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Molecular Endocrinology, doi:10.1210/me.2006-0223
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Molecular Endocrinology 20 (11): 2965-2975
Copyright © 2006 by The Endocrine Society

Targeting N-Cadherin through Fibroblast Growth Factor Receptor-4: Distinct Pathogenetic and Therapeutic Implications

Shereen Ezzat, Lei Zheng, Daniel Winer and Sylvia L. Asa

Departments of Medicine (S.E., L.Z.) and Laboratory Medicine and Pathobiology (D.W., S.L.A.), University of Toronto; The Freeman Centre for Endocrine Oncology (S.E., L.Z., D.W., S.L.A.), Mount Sinai Hospital; and Ontario Cancer Institute (S.E., L.Z., D.W., S.L.A.), University Health Network, Toronto, Ontario, Canada M5G 1X5

Address all correspondence and requests for reprints to: Dr. S. Ezzat, Ontario Cancer Institute, 610 University Avenue, 8-327, Toronto, Ontario, Canada M5G 2M9. E-mail: shereen.ezzat{at}utoronto.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several molecular aberrations have been implicated in the pathogenesis of pituitary tumors, but few have proven thus far to be of therapeutic value. Pituitary tumor-derived fibroblast growth factor receptor-4 (ptd-FGFR4) is an alternatively transcribed cytoplasmic isoform lacking most of the extracellular domain. This oncogene recapitulates the morphological features of human pituitary tumors in transgenic mice. To investigate the therapeutic potential of targeting ptd-FGFR4, we examined the impact of FGFR4 tyrosine kinase inhibition in xenografted mice. GH4 pituitary cells expressing ptd-FGFR4 develop into invasive tumors. Systemic treatment of mice bearing ptd-FGFR4 tumors with the FGFR-selective inhibitor PD173074 resulted in recovery of membranous N-cadherin staining and a significant reduction in tumor volume with less invasive growth behavior. Mutation of tyrosine Y754F in ptd-FGFR4 abrogated the effect of PD173074-mediated inhibition. The pivotal role of N-cadherin as a mediator of this pituitary cell growth was demonstrated by small interfering RNA mediated down-regulation, which promoted invasive growth in xenografted mice. To validate this model in primary human pituitary tumors, we examined the expression of ptd-FGFR4, N-cadherin, and clinical behavior. Loss of membranous N-cadherin correlated with cytoplasmic FGFR4 expression and with tumor invasiveness in surgically resected human pituitary tumors. Primary human pituitary tumor cells treated with PD173074 showed restoration of N-cadherin to the membrane with dephosphorylation of retinoblastoma protein. These data highlight the pathogenetic significance of N-cadherin misexpression and emphasize the importance of FGFR partnership in mediating its functions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PITUITARY TUMORS REPRESENT approximately 10% of all surgically excised intracranial tumors. An example of a nonmetastasizing neoplasm, they typically cause morbidity through invasive growth into surrounding brain and bony structures. Pituitary tumorigenesis rarely involves mutations of classical oncogenes or tumor suppressor genes (see Refs. 1 and 2) for review). Instead, evidence suggests that growth signals implicated in pituitary development may be relevant to the tumorigenic processes in this gland, particularly involving protein members of the bone morphogenic protein, Wnt, and fibroblast growth factor (FGF) families (3, 4).

FGF signaling is critical in pituitary development (see Ref. 5 for review). Deletion of FGF10 or its receptor, the FGF receptor (FGFR)2 IIIb isoform, leads to failure of primordial pituitary development (6). Midgestational expression of a soluble dominant-negative FGFR results in severe pituitary dysgenesis (7). FGF ligands are overexpressed in pituitary tumors. FGF-2, originally described in bovine pituitary folliculostellate cells, regulates multiple pituitary hormones (8) and is overexpressed by human pituitary adenomas (9). Estrogen administration in rats results in pituitary tumorigenesis accompanied by increased FGF-2 expression (10). Elevated circulating FGF-like immunoreactivity is found in patients with multiple endocrine neoplasia-1 and associated pituitary tumors (11) and in patients with sporadic pituitary adenomas (9). The human endogenous FGF antisense gene is expressed in the normal pituitary where it inhibits pituitary cell proliferation and is reduced in pituitary tumors (12).

We identified altered FGFR4 expression in pituitary tumors (13) due to expression of an N-terminally deleted isoform, pituitary tumor-derived FGFR4 (ptd-FGFR4) (14) generated by alternative transcription initiation utilizing a cryptic promoter (15, 16). Wild-type FGFR4 is a 110-kDa membrane-anchored protein in contrast to the cytoplasmic residence of oncogenic ptd-FGFR4 (14). The invasive tumorigenic potential of ptd-FGFR4, but not wild-type FGFR4, was demonstrated by targeted pituitary expression in transgenic mice (14). The basis for the contrasting functional differences between FGFR4 isoforms appears to be related to differences in their ability to associate with a number of cell adhesion molecules including neural cell adhesion molecule (NCAM) and engage N-cadherin (17). In particular, ptd-FGFR4 does not associate with NCAM and interferes with N-cadherin signaling to impede cell adhesion (17). In this report, we identify a distinct contribution from N-cadherin in mediating FGFR4 action and we propose a functional FGFR4-N-cadherin complex as an important therapeutic target for interrupting neoplastic cell growth and invasiveness.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tyrosine Kinase Inhibition Interrupts Growth of ptd-FGFR4 But Not Wild-Type FGFR4-Expressing Tumors
To examine the impact of FGFR4 kinase inhibition on pituitary tumor growth, GH4 pituitary cells expressing ptd-FGFR4, wild-type FGFR4, or their empty vector control were inoculated into severe combined immunodeficient (SCID) mice until distinct tumor growth was observed (Fig. 1Go). At that point, all animal groups were administered the pharmacological FGFR inhibitor PD173074 ip. As seen in Fig. 1Go, treatment with this inhibitor resulted in no appreciable reduction in tumor size in empty vector control cells (Fig. 1AGo). A modest degree of inhibition (934 ± 265 vs. 1249 ± 159 mm3) was noted in wild-type FGFR4-tranfected cells (Fig. 1BGo) after PD173074 treatment. In contrast, PD173074 treatment resulted in a more pronounced reduction (740 ± 58 vs. 1419 ± 370 mm3; P < 0.05) in tumors expressing ptd-FGFR4 (Fig. 1CGo). That this effect was related to FGFR4 inhibition was demonstrated by mutation of tyrosine Y754 in the ATP-binding site of FGFR4. Mutation of Y754 in ptd-FGFR4 resulted in tumors that failed to demonstrate an appreciable response to PD173074 treatment (Fig. 1DGo).


