BACKGROUND New therapies are required for castrate‐resistant prostate cancer (CRPC), and growth‐arrest specific 5 (GAS5) lncRNA, which riborepresses androgen receptor action, may offer novel opportunities in this regard. This lncRNA promotes the apoptosis of prostate cancer cells and its levels decline as prostate cancer cells acquire castrate‐resistance, so that enhancing GAS5 expression may improve the effectiveness of chemotherapies. Since GAS5 is a member of the 5' terminal oligopyrimidine gene family, we have examined mTOR inhibition as a strategy to increase GAS5 expression. Furthermore, we have determined if GAS5 itself mediates the action of mTOR inhibitors, as demonstrated for other chemotherapeutic agents in prostate cancer cells. METHODS The effects of mTOR inhibitors on GAS5 lncRNA levels and cell growth were determined in a range of prostate cancer cell lines. Transfection of cells with GAS5 siRNAs and plasmid constructs was performed to determine the involvement of GAS5 lncRNA in mTOR inhibitor action. RESULTS First generation mTORC1, combined mTORC1/mTORC2 and dual PI3K/mTOR inhibitors all increased cellular GAS5 levels and inhibited culture growth in androgen‐dependent (LNCaP) and androgen‐sensitive (22Rv1) cell lines, but not in androgen‐independent (PC‐3 and DU 145) cell lines. The latter exhibited low endogenous GAS5 expression, and GAS5 silencing in LNCaP and 22Rv1 cells decreased the sensitivity to mTOR inhibitors, whereas transfection of GAS5 lncRNA sensitized PC‐3 and DU 145 cells to these agents. CONCLUSION mTOR inhibition enhances GAS5 transcript levels in certain prostate cancer cell lines. This selectivity is likely to be related to endogenous GAS5 expression levels, since GAS5 lncRNA is itself required for mTOR inhibitor action in prostate cancer cells. Prostate 75:693–705, 2015. © 2015 Wiley Periodicals, Inc.

DNA methylation is one of the several epigenetic modifications that together with genetic aberrations are hallmarks of tumorigenesis including those emanating from the pituitary gland. In this study, we examined DNA methylation across 27 578 CpG sites spanning more than 14 000 genes in the major pituitary adenoma subtypes. Genome-wide changes were first determined in a discovery cohort comprising non-functioning (NF), growth hormone (GH), prolactin (PRL)-secreting and corticotroph (CT) adenoma relative to post-mortem pituitaries. Using stringent cut-off criteria, we validated increased methylation by pyrosequencing in 12 of 16 (75%) genes. Overall, these criteria identified 40 genes in NF, 21 in GH, six in PRL and two in CT that were differentially methylated relative to controls. In a larger independent cohort of adenomas, for genes in which hypermethylation had been validated, different frequencies of hypermethylation were apparent, where the KIAA1822 (HHIPL1) and TFAP2E genes were hypermethylated in 12 of 13 NF adenomas whereas the COL1A2 gene showed an increase in two of 13 adenomas. For genes showing differential methylation across and between adenoma subtypes, pyrosequencing confirmed these findings. In three of 12 genes investigated, an inverse relationship between methylation and transcript expression was observed where increased methylation of EML2, RHOD and HOXB1 is associated with significantly reduced transcript expression. This study provides the first genome-wide survey of adenoma, subtype-specific epigenomic changes and will prove useful for identification of biomarkers that perhaps predict or characterise growth patterns. The functional characterisation of identified genes will also provide insight of tumour aetiology and identification of new therapeutic targets.

