 1 IFN signaling does not regulate ß-catenin–mediated TCF-dependent transcriptional activation in aggressive fibromatosis. A, confocal microscopy images using an antibody to ß-catenin in human fibroblast cell cultures. Cell cultures are treated with control medium, Wnt-conditioned medium, lithium, IFN, or the IFN neutralizing antibody. Although Wnt and lithium treatment cause ß-catenin to localize to the nucleus, IFN treatment or blockade did not change ß-catenin localization. B, Western blot analysis for ß-catenin from human and murine cell cultures. The first set of lanes are protein lysates from human cell cultures showing increased ß-catenin protein level with Wnt treatment, but not by modulating IFN activity. The second set of lanes are protein lysates showing no difference in ß-catenin protein level between tissues from Ifnar1–/– and Infar1+/+ mice. There is a higher level of ß-catenin protein in tumors compared with normal fibrous tissue. C, TCF-dependent transcriptional activation as measured by relative ß-galactosidase activity in Tcf-reporter mice, showing no change in activity from controls with IFN treatment or blockade. Wnt and lithium treatment does cause an increase in transcriptional activity. Bars, 95% CI.

 
Type 1 IFN signaling regulates the population of mesenchymal precursor cells and the proportion of cells that exclude Hoechst dye. Because aggressive fibromatosis tumors have mesenchymal cell characteristics, we then examined the possibility that IFN signaling could regulate the populations of mesenchymal cells present, perhaps altering the number of mesenchymal precursors present or changing the proportion of cells within the tumors with precursor properties. A decrease in the number of mesenchymal progenitors in Ifnar–/– mice could result in a decrease in the number of cells present that could become tumors, and as such explain our in vivo results. There is growing evidence that solid tumors are maintained by a population of cells with stem cell–like characteristics, and an increase in cells with progenitor characteristics within the tumor (tumor-initiating cells or tumor stem cells) could enhance tumor survival. Furthermore, because Ifnar1–/– mice are known to exhibit abnormalities in the proportions of hematopoietic progenitors of the myeloid lineage (16), they might also exhibit differences in populations of mesenchymal precursors.

To explore this possibility, we compared the numbers of CFU-F which formed between Ifnar1–/– mice and Ifnar1+/+ littermates. There was a significant reduction in the number of CFU-F formed in Ifnar1–/– mice compared with littermate controls at both time points examined (106.8 versus 129.8 CFU-F for 2 months of age and 70.6 versus 96.8 CFU-F for 3 months of age; Fig. 6A ). This suggests that the type 1 IFN receptor plays a role in regulating the numbers of mesenchymal progenitors, and this could be a mechanism by which Ifnar1–/– mice develop fewer aggressive fibromatosis tumors.


Figure 6
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  		Figure 6. Type 1 IFN receptor–deficient mice have decreased numbers of mesenchymal progenitors and IFN treatment increases the proportion of cells excluding Hoechst dyes. A, there are fewer CFU-F in Ifnar1–/– mice compared with Ifnar1+/+ littermate controls at 2 and 4 mo of age. Bars, 95% CIs. B, representative flow cytometric analysis from human aggressive fibromatosis cell cultures showing that treatment with IFN increases the proportion of cells sorting to the side population (side population cells are given in the gated box in the lower left hand quadrant). Treatment with verapamil abolishes the number of cells sorting to this region. X and Y axes, Hoechst red and blue intensity, respectively.

 
To determine if IFN might effect different populations of cells within aggressive fibromatosis tumors themselves, we examined the proportion of cells which would exclude Hoechst dye and sort into a side population with IFN treatment. To confirm that the side population cells do represent cells with stem-like properties, we compared expression between the side population cells and non–side population cells using the human stem cell RT2 profiler PCR array. The side population cells showed up-regulation by at least a 3-fold margin of multiple genes on the array associated with undifferentiated stem cells, including CDH1, ABCG2, CD3D, BTRC, PPARD, and NUMB. We found that the proportion of cells sorting to this population increased with IFN treatment for either 4 or 16 h (0.32 ± 0.09 for controls, 0.75 ± 0.12 for 4-h treatment, and 1.01 ± 0.2 for 16-h treatment; P < 0.01 for both treatment groups compared with control; Fig. 6B). Taken together, these data suggest that type 1 IFN signaling regulates subpopulations of mesenchymal cells, with IFN signaling positively regulating the number of cells with progenitor characteristics.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found that genes associated with type 1 IFN signaling are expressed in human and murine aggressive fibromatoses and are regulated by ß-catenin in an IFN ligand–independent manner. Mice predisposed to aggressive fibromatosis tumor formation develop substantially fewer tumors when the type 1 IFN receptor (Ifnar1–/–) was deficient. Mouse models deficient in type 1 IFN signaling display increased tumor cell proliferation coupled with increased tumor numbers and size. Thus, our results show a surprising role for type 1 IFN signaling, promoting neoplasia.

