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Altered Expression of Fibroblast Growth Factor Receptors in Human Pituitary Adenomas

Department of Pathology, Mount Sinai Hospital, Department of Medicine (Endocrinology), Wellesley Hospital, University of Toronto, Toronto, Ontario, Canada

Address all correspondence and requests for reprints to: Dr. Shereen Ezzat, University of Toronto-The Wellesley Hospital, 160 Wellesley Street East, 134 JB, Toronto, Ontario, Canada M4Y-1J3.

	Abstract

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We have shown that basic fibroblast growth factor (FGF) is heterogeneously expressed by human pituitary adenomas and may be implicated as a growth stimulus for these tumors. There are four mammalian FGF receptor (FGFR) genes encoding a complex family of transmembrane tyrosine kinases. The prototypic receptor is composed of three Ig-like extracellular ligand-binding domains, a transmembrane domain, and a cytoplasmic split tyrosine kinase. Multiple forms of cell-bound or secretable isoforms of FGFR-1, -2, and -3 can be generated by cell- and tissue-specific alternative splicing, resulting in tissue-specific FGF function. Shifts in isoform expression accompany tumor progression in some systems.

We examined the normal human adenohypophysis and 40 pituitary adenomas to determine the pattern of FGFR expression by reverse transcription-PCR; all tumors were characterized clinically and morphologically. Ribonucleic acid (RNA) was extracted from frozen tumor tissue and primers were used to distinguish messenger RNA of the secretable first Ig-like domain (I) and those of the transmembrane and kinase domains (K) of each FGFR subtype. The normal pituitary-expressed mRNAs for FGFR-1 I and K, FGFR-2 I and K, FGFR-3 I and K, and FGFR-4 I but not FGFR-4 K; this represents the first report of a truncated isoform of FGFR-4, indicating possible alternative polyadenylation sites in this receptor. Only 3 tumors had the same pattern of expression of the 4 FGFRs as the normal gland. Although all tumors expressed FGFR-1 I, 1 tumor did not express FGFR-1 K, suggesting the production of only a secretable form of FGFR-1 by this tumor. Four tumors were negative for FGFR-2 I and K; 6 expressed the secretable form only, and 17 expressed FGFR-2 K but not I. All tumors expressed FGFR-3 I; 14 had secretable forms only, and no tumors expressed FGFR-3 K alone. As in the normal gland, 13 tumors expressed only the secretable I form of FGFR-4. Unlike the normal pituitary, however, 22 expressed FGFR-4 I and K, indicating a possible tumor-specific transmembrane receptor. Five tumors were negative for FGFR-4 I and K. Expression of FGFR proteins was confirmed by immunohistochemical localization of the C-terminal portion of FGFR-1, -2, -3, and -4; the results correlated with the RNA data in each case. There was no correlation between tumor type, size, or aggressiveness and the expression pattern of FGFRs.

Our study suggests that pituitary adenomas have altered FGFR subtype and isoform expression, which may determine their hormonal and proliferative responses to FGFs.

	Introduction

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PITUITARY ADENOMAS are common and potentially serious neoplasms. Despite their prevalence, little is known regarding their etiology. Although they are monoclonal proliferations (1, 2), the molecular events underlying their development remain unknown. Mutations that are common in other human tumors, involving ras (3, 4, 5, 6), p53 (4, 7), protein kinase C (8), and retinoblastoma (9, 10, 11) genes, are rare or absent in these neoplasms. Only a small fraction of adenomas have activating mutations of G_s (12, 13, 14), and loss of heterozygosity at the multiple endocrine neoplasm-1 gene locus is rare in sporadic adenomas (15). Other pathogenetic factors implicated in pituitary tumor formation are hypophysiotropic hormones (16, 17, 18, 19) and growth factors (17, 20, 21).

