Erica Gentilin

Everolimus as a new potential antiproliferative agent in aggressive human bronchial carcinoids.

Bronchial carcinoids (BCs) are rare tumors originating from endocrine cells dispersed in the respiratory epithelium. It has been previously demonstrated that everolimus, or RAD001, an mTOR inhibitor, has potent antiproliferative effects in human endocrine tumors. Our aim was to evaluate the possible antiproliferative effects of everolimus in human BCs in primary culture. We collected 24 BCs that were dispersed in primary cultures, treated without or with 1 nM-1 muM everolimus, 10 nM SOM230 (pasireotide, a somatostatin receptor multiligand), and/or 50 nM IGF1. Cell viability was evaluated after 48 h, and chromogranin A (CgA) as well as vascular endothelial growth factor (VEGF) secretion was assessed after 8 h incubation. Somatostatin receptors, mTOR, and AKT expression were investigated by quantitative PCR. We found that in 15 cultures (67.5%), everolimus significantly reduced cell viability (by approximately 30%; P<0.05 versus control), inhibited p70S6K activity (-30%), and blocked IGF1 proliferative effects. Everolimus also significantly reduced CgA (by approximately 20%) and VEGF (by approximately 15%) secretion. Cotreatment with SOM230 did not exert additive effects on cell viability and secretory activity. AKT expression was similar in responder and nonresponder tissues, while mTOR expression was significantly higher in the responder group, which was characterized by higher CgA plasma levels and bigger tumors with higher mitotic index and angiogenesis. Our data demonstrate that everolimus reduces VEGF secretion and cell viability in BCs with a mechanism likely involving IGF1 signaling, suggesting that it might represent a possible medical treatment for BCs.

miR-26a plays an important role in cell cycle regulation in ACTH-secreting pituitary adenomas by modulating protein kinase Cδ.

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 Cushings disease.

Therapeutic Concentrations of Mitotane (o,p′-DDD) Inhibit Thyrotroph Cell Viability and TSH Expression and Secretion in a Mouse Cell Line Model

Mitotane therapy is associated with many side effects, including thyroid function perturbations mimicking central hypothyroidism, possibly due to laboratory test interference or pituitary direct effects of mitotane. We investigated whether increasing concentrations of mitotane in the therapeutic range might interfere with thyroid hormone assays and evaluated the effects of mitotane on a mouse TSH-producing pituitary cell line. TSH, free T(4), and free T(3) levels do not significantly change in sera from hypo-, hyper-, or euthyroid patients after addition of mitotane at concentrations in the therapeutic window. In the mouse TalphaT1 cell line, mitotane inhibits both TSH expression and secretion, blocks TSH response to TRH, and reduces cell viability, inducing apoptosis at concentrations in the therapeutic window. TRH is not capable of rescuing TalphaT1 cells from the inhibitory effects of mitotane on TSH expression and secretion, which appear after short time treatment and persist over time. Our results demonstrate that mitotane does not interfere with thyroid hormone laboratory tests but directly reduces both secretory activity and cell viability on pituitary TSH-secreting mouse cells. These data represent a possible explanation of the biochemical picture consistent with central hypothyroidism in patients undergoing mitotane therapy and open new perspectives on the direct pituitary effects of this drug.

Magmas, a gene newly identified as overexpressed in human and mouse ACTH-secreting pituitary adenomas, protects pituitary cells from apoptotic stimuli.

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.

Growth hormone receptor blockade inhibits growth hormone-induced chemoresistance by restoring cytotoxic-induced apoptosis in breast cancer cells independently of estrogen receptor expression.