Figure 1
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  		Fig. 1. Effect of Pharmacological Inhibition of FGFR4 on Pituitary Tumor Cell Growth in Xenografted Mice

Two and half million GH4 cells stably transfected with empty vector (A), wild-type FGFR4 receptor (B), ptd-FGFR4 (C), or mutant ptd-FGFR4 (D) were injected into the flank of xenografted SCID mice. Pharmacological treatment with vehicle alone or with the FGFR4 inhibitor PD173074 (50 mg/kg) was commenced 5 d after cell inoculation as detailed in Materials and Methods. Shown are the tumor volumes expressed as means ± SE of measurements obtained from three independent experiments, each with five mice in each group. Statistically significant differences in tumor volume (P < 0.05) were noted after 13 d of PD173074 treatment only in tumors expressing ptd-FGFR4 (shown with *).

 
FGFR4 Kinase Inhibition Results in Distinct Effects on Invasive Growth in Vivo
To determine the mechanism underlying the different responses to tyrosine kinase inhibition of FGFR4 vs. ptd-FGFR4 in vivo, we compared the effect of PD173074 treatment on the histologic features of tumors developed by each (Fig. 2Go). As reported previously (17), the pattern of growth of cells expressing ptd-FGFR4 is more invasive than that of control cells and cells expressing FGFR4. In contrast to the strictly sc growth of tumor development by empty vector-transfected cells (Fig. 2AGo), treatment of wild-type or FGFR4-transfected cells with PD173074 resulted in unexpected invasive growth into muscle (Fig. 2EGo) compared with noninvasive untreated cells (Fig. 2BGo). PD173074 treatment of tumors expressing ptd-FGFR4 resulted in restrained growth, which was mostly contained within the sc tissue, and there was no evidence of invasion into muscle (Fig. 2FGo), in contrast with the invasive properties of untreated cells expressing ptd-FGFR4 (Fig. 2CGo).


Figure 2
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  		Fig. 2. Distinct Effects of FGFR4 Inhibition on Invasion by Pituitary Tumor Cells in Vivo

GH4 cells stably expressing ptd-FGFR4, FGFR4, or their empty-vector control cells grown in xenografted mice (as described in the legend to Fig. 1Go) were examined histologically after vehicle (upper panels) or treatment with the PD173074 FGFR inhibitor (lower panels). Control cells (left) or those transfected with FGFR4 (middle) demonstrate noninfiltrative tumor growth (Tumor) that is limited by muscle (M) and does not infiltrate dermis (Dermis). In contrast, ptd-FGFR4-expressing tumors show invasive growth through muscle (arrows). The FGFR inhibitor PD173074 (lower panels) permits infiltrative growth of cells expressing FGFR4 (arrows). In contrast, treatment with the FGFR inhibitor arrests the infiltrative behavior of cells expressing ptd-FGFR4. Shown are representative tumor samples obtained from three independent experiments, each with five mice in each group as indicated in the legend to Fig. 1Go.

 
N-Cadherin Modulates FGFR4 Action
To determine the functional basis underlying the differential effects of PD173074 on invasive growth patterns, we examined the impact of this treatment on N-cadherin. N-Cadherin was localized by immunocytochemistry in GH4 cells stably expressing ptd-FGFR4, FGFR4, or empty vector and grown in xenografted mice with or without PD173074 treatment (Fig. 3Go). Control empty vector-transfected cells (Fig. 3AGo) demonstrate mainly membranous N-cadherin staining, which is even more pronounced in FGFR4-expressing tumors (Fig. 3BGo), as was shown previously (17). In contrast, ptd-FGFR4-expressing tumors show diminished membranous N-cadherin staining (Fig. 3CGo). In the presence of the FGFR inhibitor PD173074, empty vector control cells (pcDNA3.1; Fig. 3DGo) demonstrate loss of membrane N-cadherin staining, consistent with interruption of the effect of endogenous FGFR4. This effect was even more pronounced in cells overexpressing FGFR4 (Fig. 3EGo). In contrast, treatment of ptd-FGFR4 tumors with PD173074 results in recruitment of N-cadherin back to the cell membrane (Fig. 3FGo).


Figure 3
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  		Fig. 3. N-Cadherin as a Target of FGFR4 Action

GH4 cells stably expressing ptd-FGFR4, FGFR4, or their empty-vector control cells grown in xenografted mice (as described in the legend to Fig. 1Go) were examined by immunocytochemistry for N-cadherin. Control empty vector-transfected cells (left) demonstrate mainly membranous N-cadherin staining, which is more pronounced in FGFR4-expressing tumors (middle panel). In contrast, ptd-FGFR4-expressing tumors show diminished membranous N-cadherin staining. In the presence of the FGFR inhibitor PD173074 (lower panels), empty vector control (pcDNA3.1) and FGFR4 cells demonstrate loss of N-cadherin staining. In contrast, treatment of ptd-FGFR4 tumors with PD173074 results in N-cadherin recruitment to the cell membrane. The results shown are representative of findings obtained in three independent experiments, each with five animals in each group.