Fibroblast growth factor-2 [FGF-2; basic FGF (bFGF)] appears to be important for pituitary regulation. It is one of nine members of the FGF family that have mitogenic, angiogenic, and hormone regulatory functions (22, 23). bFGF was originally isolated from bovine pituitary and regulates pituitary hormone production (24, 25). We have documented varying levels of bFGF messenger ribonucleic acid (mRNA) expression in human pituitary adenomas, release in vitro by human pituitary adenoma cells and circulation in vivo in patients with pituitary adenomas (26); the highest bFGF mRNA and blood levels were associated with the most aggressive tumors (27). Although mutated p53 can induce bFGF expression (28), p53 is intact in pituitary tumors (7), excluding this explanation. An NH₂-terminally extended form with a putative nuclear localization sequence is also present in pituitary tumor cells (29). In contrast to these suggestions of a mitogenic role, bFGF has been reported to inhibit DNA synthesis (30, 31, 32, 33), suggesting that some forms of FGF or its receptor may act as growth inhibitors.

There are four mammalian FGF receptor (FGFR) genes encoding a complex family of transmembrane receptor tyrosine kinases (34). Each receptor is composed of three Ig-like extracellular domains, two of which are involved in ligand binding, a single transmembrane domain with a long juxtamembrane region, a split tyrosine kinase cytoplasmic domain, and a COOH-terminal tail that contains tyrosines that are phosphorylated upon ligand binding and recruit intracellular signaling proteins (34). Ligand binding appears to depend on the interaction of FGFs with cell surface heparan sulfate proteoglycans (35). Multiple forms of cell-bound or secreted receptors are produced by the same gene. Tissue-specific alternative splicing, variable polyadenylation sites, and alternative initiation of translation result in truncated receptor forms (36, 37). The spatial and temporal expression pattern and binding specificities of these receptor subtypes and their isoforms may play a key role in regulating tissue-specific FGF function and may explain the different effects of FGF in different cell proliferation assays.

FGFRs are expressed in human gliomas (38, 39), breast (40), and ovarian carcinomas (41, 42). FGFR mRNA has been reported in human pituitary adenomas (29), but the exact type of receptor(s) in these tumors has not been characterized. It is essential to clarify this issue because the FGFR type and isoform expressed in any given tissue may determine the hormonal and proliferative responses to FGFs.

	Materials and Methods

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Human pituitary tissues and tumors

Pituitary adenomas were collected after transsphenoidal surgery from patients after full endocrine preoperative evaluation. Tissue was divided into four parts, as described previously, for histological and immunocytochemical studies, electron microscopy, tissue culture, and molecular analysis (26, 43, 44).

Normal human adenohypophyses were obtained at autopsy from patients with no evidence of endocrine abnormality; they were examined histologically and using immunocytochemistry to exclude the possibility of incidental pathology.

The MCF-7 cell line, which is known to express all four FGFRs (45), was used as a positive control for all experiments.

Reverse transcription-PCR (RT-PCR) for mRNA analysis

Total RNA was extracted by the guanidinium isothiocyanate method. One microgram was reverse transcribed using 2.5 U murine leukemia virus reverse transcriptase. The oligonucleotide primers used to identify the first Ig-like domain and the third Ig-like domain of FGFR-1, -2, -3, and -4 are shown in Table 1. All primer sets span at least one putative intron to allow distinction of genomic contamination. PCR conditions were optimized using mRNA from MCF-7 cells. PCR reactions were performed in a final volume of 25 µL containing 2 mmol/L MgCl₂, 500 mmol/L KCl, 100 mmol/L Tris-HCl, and 2.5 U Taq polymerase. Primers were added at a final concentration of 0.5 µmol/L. Reaction conditions were as follows: an initial denaturation at 95 C for 2 min, followed by 35 cycles of 95 C for 30 s, annealing at 60–62 C for 30 s, extension at 72 C for 45 s for each cycle, and a final cycle of 72 C for 7 min.

