Chapter 8 Folate Receptor Expression in Pituitary Adenomas

Abstract

Clinically nonfunctional pituitary adenomas cause hypopituitarism or compression of regional structures. Unlike functional tumors, there is no available medical treatment or specific imaging technique for these tumors. We have recently discovered that both folate receptor (FR)α mRNA and protein are uniquely overexpressed in nonfunctional pituitary tumors, but not in functional adenomas. We hypothesized that FRα may hold significant promise for medical treatment by enabling novel molecular imaging and targeted therapy. Here, we used murine pituitary tumor cell line αT3‐1 as a model to investigate the biological significance of FRα and its mutant FR67. We demonstrate that overexpression of FR facilitated tumor cell growth and anchorage‐independent growth in soft agar. More colonies were observed in FR overexpressing cells than in mutant FR67 clones in soft agar. Cell proliferation rate was increased, the percentage of cells in S‐phase was increased, and high PCNA staining was detected in cells overexpressing the receptor. In αT3‐1 cells transfected with mutant FR67, cell proliferation rate was reduced, the percentage of cells residing in S‐phase was slightly decreased, and low PCNA staining was observed. By real‐time quantitative PCR, the genes involved in NOTCH3 pathway including NOTCH3, HES‐1, and TLE2 were altered; the mRNA expression of FGFR1 was upregulated, and ERβ mRNA was downregulated in FR overexpressing cells. Our findings suggest that FRα plays a role in pituitary tumor formation, and this effect may in part be due to its regulation of the NOTCH3 pathway.

Introduction

Pituitary tumors are mostly benign adenomas arising from adenohypophyseal cells in the anterior pituitary. They comprise 10% of all brain tumors and occur in ∼20% of the population. They cause significant morbidity by compression of regional structures and the inappropriate expression of pituitary hormones (Asa, 1998, Greenman and Melmed, 1996). Functional tumors, such as GH and ACTH adenomas, give rise to severe life‐threatening clinical syndromes, such as Acromegaly or Cushing's disease, and PRL adenomas result in impaired reproduction. However, ∼30% of all anterior pituitary adenomas are termed nonfunctional (NF) pituitary adenomas due to their lack of clinical hormone hypersecretion (Asa and Kovacs, 1992). Clinically, NF tumors manifest as hypopituitarism or visual field defects due to regional compression of the optic chiasm (Asa and Ezzat, 1998, Asa and Kovacs, 1992, Black et al., 1987, Katznelson et al., 1993). The NF tumors are uniquely heterogeneous (Table 8.1). They typically are quite large and cause hypopituitarism or blindness from regional compression (Greenman and Melmed, 1996). Despite the lack of clinical hormone hypersecretion, immunocytochemical staining for hormones reveals evidence for hormone expression in ≤79% of these tumors, and we refer to these as immunohistochemically positive (NF+). The remainder is negative for hormone expression (Asa et al., 1992, Katznelson et al., 1993) and these are referred to as immunohistochemically negative (NF−).

In a previous study (Evans et al., 2001), we used cDNA microarray analysis and real‐time quantitative PCR (RT‐qPCR) to compare expression profiles of 7075 genes in the normal pituitary with that in different adenomas, including NF−, PRL‐producing adenomas, GH‐producing adenomas, and ACTH‐secreting adenomas. In those experiments, we found that the folate receptor (FR)α gene was significantly overexpressed in NF− adenomas. Next, we have discovered that both FRα mRNA and protein are uniquely overexpressed in NF tumors, but not in functional adenomas (Evans et al., 2003, Moreno et al., 2005). But whether or how FRα plays a role in pituitary tumorigenesis is unclear. The FR genes are located on chromosome 11q13.3–13.5, a region commonly amplified in carcinomas of the head and neck and breast (Rijnboutt et al., 1996). There are three isoforms of FR (FRα, FRβ, and FRγ) that vary in sequence, ligand preference, and tissue distribution (Miotti et al., 1987, Shen et al., 1994). FRα (GenBank U20391) is the major isoform mediating folate transport and is the subject of this study. The FRα receptor is absent or weakly expressed in most normal tissues. FRα is vastly overexpressed in tumors such as ovarian, renal, breast, and colorectal carcinomas, as well as anaplastic ependymomas, choroid plexus, and pituitary tumors (Evans et al., 2001, Kane et al., 1986, Mathias and Green, 1998, Mathias et al., 1998, Miotti et al., 1987).

It is suggested that elevated levels of FRα induce cell proliferation not only by mediating folate uptake but also by generating other regulatory signals. In vitro studies demonstrate that cellular overexpression of the FRα results in enhanced proliferation and survival by providing enhanced folic acid uptake (Antony, 1992, Chung et al., 1993, Kane et al., 1986, Luhrs et al., 1992, Ross et al., 1994).

Cell culture studies also demonstrate that some NF tumors secrete hormone in vitro (Yamada et al., 1989). Unlike functional tumors, currently, there is no available medical treatment or specific imaging technique for these NF tumors.

In order to study the possible influence of FRα overexpression in NF pituitary tumors cell growth, we analyzed the biochemical and the biological characteristics of the murine αT3‐1 cell transfected with FRα cDNA and a mutant FR67 cDNA. We took advantage of this mutant FR67 in our current study because it significantly inhibited folate binding and uptake in one tumor cell line in other studies (Orr and Kamen, 1994, Orr and Kamen, 1995). We also examined molecules that might physically and functionally associate with FRα in order to elucidate the involvement of the receptor in signal transduction of pituitary adenoma pathogenesis.