Targeted expression of a human pituitary tumor-derived isoform of FGF receptor-4 recapitulates pituitary tumorigenesis

Abstract

It is estimated that up to one in five individuals develop pituitary gland tumors. Despite the common occurrence of these tumors, the pathogenetic mechanisms underlying their development remain largely unknown. We report the identification of a novel pituitary tumor-derived, N-terminally truncated isoform of FGF receptor-4 (ptd-FGFR4). The corresponding mRNA results from alternative transcription initiation and encodes a polypeptide that lacks a signal peptide and the first two extracellular Ig-like domains. ptd-FGFR4 has a distinctive cytoplasmic residence, is constitutively phosphorylated, and is transforming in vitro and in vivo. Here we show that targeted expression of ptd-FGFR4, but not FGFR4, results in pituitary tumors that morphologically recapitulate the human disease.

Figure 1

FGFR4 gene and protein structure, and RNA analysis. (a) The gene (top) contains 18 exons. The protein (bottom) is a transmembrane tyrosine kinase with three extracellular Ig-like loops. Exon 1 is untranslated. Exon 2 encodes signal peptide (SP). Exon 3 encodes first Ig-like domain (I₁). Exon 4 encodes acid box (AB). Exons 5 and 6 encode second Ig-like domain (I₂). Exons 7 and 8 encode third Ig-like domain (I₃). Exon 9 encodes trans-membrane domain (TM). Exons 10–12 and 14–17 encode split kinase (K1 and K2). (b) RT-PCR of RNA from human pituitary tumors and normal pituitaries (N), identified an 818-bp product (exons 7–12) of FGFR4 in five of seven tumors, but not in normals. RNA integrity is indicated by RT-PCR for PGK-1 (bottom). Negative controls omitted reverse transcriptase (–). (c) Although the predicted 1,500-bp mRNA encoding full-length FGFR4 was present in MCF-7 breast cancer cells (+), it is undetectable in normal pituitary and tumors. PCR on the same samples, using corresponding primers in signal peptide and kinase domains of FGFR1, resulted in products of the predicted 1,700-bp size (bottom). (d) Northern blot analysis of hFGFR4 expression. A 2.3 kb band is identified in RNA from MCF-7 cells (+). Normal pituitary (N) is negative; two pituitary tumors (T1 and T2) contain smaller bands of approximately 1.7 kb. Hybridization with PGK-1 cDNA identifies a 1.8-kb species (bottom). (e) RACE. Arrow in the exon 5/6 sequence indicates the farthest 5′ transcription initiation site in 5′ RACE-generated products from pituitary tumors. The putative in-frame ATG translational start site is indicated in bold italics. (Sequence 492–782, following GenBank FGFR4 cDNA sequence accession no. X57205).

Figure 2

Characterization of ptd-FGFR4 protein. (a) Immunoprecipitation of normal and tumorous pituitary protein with anti-FGFR4, and immunoblotting with C-terminal anti-FGFR4, identifies no product at the size expected for full-length protein (approximately 110 kDa); instead there is a band that migrates at greater than 64 kDa that is found in tumor samples (T1 and T2), but not in normal pituitary samples (N). Equal amounts of protein that were immunoprecipitated and immunoblotted for actin are shown immediately below. (b) Immunohistochemical localization of FGFR4 reactivity in a human pituitary tumor. C-terminal FGFR4 immunoreactivity is seen in the cytoplasm of tumor (T) cells (arrow), including those infiltrating around adjacent nontumorous (NT) pituitary cells that are immunonegative (left); absorption of primary antibody eliminates reactivity (right), proving the specificity of the reaction. (c) Using confocal microscopy, cells transfected with full-length FGFR4 (left) exhibit strong membrane-specific localization of immunoreactivity and irregular nuclear membranes, a phenomenon known as membrane ruffling. In contrast, cells transfected with ptd-FGFR4 (right) exhibit a diffuse cytoplasmic staining pattern. (d) Transfected full-length FGFR4 is detected as a 110-kDa protein, predominantly in membrane fractions (M); transfected ptd-FGFR4 migrates at 65 kDa in cytoplasmic fractions (C) as well as in membrane fractions. Cells transfected with empty vector (pcDNA) are negative, and nuclear fractions (N) are negative.

