Effects of two mTOR inhibitors on in-vitro models of GH-secreting pituitary adenomas

Background: Gigantism and acromegaly are the main consequences of GH excess, mainly due to a pituitary adenoma. Surgery is the first therapeutic option, but also medical therapy is employed, being represented mostly by somatostatin analogues (SSA), that reduce both tumour mass and GH hypersecretion. However about 10% of patients is resistant to SSA. PI3K/Akt/mTOR pathway, activated by growth-factors such as IGF1, is important in regulating many cellular processes (viability, apoptosis and cell cycle), and it is deregulated in several neoplasms including GH-secreting pituitary tumours. Aim of the study: To understand how PI3K/Akt/mTOR pathway can influence viability and GH secretion in pituitary adenomas, we employed two inhibitors (Everolimus, mTOR inhibitor, and NVP-BEZ235, mTOR and PI3K inhibitor), evaluating their effects in presence or in absence of IGF1. Material and methods: We employed two GH-secreting pituitary adenoma rat cell lines and we assessed cell viability. Culture medium was collected to evaluate GH secretion by ELISA. Results: Both compounds (10–500 nM) caused a significant reduction in cell viability up to 60% in the two cell lines. On the other hand, IGF-1 induced cell viability by 60% as compared to control cells in GH3 cells and by 40% in GH4C1 cells. This effect was efficiently counteracted in both lines by Everolimus and NVP-BEZ235, indicating that these compounds could act, at least in part, on IGF-1 activated pathways. GH secretion was reduced by IGF1 in both cell lines; this effect was enhanced by Everolimus, but not by NVP-BEZ235 in both cell lines. Conclusions: Our results suggest that both compounds interfere with IGF1 signalling in GH-secreting rat cell lines, that could be used as a model to identify alternative pharmacological targets for GH-secreting pituitary adenomas resistant to SSA therapy

ctivation of EGFR promotes ACC cell proliferation by inducing VEGF autocrine secretion

Adrenocortical cancer (ACC) is a rare and aggressive malignancy. Currently the main therapeutic option is surgery, but due to difficult and delayed diagnosis and to the onset of metastases, medical therapy is often tried. ACC treatment is mainly represented by Mitotane alone or in association with chemotherapy, with variable results. Understanding the molecular mechanisms that regulate ACC proliferation could be useful to identify new therapeutic options. Aim of our study is to identify growth factors that may regulate ACC proliferation, using two human ACC cell lines, the SW13 and the NCI-H295 cells. Our data show that epidermal growth factor (EGF) and transforming growth factor (TGF)-α enhance SW13 cell proliferation and reduced apoptosis, while had modest effects on NCI-H295 cells. Sunitinib, an EGF receptor (EGFR) inhibitor, and NVP-BEZ235, a PI3K/mTOR inhibitor, reduced cell viability in both cell lines, being counteracted by both EGF and TGF-α in SW13 cells. Since in other settings EGF regulates cell proliferation by inducing VEGF, we investigated VEGF secretion by the two cells lines. EGF and TGF-α enhanced VEGF secretion only in SW13 cells while had no effects on NCI-H295. In addition, a VEGF receptor blocking antibody significantly reduced EGF and TGF-α induced cell proliferation. We investigated in both cell lines the expression of EGFR, which is higher and ubiquitous in SW13 cells, while it is weaker and sparse in NCI-H295 cells, where it is present only on the membrane. These data demonstrate that EGF and TGF-α are important in regulating Sw13 cell proliferation, also by modulating VEGF secretion. In conclusion our data suggest that EGF pathway could represent a new molecular target in drug design for treatment of ACC that display enhanced EGFR expression

Epidermal growth factor pathway as a possible target in the medical therapy of bronchial carcinoids

Bronchial carcinoids (BC) are rare tumors originating from endocrine cells dispersed in the respiratory epithelium. Currently, the main BC treatment is surgery, that can be curative in most of the cases, but is not feasible for large, infiltrating and metastatic disease. In these settings, medical therapy is often tried, being mainly represented by chemotherapy and radiation in the attempt to reduce tumor mass, while somatostatin analogues are employed for symptomatic control. Therefore it is important to identify new therapeutic targets and new molecules capable of providing adequate medical treatment for patients with BC for which surgical removal is not feasible. Growth factors which are important in experimental models of neuroendocrine tumors include epidermal growth factor (EGF), transforming growth factor (TGF) α, TGFβ. EGF and TGFα bind to the EGF receptor to stimulate the PI3K/RAS/RAF/MAPK pathway, leading to the transcription of genes associated with cell proliferation, invasion and metastasis. Our aim is to evaluate the effects of Sunitinib, a multi-targeted receptor tyrosine kinase (RTK) inhibitor, and NVP-BEZ235, a PI3K/mTOR inhibitor, on human primary BC cells cultures in order to verify the involvement of the EGF pathway in regulating crucial cellular processes. Human BC primary cultures were treated with Sunitinib or NVP-BEZ235, alone or in combination with EGF. EGFR expression, cell viability and caspase 3/7 activation were evaluated. By immunofluorescences we found that EGFR is expressed in all primary cultures. In addition, 100 nM NVP-BEZ235 and 10 μM Sunitinib inhibit cell viability by 30 and 20% (P<0.01), respectively. Both NVP-BEZ 235 and Sunitinib promote apoptosis (100%). 100 ng/ml EGF impairs the antiproliferative and pro-apoptotic effects of both Sunitinib and NVP-BEZ 235. These data suggest a possible role for EGFR pathway as molecular target in the medical treatment of BC. Further studies are necessary to understand the molecular basis of this mechanism

