Gonadotrope and null cell pituitary tumors cause significant morbidity, often presenting with signs of hypogonadism together with visual disturbances due to mass effects. Surgery and radiation are the only therapeutic options to date. To identify dysregulated genes and pathways that may play a role in tumorigenesis and/or progression, molecular profiling was performed on 14 gonadotrope tumors, with nine normal human pituitaries obtained at autopsy serving as controls. Bioinformatic analysis identified putative downstream effectors of tumor protein 53 (p53) that were consistently repressed in gonadotrope pituitary tumors, including RPRM, P21, and PMAIP1, with concomitant inhibition of the upstream p53 regulator, PLAGL1(Zac1). Further analysis of the growth arrest and DNA damage-inducible (GADD45) family revealed no change in the p53 target, GADD45α, but identified repression of GADD45β in pituitary tumors in addition to the previously reported inhibition of GADD45γ. Overexpression of GADD45β in LβT2 mouse gonadotrope cells blocked tumor cell proliferation and increased rates of apoptosis in response to growth factor withdrawal. Stable gonadotrope cell transfectants expressing increased GADD45β showed decreased colony formation in soft agar, confirming its normal role as a tumor suppressor. Unlike previous studies of GADD45γ in pituitary tumors and α and β in other tumors, bisulfite sequencing showed no evidence of hypermethylation of the GADD45β promoter in human pituitary tumor samples to explain the repression of its expression. Thus, GADD45β is a novel pituitary tumor suppressor whose reexpression blocks proliferation, survival, and tumorigenesis. Together these studies identify new targets and mechanisms to explore in pituitary tumor initiation and progression.

Reprimo (RPRM), initially identified as a downstream effector of p53-induced cell cycle arrest at G(2)/M, is a putative tumor suppressor silenced in some types of cancer. In microarrays, the RPRM transcript was repressed 26-fold in gonadotrope (null cell) human pituitary tumors compared with normal pituitary but in the absence of changes in p53. Inhibition of RPRM mRNA was confirmed by RT-PCR in all gonadotrope tumors, most GH samples, and variably in other tumor types. Human pituitary tumors showed no evidence of abnormal promoter hypermethylation as a mechanism of RPRM repression. RPRM stable expression in gonadotrope (LβT2) and GH (GH3) pituitary cells resulted in decreased rates of cell proliferation by 55 and 30%, respectively; however, RPRM reexpression did not alter G(2)/M transition. In addition, RPRM increased rates of apoptosis in response to growth factor deprivation as assessed by caspase-3 cleavage and nuclear condensation. Clonagenic assays showed a 5.3- and 3.7-fold suppression of colony growth in RPRM-overexpressing LβT2 and GH3 cells, respectively, supporting its role as a tumor suppressor. In cells stably expressing RPRM mRNA, protein levels were actively suppressed due to rapid degradation through ubiquitination and proteasomal targeting. Growth factor withdrawal, as a model of cellular stress, stabilized RPRM protein levels. Together these data suggest that RPRM is transiently up-regulated at a posttranscriptional level in times of cellular stress to restrict cell survival, proliferation, and tumor formation. When RPRM is silenced as in human pituitary tumors, unrestrained growth and tumor progression may occur.

Based on prior work showing that human pituitary tumors overexpress epidermal and fibroblast growth factor receptors, we hypothesized that downstream components of growth factor signaling pathways may also be dysregulated. Epidermal growth factor pathway substrate number 8 (Eps8) was identified as a transcript overexpressed (5.9-fold) in human pituitary tumors compared with normal pituitary by DNA microarrays. Eps8 mRNA up-regulation was confirmed by semiquantitative RT-PCR. Immunoblot analysis showed that Eps8 protein levels and its downstream target phosphorylated ERK were also up-regulated in human pituitary tumors. Stable overexpression of Eps8 in LbetaT2 gonadotrope pituitary cells augmented colony formation in soft agar at d 21. Eps8 cells proliferated more robustly compared with controls in growth factor replete as well as growth-restricted conditions. In addition, the Eps8 overexpressing cells were protected from serum withdrawal-induced apoptosis compared with controls as assessed by caspase-3 cleavage. Epidermal growth factor activated a robust amplification of ERK and modest up-regulation of Akt in Eps8-overexpressing pituitary cells compared with vector controls. MAPK kinase inhibition or silencing of Eps8 blunted the proliferation of the cells in response to growth factor stimulation. Blockade of the phosphatidylinositol 3-kinase pathway or silencing of Eps8 resulted in a loss of the Eps8 protection from growth factor withdrawal-induced apoptosis. Together these data support a role of Eps8 in amplifying growth factor receptor signaling in human pituitary tumors to promote proliferation and cell survival.