Figure 3

Phosphotyrosine activity of FGFR4 and ptd-FGFR4. Transiently transfected HEK 293 cells (a) and stably transfected NIH 3T3 cells (b and c) were exposed to FGF-1 (50 ng/ml; a and c). Equal amounts of protein were immunoprecipitated with an antibody directed against the C-terminus of FGFR4 and electrophoresed. (a) In HEK 293 cells, immunoblotting with anti-phosphotyrosine identifies a 110-kDa fragment only after exposure to FGF-1 in cells transfected with full-length FGFR4 (upper arrow). Cells transfected with empty vector (pcDNA) are negative. Cells expressing ptd-FGFR4 exhibit phosphorylation of the 65-kDa isoform with and without FGF-1 treatment, consistent with constitutive phosphorylation of the truncated receptor (lower arrow). (b) NIH 3T3 cells stably transfected with FGFR4 express large amounts of the 110/90-kDa protein (upper arrow), and those stably transfected with ptd-FGFR4 express the 65-kDa protein (lower arrow); cells transfected with empty vector are negative. (c) NIH 3T3 cells transfected with empty vector (pcDNA, right) express endogenous FGFR4 that migrates at 110 kDa and is detected with phosphotyrosine (pTYR) antibody after exposure to FGF-1 (upper arrow). Cells transfected with FGFR4 (left) exhibit the same pattern, but with greater intensity (documented by densitometry). Cells transfected with ptd-FGFR4 (middle) express a 65-kDa protein that is phosphorylated in the presence and absence of FGF-1 (lower arrow); these cells also express the 110-kDa protein, and phosphorylation of this endogenous receptor is enhanced in the presence of FGF-1 to a greater degree than in control cells. These transfected NIH 3T3 cells had FGFR4 immunoreactivity that comigrated with the pTYR-immunoreactive bands.

Figure 4

ptd-FGFR4 transforming ability in vitro and in vivo. (a) Anchorage independence in culture. NIH 3T3 cells stably transfected with ptd-FGFR4 lose contact inhibition. (b) Growth in soft agar. Cells stably transfected with FGFR4 rarely formed colonies (left), whereas those transfected with ptd-FGFR4 (right) formed numerous colonies greater than 50 μm in diameter. (c) NIH 3T3 cells stably transfected with ptd-FGFR4 formed tumors within 2 weeks of injection. Cells transfected with empty vector or full-length FGFR4 did not form tumors.

Figure 5

Pituitary morphology of PRL–ptd-FGFR4 and PRL-FGFR4 transgenic mice. (a) The pituitary of an 11-month-old PRL–ptd-FGFR4 transgenic female mouse (left) is large, with nodular distortion; compare with nontransgenic female littermate (right). (b) The pituitary of an 11-month-old PRL–ptd-FGFR4 transgenic female mouse (left) is enlarged, with a tumor in the right lobe and a larger tumor replacing the left lobe. Normal pituitary of a nontransgenic female littermate (right). (c) Western blotting of mouse serum reveals higher levels of circulating PRL in transgenic mice (+) than in nontransgenic mice (–). The two left lanes are from males (M); the four right lanes are from females (F). Basal PRL is lower in the nontransgenic male than in the nontransgenic females. The degree of elevation in transgenic mice correlated with size of tumor. Albumen (Alb) control indicates protein loading (lower). (d) The pituitary tumor (T) of an 11-month-old PRL–ptd-FGFR4 transgenic female mouse invades the hypothalamus (H). (e) The pituitary of an 11-month-old PRL–ptd-FGFR4 transgenic male mouse contains a tumor composed of small cells with chromophobic cytoplasm and prominent nuclei. There is loss of acinar architecture and increased vascularity. (f) Tumor cells contain PRL in PRL–ptd-FGFR4 transgenic mice. (g) Tumor cells in a PRL–ptd-FGFR4 transgenic mouse contain variable amounts of cytoplasmic FGFR4, similar to that seen in human tumors (Figure 2b). (h) The pituitary of a 12-month-old mouse transgenic for PRL-FGFR4 exhibits normal architecture and distribution of the various pituitary cell types. (i) Wild-type FGFR4 in a 12-month-old PRL-FGFR4 transgenic mouse has a membrane-staining pattern (arrows), and is found in scattered cells that correspond to lactotrophs.