The products were visualized on 1% agarose gel electrophoresis with ethidium bromide staining and verified by Southern hybridization. The complementary DNAs (cDNAs) of human FGFR-1 (provided by Dr. M. Jaye, Rhône-Poulenc-Rorer, Collegeville, PA); FGFR-2 (donated by Dr. R. Breathnach, Nantes, France); and FGFR-3, FGFR-4, and PGK-1 (American Type Culture Collection, Rockville, MD) were labeled with digoxigenin coupled to deoxy-UTP. The blots were prehybridized for 2 h, then hybridized for 18 h at 42°C and washed at high stringency [0.1 x SSC (standard saline citrate) at 68 C], and the reaction was visualized with antidigoxigenin and alkaline phosphatase detection. As the first Ig-like domain of FGFR-3 was not included in the cDNA, the 202-bp fragment generated was digested with the restriction endonucleases PstI and HinfI; the resulting products were visualized by electrophoresis with ethidium bromide staining. Negative controls included mock reverse transcription without reverse transcriptase and RT-PCR reactions without RNA or cDNA to determine amplification of contaminating cDNA from another sample. Positive controls were provided by RNA derived from MCF-7 cells (45).

Immunocytochemical localization

The streptavidin-biotin-peroxidase complex technique was applied to paraffin sections using polyclonal antisera against cytoplasmic tails of FGFR-1, FGFR-2, FGFR-3, and FGFR-4 (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:400. Sections were subjected to microwave antigen retrieval before staining. The specificity of the reaction was verified by preabsorption of the primary antiserum with homologous and heterologous antigens, and positivity was accepted only when staining was abolished by preabsorption with homologous antigen; absorption with heterologous antigens, including pituitary hormones, did not alter the results. The analysis of immunostaining was performed in a blinded fashion, so that the results of PCR on the tumors would not alter interpretation.

Double staining for each of the FGFRs and pituitary hormones was performed to identify the cell types containing each FGFR in the nontumorous adenohypophysis (47, 48). FGFRs were localized with streptavidin-biotin-peroxidase using cobalt blue (49). Adenohypophysial hormones were identified using the peroxidase-antiperoxidase method and visualized with 3,3'-diaminobenzidine using polyclonal antisera against GH and ACTH (Dako Corp., Carpenteria, CA; prediluted 1:15 and 1:1500, respectively) and monoclonal antibodies against PRL (Biomeda Corp., Foster City, CA; prediluted), TSHß, LHß, and FSHß (Amac, Westbrook, ME; diluted 1:500, 1:400, and 1:400, respectively). Controls were as described above.

	Results

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RT-PCR

A total of 40 pituitary adenomas were considered informative for this study because they had intact PGK-1 mRNA, and there was no evidence of contamination by nontumorous adenohypophysis using PCR for Pit-1 or SF-1 (44, 46). These included 8 sparsely granulated lactotroph adenomas, 6 sparsely granulated somatotroph adenomas, 3 densely granulated somatotroph adenomas, 2 mammosomatotroph adenomas, 7 corticotroph adenomas, 12 gonadotroph adenomas, and 2 plurihormonal silent subtype III adenomas. Ten morphologically normal adenohypophyses were obtained and were confirmed to have intact PGK-1 mRNA, as shown by RT-PCR.

The results of RT-PCR of representative tissues are illustrated in Fig. 1. The normal pituitary expressed mRNAs for FGFR-1, -2, and -3, including both the first Ig-like domain (I) and the transmembrane and kinase domains (K) of each FGFR subtype. Transcripts were also found for FGFR-4 I, but not FGFR-4 K, suggesting expression of a secretable Ig-like domain only of this receptor. Southern hybridization confirmed the identity of the products visualized. At least one smaller PCR product of each reaction for FGFR-1, -2, and -3 hybridized, suggesting the presence of isoforms of these receptors. Restriction enzyme digestion of the PCR product using primers for FGFR-3 I yielded the expected 74- and 128-bp bands after digestion with PstI, and the appropriate 101-bp band after digestion with HinfI (Fig. 2).