 
To determine the functional basis underlying the reciprocal effects of PD173074 on N-cadherin responses, we examined the effect of small interfering RNA (siRNA)-mediated down-regulation of this adhesion molecule. Stable GH4 clones expressing N-cadherin siRNA exhibited down-regulation of N-cadherin (Fig. 4AGo) and its putative downstream effector ß-catenin (Fig. 4AGo). Fluorescence-activated cell sorting analysis of N-cadherin knocked-down cells showed accelerated progression into S-phase (15.2 ± 1.0% vs. 8.2 ± 0.2%; n =9; P = 0.03) compared with control cells.


Figure 4
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  		Fig. 4. N-Cadherin Down-Regulation Promotes Pituitary Tumor Invasiveness in Xenografted Mice

A, GH4 cells stably expressing siRNA targeting the N-cadherin sequence or a scrambled sequence (Control) were examined by Western blotting for N-cadherin, its downstream target ß-catenin, or actin (as indicated). Each lane represents an independent stably transfected clone as detailed in Materials and Methods. B, Cells expressing N-cadherin siRNA and control scrambled sequence were xenografted into SCID mice as described in the legend to Fig. 1Go. Volumetric assessments of mean ± SE tumor size obtained from two independent experiments, each with three to five animals in each group, are shown. Statistically significant change (P < 0.003) in tumor volume is noted at d 16–21 after cell inoculation. C and D, Light microscopy demonstrates invasive tumor growth (arrows) in N-cadherin siRNA-transfected cells (D) compared with control cells (C). The features are similar to those of cells expressing ptd-FGFR4 compared with controls (see Fig. 2Go).

 
Xenografts of control cells carrying scrambled sequences demonstrated noninvasive growth (Fig. 4Go), as demonstrated earlier. In contrast, xenografts of N-cadherin down-regulated GH4 cells demonstrated accelerated growth (Fig. 4BGo) with histological features of invasion similar to those noted in ptd-FGFR4-expressing cells (Fig. 4Go, C and D).

N-Cadherin Expression Predicts Invasive Human Pituitary Tumorous Growth
To determine whether N-cadherin represents a signaling target of ptd-FGFR4 action in human tumorigenesis, we examined the relationship between these factors in primary human pituitary tumors. Seventy-eight surgical human pituitaries, including seventy-three adenomas and five normal glands (Table 1Go), were collected and stained by immunohistochemistry for FGFR4, N-cadherin, and ß-catenin. Complete membrane, cytoplasmic, or nuclear staining was then scored for each case (Table 2Go). Cytoplasmic FGFR4, the hallmark of ptd-FGFR4 (18), was expressed in a variety of pituitary adenomas but primarily in gonadotroph and null cell tumors (Table 2Go). The presence of cytoplasmic FGFR4 correlated with increased tendency for tumor invasiveness (24 of 29 or 83% of invasive adenomas expressed moderate to strong cytoplasmic FGFR4 compared with 23 of 44 or 52% noninvasive adenomas, P = 0.012).


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  		Table 1. Clinical Characteristics of Normal Adenohypophysis and 73 Pituitary Adenomas

 

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  		Table 2. Expression of Moderate to Strong1 FGFR4, N-Cadherin, and ß-Catenin in Normal Pituitary and Major Tumor Types

 
Membranous staining of N-cadherin was seen around some, but not all, cells in the normal pituitary. In particular, normal lactotrophs demonstrated minimal, if any, N-cadherin reactivity, and somatotrophs were rarely positive (data not shown). This finding was reflected by a marked decrease in membrane expression of N-cadherin in somatotroph (P = 0.0004) and lactotroph (P = 0.0085) adenomas, compared with all other types of adenomas. In all groups and even after adjusting for this cell-specific variation, cytoplasmic FGFR4 expression in a variety of pituitary tumors was associated with altered expression of N-cadherin. Strong ptd-FGFR4 expression was associated with increased cytoplasmic N-cadherin staining and focal loss of N-cadherin membranous staining: 24 of 48 tumors (50%) with intense ptd-FGFR4 reactivity demonstrated cytoplasmic dislocation of N-cadherin staining compared with only three of 25 tumors (12%) that were negative or weakly positive for ptd-FGFR4 (P = 0.0019). Representative examples are shown in Fig. 5Go. Because normal and neoplastic human prolactin and GH-producing pituitary cells expressed little N-cadherin, prolactinomas and somatotroph adenomas were excluded from analysis. Reduced membranous N-cadherin in gonadotroph and null cell tumors correlated with tumor invasiveness: 17 of 20 noninvasive tumors (85%) showed moderate to strong membranous N-cadherin compared with nine of 20 invasive tumors (45%) with this pattern (P = 0.019). Despite this pathological loss of N-cadherin, however, we found no evidence of nuclear ß-catenin accumulation.


Figure 5
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  		Fig. 5. ptd-FGFR4 Expression Correlates with N-Cadherin Misexpression in Primary Human Pituitary Tumors

A, A pituitary gonadotroph adenoma that had no detectable ptd-FGFR4 expression shows predominantly well-delineated N-cadherin membrane staining and little cytoplasmic N-cadherin expression (original magnification, x400). B, A gonadotroph adenoma that had strong ptd-FGFR4 expression shows patchy membranous N-cadherin staining and areas of increased cytoplasmic expression (original magnification, x400).