The cytotoxic effect of sunitinib on human bronchial carcinoid cell lines and primary cultures is counteracted by EGF and IGF-1 but not by VEGF

Background: Bronchial carcinoids (BC) are rare tumors originating from endocrine cells dispersed in the respiratory epithelium. The main BC treatment is surgery, which is not feasible for large, infiltrating and metastatic disease. In these settings, medical therapy is often tried. Therefore it is important to identify new therapeutic targets and new molecules capable of providing adequate medical treatment for patients with BC. Sunitinib, is an oral, small-molecule, multi-targeted receptor tyrosine kinase inhibitor (TKI). Aim: To verify if sunitinib is active in inhibiting cell viability of human BC and what are its targets in these cells. Methods: Human BC cell lines (NCH-727 and NCI-H727 cells) and human BC primary cultures were treated with sunitinib 5 μM and/or EGF 30 nM, IGF1 50 nM, or VEGF 10 nM. Cell viability and caspase 3/7 activation were measured after 48 h of treatment. Results: Sunitinib is capable of inhibiting cell viability of BC cell lines and primary cultures (by 20–50% vs control); moreover sunitinib activates caspase 3/7 (by 20–100% vs control). Both events are counteracted by EGF and IGF-1 at concentrations similar to those found in plasma, but not by VEGF despite its receptors are usually considered the main target of sunitinib. Conclusion: These data indicate that sunitinib is a potential therapeutic agent for treatment of BC, and that its mechanism of action could be mediated, at least in part, by EGFR and IGF1R. Further experiments are needed to deeply understand this issue

VEGF autocrine secretion is enhanced by EGFR activation trough ERK1/2 phosphorylation in human adrenocortical carcinoma cell lines

Adrenocortical cancer (ACC) is still orphan of medical treatment. Our preliminary data show that EGF induces ACC cell lines proliferation (+20 and +10% vs control in SW13 and NCI-H295 cell lines respectively). EGF receptor (EGFR) expression is higher and ubiquitous in SW13 cells, while it is weaker in NCI-H295 cells, where it is present only on the membrane. Aim of our study is to analyze EGFR downstream signalling in ACC cell lines. EGF induces VEGF synthesis, therefore we investigated EGF-induced VEGF secretion in ACC cells. EGF enhanced VEGF secretion only in SW13 cells while had weak effects on NCI-H295. In addition, a VEGF receptor (VEGFR) blocking antibody significantly reduced EGF effects on SW13 cells proliferation, while it had negligible effects on NCI-H295 cells. VEGF synthesis and secretion is controlled by PKCα, PKC β2 and ERK1/2, that are involved in EGFR downstream signalling. PKCα and PKC β2 were not modulated by treatment with EGF or with Sunitinib (an EGFR inhibitor), nor by the combination of EGF and Sunitinib. ERK1/2 phosphorylation was strongly enhanced by EGF, an effect slightly counteracted by Sunitinib. These effects were more evident in SW13 as compared to NCI-H295 cells. These data demonstrate a crosstalk between EGF and VEGF signalling pathways that is, at least in part, mediated by ERK 1/2, and could indicate novel molecular targets possibly useful in the future design of ACC medical therapy. Further studies are needed to deeply understand these pathways in ACC

mTOR, AKT, p70S6K and ERK1/2 levels predict sensitivity to mTOR and PI3K/mTOR inhibitors in human bronchial carcinoids

Background: Bronchial carcinoids (BCs) are rare neuroendocrine tumors that are still orphan 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. Aim: To assess whether the novel dual PI3K/mTOR inhibitor, NVP-BEZ235, may be effective in everolimus-resistant human BC tissues and cell lines. In addition, we search for possible markers of mTOR inhibitors efficacy, that may help in identifying the patients that may benefit from mTOR inhibitors treatment, sparing them from ineffective therapy. Results: 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 by-pass cyclin D1 down-regulation and escape G0/G1 blockade. Rebound AKT activation was not observed in response to treatment with either mTOR inhibitor in ‘resistant’ BC cells. We also show that, in addition to total mTOR levels, putative markers of BC sensitivity to mTOR inhibitors are represented by higher AKT, p70S6K and ERK1/2 protein levels. Conclusion: These data indicate that the dual PI3K/mTOR inhibitor NVP-BEZ235 is more potent than everolimus in reducing human BC cell proliferation. ‘Resistant’ cells display lower levels of mTOR, AKT, p70S6K and ERK1/2, indicating that these proteins may be useful as predictive markers of resistance to mTOR and PI3K/mTOR inhibitors in human BC

Early onset acromegaly associated with a novel deletion in CDKN1B 5′UTR region

Genetic alterations frequently are involved in the development of a pituitary adenoma in young age. We here characterize the functional role of a deletion in CDKN1B 5'-UTR region (c.-29_-26delAGAG) identified in an acromegalic patient that developed a growth hormone in pituitary adenoma during childhood. Our results show that the identified novel heterozygous deletion in the CDKN1B 5'-UTR region associates with a reduction in CDKN1B mRNA levels, a predicted altered secondary mRNA structure, and a reduced CDKN1B 5'-UTR transcriptional activity in vitro. The patient displayed loss of heterozygosity in the same CDKN1B 5'-UTR region at tissue level and the 5'UTR region containing the deleted sequence encompasses a GRE. These findings indicate that the identification of functional alterations of newly discovered genetic derangements need to be fully characterized and always correlated with the clinical manifestations