View larger version (108K): [in this window] [in a new window]   Figure 1. a, RT-PCR and Southern hybridization analysis of FGFR-1 mRNA in human pituitaries and pituitary adenomas. b, RT-PCR and Southern hybridization analysis of FGFR-2 mRNA in human pituitaries and pituitary adenomas. c, RT-PCR and Southern hybridization analysis of FGFR-3 mRNA in human pituitaries and pituitary adenomas. d, RT-PCR and Southern hybridization analysis of FGFR-4 mRNA in human pituitaries and pituitary adenomas. Lane 1 contains the DNA ladder. The MCF-7 breast cancer cell line was a positive control for all reactions (lane 2 and -rt in lane 3). Two nontumorous pituitaries (lanes 4 and 6) are representative of the 10 examined. They consistently express both I and K domains of FGFR-1, -2, and -3, but only the I domain of FGFR-4 (lanes 5 and 7, -RT). The 16 pituitary adenomas in lanes 8–23 are representative of the 40 examined. They have a highly variable pattern of expression of the I and K domains of FGFR-1, -2, and -3, and 22 expressed the K domain of FGFR-4, indicating the presence of a novel tumor-specific transmembrane kinase (lanes 24–27, -RT).

View larger version (30K): [in this window] [in a new window]   Figure 2. Restriction enzyme analysis of FGFR-3 I PCR product. Digestion of a 202-bp PCR product with PstI produced the expected 74- and 128-bp bands; digestion with HinfI, which has a restriction site exactly at the middle of our PCR product, produced a 101-bp band.

RT-PCR with omission of reverse transcriptase and with water replacing template were both negative.

No correlation was identified between the cell type, clinical tumor size, or aggressiveness of the tumors and the pattern of FGFR expression.

Immunocytochemical localization

Normal adenohypophysis showed a focal pattern of immunoreactivity for the C-terminal tails of FGFR-1, -2, and -3. Positivity was found in many cells throughout the gland (Fig. 3, a and b), but was not homogeneous in all cells. In general, the pattern of reactivity was cytoplasmic, but there was also a suggestion of membrane localization in well fixed tissues. Double staining characterized the positivity for FGFR-1, -2, and -3 in cells containing cytoplasmic reactivity for all pituitary hormones; therefore, the expression of these receptors is not cell type specific (data not shown). Preabsorption of antibodies with antigen yielded an appropriate curve, and staining was abolished with 2 µg/mL of a 15-amino acid C-terminal fragment of FGFR-1, 1 µg/mL of a 17-amino acid C-terminal fragment of FGFR-2, and 0.125 µg/mL of a 15-amino acid C-terminal fragment of FGFR-3. Staining for FGFR-4 yielded no convincing positivity that could be abolished with excess antigen.

View larger version (107K): [in this window] [in a new window]   Figure 3. a, A pituitary adenoma does not stain for FGFR-2, whereas the majority of surrounding nontumorous cells have diffuse cytoplasmic positivity. b, This pituitary adenoma exhibits strong cytoplasmic reactivity for FGFR-3, as do the adjacent nontumorous cells. c, Neoplastic pituitary cells stain for FGFR-4, whereas the nontumorous cells are negative. In a, b, and c, the streptavidin-biotin peroxidase method with hematoxylin counterstain was used.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
FGFR mRNA has been reported in pituitary adenomas (29), but the types of receptors in these tumors have not previously been characterized. Clearly, the discrepant effects of the various FGFs and the numerous potential FGFR isoforms indicate that this issue warranted clarification.

We report that normal pituitary expresses FGFR-1, -2, and -3. The presence of both I and K mRNAs and the presence of a C-terminal tail by immunocytochemistry are consistent with intact transmembrane kinases. In addition, the production of isoforms, as previously described (34), can explain the presence of multiple specific PCR products of different sizes. It remains possible that alternative splicing or variable polyadenylation results in the secretion of one or several extracellular Ig-like domains. The production of secretable isoforms of FGFRs raises the possibility that these receptors bind ligand and remove it from the cell surface to alter FGF-induced responses. This possibility is perhaps more relevant in the tumors, where the absence of the kinase region almost certainly results in secretable isoforms. Further studies are required to clarify the isoforms of FGFR-1, -2, and -3 expressed by normal pituitary tissue and to confirm that secretable forms are released by tumors that express I but not K. The structure and functional significance of kinase forms in tumors lacking the first Ig-like domain remain to be identified.