 
FGFR4 Tyrosine Kinase Inhibition Restores N-Cadherin in Primary Human Pituitary Tumors
To determine whether the actions of FGFR inhibition on N-cadherin may be of potential therapeutic significance, we treated dispersed cells from eight primary human pituitary adenomas that express ptd-FGFR4 with PD173074. After 2 d of drug exposure, tumor cells demonstrated a marked increase in N-cadherin staining, which was membranous in localization (Fig. 6AGo). This was not evident in vehicle-treated control cells. The change in staining pattern induced by PD173074 was associated with clustering of cells in vitro, a feature not seen in vehicle-treated cells of the same tumors under the same conditions, suggesting that the enhanced expression of membrane-localized N-cadherin was associated with increased cell-cell adhesion. Because these primary tumor cells do not replicate in vitro, direct measures of cell proliferation including MIB-1 index was too low for measurement. PD173074-treated cells, however, showed dephosphorylation of the retinoblastoma protein, suggestive of reduced cell cycle entry (Fig. 6BGo) and providing ancillary evidence for potential antitumor activity.


Figure 6
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  		Fig. 6. FGFR4 Tyrosine Kinase Inhibition Restores N-Cadherin in Primary Human Pituitary Tumor Cells

A, Two separate examples of primary human pituitary tumors were dispersed and grown in serum-free defined media as detailed in Materials and Methods. Top, After 48 h of exposure to PD173074 (right), tumor cells demonstrate an obvious increase in N-cadherin staining, which is mainly membranous in localization. This effect was not evident in vehicle-treated control cells (left). Bottom, Another tumor shows accumulation of membrane N-cadherin staining associated with formation of cell clusters, compared with the dispersed pattern of cells exposed to vehicle control (left). The changes are more pronounced after incubation in 5 µM PD173074 than after incubation in 100 nM PD173074. B, PD173074 treatment of tumor cells also resulted in dephosphorylation of retinoblastoma compared with control cells, consistent with reduced cell cycle entry.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Progression to neoplasia involves changes in cell capabilities to adhere with neighboring cells and with their extracellular matrix environment (19). Correlative studies in human carcinomas and functional studies in mouse models have demonstrated that loss of and/or impaired cell adhesion are important determinants in epithelial neoplasia (20). In this study, we demonstrate that an important function of the wild-type FGFR4 tyrosine kinase is to orchestrate signaling events that are critical for sustained normal cell adhesiveness. Interruption of wild-type FGFR4 interaction with NCAM and N-cadherin by ptd-FGFR4 impairs pituitary cell adhesion and accelerates invasive growth (17). In contrast, we show in this report that pharmacological targeting of this kinase in cells expressing the tumor-derived ptd-FGFR4 isoform results in selective inhibition of invasive growth.

Pituitary tumors are usually classified based on their cell of origin and degree of invasiveness into surrounding structures (21). Hormonally active forms may be amenable to treatment with hypophysiotropic hormone analogs; for example, lactotroph adenomas are treated with dopamine agonists to suppress prolactin secretion, and this results in tumor shrinkage (21). GH-producing adenomas respond to somatostatin with suppression of hormone hypersecretion, but this is not usually associated with control of tumor size or invasiveness (22). Thus far, there are no effective forms of medical therapy for the hormonally inactive inoperable tumors. This is of major clinical significance, because these comprise nearly half of all human pituitary tumors (2, 21). Despite the well-known abundance of receptor tyrosine kinase (RTK) expression in human pituitary tumors (23), little is known about the potential utility of pharmacological RTK inhibitors in the management of patients with invasive tumors. In one previous study, the nonselective RTK inhibitor genestein was shown to reduce thymidine uptake into primary human pituitary tumor cells (24). The growth-stimulatory effect of conditioned medium from rodent GH3 pituitary cells was also inhibited by genestein, providing additional evidence that pituitary tumors are a rich source of growth factors and their receptors (23).

The current study demonstrates that selective targeting of the FGF receptor with PD173074, a synthetic compound of the pyrido[2,3-d]pyrimidine class, can retard pituitary tumor progression. The crystal structure of PD173074 has confirmed this compound to be in complex within the ATP-binding pocket of the tyrosine kinase domain of FGFRs (25). Systemic administration of this compound effectively blocks FGF-induced angiogenesis (25) and neurotrophic actions (26). In addition to its effects on the various FGFRs and VEGF, PD173074 efficiently inhibits FGFR-4 in human breast (27) and thyroid carcinoma epithelial cells (28). The FGFR specificity of the growth inhibition mediated by this compound was further demonstrated in our analysis by mutational studies in which disruption of the putative Y754 in the ATP-binding pocket in the FGFR4 kinase resulted in loss of response to PD173074.

Our current findings underscore the significance of N-cadherin as an adhesive molecule that is pivotal in modulating the response to FGFR4 signaling interruption. In particular, we demonstrate that N-cadherin membranous residence is a critical component of cell adhesion regulation. Inhibition of wild-type FGFR4 resulted in loss of N-cadherin staining, a feature that was associated with relative insensitivity to tumor inhibition by PD173074. In contrast, inhibition of kinase activity of ptd-FGFR4 resulted in restitution of membrane localization of N-cadherin and successful and selective arrest of invasive growth. That N-cadherin was pivotal in determining these divergent responses was demonstrated by siRNA down-regulation of N-cadherin. Loss of N-cadherin resulted in dramatic loss of its coupled protein ß-catenin and accelerated tumor progression in xenografts. Uncoupling of cadherins from the cytoskeleton is becoming appreciated as an essential aspect of neuroendocrine cell migration.

N-Cadherin-mediated neurite outgrowth depends on inactivation of N-cadherin-mediated adhesions at pathway boundaries (29). Here we show that tumor-derived ptd-FGFR4-mediated tumor growth is closely associated with displacement of N-cadherin residence from the cell membrane. The clinical significance of our findings was further validated in a panel of primary human pituitary tumor samples, in which loss of membranous N-cadherin was seen in some tumors showing increased tumor invasiveness.

