Functional characterization of a CDKN1B mutation in a Sardinian kindred with multiple endocrine neoplasia type 4 (MEN4)

Inactivating germline mutations of the CDKN1B gene, encoding for the nuclear cyclin-dependent kinase inhibitor p27kip1 protein, have been reported in patients with multiple endocrine neoplasia type 4 (MEN4), a MEN1-like phenotype without MEN1 mutations. The aim of this study was to in vitro characterize the germline CDKN1B mutation c.374_375delCT (S125X) we detected in a patient with MEN4. The proband was affected by multiglandular primary hyperparathyroidism and gastro-entero-pancreatic tumors. We carried out subcellular localization experiments transfecting into eukaryotic HeLa and GH3 cell lines plasmid vectors expressing the CDKN1B wild type (wt) or mutant cDNA. Western blot studies showed that fusion proteins were expressed at equal levels. The mutated protein was shorter compared to the wt protein and lacked the highly conserved C-terminal domain, which includes the bipartite nuclear localization signal at amino acids 152/153 and 166/168. In HeLa and GH3 cells wt p27 localized in the nucleus whereas the p27_S125X protein was retained in the cytoplasm predicting the loss of tumor suppressive function. The proband's tumoral parathyroid tissue did not show allelic loss, since wt and mutant alleles were both present by sequencing the somatic DNA. Immunohistochemistry showed a complete loss of nuclear p27 expression in the parathyroid adenoma removed by the patient at the second surgery. In conclusion, our study confirms the pathogenic role of the c.374_375delCT CDKN1B germline mutation in a patient with MEN4

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

Cyclin D1 levels are involved in the resistance to m-TOR inhibitors in human bronchial carcinoids

Background: Bronchial carcinoids (BC) are still orphan of medical therapy. We previously demonstrated that the typical BC human cell line NCI-H727 is sensitive to Everolimus, in terms of cell viability reduction, while the atypical human BC cell line NCI-H720 is not. However, the mechanisms underlying this phenomenon have not been fully clarified. Aim: The aim of our study is to investigate the mechanisms of resistance to mTOR inhibitors in BC cells. Methods: Cell cycle protein profiling was performed throughout G0/G1/S phases evaluating important complexes regulated during these transition phases at different cell-cycle times, such as CDK2/Cyclin E, CDK4/CyclinD1 and p27Kip1. Results: The two human BC cell lines, NCI-720 and NCI-727 cells, showed different levels of cell cycle-regulating proteins during cycle progression. We found that under starvation the resistant cell line (NCI-720 cells) still expressed most of the cell cycle regulating protein, while in the sensitive cell line (NCI-727 cells), proteins as p27, cyclin D1 and E were highly down regulated. In addition we observed that, during cell cycle progression, CyclinE/CDK2 complex seems to be more expressed in resistant NCI-H720 cells as compared to NCI-727 cells. In contrast, CyckinD1/CDK4 is more expressed in the sensitive NCI-H727 cell line, while p27 does not show a different expression pattern during cell cycle progression in the two cell lines. Conclusion: The pattern of proteins involved in cell cycle regulation is clearly different in the two cell lines, suggesting a possible involvement of these molecules in the mechanism of mTOR inhibitors resistance

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

Functional characterization of a new deletion in CDKN1B 5'-UTR region

Introduction: CDKN1B gene, which encodes a cyclin-dependent kinase (Cdk) inhibitor, regulates the progression throughout G1 to S cell cycle progression. CDKN1B loss-of-function germinal mutations cause the multiple endocrine neoplasia type 4 syndrome (MEN4). Objective: The aim of the study is the functional characterization of a new 4 bp deletion in CDKN1B 5′-UTR region, identified in an acromegalic patient. Materials and methods: We assessed a functional in vitro study, based on firefly luciferase reporter gene, on the deleted promoter of human (MCF-7), murine (AtT20/D16V-F2) and rat (GH3) cell lines. Total mRNA and proteins were extracted from peripheral blood of the patient and control subjects to analyze CDKN1B/p27Kip1 expression. In addition, we evaluated p27Kip1 expression in tissue sections of the patient’s GH-secreting pituitary adenoma by immunohistochemistry. Results: A novel heterozygous deletion in CDKN1B 5′-UTR region was identified in an acromegalic patient. The deletion extends from nucleotide −29 to −26 from the translation start site. Transcriptional activity of the deleted promoter is significantly decreased (~30–60% P<0.01). CDKN1B/p27Kip1 expression analysis of patient’s circulating leukocytes showed a significant reduction (~72%) in mRNA levels, while p27Kip1 protein levels are similar to WT controls. Immunohistochemistry shows a reduced p27Kip1 expression in the patient’s pituitary adenoma compared to the control. Conclusions: Our data show that the identified deletion causes a significant reduction in promoter transcriptional activity and in CDKN1B mRNA expression, indicating a putative role of the deletion in the patient’s disease. Further studies are needed order to understand whether the deletion could impact protein function

Protein Kinase C Delta restrains growth in ACTH-secreting pituitary adenoma cells

Protein Kinase C Delta (PRKCD) has been highlighted among disrupted pathways in corticotroph adenomas. PRKCD is expressed at low level in human corticotroph adenomas and controls cell cycle in vitro. Therefore, PRKCD may play an important role in the development/progression of corticotroph adenomas, warranting further studies to understand the role of PRKCD and related pathways in restraining pituitary cell growth. We evaluated PRKCD role in influencing cell behavior in terms of cell viability, hormone expression and protein expression profile, by silencing PRKCD in AtT-20/D16v-F2 cells. PRKCD silencing increases cell viability, enhances hormone expression and induces morphological changes associated with deregulation of adhesion molecules. PRKCD silencing is associated with an increase in Epithelial Growth Factor Receptor (EGFR) expression, a marker of tumor aggressive behavior, and sensitivity to anti-EGFR molecules. PRKCD might restrain corticotroph adenoma cells from acquiring an aggressive behavior, candidating PRKCD as a possible molecular target for the treatment of corticotroph adenomas

Inhibition of epithelial growth factor receptor can play an important role in reducing cell growth and survival in adrenocortical tumors

Medical treatment of adrenocortical carcinoma (ACC) is still far from optimal, since even molecular targeted therapy failed to demonstrate striking results. Clinical trials enrolling ACC patients with high tissue vascular endothelial growth factor receptor (VEGFR) expression levels showed controversial results after treatment with Sunitinib, possibly due to variability in the expression of drug targets, which include epidermal growth factor receptor (EGFR). To better clarify this issue, we evaluated whether VEGFR may play a crucial role in ACC responsiveness to Sunitinib and whether EGFR may represent an alternative target in ACC medical treatment, by employing two ACC cell lines, the NCI-H295 and SW13 cells lines, and adrenocortical tissues primary cultures. Our data show that VEGF/VEGFR system may not be crucial in modulating ACC proliferation and responsiveness to Sunitinib. In addition, by cell viability, proliferation and caspase activation assays we found that Sunitinib inhibits adrenocortical cell viability acting, at least in part, through EGFR, that, in turn, is crucial for EGF proliferative effect on adrenocortical cells. The latter depends, at least in part, on ERK 1/2 activation. An EGFR selective inhibitor was highly effective in reducing cell viability in an adrenocortical tumor primary culture and in the SW13 cells, which express high EGFR levels. Our results suggest that EGFR inhibitors could represent effective therapeutic tools in ACC patients whose tumors express high EGFR levels, that, in turn, may be considered a predictive factor of response. Accurate molecular tumor profiling is crucial to predict drug efficacy and to tailor ACC patients therapeutic approach

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

Abstract 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 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