Isolation and characterization of novel pituitary tumor related genes: a cDNA representational difference approach

Abstract

Recently, progress has been made in understanding human pituitary tumor pathogenesis by the investigation of differences in gene expression between normal pituitary tissue and pituitary tumors. A number of approaches, including differential display (DD), representational difference analysis (RDA), and microarray analysis have been used and several molecular targets potentially associated with pituitary tumor development and invasion have been identified. We have used RDA to compare gene expression patterns between normal human pituitary and clinically non-functioning pituitary adenomas, and identified genes with growth suppression function which are expressed in the normal pituitary but not in pituitary tumors. In particular, we have focused on an imprinted gene, Maternally Expressed Gene 3 (MEG3), which is specifically associated with clinically non-functioning pituitary adenomas of a gonadotroph lineage. MEG3 functions to suppress tumor cell growth, increase protein expression of the tumor suppressor p53, and selectively activate p53 target genes. Interestingly, MEG3 does not encode a protein but a non-coding RNA. Therefore, these studies have revealed novel mechanisms for the function of a non-coding RNA in pituitary physiology and tumorigenesis.

Fig 1

RT-PCR showing MEG3 RNA expression in all cases of functioning pituitary adenomas (A, GH-secreting tumors; B, PRL-secreting tumors; and C, ACTH-secreting tumors) and normal human pituitaries (D). Some prolactinomas have a weaker expression (in two cases) (B). In contrast, no MEG3 RNA is detected in any of the 17 clinically non-functioning tumors (NFT) (E). NP: positive control with normal pituitary cDNA; C: negative control without cDNA template. (Copyright 2008, The Endocrine Society)

Fig 2

MEG3 suppresses tumor cell growth. (A) H4 cells were transfected with the blank vector pCI-neo (control) or expression vector for LacZ, MEG3, or GADD45-γ. After 2 weeks of neomycin selection, the plates were fixed and stained with crystal violet solution. (B) Viable colonies of HeLa cells, MCF-7, and H4 cells in similar experiments, were counted and normalized to control. The data are represented as mean ± SD for counts from at least three independent experiments. (Copyright 2003, The Endocrine Society)

Fig 3

MEG3 activates p53 and downregulates MDM2. HCT116 cells were transfected with constructs expressing p14^ARF or MEG3 as indicated. Expression of p53 and MDM2 were detected by Western blotting. β-Actin was probed as an equal protein loading control. (Copyright 2007, The American Society for Biochemistry and Molecular Biology)

Figure 4

MEG3 interacts with both p53 and Rb pathways. In the p53 pathway, MEG3 may activate p53 directly by RNA-protein interaction or indirectly by suppressing MDM2, resulting in selective activation of p53 downstream targets such as GDF-15 with both anti-proliferative and tumor suppressive functions. In the Rb pathway, MEG3 may activate Rb directly by RNA-protein interactions or indirectly by activating the positive regulator p16^INK4a, which in turn activates Rb pathway to suppress cell proliferation and tumor formation.