N-Cadherin is typically considered as a mediator of the epithelial-to-mesenchymal neoplastic transition (20). However, N-cadherin is a member of the classical cadherin family of cell-cell adhesion molecules of particular importance in normal neuroendocrine cell function (30). It is the predominant member expressed in the nervous system and interacts directly with ß-catenin and in turn with {alpha}-catenin as the intermediate link to the actin cytoskeleton. This cytoskeleton linkage is critical in stabilizing cadherin clusters at the cell surface and strengthening cell-cell adhesion. Estradiol, a recognized promoter of rodent pituitary tumor growth, reduces membranous N-cadherin expression to barely detectable levels, whereas antiestrogen treatment reverses this effect (31). Consistent with these findings, forced ptd-FGFR4-expression results in diminished and ectopic cytoplasmic expression of N-cadherin (17), a relationship we also show here to hold true in primary human pituitary tumors. Despite N-cadherin cytoplasmic misexpression, however, we and others (18) found only reduced ß-catenin expression in pituitary tumors. The lack of nuclear ß-catenin translocation is likely the result of intact APC-mediated degradation (32) and is biologically consistent with the nonmetastasizing nature of pituitary tumors (2).

Another feature contrasting our current findings and other forms of epithelial neoplasia can be seen in breast carcinoma where N-cadherin has been shown to promote metastatic growth (33). The mechanism for the latter finding has been suggested to relate to the ability of N-cadherin to protect FGFR1 from ligand-induced down-regulation (33). The contrasting actions of N-cadherin in breast and pituitary neoplasia support a model wherein the functions of this adhesive molecule are dictated by the cellular context and/or the FGFR partner with which it interacts.

In summary, we have demonstrated contrasting patterns in the effects of FGFR4 kinase inhibition on tumor progression. Interruption of the wild-type receptor resulted in minimal if any impact on invasive growth. In contrast, interruption of the tumor-derived ptd-FGFR4 receptor isoform significantly abrogated tumor invasiveness. A proposed model for this multiprotein complex and its role in cell adhesion and tumor invasion is provided in Fig. 7Go. Another previously described alteration in pituitary tumors is expression of the polysialated form of NCAM that correlates with tumor growth and invasiveness (34); polysialation results in steric inhibition of membrane-membrane apposition and cell adhesiveness and therefore probably disrupts the multiprotein complex as does ptd-FGFR4. Our data emphasize the critical nature of membranous N-cadherin in determining FGFR4 action. The extent to which other adhesion molecules play a role in governing FGFR action will prove to be critical in refining pharmacotherapuetic approaches.


Figure 7
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  		Fig. 7. Proposed Model of FGFR4/N-Cadherin Interaction

Intact NCAM physical association with FGFR4 maintains N-cadherin at the membrane that serves to link ß-catenin to the cell surface. This interaction is necessary to maintain normal neuroendocrine cell architecture and stromal interaction. Tumorigenic signals such as introduced by ptd-FGFR4 or possibly by polysialated forms of NCAM (PSA-NCAM) disrupt N-cadherin residence at the cell membrane with consequent loss of catenin-actin mediated cytoskeletal integrity. Alternatively, pharmacological inhibition of wild-type FGFR4 can disrupt normal interaction, resulting in N-cadherin displacement and impaired cell-matrix adhesiveness.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Lines and Cultures
Rat pituitary GH4 cells were propagated in DMEM (Life Technologies, Inc., Burlington, Ontario, Canada) with high glucose, 10% fetal bovine serum, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin.

Plasmid Constructions and Transfections
Plasmids containing the coding regions of FGFR4 or the tumor derived ptd-FGFR4 in tagged pcDNA3.1 were generated and stably transfected into GH4 cells as previously described (14). Mutation of tyrosine 754 in the cytoplasmic ATP binding pocket of FGFR4 (Y754F) was introduced by site-directed mutagenesis according to the manufacturer’s protocol (Clontech Laboratories, Palo Alto, CA). The mutation was confirmed by DNA sequencing.

N-Cadherin Silencing
The hairpin siRNAs corresponding to the rat N-cadherin were designed using Ambion software for cloning into the pSilencer 2.1-U6 neo vector (Ambion Inc., Austin, TX). The N-cadherin target sequence was as follows: 5'- GACTGGATTTCCTGAAGAT-3' (corresponding to position 97) and antisense strand 5'-ATCTTCAGGAAATCCAGTC-3'. The double-stranded oligonucleotide templates containing a 21-mer siRNA sequence were synthesized as follows. Top strand oligo template:

5'-GATCCGACTGGATTTCCTGAAGATTTCAAGAGAATCTTCAGGAAATCCAGTCTTTTTTGGAAA-3'; and bottom strand oligo template: 5'-AGCTTTTCCAAAAAAGACTGGATTTCCTGAAGATTCTCTTGAATCTTCAGGAAATCCAGTCG-3'.

The expression vector pSilencer 2.1-U6 neo-siN-cadherin was constructed according to the manufacturer’s (Ambion) instructions. Ligation of the vector and oligo was carried out in a reaction consisting of 1 µl of vector, 1 µl of oligo, 2 µl of 10x T4 DNA ligase buffer, and 3 µl of T4 DNA ligase for 5 min at room temperature. An aliquot of 3 µl of ligation reaction was then used to transform DH5{alpha} Escherichia coli. Clones containing the oligonucleotide inserts were identified by PstI restriction digestion and verified by DNA sequencing. Plasmid DNA for transfection was prepared using QIApre Spin Maxiprep Kit (QIAGEN, Valencia, CA).