Although multiple isoforms of FGFR-1, -2, and -3 have been reported, there has not yet been any evidence of alternative splicing or variable polyadenylation of FGFR-4. We report expression of the first Ig-like domain only by nontumorous pituitary. This potentially secretable form of the receptor suggests the presence of variable polyadenylation sites in FGFR-4, similar to those reported in FGFR-1, -2, and -3 (34).

We also have identified expression of the transmembrane and kinase domains of FGFR-4 uniquely in pituitary tumors. Immunostaining confirmed the presence of a potentially functional molecule with a C-terminal tail. It remains to be seen whether this reflects the production of an intact molecule, as the K domain was not expressed in any tumors without the I domain. Intact FGFR-4 binds bFGF (50) and aFGF with 10-fold higher affinity (51); K-FGF/hst-1 binding is controversial (50, 51). Although the prototypic receptor has three Ig-like domains, isoforms of other FGFRs that lack only the first Ig-like domain are still efficiently activated by FGFs (52), as the second and third domains are implicated in ligand binding; it has been suggested that the first domain may even decrease the binding affinity of FGFs and heparin. Therefore, even if the tumors release the first Ig-like domain as does the normal gland, the transmembrane receptor could still be implicated as a functional tumor-specific kinase. The actions of FGFR-4 remain poorly defined. Although initially FGFR-4 was thought to be only weakly mitogenic, recent data suggest that it increases DNA synthesis as effectively as FGFR-1 (53), albeit by what appears to be a different mechanism (51, 53, 54, 55). Moreover, FGFR-4 has been implicated as a unique mediator of membrane ruffling in breast cancer cells (56); although the significance of this phenomenon is unclear, it indicates potential differentiation activity of this receptor.

A variant of FGFR-1, the -form, contains Ig domains 2 and 3 and the cytoplasmic kinase, but lacks a signal peptide and the transmembrane domain; this variant may be important in intracellular signaling by nonsecreted ligands, such as the nuclear-localizing NH₂-terminally extended form of bFGF (29), which has been shown to be abundant in some tumor cells (36). Our primers for FGFR-4 K detected a slightly smaller variant in a few tumors that could correspond to a -like form of this receptor.

In conclusion, we have identified mRNAs for FGFR-1, -2, and -3 as well as a novel secretable isoform of FGFR-4 in the nontumorous human pituitary. We have documented altered expression of FGFR-1, -2, and -3 in some pituitary tumors as well as the appearance of a unique transmembrane FGFR-4 kinase in a majority of human pituitary adenomas. Further studies will determine whether this tumor-specific receptor can alter the hormonal and proliferative responses to FGFs by pituitary adenomas. These data suggest that dysregulated FGF/FGFR function plays a role in pituitary tumorigenesis.

	Acknowledgments

 
The authors acknowledge the technical assistance of Ms. Lily Ramyar, Dr. Lei Zheng, and Mr. Kelvin So. Dr. H. S. Smyth (Wellesley Hospital) provided tissue for analysis, and Dr. K. Kovacs (St. Michael’s Hospital) assisted with morphological characterization of the tumors. We thank Dr. M. Jaye (Rhône-Poulenc-Rorer Research and Development, Collegeville, PA) and Dr. R. Breathnach (Faculté des Sciences et des Techniques, Université de Nantes and INSERM U-211, Nantes, France) for providing the cDNAs of FGFR-1 and FGFR-2, respectively.

Received October 16, 1996.

Revised December 12, 1996.

Accepted January 2, 1997.

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