Pituitary tumors are mostly benign, being locally invasive in 5-35% of cases. Deregulation of several genes has been suggested as a possible alteration underlying the development and progression of pituitary tumors. We here report the identification of a cDNA, corresponding to Magmas gene (mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction), which is highly expressed in two different ACTH-secreting mouse pituitary adenoma cell lines as compared with normal pituitary as well as in two thirds of 64 examined pituitary adenomas as compared with human normal pituitary. Tim 16, the mitochondrial protein encoded by Magmas, was indeed expressed in a mouse ACTH-secreting pituitary adenoma cell line, AtT-20 D16v-F2 cells, in a subcellular compartment likely corresponding to mitochondria. Magmas silencing determined a reduced rate of DNA synthesis, an accumulation in G1 phase, and a concomitant decrease in S phase in At-T20 D16v-F2 cells. Moreover, Magmas-silenced cells displayed basal caspase 3/7 activity and DNA fragmentation levels similar to control cells, which both increased under proapoptotic stimuli. Our data demonstrate that Magmas is overexpressed in mouse and human ACTH-secreting pituitary adenomas. Moreover, our results show that Magmas protects pituitary cells from apoptosis, suggesting its possible involvement in neoplastic transformation.

The functional aftermath of microRNA (miRNA) dysregulation in ACTH-secreting pituitary adenomas has not been demonstrated. miRNAs represent diagnostic and prognostic biomarkers as well as putative therapeutic targets; their investigation may shed light on the mechanisms that underpin pituitary adenoma development and progression. Drugs interacting with such pathways may help in achieving disease control also in the settings of ACTH-secreting pituitary adenomas. We investigated the expression of 10 miRNAs among those that were found as most dysregulated in human pituitary adenoma tissues in the settings of a murine ACTH-secreting pituitary adenoma cell line, AtT20/D16v-F2. The selected miRNAs to be submitted to further investigation in AtT20/D16v-F2 cells represent an expression panel including 5 up-regulated and 5 down-regulated miRNAs. Among these, we selected the most dysregulated mouse miRNA and searched for miRNA targets and their biological function. We found that AtT20/D16v-F2 cells have a specific miRNA expression profile and that miR-26a is the most dysregulated miRNA. The latter is overexpressed in human pituitary adenomas and can control viable cell number in the in vitro model without involving caspase 3/7-mediated apoptosis. We demonstrated that protein kinase Cδ (PRKCD) is a direct target of miR-26a and that miR26a inhibition delays the cell cycle in G1 phase. This effect involves down-regulation of cyclin E and cyclin A expression via PRKCD modulation. miR-26a and related pathways, such as PRKCD, play an important role in cell cycle control of ACTH pituitary cells, opening new therapeutic possibilities for the treatment of persistent/recurrent Cushing's disease.

Bronchial carcinoids (BCs) are rare neuroendocrine tumors that are still orphans of medical treatment. Human BC primary cultures may display resistance to everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), in terms of cell viability reduction. Our aim was to assess whether the novel dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor NVP-BEZ235 is effective in everolimus-resistant human BC tissues and cell lines. In addition, we searched for possible markers of the efficacy of mTOR inhibitors that may help in identifying the patients who may benefit from treatment with mTOR inhibitors, sparing them from ineffective therapy. We found that NVP-BEZ235 is twice as potent as everolimus in reducing cell viability and activating apoptosis in human BC tissues that display sensitivity to mTOR inhibitors, but is not effective in everolimus-resistant BC tissues and cell lines that bypass cyclin D1 downregulation and escape G0/G1 blockade. Rebound AKT activation was not observed in response to treatment with either mTOR inhibitor in the 'resistant' BC cells. In addition to total mTOR levels, putative markers of the sensitivity of BCs to mTOR inhibitors are represented by AKT, p70S6K (RPS6KB2), and ERK1/2 (MAPK3/1) protein levels. Finally, we validated these markers in an independent BC group. These data indicate that the dual PI3K/mTOR inhibitor NVP-BEZ235 is more potent than everolimus in reducing the proliferation of human BC cells. 'Resistant' cells display lower levels of mTOR, p70S6K, AKT, and ERK1/2, indicating that these proteins may be useful as predictive markers of resistance to mTOR and PI3K/mTOR inhibitors in human BCs.

In human BC cell lines, GH directly promotes resistance to apoptosis induced by chemotherapeutic drugs independently of ER expression by modulating JNK, further broadening the concept that GH excess may hamper cytotoxic BC treatment. These findings support the hypothesis that blocking GH receptor may be viewed as a potential new therapeutic approach to overcome chemoresistance, especially in ER-negative BC.

Magmas is a nuclear gene that encodes for the mitochondrial import inner membrane translocase subunit Tim16. Magmas is overexpressed in the majority of human pituitary adenomas and in a mouse ACTH-secreting pituitary adenoma cell line. Here we report that Magmas is highly expressed in two out of four rat pituitary adenoma cell lines and its expression levels inversely correlate to the extent of cellular response to staurosporine in terms of apoptosis activation and cell viability. Magmas over-expression in rat GH/PRL-secreting pituitary adenoma GH4C1 cells leads to an increase in cell viability and to a reduction in staurosporine-induced apoptosis and DNA fragmentation, in parallel with the increase in Magmas protein expression. These results indicate that Magmas plays a pivotal role in response to pro-apoptotic stimuli and confirm and extend the finding that Magmas protects pituitary cells from staurosporine-induced apoptosis, suggesting its possible involvement in pituitary adenoma development.