Western Blotting
Cells were lysed in lysis buffer (0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, and 1x PBS) containing proteinase inhibitors [100 µg/ml phenylmethylsulfonyl fluoride, 13.8 µg/ml aprotinin (Sigma, St. Louis, MO), and 1 mM sodium orthovanadate (Sigma)]. Total cell lysates were incubated on ice for 30 min, followed by microcentrifugation at 10,000 x g for 10 min at 4 C. Protein concentrations of the supernatants were determined by the Bio-Rad (Hercules, CA) method. Equal amounts of protein (10 µg) were mixed with 2x sodium dodecyl sulfate sample buffer, boiled for 5 min and separated by 15% SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (0.45 µm; Millipore, Bedford, MA). Blots were incubated with polyclonal affinity-purified rabbit antiserum against the carboxy terminus of FGFR4 (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoblotting was performed using anti-FGFR4 (Santa Cruz Biotechnology) or a monoclonal antibody to the V5-tag (Invitrogen, Burlington, Ontario, Canada). Antisera for pERK (1:1000), ERK (1:1000), N-cadherin (1:2500), and ß-catenin (1:500) were purchased from Pharmingen (Mississauga, Ontario, Canada). Loading was monitored by detection of actin (1:500; Sigma) as a control. Nonspecific binding was blocked with 5% nonfat milk in 1x TBST (Tris-buffered saline with 0.1% Tween 20). After washing for three times for 10 min in 1x TBST, blots were exposed to the secondary antibody (antimouse or -rabbit IgG-HRP; Santa Cruz Biotechnology) at a dilution of 1:2000 and were visualized using ECL chemiluminescence detection system (Amersham, Piscataway, NJ).

In Vivo Tumor Growth Assay
Female immunodeficient SCID mice 6–7 wk of age were purchased from the animal facility of the Ontario Cancer Institute (Toronto, Canada) and maintained under specific pathogen-free conditions. Five x 106 GH4 cells (transfected as indicated) were injected sc into the flank. Following institutional guidelines, experiments ended when tumor volumes exceeded 100 mm3 in volume, which typically occurred 10 d after implantation. Tumor dimensions were measured using a vernier caliper (Fisher Scientific, Ltd., Ontario, Canada). Tumor volumes were calculated as (length x width x depth)/2. Complete autopsies were done at the time of the mice were killed; tissues were fixed in formalin and embedded in paraffin for histological and immunohistochemical analysis. Portions of tumors were snap-frozen in liquid nitrogen and stored at –70 C.

Pharmacological FGFR Inhibition
For in vitro studies, GH4 cells were seeded at a density of 8000 cells per well in 96-well plates. Cells were treated at different concentrations (0–50 µmol/liter) with the FGFR tyrosine kinase inhibitor PD173074 (Pfizer, Groton, CT), which selectively inhibits FGFR tyrosine kinase activity and autophosphorylation (27) for 72 h. For in vivo studies, each mouse was injected ip with PD173074 at a concentration of 1 mg/mouse (50 mg/kg) suspended in DMSO. The latter served as the vehicle control. Treatment was administered every other day for 5 d.

Measures of Cell Proliferation
Cells were labeled with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (Sigma) as a measure of cell proliferation. Absorbance was measured with a Thermo max microplate reader (Molecular Devices, Sunnyvale, CA) at 570 nm and reference wavelength of 690 nm.

For fluorescence-activated cell sorting analysis of cell cycle, cells were seeded to a density of 2 x 106 in 100-mm plates and incubated at 37 C in 5% CO2 for 72 h. Cells were harvested, washed in PBS, and fixed in ice-cold 80% ethanol for 1 h, washed twice in calcium/magnesium-free PBS and resuspended in staining buffer (0.2% Triton X-100, 1 mmol EDTA in calcium/magnesium-free PBS) at room temperature. Cells were then centrifuged at 1500 rpm and resuspended in a staining buffer containing 50 µg/ml of deoxyribonuclease-free ribonuclease (Sigma, Oakville, Ontario). Propidium iodide was then added for 2 h at room temperature in the dark. Each sample was filtered through a 50-µmol/liter nylon mesh to remove large aggregates. Samples were run on flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Data were analyzed by the ModFit LT Software (Verity Software House, Inc., Topsham, ME).

In mouse xenografts, cell proliferation was also analyzed by labeling for the Ki-67 antigen using the mouse-specific MIB-5 antibody. Immunohistochemical detection was performed with the avidin-biotin complex technique and scored as the number of positive nuclei per 100 nuclei in five representative fields.

Human Pituitary Tumor Specimens
Primary human pituitary tumors were obtained at the time of surgery after informed consent and institutional review. The pathology of pituitary adenomas was examined using immunohistochemistry for all cases, and tumors were classified histologically according to the accepted Armed Forces Institute of Pathology and World Health Organization criteria (21, 35). A compilation of tumors examined is shown in Table 1Go. Tumor size and invasiveness were defined on the basis of preoperative radiological findings and operative findings using the modified Hardy’s classification (36).

Human Pituitary Tumor Cell Treatment
Primary human pituitary tumors were obtained after informed consent and approval of the University Health Network Research Ethics Board. Samples were collected at the time of surgery under sterile conditions and immediately placed in DMEM containing antibiotics for transport to the laboratory. Cells were dispersed for culture by mechanical agitation and incubation in 1 mg/ml of collagenase for 30 min at 37 C. Cells were pelleted and resuspended in serum-free defined media consisting of DMEM containing 30 µg/ml putresine, 1 x 10–6 M hydrocortisone, 1 x 10–11 M T3, 0.01 mg/ml insulin, transferrin, and 0.375% albumin bovine factor V.