CONTEXT GH and IGF-I play a role in breast cancer (BC) development. We previously demonstrated that GH protects the estrogen receptor (ER) positive BC-derived MCF7 cell line toward the cytotoxic effects of doxorubicin (D), independently of IGF-I. This issue may be important in ER negative BC cells that are more aggressive and more likely to develop chemoresistance. AIM OF THE STUDY The aim of this study was to evaluate whether GH may impact chemoresistance phenotype of ER-negative BC-derived MDA-MB-231 cell line and investigate the possible mechanisms implicated in the protective action of GH toward the cytotoxic effects of D in both ER-positive and ER-negative BC-derived cell lines. RESULTS GH protects ER-negative MDA-MB-231 cells from the cytotoxic effects of D and GH receptor antagonist pegvisomant reduces GH-induced DNA synthesis also in these cells. In both MDA-MB-231 and MCF7 cells, GH does not revert D-induced G2/M accumulation but significantly reduces basal and D-induced apoptosis, an effect blocked by pegvisomant. Glutathione S-transferase activity is not implicated in the protective effects of GH, whereas D-induced apoptosis depends on c-Jun N terminal kinase (JNK) activation. GH reduces both basal and D-stimulated JNK transcriptional activity and phosphorylation. CONCLUSIONS 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.

Mitotane enhances doxorubicin cytotoxic activity by inhibiting P-gp in human adrenocortical carcinoma cells

Mitotane is currently employed as adjuvant therapy as well as in the medical treatment of adrenocortical carcinoma (ACC), alone or in combination with chemotherapeutic agents. It was previously demonstrated that mitotane potentiates chemotherapeutic drugs cytotoxicity in cancer cells displaying chemoresistance due to P-glycoprotein (P-gp), an efflux pump involved in cancer multidrug resistance. The majority of ACC expresses high levels of P-gp and is highly chemoresistent. The aim of our study was to explore in vitro whether mitotane, at concentrations lower than those currently reached in vivo, may sensitize ACC cells to the cytotoxic effects of doxorubicin and whether this effect is due to a direct action on P-gp. NCI-H295 and SW13 cell lines as well as 4 adrenocortical neoplasia primary cultures were treated with mitotane and doxorubicin, and cell viability was measured by MTT assay. P-gp activity was measured by calcein and P-gp-Glo assays. P-gp expression was evaluated by Western blot. We found that very low mitotane concentrations sensitize ACC cells to the cytotoxic effects of doxorubicin, depending on P-gp expression. In addition, mitotane directly inhibits P-gp detoxifying function, allowing doxorubicin cytotoxic activity. These data provide the basis for the greater efficacy of combination therapy (mitotane plus chemotherapeutic drugs) on ACC patients. Shedding light on mitotane mechanisms of action could result in an improved design of drug therapy for patients with ACC.

Mitotane reduces human and mouse ACTH-secreting pituitary cell viability and function

Medical therapy for Cushings disease (CD) is currently based on agents mainly targeting adrenocortical function. Lately, pituitary-directed drugs have been developed, with limited efficacy. Mitotane, a potent adrenolytic drug, has been recently investigated for the treatment of CD, but the direct pituitary effects have not been clarified so far. The aim of our study was to investigate whether mitotane may affect corticotroph function and cell survival in the mouse pituitary cell line AtT20/D16v-F2 and in the primary cultures of human ACTH-secreting pituitary adenomas, as an in vitro model of pituitary corticotrophs. We found that in the AtT20/D16v-F2 cell line and in primary cultures, mitotane reduces cell viability by inducing caspase-mediated apoptosis and reduces ACTH secretion. In the AtT20/D16v-F2 cell line, mitotane reduces Pomc expression and blocks the stimulatory effects of corticotropin-releasing hormone on cell viability, ACTH secretion, and Pomc expression. These effects were apparent at mitotane doses greater than those usually necessary for reducing cortisol secretion in Cushings syndrome, but still in the therapeutic window for adrenocortical carcinoma treatment. In conclusion, our results demonstrate that mitotane affects cell viability and function of human and mouse ACTH-secreting pituitary adenoma cells. These data indicate that mitotane could have direct pituitary effects on corticotroph cells.