Because aggressive fibromatosis is caused by mutations resulting in ß-catenin stabilization and TCF-dependent transcriptional activation (7), it is likely that the various IFN responses are regulated directly or indirectly by ß-catenin–mediated TCF-dependent transcription. Our data showing that Wnt3A or lithium treatment of normal fibroblasts caused an increase in expression of the IFN response genes, even in the absence of IFN ligand activity, support this concept. Although we did not examine the expression of all IFN response genes, we did verify the expression of the genes that were identified as up-regulated in aggressive fibromatosis in previous gene profiling work from our group and others (14, 28). The specific genes we found to be up-regulated are activated primarily by type 1 IFN signaling. Colonic tumors, also exhibiting ß-catenin–mediated TCF-dependent transcription, show increased expression of another set of IFN-related genes, members of the IFITM family (29), raising the possibility that up-regulation of IFN response genes is a common occurrence in ß-catenin–driven neoplasia.

MxA/Mx1 and MxB/Mx2 are activated by type 1 IFN signaling and play a role in mediating antiproliferative effects in cells (30). Indeed, we found that type 1 IFN activation has an antiproliferative effect on aggressive fibromatosis and normal fibroblasts. This correlates well with the antiproliferative activities of IFNs that have been observed in several tumor types and cell types (31, 32). Intriguingly, however, our observation that substantially fewer tumors develop when the type 1 IFN receptor (Ifnar1–/–) was deficient in mice cannot be explained by changes in cell proliferation. One possible explanation for this is that IFN signaling regulates ß-catenin, perhaps acting in a positive feedback loop. In support of this possibility, the expression of the IFN target gene, STAT3, in colorectal tumors resulted in prolonged accumulation of nuclear ß-catenin (33). However, we did not observe any change in ß-catenin with IFN stimulation or blockade in aggressive fibromatosis or fibroblast cultures. This finding is in agreement with data from neural origin cell lines, in which IFN-ß had no effect on ß-catenin (34). Whereas IFN commonly regulates cell proliferation, it can also modulate cell apoptosis in some situations (35, 36), and this is another possible explanation for our in vivo findings. However, treatment of human and murine aggressive fibromatosis tumor cultures with IFN-ß for various periods of time did not result in any change in the rate of cellular apoptosis as measured by Annexin V staining, an early marker of apoptosis. Thus, the regulation of proliferation, apoptosis, or ß-catenin levels by IFN-ß could not explain our in vivo findings.

Another possible explanation for our observation that Ifnar1–/– mice develop smaller numbers of tumors is that Ifnar1 deficiency alters the population of mesenchymal precursors present. Decreasing the number of mesenchymal precursors might decrease the number of aggressive fibromatosis tumors that can form. Because IFNs play a role in regulating hematopoietic progenitor development (11, 27, 37, 38) and cell self-renewal (39), they could play a similar role in mesenchymal cells. Our finding that Ifnar1 deficiency increases the number of CFU-F present supports this hypothesis. It is important to note, however, that CFU-F is a gross measure of mesenchymal progenitors, and changes can also be reflective of changes in the population of other progenitor cell types found in the bone marrow.

Solid tumors are composed of a heterogeneous population of cells with different proliferative capacities, of which only a minority have the ability to initiate tumor formation in immunodeficient mice. This finding suggests that there is a small subpopulation of cells within tumors that act as tumor-initiating cells or cancer stem cells (40, 41). These tumor-initiating cells may be resistant to chemotherapies, and they may be responsible for resistance to treatment. Whereas IFN decreases cell proliferation in the entire population of aggressive fibromatosis cells, it is possible that tumor-initiating cells are relatively resistant to IFN and might not be affected by treatment. In this case, the proportion of tumor-initiating cells present would increase with IFN treatment and would continue to drive neoplasia by selectively maintaining tumor cells with self-renewal capacity. One property of tumor-initiating cells is the ability to efflux chemotherapeutic drugs and certain dyes such as Hoechst 33342 (42, 43). Cells that efflux this dye will fall to the "side" of the majority during flow cytometry; hence, they are commonly referred to as side population cells (26). They are enriched with progenitor cells in a variety of tissue types and with tumor-initiating cells in a variety of cancers (33, 44–46). Our finding that IFN treatment increases the percentage of cells sorting to the side population supports this possibility in aggressive fibromatosis. We certainly have not shown that these side population cells are indeed tumor-initiating cells in aggressive fibromatosis. Furthermore, the side population cells may instead represent a chemoresistant cell population because the same mechanism that excludes dyes could exclude chemotherapeutic agents. Despite these limitations, we found a reproducible change in the proportion of cells sorting to the side population with IFN treatment, showing that, at the very least, IFN treatment changes the proportion of various cell populations present in aggressive fibromatosis.

We identified a novel function for type 1 IFN signaling in mesenchymal neoplasia, modulating the numbers of progenitor cells and tumor-initiating cells. Such a function can explain the disappointing results of some clinical trials of IFN despite suggestive in vitro data. Treatment of aggressive fibromatosis tumors with IFN has been reported in isolated cases (47–50) with variable results. Our data show disparate roles for IFN signaling in aggressive fibromatosis, a finding which explains the variable clinical results in these case reports. Because IFN treatment can have undesirable effects in this tumor type, it is a less than optimal therapeutic approach.


   Acknowledgments
 
Grant support: National Cancer Institute of Canada, Canada Research Chairs Program (B.A. Alman), and the Ontario Graduate Scholarship in Science and Technology and University of Toronto Fellowship Program at the University of Toronto (S. Tjandra).

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.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 2/21/07. Revised 5/ 2/07. Accepted 5/30/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Correction: IFN Signaling in Aggressive Fibromatosis
Cancer Res., February 1, 2008; 68(3): 956 - 956.
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