Cells were plated in 0.01% poly-L-lysine-treated chamber slides for immunohistochemical analysis. They were rinsed once in PBS and treated for 48–96 h with varying doses of PD173074 as indicated. Upon completion of treatment, cells were rinsed twice in PBS, fixed in 10% formalin for 15 min, and rinsed in PBS. Slides were allowed to air-dry and were then used for immunohistochemical studies.

Immunohistochemistry
FGFR4 localization was performed using a polyclonal antiserum directed against the C terminus (Santa Cruz Biotechnology) at a dilution of 1:500 as previously described (18). A polyclonal antiserum to N-cadherin and a monoclonal antibody to ß-catenin (both from BD Biosciences Pharmingen) were applied at dilutions of 1:200 and 1:500, respectively. Phosphorylated retinoblastoma was localized using anti-Ser 807/811 (Cell Signaling Technology, Beverly, MA). Reactions were detected with the ABC Elite kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine. All samples were evaluated by two independent observers (D.W., S.L.A.) who were blinded to independent parameters at the time of evaluation and scoring. Membrane staining for human surgical specimens was scored based on complete rimming of the cytoplasm. Staining score for membranous, cytoplasmic, and nuclear expression was based on percentage of cells staining as follows: none, 0% cells staining; weak; 1- 25% cells staining; moderate, 26–60%; strong, 61–100%. Colocalization by double staining was performed for N-cadherin with pituitary hormones as previously described (12).

Statistical Analysis
Data are presented as mean ± SD. In the experimental models, differences were assessed by the unpaired, two-sided t test. P < 0.05 was considered statistically significant. In the analysis of surgical human tumor specimens, we applied Fisher’s exact test.


   ACKNOWLEDGMENTS
 
The authors thank Kelvin So for technical assistance.


   FOOTNOTES
 
This work was supported by the Canadian Institutes of Health Research (Grants MT-14404) and the Toronto Medical Laboratories.

Disclosure summary: The authors have nothing to disclose.

First Published Online July 20, 2006

Abbreviations: FGF, Fibroblast growth factor; FGFR, FGF receptor; NCAM, neural cell adhesion molecule; ptd-FGFR4, pituitary tumor-derived FGFR4 isoform; RTK, receptor tyrosine kinase; SCID, severe combined immunodeficient; siRNA, small interfering RNA.