Protein Kinase C: A Putative New Target for the Control of Human Medullary Thyroid Carcinoma Cell Proliferation in Vitro

We investigate the role of protein kinase C (PKC) in the control of medullary thyroid carcinoma (MTC) cell proliferation by a PKC inhibitor, Enzastaurin, in human MTC primary cultures and in the TT cell line. We found that PKC inhibition reduces cell proliferation by inducing caspase-mediated apoptosis and blocks the stimulatory effect of IGF-I on calcitonin secretion. Enzastaurin reduces PKCβII (Thr500) phosphorylation, indicating a direct involvement of this isoform as well as the phosphorylated levels of Akt (Ser 473) and glycogen synthase kinase (Ser9), PKC pathway downstream targets and pharmacodynamic markers for PKC inhibition. PKCβII and PKCδ enzyme isoforms expression and localization were investigated. These data indicate that in vitro PKC is involved in the control of human MTC proliferation and survival by modulating apoptosis, with a mechanism that implicates PKCβII inhibition and translocation in different subcellular compartments. Targeting PKC may represent a useful therapeutic approach for controlling MTC proliferation.

Targeting protein kinase C by Enzastaurin restrains proliferation and secretion in human pancreatic endocrine tumors

Dysregulation of the protein kinase C (PKC) signaling pathway has been implicated in tumor progression. In this study, we investigate the effects of a PKC inhibitor, Enzastaurin, in human pancreatic neuroendocrine neoplasms (PNN) primary cultures and in the human pancreatic endocrine cancer cell line, BON1. To this aim six human PNN dispersed in primary cultures and BON1 cells were treated without or with 1-10 μM Enzastaurin and/or 100 nM IGF1 in the presence or absence of serum. Cell viability and apoptosis were evaluated after 48-72 h; Chromogranin A (CgA) and/or insulin secretion was assessed after 6 h of incubation. PKC expression was investigated by immunofluorescence and western blot. We found that Enzastaurin significantly reduced human PNN primary culture cell viability, as well as CgA and insulin secretion. Moreover, in the BON1 cell line Enzastaurin inhibited cell proliferation at 5 and 10 μM by inducing caspase-mediated apoptosis, and reduced phosphorylation of glycogen synthetase kinase 3β (GSK3β) and of Akt, both downstream targets of PKC pathway and pharmacodynamic markers for Enzastaurin. In addition, Enzastaurin blocked the stimulatory effect of IGF1 on cell proliferation, and reduced CgA expression and secretion in BON1 cells. Two different PKC isoforms are expressed at different levels and have partially different subcellular localization in BON1 cells. In conclusion, Enzastaurin reduces cell proliferation by inducing apoptosis, with a mechanism likely involving GSK3β signaling, and inhibits secretory activity in PNN in vitro models, suggesting that Enzastaurin might represent a possible medical treatment of human PNN.

The expression of the truncated isoform of somatostatin receptor subtype 5 associates with aggressiveness in medullary thyroid carcinoma cells

The truncated somatostatin receptor variant sst5TMD4 associates with increased invasiveness and aggressiveness in breast cancer. We previously found that sst5 activation may counteract sst2 selective agonist effects in a medullary thyroid carcinoma (MTC) cell line, the TT cells, and that sst5TMD4 is overexpressed in poorly differentiated thyroid cancers. The purpose of this study is to evaluate sst5TMD4 expression in a series of human MTC and to explore the functional role of sst5TMD4 in TT cells. We evaluated sst5TMD4 and sst5 expression in 36 MTC samples. Moreover, we investigated the role of sst5TMD4 in TT cells evaluating cell number, DNA synthesis, free cytosolic calcium concentration ([Ca2+]i), calcitonin and vascular endothelial growth factor levels, cell morphology, protein expression, and invasion. We found that in MTC the balance between sst5TMD4 and sst5 expression influences disease stage. sst5TMD4 overexpression in TT cells confers a greater growth capacity, blocks sst2 agonist-induced antiproliferative effects, modifies the cell phenotype, decreases E-cadherin and phosphorylated β-catenin levels, increases vimentin, total β-catenin and phosphorylated GSK3B levels (in keeping with the development of epithelial to mesenchymal transition), and confers a greater invasion capacity. This is the first evidence indicating that sst5TMD4 is expressed in human MTC cells, where it associates with more aggressive behavior, suggesting that sst5TMD4 might play a functionally relevant role.