Dopamine (DA)-agonist targeting of the DA D 2 receptor (D2R) in prolactinomas is the first-line treatment choice for suppression of prolactin and induction of tumor shrinkage. Resistance to DA agonists seems to be related to receptor number. Using the MMQ and GH3 pituitary cell lines, that either do or do not express D2R, respectively, we explored the epigenetic profile associated with the presence or absence of D2R in these cells lines. These studies led us to explore pharmacological strategies designed to restore receptor expression and thereby potentially augment DA agonistmediated apoptosis. We show in GH3 cells that the D2R harbors increased CpG island-associated methylation and enrichment for histone H3K27me3. Conversely, MMQ cells and normal pituitaries show enrichment for H3K9Ac and barely detectable H3K27me3. Coculture of GH3 cells with the demethylating agent zebularine and the histone deacetylase inhibitor trichostatin A was responsible for a decrease in CpG island methylation and enrichment for the histone H3K9Ac mark. In addition, challenge of GH3 cells with zebularine alone or coculture with both agents led to expression of endogenous D2R in these cells. Induced expression D2R in GH3 cells was associated with a significant increase in apoptosis indices to challenge with either DA or bromocriptine. Specificity of a receptor-mediated response was established in coincubations with specific D2R antagonist and siRNA approaches in GH3 cell and D2R expressing MMQ cell lines. These studies point to the potential efficacy of combined treatment with epigenetic drugs and DA agonists for the medical management of different pituitary tumor subtypes, resistant to conventional therapies. (Endocrinology 152: 364 -373, 2011) D opamine (DA) agonists are considered a first-line treatment choice for patients with pituitary prolactinomas where they not only effectively suppress prolactin (PRL) but also reduce tumor size (1, 2). Despite their success, a small proportion of patients are intolerant to DA agonist therapy (3, 4), and in cases where resistance is apparent this seems to be related to the number of DA D2 receptors (D2R) expressed by the adenoma (5). Activation of the D2R by DA and DA agonists leads to reduced cAMP production through interaction with Gi/Go proteins (6).The reduction in cAMP levels in normal and tumoral lactotrophs is considered integral to the inhibition of PRL synthesis and release (1, 6 -9).In contrast to our understanding of pathways regulating PRL release within the lactotroph our understanding of receptor(s) and their intracellular pathways responsible for tumor shrinkage and or apoptosis are less clear. Early studies suggested that resistance to DA but not bromocriptine (BC)-mediated apoptosis was a consequence of these cells not expressing a D2R (10). However, more recent

Bone morphogenetic protein (BMP)-4 is a key mediator of anterior pituitary organogenesis. However, through inappropriate expression patterns, BMP-4 is also pathogenic in a pituitary adenoma subtype-specific context. In these cases, increase or decrease in BMP-4 in lactotroph-and corticotroph-derived adenomas, respectively, is consistent with a bifunction role for this protein toward either promotion or inhibition of cell proliferation and hormone secretion. To gain insight into the aberrations responsible for differential expression, we examined BMP-4 transcript and protein expression patterns in the major adenomas subtypes. BMP-4 transcript and protein are differentially expressed and show increase in the majority of prolactinomas relative to normal pituitary, whereas the majority of other adenoma subtypes show reduced expression relative to both prolactinoma and normal pituitaries. Reduced expression of BMP-4 is not associated with change in CpG island methylation status. However, histone tail modifications are apparent, as enrichment for a modification associated with silent genes, H3K27me3, and depletion of a modification associated with active genes, H3K9Ac. In pituitary cell lines, reduced BMP-4 expression is also associated with similar histone tail modifications and contemporaneous increase in CpG island methylation. In these cells, coincubation with the demethylating agent zebularine and histone deacetylase inhibitor, trichostatin A, reversed epigenetic changes and restored expression of BMP-4. These studies show that, in contrast to prolactinomas, other adenoma subtypes show reduced expression of BMP-4 where epidrug induced reexpression, alone or in combination with conventional therapies, may offer new treatment strategies. (Endocrinology 153: 3603-3612, 2012)

Global and gene-specific changes in the epigenome are hallmarks of most tumour types, including those of pituitary origin. In contrast to genetic mutations, epigenetic changes (aberrant DNA methylation and histone modifications) are potentially reversible. Drugs that specifically target or inhibit DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) can be used to restore the expression of epigenetically silenced genes. These drugs can potentially increase the sensitivity of tumour cells to conventional treatment modalities, such as chemotherapy and radiotherapy. Drug-induced reversal of transcriptional silencing can also be used to restore dopamine-D(2)-receptor-negative, hormone-refractory tumours to their previous receptor-positive, hormone-responsive status. Synergy between HDAC and DNMT inhibitors makes these pharmacological agents more therapeutically effective when administered in combination than when used alone. Studies in pituitary tumour cell lines show that drug-induced re-expression of the epigenetically silenced dopamine D(2) receptor leads to an increase in apoptosis mediated by a receptor agonist. Collectively, the use of drugs to directly or indirectly reverse gene-specific epigenetic changes, in combination with conventional therapeutic interventions, has potential for the clinical management of multiple tumour types-including those of pituitary origin. Furthermore, these drugs can be used to identify epigenetically regulated genes that could be novel, tumour-specific therapeutic targets.