Received for publication May 24, 2006. Accepted for publication July 12, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Asa SL, Ezzat S 1998 The cytogenesis and pathogenesis of pituitary adenomas. Endocr Rev 19:798–827[Abstract/Free Full Text]
  2. Asa SL, Ezzat S 2002 The pathogenesis of pituitary tumours. Nat Rev Cancer 2:836–849[CrossRef][Medline]
  3. Treier M, Gleiberman AS, O’Connell SM, Szeto DP, McMahon JA, McMahon AP, Rosenfeld MG 1998 Multistep signaling requirements for pituitary organogenesis in vivo. Genes Dev 12:1691–1704[Abstract/Free Full Text]
  4. Paez-Pereda M, Giacomini D, Refojo D, Nagashima AC, Hopfner U, Grubler Y, Chervin A, Goldberg V, Goya R, Hentges ST, Low MJ, Holsboer F, Stalla GK, Arzt E 2003 Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk. Proc Natl Acad Sci USA 100:1034–1039[Abstract/Free Full Text]
  5. Itoh N, Ornitz DM 2004 Evolution of the Fgf and Fgfr gene families. Trends Genet 20:563–569[CrossRef][Medline]
  6. De Moerlooze L, Spencer-Dene B, Revest J, Hajihosseini M, Rosewell I, Dickson C 2000 An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signalling during mouse organogenesis. Development 127:483–492[Abstract]
  7. Celli G, LaRochelle WJ, Mackem S, Sharp R, Merlino G 1998 Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning. EMBO J 17:1642–1655[CrossRef][Medline]
  8. Gospodarowicz D, Abraham JA, Schilling J 1989 Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells. Proc Natl Acad Sci USA 86:7311–7315[Abstract/Free Full Text]
  9. Ezzat S, Smyth HS, Ramyar L, Asa SL 1995 Heterogeneous in vivo and in vitro expression of basic fibroblast growth factor by human pituitary adenomas. J Clin Endocrinol Metab 80:878–884[Abstract]
  10. Heaney AP, Horwitz GA, Wang Z, Singson R, Melmed S 1999 Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis. Nat Med 5:1317–1321[CrossRef][Medline]
  11. Zimering MB, Katsumata N, Sato Y, Brandi ML, Aurbach GD, Marx SJ, Friesen HG 1993 Increased basic fibroblast growth factor in plasma from multiple endocrine neoplasia type 1: relation to pituitary tumor. J Clin Endocrinol Metab 76:1182–1187[Abstract]
  12. Asa SL, Ramyar L, Murphy PR, Li AW, Ezzat S 2001 The endogenous fibroblast growth factor-2 antisense gene product regulates pituitary cell growth and hormone production. Mol Endocrinol 15:589–599[Abstract/Free Full Text]
  13. Abbass SAA, Asa SL, Ezzat S 1997 Altered expression of fibroblast growth factor receptors in human pituitary adenomas. J Clin Endocrinol Metab 82:1160–1166[Abstract/Free Full Text]
  14. Ezzat S, Zheng L, Zhu XF, Wu GE, Asa SL 2002 Targeted expression of a human pituitary tumor-derived isoform of FGF receptor-4 recapitulates pituitary tumorigenesis. J Clin Invest 109:69–78[CrossRef][Medline]
  15. Ezzat S, Yu S, Asa SL 2003 Ikaros isoforms in human pituitary tumors: distinct localization, histone acetylation, and activation of the 5' fibroblast growth factor receptor-4 promoter. Am J Pathol 163:1177–1184[Abstract/Free Full Text]
  16. Yu S, Asa SL, Weigel RJ, Ezzat S 2003 Pituitary tumor AP-2{alpha} recognizes a cryptic promoter in intron 4 of fibroblast growth factor receptor 4. J Biol Chem 278:19597–19602[Abstract/Free Full Text]
  17. Ezzat S, Zheng L, Asa SL 2004 Pituitary tumor-derived fibroblast growth factor receptor 4 isoform disrupts neural cell-adhesion molecule/N-cadherin signaling to diminish cell adhesiveness: a mechanism underlying pituitary neoplasia. Mol Endocrinol 18:2543–2552[Abstract/Free Full Text]
  18. Qian ZR, Li CC, Yamasaki H, Mizusawa N, Yoshimoto K, Yamada S, Tashiro T, Horiguchi H, Wakatsuki S, Hirokawa M, Sano T 2002 Role of E-cadherin, {alpha}-, ß-, and {gamma}-catenins, and p120 (cell adhesion molecules) in prolactinoma behavior. Mod Pathol 15:1357–1365[CrossRef][Medline]
  19. Hirohashi S, Kanai Y 2003 Cell adhesion system and human cancer morphogenesis. Cancer Sci 94:575–581[CrossRef]
  20. Cavallaro U, Schaffhauser B, Christofori G 2002 Cadherins and the tumour progression: is it all in a switch? Cancer Lett 176:123–128[CrossRef][Medline]
  21. Asa SL 1998 Tumors of the pituitary gland. Atlas of tumor pathology, third series, fascicle 22. Washington, D.C.: Armed Forces Institute of Pathology
  22. Ezzat S, Snyder PJ, Young WF, Boyajy LD, Newman C, Klibanski A, Molitch ME, Boyd AE, Sheeler L, Cook DM, Malarkey WB, Jackson I, Vance ML, Thorner MO, Barkan AL, Frohman LA, Melmed S 1992 Octreotide treatment of acromegaly. A randomized, multicenter study. Ann Intern Med 117:711–718[CrossRef][Medline]
  23. Ezzat S 2001 The role of hormones, growth factors and their receptors in pituitary tumorigenesis. Brain Pathol 11:356–370[Medline]
  24. Jones TH, Justice SK, Price A 1997 Suppression of tyrosine kinase activity inhibits [3H]thymidine uptake in cultured human pituitary tumor cells. J Clin Endocrinol Metab 82:2143–2147[Abstract/Free Full Text]
  25. Mohammadi M, Froum S, Hamby JM, Schroeder MC, Panek RL, Lu GH, Eliseenkova AV, Green D, Schlessinger J, Hubbard SR 1998 Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain. EMBO J 17:5896–5904[CrossRef][Medline]
  26. Skaper SD, Kee WJ, Facci L, Macdonald G, Doherty P, Walsh FS 2000 The FGFR1 inhibitor PD 173074 selectively and potently antagonizes FGF-2 neurotrophic and neurotropic effects. J Neurochem 75:1520–1527[CrossRef][Medline]
  27. Koziczak M, Holbro T, Hynes NE 2004 Blocking of FGFR signaling inhibits breast cancer cell proliferation through downregulation of D-type cyclins. Oncogene 23:3501–3508[CrossRef][Medline]
  28. St Bernard R, Zheng L, Liu W, Winer D, Asa SL, Ezzat S 2005 Fibroblast growth factor receptors as molecular targets in thyroid carcinoma. Endocrinology 146:1145–1153[Abstract/Free Full Text]
  29. Lilien J, Arregui C, Li H, Balsamo J 1999 The juxtamembrane domain of cadherin regulates integrin-mediated adhesion and neurite outgrowth. J Neurosci Res 58:727–734[CrossRef][Medline]
  30. Conacci-Sorrell M, Zhurinsky J, Ben Ze’ev A 2002 The cadherin-catenin adhesion system in signaling and cancer. J Clin Invest 109:987–991[CrossRef][Medline]
  31. Heinrich CA, Lail-Trecker MR, Peluso JJ, White BA 1999 Negative regulation of N-cadherin-mediated cell-cell adhesion by the estrogen receptor signaling pathway in rat pituitary GH3 cells. Endocrine 10:67–76[CrossRef][Medline]
  32. Sun C, Yamato T, Kondo E, Furukawa T, Ikeda H, Horii A 2005 Infrequent mutation of APC, AXIN1, and GSK3B in human pituitary adenomas with abnormal accumulation of CTNNB1. J Neurooncol 73:131–134[CrossRef][Medline]
  33. Suyama K, Shapiro I, Guttman M, Hazan RB 2002 A signaling pathway leading to metastasis is controlled by N-cadherin and the FGF receptor. Cancer Cell 2:301–314[CrossRef][Medline]
  34. Daniel L, Trouillas J, Renaud W, Chevallier P, Gouvernet J, Rougon G, Figarella-Branger D 2000 Polysialylated-neural cell adhesion molecule expression in rat pituitary transplantable tumors (spontaneous mammotropic transplantable tumor in Wistar-Furth rats) is related to growth rate and malignancy. Cancer Res 60:80–85[Abstract/Free Full Text]
  35. Tumours of endocrine organs. In: DeLellis RA, Lloyd RV, Heitz PU, Eng C, Kleihues P, eds. 2004 World Health Organization classification of tumours. Lyons, France: International Agency for Research on Cancer (IARC) Press
  36. Hardy J 1973 Transsphenoidal surgery of hypersecreting pituitary tumors. In: Kohler PO, Ross GT, eds. Diagnosis and treatment of pituitary tumors. International Congress Series No. 303. Amsterdam: Exerpta Medica; 179–198



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